AU686291B2

AU686291B2 – Promoter
– Google Patents

AU686291B2 – Promoter
– Google Patents
Promoter

Download PDF
Info

Publication number
AU686291B2

AU686291B2
AU12506/95A
AU1250695A
AU686291B2
AU 686291 B2
AU686291 B2
AU 686291B2
AU 12506/95 A
AU12506/95 A
AU 12506/95A
AU 1250695 A
AU1250695 A
AU 1250695A
AU 686291 B2
AU686291 B2
AU 686291B2
Authority
AU
Australia
Prior art keywords
promoter
ltp2
aleurone
goi
gene
Prior art date
1993-12-02
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Ceased

Application number
AU12506/95A
Other versions

AU1250695A
(en

Inventor
Roger Kalla
Odd-Arne Olsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

ODD ARNE OLSEN

Original Assignee
ODD ARNE OLSEN
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1993-12-02
Filing date
1994-11-24
Publication date
1998-02-05

1994-11-24
Application filed by ODD ARNE OLSEN
filed
Critical
ODD ARNE OLSEN

1995-06-19
Publication of AU1250695A
publication
Critical
patent/AU1250695A/en

1998-02-05
Application granted
granted
Critical

1998-02-05
Publication of AU686291B2
publication
Critical
patent/AU686291B2/en

2014-11-24
Anticipated expiration
legal-status
Critical

Status
Ceased
legal-status
Critical
Current

Links

Espacenet

Global Dossier

Discuss

Classifications

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

C12N15/09—Recombinant DNA-technology

C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation

C12N15/823—Reproductive tissue-specific promoters

C12N15/8234—Seed-specific, e.g. embryo, endosperm

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Abstract

The in vivo expression in the aleurone cells of a cereal of a conjugate is described. The conjugate comprises a GOI (gene of interest) and a particular Ltp (lipid transfer protein) promoter-namely the Ltp2 gene promoter. The conjugate is stably integrated within the cereal’s genomic DNA.

Description

WO 95/15389 PCTINO94/00190 1
PROMOTER
The present invention relates to a promoter and to a conjugate comprising the same. The present invention also relates to the use of the promoter for stage- and tissue- specific expression of a gene of interest (GOI). The present invention also relates to the genomic nucleotide sequence of, and isolation of, the promoter.
In particular the present invention relates to a promoter for a lipid transfer protein (Ltp) gene known as the Ltp2 gene. The present invention also relates to the application of this Ltp2 gene promoter to express a GOI specifically in the aleurone layer of a monocotyledon especially a transgenic cereal seed more especially a developing transgenic cereal seed.
A mature cereal seed contains two distinct organs: the embryo which gives rise to the vegetative plant and the endosperm which supports the growth of the emerging seedling during a short period of time after germination. The endosperm, which is the site of deposition of different storage products such as starch and proteins, is further sub-divisible into a peripheral layer of living aleurone cells surrounding a central mass of non-living starchy endosperm cells.
The aleurone cells differentiate from primary endosperm cells early during seed development or between 10 to 21 days after fertilization. The aleurone layer and embryo share many similarities in their gene expression programmes. They are the only cereal seed tissues that survive the desiccation process during seed maturation and they both have active gene transcription during seed germination.
The aleurone layer of cereal seeds comprises specialized cells that surround the central starchy endosperm, i.e. the site for starch and protein accumulation in the developing seed (Bosnes et al., 1992, Olsen et al., 1992). During seed germination, the cells of the aleurone layer produce amylolytic and proteolytic enzymes that degrade the storage compounds into metabolites that are taken up and are used by the growing embryo. Two aspects of aleurone cell biology that have been intensively studied are the genetics of anthocyanin pigmentation of aleurone cells in maize (McClintock, 1987) and the hormonal regulation of gene transcription in the aleurone layer of germinating barley seeds (Fincher, 1989).
WO 95/15389 PCT/NO94/00190 2 Using transposon tagging, several structural and regulatory genes in the anthocyanin synthesis pathway have been isolated and characterized (Paz-Ares et al., 1987; Dellaporta et al., 1988). In barley, alpha-amylase and beta-glucanase genes that are expressed both in the aleurone layer and embryos of mature germinating seeds have been identified (Karrer et al., 1991; Slakeski and Fincher, 1992). In addition, two other cDNAs representing transcripts that are differentially expressed in the aleurone layers of developing barley grains have been isolated. These are CHI26 (Lea et al.,1991) and pZE40 (Smith et al., 1992). For none of these gene products has it been shown in transgenic cereal plants that the promoter directs expression in just the aleurone layer of developing grains.
Non-specific lipid transfer proteins (nsLtp’s) have the ability to mediate in vitro transfer of radiolabelled phospholipids from liposomal donor membranes to mitochondrial acceptor membranes (Kader et al., 1984; Watanabe and Yamada, 1986). Although their in vivo function remains unclear, nsLTPs from plants have recently received much attention due to their recurrent isolation as cDNA clones representing developmentally regulated transcripts expressed in several different tissues. A common feature is that, at some point in development, they are highly expressed in tissues producing an extracellular layer rich in lipids. Thus, transcripts corresponding to cDNAs encoding 10 kDa nsLTPs have been characterized in the tapetum cells of anthers as well as the epidermal layers of leafs and shoots in tobacco (Koltunow et al., 1990; Fleming et al., 1992), and barley aleurone layers (Mundy and Rogers, 1986; Jakobsen et al., 1989).
In addition, a 10 kDa nsLTP was discovered to be one of the proteins secreted from auxintreated somatic carrot embryos into the tissue culture medium (Sterk et al., 1991). Based on in situ data demonstrating that the Ltp transcripts are localized in the protoderm cells of the somatic and zygotic carrot embryo and in the epithelial layer of the maize embryonic scutellum, it was suggested that in vivo nsLTPs are involved in either cutin biosynthesis or in the biogenesis and degradation of storage lipids (Sossountzov et al., 1991; Sterk et al., 1991).
A nsLTP in Arabidopsis has been localized to the cell walls lending further support to an extracellular function if this class of proteins (Thoma et al., 1993).
WO 95/15389 PCT/NO94/00190 3 PCT WO 90/01551 mentions the use of the aleurone cells of mature, germinating seeds to produce proteins from GOIs under the control of an alpha-amylase promoter. This promoter is active only in germinating seeds.
Recently, using a standard in vitro Ltp assay, two 10 kDa and one member of a novel class of 7 kDa nsLtp’s were isolated from wheat seeds (Monnet, 1990; Dieryck et al., 1992). The sequence of this 7 kDa wheat nsLtp protein shows a high degree of similarity with the predicted protein from the open reading frame (ORF) of the Bzl 1E cDNA, which had been isolated in a differential screening for barley aleurone specific transcripts (Jakobsen et al., 1989). However, the amino acid sequence of this polype-tide showed only limited sequence identities with the previously sequenced 10 kDa proteins. In sub-cellular localisation studies using gold labelled antibodies one 10 kDa protein from Arabidopsis was localised to the cell wall of epidermal leaf cells. The presence of a signal peptide domain in the N-terminus of the open reading frames of all characterised plant ns-LTP cDNAs, also suggests that these are proteins destined for the secretory pathway with a possible extracellular function.
Olsen et al. in a paper titled «Molecular Strategies For Improving Pre-Harvest Sprouting Resistance In Cereals» published in 1990 in the published extracts from the Fifth International Symposium On Pre-Harvest Sprouting In Cereals (Westview Press Inc.) describe three different strategies for expressing different «effector» genes in the aleurone layer and the scutellum in developing grains of transgenic plants. This document mentions 4 promoter systems including a system called Bl1E (which is now recognised as being the same as the Ltp2 gene promoter). There is no sequence listing for B11E given in this document.
Kalla et al. (1993) in a paper titled «Characterisation of Promoter Elements Of Aleurone Specific Genes From Barley» describe the possibility of the expression of anti-sense genes by the use of promoters of the aleurone genes B22E, B23D, B14D, and B11E (which is now shown to be the same as Ltp2).
The Kalla et al. (1993) paper gives a very general map of the Ltp2 gene promoter. The transient expression results showed very low levels of expression of the reporter gene.
WO 95/15389 PCT/NO94/00190 4 A sequence listing of the Ltp2 gene was available as of 23 December 1992 on the EMBL database.
One of the major limitations to the molecular breeding of new varieties of crop plants with aleurone cells expressing GOIs is the lack of a suitable aleurone specific promoter.
At present, the available promoters such as the CaMV 35S, rice actin and maize alcohol dehydrogenase all are constitutive. In this regard, they are non-specific in target site or stage development as they drive expression in most cell types in the plants.
Another problem is how to achieve expression of a product coded for by a GOI in the aleurone layer of the endosperm that gives minimal interference with the developing embryo and seedling.
It is therefore desirable to provide aleurone specific expression of GOIs in cereal such as rice, maize, wheat, barley and other transgenic cereal plants.
Moreover it is desirable to provide aleurone specific expression that does not lead to the detriment of the developing embryo and seedling.
According to a first aspect of the present invention there is provided a Ltp2 gene promoter comprising: the sequence shown as SEQ. I.D. 1, or a sequence that has substantial homology with that of SEQ. I.D. 1, or a variant thereof.
According to a second aspect of the present invention there is provided a conjugate comprising a GOI and a Ltp2 gene promoter as just defined.
WO 95/15389 PCT/NO94/00190 According to a third aspect of the present invention there is provided an in vivo expression system comprising a conjugate comprising a GOI and a Ltp2 gene promoter as just defined wherein the conjugate is integrated, preferably stably integrated, within a monocotyledon’s (preferably a cereal’s) genomic DNA.
According to a fourth aspect of the present invention there is provided a transgenic cereal comprising a conjugate comprising a GOI and a Ltp2 gene promoter as just defined wherein the conjugate is integrated, preferably stably integrated, within a cereal’s genomic DNA.
According to a fifth aspect of the present invention there is provided the in vivo expression in the aleurone cells of a monocotyledon (preferably a cereal) of a conjugate comprising a GOI and a Ltp2 gene promoter as just defined; wherein the conjugate is integrated, preferably stably integrated, within the monocotyledon’s genomic DNA.
According to a sixth aspect of the present invention there is provided a method of enhancing the in vivo expression of a GOI in just the aleurone cells of a monocotyledon (preferably a cereal) which comprises stabiy inserting into the genome of those cells a DNA conjugate comprising a Ltp2 gene promoter as just defined and a GOI; wherein in the formation of the conjugate the Ltp2 gene promoter is ligated to the GOI in such a manner that each of the myb site and the myc site in the Ltp2 gene promoter is maintained substantially intact.
According to a seventh aspect of the present invention there is provided the use of a myb site and a myc site in an Ltp2 gene promoter to enhance in vivo expression of a GOI in just in the aleurone cells of a monocotyledon (preferably a cereal) wherein the Ltp2 gene promoter and the GOI are integrated into the genome of the monocotyledon.
According to an eighth aspect of the present invention there is provided a method of enhancing the in vivo expression of a GOI in just the aleurone cells of a monocotyledon (preferably a cereal) which comprises stably inserting into the genome of those cells a DNA conjugate comprising a Ltp2 gene promoter as just defined and a GOI; wherein in the formation of the conjugate the Ltp2 gene promoter is ligated to the GOI in such a manner that any one of the Sphl site, the AL site or the DS site in the Ltp2 gene promoter is (are) WO 95/15389 PCT/NO94/00190 6 maintained substantially intact. The Sphl site, the AL site and the DS site are defined later, Preferably the promoter is a barley aleurone specific promoter.
Preferably the promoter is for a 7 kDa lipid transfer protein.
Preferably the promoter is used for expression of a GOI in a cereal seed.
Preferably the promoter is used for expression of a GOI in a monocotyledonous species, including a grass preferably a transgenic cereal seed.
Preferably the cereal seea is anyone of a rice, maize, wheat, or barley seed.
Preferably the promoter is the promoter for Ltp2 of Hordeum vulgare.
Preferably at least one additional sequence is attached to the promoter gene or is present in the conjugate to increase expression of a GOI or the GOI.
The additional sequence may be one or more repeats tandem repeats) of the promoter upstream box(es) which are responsible for the aleurone specific pattern of expression of Ltp2. The additional sequence may even be a Shl-intron.
The term «GOI» with reference to the present invention means any gene of interest but not the remainder of the natural Ltp2 gene for the cereal in question. A GOI can be any gene that is either foreign or natural to the cereal in question.
Typical examples of a GOI include genes encoding for proteins giving for example added nutritional value to the seed as a food or crop or for example increasing pathogen resistance.
The GOI may even be an antisense construct for modifiying the expression of natural transcripts present in the relevant tissues.
I
WO 95/15389 7 Preferably the GOI is a gene encoding for any one of a protein having a high nutritional value, a Bacillus thuringensis insect toxin, or an alpha- or beta- amylase or germination induced protease antisense transcript.
The term «a variant thereof» with reference to the present invention means any substitution of, variation of, modification of, replacement of, deletion of or the addition of one or more nucleic acid(s) from or to the listed promoter sequence providing the resultant sequence exhibits aleurone specific expression.
The term «substantial homology» covers homology with respect to at least the essential nucleic acids of the listed promoter sequence providing the homologous sequence exhibits aleurone specific expression. Preferably there is at least 80% homology, more preferably at least 90% homology, and even more preferably there is at least 95% homology with the listed promoter sequence.
The term «maintained substantially intact» means that at least the essential components of each of the myb site and the myc site remain in the conjugate to ensure aleurone specific expression of a GOI. Preferably at least about 75%, more preferably at least about of the myb or myc site is left intact.
The term «conjugate», which is synonymous with the terms «construct» and «hybrid», covers a GOI directly or indirectly attached to the promoter gene to from a Ltp2-GOI cassette. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence, such as the Shl-intron, intermediate the promoter and the GOI.
The present invention therefore provides the novel and inventive use of an aleurone specific promoter namely the use of the Ltp2 gene promoter, preferably the Ltp2 gene promoter from barley.
The main advantage of the present invention is that the use of the Ltp2 gene promoter results in specific aleurone expression of a GOI in the aleurone layer(s) of cereals such as rice, maize, wheat, barley and other transgenic cereal seeds, preferably maize seed.
i WO 95/15389 I’CT/NO94/0190 8 It is particularly advantageous that the expression is both stage- and tissue- specific.
A further advantage is that the expression of the product coded for by a GOI in the aleurone layer of the endosperm gives minimal interference with the developing embryo and seedling.
This is in direct contrast to constitutive promoters which give high levels of expression in the developing seedling and mature plant tissues which severely affect normal plant development.
The present invention is particularly useful for expressing GOI in the aleurone layer of developing grains such as cereal seeds.
With regard to the present invention it is to be noted the EMBL database sequence listing (ibid) does not suggest that the Ltp2 gene promoter could be used to express a GOI in a stage- and tissue- specific manner. Also the database extract does not mention the importance of the myb gene segment or the myc gene segment.
It is also to be noted the paper titled «Molecular Strategies For Improving Pre-Harvest Sprouting Resistance In Cereals» (ibid) does not give any specific sequence listing information for the Ltp2 gene promoter. Also there is no explicit mention in this paper of using just the Ltp2 gene promoter to induce expression in just aleurone cells. Moreover, there is no mention in this paper of an Ltp2 GOI conjugate being formed. Also there is no mention in this paper of the importance of the myb site or the myc site.
It is also to be noted that in the paper titled «Characterisation of Promoter Elements Of Aleurone Specific Genes From Barley» (ibid) there is no mention of an Ltp2 GOI conjuagte stably integrated into genomic DNA of a cereal. Also there is no explicit disclosure of an in vivo expression system. Moreover, there is no full sequence listing in this paper for the Ltp2 gene promoter. Also there is no explicit mention in this paper of the importance of the myb site or the myc site of Ltp2 gene promoter for in vivo GOI expression.
I
WO 95/15389 PCITNO94/00190 9 In contrast to the work disclosed in PCT WO 90/01551, the Ltp2 gene promoter (which is not disclosed in PCT WO 90/01551) the Ltp2 gene promoter results in aleurone specific expression in developing grains.
In general, therefore, the present invention relates to a promoter for a Ltp2 gene encoding a 7 kDa nsLTP. In situ hybridization analysis demonstrates that the Ltp2 transcript is expressed exclusively in aleurone cells from the beginning of the differentiation stage and half-way into the maturation stage. Further commentary on the maturation stages is provided by Bosnes et al., 1992.
The Ltp2 gene promoter may be inserted into a plasmid. For example, the Ltp2 BglII 0.84 kb fragment can be inserted into the BamHI site of Bluescript. A GOI, such as GUS, can then be inserted into this conjugate (construct). Furthermore, a Shl intron can then be inserted into the SmaI site of this conjugate.
Stable integration may be achieved by using the method of Shimamoto (1989). Another way is by bombardment of an embryonic suspension of cells maize cells). Another way is by bombardment of immature embryos barley embryos).
With the present invention, it can be shown by using particle bombardments that the -807 bp Ltp2 gene promoter fused to a beta-glucuronidase (GUS) reporter gene (which serves as a GOI) is active in the aleurone layer of developing barley seeds, giving 5 of the activity of the strong constitutive actin-promoter from rice. Also, in transgenic rice plants, the barley Ltp2-promoter directs strong expression of the GUS-reporter gene exclusively in the aleurone layer of developing seeds, suggesting the presence of conserved mechanisms for aleurone cell gene expression in the cereals.
In a preferred embodiment, the Ltp2 gene encodes a 7kDa barley seed nsLTP and has about identity to the wheat 7kDa protein.
I I WO 95/15389 WO 95/15389 CTINO94/001 The transcript of the Ltp2 gene is detectable in the earliest morphologically distinguishable aleurone cells and accumulates during the differentiation stage to decline finally during seed maturation. It can also serve as a molecular marker for the differentiating aleurone cells as shown in situ hybridisation experiments where the spatial distribution of the transcript is to be examined.
In the present invention, a genomic clone was isolated using the cDNA insert of previously isolated cDNA clone pBzl E and characterised by DNA sequencing.
The sequence of the cDNA and isolated genomic clone was found to be identical in the overlapping region. It was found the Ltp2 gene does not contain any intron.
To prove that this is an active gene, the 5′ region carried on a 845 bp DNA fragment delineated by two Bgl I restriction sites was fused to the GUS gene (following Jefferson 1987) and the construct was introduced into barley aleurone layers using micro projectile bombardment. Aleurone cells expressing GUS activity were detected proving that the gene promoter was indeed capable of driving the expression of the GOI in the relevant tissue.
By comparing the DNA sequence of this active promoter sequences several putative cis-acting elements with the potential of binding known transcriptional factors present in cereal aleurone layers were detected. They include the binding sites for transcriptional factors of the myb and myc class, namely TAACTG and CANNTG respectively. Our experiments showed that the myb and myc sites were important for good levels of expression.
Gel retardation experiments showed that the Ltp2 gene promoter has a myb site that is recognised by a MYB protein from chicken).
In the present invention, mature fertile rice plants were regenerated from transformed cultured rice protopiasts. The developing seeds of these primary transformants were analysed for the expression of GUS. It was found that the barley seed Ltp2 gene promoter confers aleurone specific expression in transgenic rice plants. This is the first example of an aleurone specific promoter in developing seeds of a transgenic cereal.
WO 95115389 PCTIN094(0190 11 The following were deposited in accordance with the Budapest Treaty at the recognised depositary The National Collections of Industrial and Marine Bacteria Limited (NCIMB) at 23 St Machar Drive, Aberdeen, Scotland, UK, AB2 1RY, on 22 November 1993: An E. Coli K12 bacterial stock containing the plasmid pLtp2pr i.e. Bluescript containing the Ltp2 gene promoter (Deposit Number NCIMB 40598).
[To form pLtp2pr, the Ltp2 promoter of Figure 2b (see later) contained on a BgIII fragment was inserted in the Bluescript KS vector into the BamHI site.] (ii) An E. Coli K12 bacterial stock containing the plasmid pLtp2/GN i.e. Bluescript containing a Ltp2 gene promoter GUS conjugate (Deposit Number NCIMB 40599).
[To form pLtp2/GN, the GUS-reporter gene cassette (GN) contained on the Smal-EcoRI fragment of the commercially available vector pBI101 (Clontech Inc.) was cloned directionally into the Smal and EcoRI sites of pLtp2pr.] (iii) An E. Coli K12 bacterial stock containing the plasmid pLtp2BCIGN i.e. Bluescript containing an Ltp2 gene promoter with a deletion spanning the myb and myc sites GUS conjugate (Deposit Number NCIMB 40601).
[To form pLtp2ABCIGN, the Ltp2 promoter and the GN gene was inserted as described for pLtp2pr and pLtp2/GN except for the use of Bluescript SK and that the Ltp2 promoter was deleted in the myb-myc region (using a PCR strategy) as explained in the legend of Figure 7 (see later).] (iv) An E. Coli K12 bacterial stock containing the plasmid pLtp2Shl/GN i.e. Bluescript containing an Ltp2 gene promoter-Shl intron-GUS conjugate (Deposit No. NCIMB 40600).
[To form pLtp2shl/GN, the Ltp2 promoter and the GN gene was inserted as described for pLtp2pr and pLtp2/GN except for the use of Bluescript SK. The Shl intron from maize contained on a Hincll restriction fragment was inserted into the Smal site of this construct.] WO ON/ I16,H1 12 Other embodiments and aspects of the present invention include: A transformed host having the capability of expressing a GOI in just the aleurone layer; A vector incorporating a conjugate as hereinbefore described or any part thereof; A plasmid comprising a conjugate as hereinbefore described or any part thereof; A cellular organism or cell line transformed with such a vector; A monocotylenedonous plant comprising any one of the same; A developping seed comprising any of the same; and A method of expressing any one of the same.
The present invention will now be described only by way of examples in which reference shall be made to the accompanying Figures in which: Figure 1 is a nucleotide sequence of the Ltp2 gene; Figure 2a is a nucleotide sequence of the Ltp2 gene promoter; Figure 2b is a nucleotide sequence of the Ltp2 gene promoter with an additional 39 nucleotides for fusion to a GUS gene for transgenic rice and transient assay studies; Figure 3 shows transverse sections from the mid-region of barley seeds and steady state levels of the Ltp2 mRNA in different tissue fractions of developing barley endosperm; Figure 4 shows the results for an in situ hybridization expe iment; Figure 5 is the result of a Southern blot experiment using DNA from transgenic rice plants; Figure 6 shows the expression of a GusA-reporter gene driven by the Ltp2 gene promoter in the aleurone layer of developing transgenic rice seeds; and Figure 7 shows the position of the myb and myc binding sites in the barley Ltp2 gene promoter.
ftn ,PI I WO 95/15389 9CT/NO9410()190 13 A. METHODS i. Plant material Seeds of Barley (Hordeum vulgare cv. Bomi) were collected from plants grown in a phytotron as described before (Kvaale and Olsen, 1986). The plants were emasculated and pollinated by hand and isolated in order to ensure accurate determination of seed age.
ii. cDNA aud genomic clones The isolation and sequencing of the aleurone specific cDNA clone pBzllE (which is the same as Ltp2) was conducted as described by Jakobsen et al. (1989).
A barley, cv. Bomi genomic library was constructed by partial MboI digestion of total genomic DNA and subsequent ligation of the 10-20 kilo basepair (kb) size fraction with BamHI digested lambda EMBL3 DNA (Clontech Labs, Palo Alto, Ca, USA). Out of a total of 2 x 106 plaques screened, using the BzllE cDNA insert as a template for probe synthesis with a random labelling kit (Boehringer-Mannheim), four positive clones (gHv29-101, gHv38-201, gHv53-201 and gHv59-101) were identified after repeated rounds of plaque hybridization. DNA purified from these clones were restricted with several enzymes and characterized by Southern blot analysis.
The restriction maps of the four clones showed extensive overlap. One clone, gHv53-201, containing an insert of around 12 kb, was chosen for further analysis. A 6 kb PstI fragment contained within the insert -that hybridized to the cDNA probe was subcloned into Bluescript (Stratagene) giving the subclone BL53Psl7. A NheI restriction fragment of 0.7 kb covering the coding region of the Ltp2 gene was cloned into the XbaI site of M13mpl8 and sequenced using the Sequenase protocol (USB) after isolation of ssDNA template using PCR amplification and magnetic beads (Dynabeads M280- Streptavidin, Dynal).
I
II WO 95115389 PCINO94/00190 14 In order to characterize the 5′ and 3′ sequences of the Ltp2 gene, the following DNA fragments were generated by PCR amplification: i) a 1.2 kb fragment covering the 5′ end from a vector primer (KS) to the PLT11 primer located within the 5’end of the cDNA; and ii) a 0.3 kb PCR product generated by amplification directed by the primers LTP13 and of which the latter is based upon sequence information from the cDNA clone Bzl4A, which is overlapping and identical with the Bzl1E cDNA but contains some additional base pairs after the polyadenylation site indicated at position 490 in Figure 1.
The sequences are: KS: 5′ CGAGGTCGAC GGTATCG PLT11: 5′ LTP13: 5′ 5′
TACGGTGATC
ACGAAGCCGA
GGACTAAAAA
TACTCGGCTA
GCGGCGAGT
AAAAGTTGCA ACACAAATTT C The PLT11 sequence contains one base substitution (shown in bold and underlined) creating a BglII restriction site.
The 1.2 kb PCR product containing the 5′ end was restricted with Bgll which gave a 0.84 kb fragment with BamHI compatible sticky ends that was subsequently cloned into the BamHI site of pBluescript.
The 0.3 kb PCR product of the 3′ end was treated with T4 DNA polymerase (Sambrook et al., 1989) and subsequently cloned into the SmaI site of M13mpl8.
The sequences of the PCR products were determined as described above.
I WO 95/15389 PCTIN094/00190 iii. Northern analyses Total RNA was extracted from barley seed tissues of 10 DAP and older plant material essentially as described by Logemann et al. (1987), except that LiCI precipitation was used in place of ethanol precipitation. The RNA was denatured using formaldehyde and separated on 1.2% agarose gels as described by Selden (1987) and blotted onto GeneScreen (NEN) membranes using a Stratagene posiblotter apparatus according to supplier’s instructions.
Hybridization was according to GeneScreen instruction manual (NEN) using radioactively labelled DNA strands complementary to the pBzl1E cDNA insert generated with a random primed DNA labeling kit (Boehringer Mannheim).
iv. In situ hybridization For in vitro transcription of antisense RNA, the plasmid pBzl 1E (Jakobsen et al., 1989) was linearized with PstI and transcribed with T7 RNA polymerase by using MAXIscript (Ambion) and [5,6-3H]-Uridine5′-triphosphate (40-60 Ci mmol-1) (Amersham International) according to the specifications of the suppliers. The probe was hydrolyzed to fragments of about 100 bp as described by Somssich et al. (1988). Seed tissues were fixed in 1% glutaraldehyde, 100 mM sodium phosphate (pH 7.0) for 2 hours and embedded in Histoplast (Histolab, G6teborg, Sweden).
Sections of 10 pm were pretreated with pronase (Calbiochem) as described by (Schmelzer et al., 1988) and hybridized with 25 ml of hybridization mix (200 ng probe ml-1, formamide, 10% dextran sulphate, 0.3 M NaC1, 10 mM Tris-HCl, 1 mM EDTA (pH 0.02% polyvinyl- pyrrolidone, 0.2% Ficoll, 0.02% bovine serum albumin) for 15 hours at 50 oC.
Posthybridization was carried out according to Somssich et al. (1988) and autoradiography was done as described by Schmelzer et al. (1988), except that sections were exposed for weeks.
i I WO 95/15389 PCT/NO94/00190 16 v. Constructs for transient expression analysis For the microprojectile bombardment experiments, the following constructs were used: CONTROL A: CONTROL B: CONTROL C: CONTROL D: pActlf-GUS containing the rice Actin 1 promoter fused to the uidA reporter gene encoding the GUS enzyme (McElroy et al., 1990); pRT101-ex/s-int/s-LUC containing the 35S CaMV promoter-Shl first exon/intron fused to the firefly luciferase gene (Maas et al., 1991); and pRT101C1 containing the Cl cDNA downstream of the 35S CaMV promoter (Paz-Ares et al., 1987); pMF6Lc(R) containing the Lc cDNA corresponding to one R gene allele coupled to the 35S CaMV promoter-Adhl intron (Ludwig et al., 1989).
For the transient expression studies in barley aleurone the first intron of the maize Shl gene carried on a 1.1 kb HincI fragment (Maas et al., 1991) was inserted into the SmaI site of the promoter-reporter gene constructs according to the present invention. The Ltp2 gene promoter is contained on the 0.84 kb BgllI fragment (sequence is presented in Figure 2) and was inserted into the BamHI site of pBluescript. Thereafter the structural uidA gene encoding the beta-glucuronidase (Gus) enzyme was fused to the Ltp2 gene promoter.
The following conjugates according to the present invention were studied: Ltp2/GN: (ii) Ltp2Shl/GN: A Ltp2 gene promoter GUS conjugate (same as conjugate in pLtp2/GN see earlier); A Ltp2 gene promoter Shl intron GUS conjugate (same as conjugate in pLtp2Shl/GN see earlier).
WO 95/15389 PCT/NO94/00190 17 Isolated plasmid DNA was used in the bombardment studies. The same conditions were used for the control conjugates and for the conjugates of the present invention.
For transient assay studies with rice protoplasts, the following conjugates according to the present invention were studied: Ltp2/GN: As above; and (iii) Ltp2ABCIGN: A Ltp2 gene promoter {with a deletion spanning the myb and myc sites} GUS conjugate (same as conjugate in pLtp2BCIGN see earlier).
vi. Transformation of barley aleurone layers by particle bombardment Barley seeds were harvested at 25 DAP, surface sterilized in 1% sodium hypochlorite for min and then washed 4 times in sterile distilled water. The maternal tissues were removed to expose the aleurone layer and the seed was then divided into two, longitudinally along the crease. The pieces of tissue were then placed, endosperm down, onto MS (Murashige Skoog 1962) media with 10 g/l sucrose solidified with 10 g/l agar in plastic petri dishes (in two rows of 4 endosperm halves per dish).
Single bombardments were performed in a DuPont PDS 1000 device, with M-17 tungsten pellets (approx. 1 pm in diameter) coated with DNA as described by Gordon-Kamm et al.
(1990) and using a 100 mm mesh 2 cm below the stopping plate. Equal amounts (25 pg per preparation) of the GUS (promoter-reporter gene) and LUC (internal standard) plasmids were mixed before adding the microprojectiles. One tenth of this amount, 2.5 pg, was used for the Lc and Cl cDNA constructs. Bombarded tissue was incubated at 24 C for 3-4 days before extraction and measurement of GUS and LUC activities. Anthocyanin pigmentation could be observed in the bombarded aleurones directly without further treatment.
Histochemical staining for GUS expression was performed with X-Gluc (5-bromo,4-chloro,3indolyl,f-D,Glucuronic acid) as described by Jefferson (1987) at 37*C for 2 days. Extraction of cellular proteins for quantitative analysis was performed by grinding 4-8 half seeds in a mortar and pestle with 0.5 ml of extraction buffer (50 mM Na-phosphate pH, 1 mM DTT, pH II WO 95/15389 PCT/N094Q0 0190 18 After grinding, a further 0.5 ml was added and two 400 /l aliquots were taken. To one of these, 100 1 tl of 5 x Luciferase cell lysis buffer (Promega) was added and the sample vortexed before clearing by centrifugation at 10,000 rpm. A 20 /l aliquot was then measured for LUC activity in a scintillation counter (Tri-Carb 4000), using the luciferase assay system of Promega (E1500). To the other 400 t1 aliquot, 100 !l of 5x lysis buffer (500 mM Na phosphate pH 7.0, 50 mM EDTA, 10 mM DIT, 0.5% Sarcosyl, 0.5% Triton X-100) was added, the mixture vortexed and cleared as above and assayed for GUS activity using 4methylumbelliferone, B-D,glucuronide as described by Jefferson (1987) modified to include methanol in the reaction mixture (Kosugi et al., 1990).
Production of 4-methylumbelliferone (MU) was measured after 1 and 4 h using a TKO 1000 Mini-Floorimeter (Hoefer Scientific Instruments). In the analysis of promoter activities, the GUS readings (MU produced per hr) were standardized by division with the LUC value (photons produced over 30 s, beginning 60 s after mixing) from the same extract.
vii. Transient assay of rice protoplasts In this experiment, the same type of protoplasts as used for stable transformation of rice plants was transiently transformed with constructs and (iii) (see above) and then assayed for GUS activity.
viii. Rice transformation Southern blot analysis of transgenic rice plants Total genomic DNA was isolated from mature leaves, digested with Xba I and then transferred to a nylon membrane (Amersham). The coding region of the GUS gene was labelled and amplified with digoxigenin 11-dUTP by polymerase chain reaction and used for probing the Ltp2 GUS gene. Hybridization and chemiluminesence signal detection were performed according to manufacturers specifications (Boeringen Mannheim).
M
1 3 1 WO 95/15389 PCT/NO94/00190 19 B. RESULTS WITH REFERENCE TO THE FIGURES i. Figure 1 is a nucleotide sequence of the Ltp2 gene. A transcription start site has been assigned as The TATA consensus sequence is boxed. Consensus myb and myc binding sites and the SphI element (Hattori et al., 1992) found in the Cl promoter sequence are shown in bold italics.
In the ORF (open reading frame), the nucleotides are shown in bold letters, starting with the first ATG codon and ending with the TAG stop codon. The single base substitution introduced at position +41 A T) creates a BglII restriction site which delimits the 3′ end of the fragment covering the Ltp2 gene promoter. The positions of the 5′ end and polyadenylation site of the corresponding cDNA, BzllE (Jakobsen et al., 1989), are shown by arrows. Two putative polyadenylation signals are underlined.
ii. Figure 2a is a nucleotide sequence for the Ltp2 gene promoter. Figure 2b is a nucleotide sequence for the Ltp2 gene promoter with an additional number of nucleotides for fusion to a GUS gene.
iii. Figure 3 shows transverse sections from the mid-region of barley seeds and steady state levels of the Ltp2 mRNA in different tissue fractions of developing barley endosperm Figure 3 can be analysed as follows: Ten DAP (days after pollination) endosperm isolated from the surrounding maternal tissues by manual extrusion. For maternal tissues, see The extruded endosperm consists of the central starchy endosperm cells, a group of modified aleurone cells over the crease area (arrow) and one layer of highly vacuolated peripheral aleurone cells (arrowhead).
Enlargement showing vacuolated peripheral aleurone cells (AC) and starchy endosperm cells (SE) in area of marked with arrowhead.
I I WO 95/15389 W CT/N0 94/00190 Pericarp of 10 DAP seed after extrusion of the endosperm with the nucellus epidermal layer (NE) facing the endosperm cavity, which contained the endosperm in before extrusion.
Extruded 15 DAP endosperm with central starchy endosperm cells and modified aleurone cells (arrow), but without peripheral aleurone cells.
15 DAP pericarp with adhering aleurone layer after extrusion of the endosperm (in D).
Northern blot showing the steady state level of Ltp2 mRNA in the extruded endosperm fraction and the pericarp fraction in the interval from 10 to 13 DAP. For this blot, 10 ug of total RNA was loaded in each lane. The gel was blotted and hybridized with randomly primed Ltp2 cDNA.
iv. Figure 4 shows the results for an in situ hybridization of 3 H-labelled Ltp2 antisense probe to transverse sections of barley endosperm (A and B) and transient gene expression analysis of different promoter-reporter gene constructs in developing barley aleurone layers after particle bombardment D and Figure 4 can be analysed as follows: Dark field microphotograph of 13 DAP endosperm showing hybridization of the Ltp2 probe to the peripheral aleurone cells (AL) ventrally and laterally, but not to aleurone cells on the dorsal side of the grain nor to the modified aleurone cells over the crease area (MA).
Magnification of peripheral endosperm (frame in A) showing gradient of in situ hybridization signal towards the dorsal side of the seed containing undifferentiated aleurone cells.
Colourless barley aleurone layer co-bombarded with the 35S-C1 and cDNA constructs. Single aleurone cells synthesizing anthocyanin pigment appear as red spots.
II WO 95/15389 PCT/N94/00190 21 Exposed aleurone layer of 25 DAP barley seeds bombarded with the Ltp2/Shl int/GUS construct. The transfected seed was stained for detection of GUS activity.
Exposed aleurone layer of barley seed of the same stage bombarded with pActlf-GUS construct and histochemically stained as in Ventral crease area.
v. Figure 5 is the result of a Southern blot experiment of DNA from transgenic rice plants harbouring the Ltp2-GUS construct. Figure 5 can be analysed as follows: Lane P: plasmid Ltp2-GUS.
Lane C: untransformed control plants.
Lane 1: transgenic line 3-15.
Lane 2: transgenic line 4-13.
Lane 3: transgenic line 2-6.
Lane 4: transgenic line 4-6.
vi. Figure 6 shows the expression of a GUS-reporter gene driven by the Ltp2-wildtype promoter in the aleurone layer of developing transgenic rice seeds. Figure 6 can be analysed as follows: Longitudinal section of 20 DAP seed showing GUS staining exclusively in the aleurone layer but not in the embryo, starchy endosperm (SE) or in the maternal tissue Transverse section from the mid-.region of 20 DAP seed.
i I I WO 95115389 PCT/NO94/00190 22 Enlargement of dorsal side of seed shown in Non-transgenic control seed, same age as in A 5 day-old seedling transformed with the Ltp2 GUS gene.
A 5 day-old seedling transformed with the CaMV35S GUS gene. (Terada and Shimamoto 1990). Arrows indicate regions of GUS expression. Bars in (A,B and D) are 10 mm and in 2.5 mm.
vii. Figure 7 shows the position of the myb and myc sites in the barley Ltp2 gene promoter. The distance from the 3′ end of the myc site to the TATA box is given in nucleotides. The following nucleotides from and between the myb and myc sites were deleted to form the conjugate containing the deletion in the Ltp2 gene promoter gene:
CAACTACCATCGGCGAACGACCCAGC.
C. CONCLUSIONS 1. The barley Ltp2 gene encodes a protein homologous to the 7 kDa wheat lipid transfer protein Using the BzllE cDNA (Jakobsen et al., 1989) as a probe, the corresponding barley cv.
Bomi genomic clone was isolated. The sequences of the genomic clone and that of the Bzl 1E cDNA are identical in overlapping regions and no intervening sequences were detected (Figure 1) accordingly this gene is Ltp2. The ATG codon initiating the longest open reading frame (ORF) in the Ltp2 sequence is located 64 bp downstream of the putative transciiptional start site at nucleotide number 1 (Figure The ORF contains eight potential translation start codons between nucleotides 64 and 127. Two polyadenylation signals, which conform to the plant consensus sequence (Joshi, 1987) are found in the 3′ end of the genomic sequence. In the BzllE cDNA the polyA tail extends after the G at position 491 (Figure 1 and Figure 2).
1 WO 95/15389 PCTINO94/00190 23 2. The Ltp2 transcript can be a molecular marker for peripheral aleurone cell diferentiation In the developing seed at approximately 8 days after pollination (DAP), aleurone cell differentiation is initiated over the ventral crease area resulting in the formation of the modified aleurone cells (Figure 3A and Bosnes et al., 1992). Shortly after, at 9 DAP, the second type of aleurone cells, characterized by their extensive vacuolation (Figure 3B), appears in the peripheral endosperm close to the crease area, spreading first laterally and then to the dorsal side of the seed (see Figure 3A). At this stage, when whole deembryonated seeds are squeezed, the extruded endosperm consists of the starchy endosperm, the peripheral and the modified aleurone cells (Figure 3 This is in contrast to later developmental stages, where the extruded endosperm consists only of the starchy endosperm and the modified aleurone cells (Figure 3D). The reason for this is that the cells of the aleurone layer adhere to the maternal pericarp (Figure 3E). Aleurone cell formation is completed at 21 DAP, when cell division stops (Kvaale and Olsen, 1986). Using the Ltp2 probe on Northern blots with total RNA, the signal obtained gradually becomes stronger in the pericarp, compared to the extruded endosperm, confirming the relocation of the aleurone cells from the endosperm fraction to the pericarp fraction in the interval between 10 and 13 DAP (Figure 3F).
From the experimental results presented in Figure 3 it is concluded that the Ltp2 transcript is a potential marker for aleurone cell differentiation. To corroborate the usefulness of the Ltp2 transcript as a molecular marker for aleurone cell differentiation, in situ analysis was carned out on transverse sections of 13 DAP seeds. The rationale for using seeds from this stage was the earlier observed gradual differentiation of the peripheral aleurone cells, starting near the crease area and spreading to the dorsal side (Bosnes et al., 1992).
Using 3 H-labelled antisense transcript as probe, a positive signal is clearly visible in the peripheral aleurone cells in the ventral part adjacent to the crease area as well as laterally up towards the dorsal side of the grain (Figure 4A). However, no signal is present in the dorsal region of the seed, nor over the modified aleurone cells.
I II I WO 95/15389 WPCT/NO94/O001J9 24 Focusing on the most dorsal aleurone cells showing a positive signal in the in situ analysis (Figure 4B), the morphology of these cells corresponds to that of the highly vacuolated peripheral aleurone cella in 10 DAP endosperm (Figure 3B).
The Ltp2 transcript therefore represents a highly tissue specific molecular marker for aleurone cell differentiation.
3. The Ltp2 gene promoter is transiently expressed in developing barley aleurone cells after particle bombardment The Ltp2 gene promoter contained on a 842 bp BglII restriction fragment (from nt -807 to nt +35 in Fig.l) was fused to the GUS-reporter gene and introduced into the exposed aleurone layers of 25 DAP whol barley seeds by the biolistic method. In the first st of experiments, Ltp2 gene promoter activity was assayed visually after histochemical staining with X-Gluc. Due to the large variation between individual experiments with the biolistic method, plasmid DNA containing the Lc and C1 cDNAs from maize under the control of the CaMV promoter was co-bombarded with the Ltp2 construct to monitor shooting efficacy.
In combination, but not individually, the latter two cDNAs give righ numbers of red anthocyanin spots in the barley aleurone cells without any treatment after 1 to 2 days of incubation of the seeds on solid nutrient medium (Figure 4C). Compared to the number of red spots, the Ltp2-GUS construct consistently gave very few spots after histochemical staining in co-bombardment experiments.
Based on previous reports that insertion of introns in promoter construct enhance the level of transient expression (Maas et al., 1991) without interfering with the tissue specificity of the promoters, the intron from the maize Shrunken-1 gene was inserted into the Ltp2-GUS construct after the promoter. Using this construct the number of spots in immature aleurone layers increased (Figure 5D). Still, however, compared to aleurone layers bombarded with the pActlf-GUS construct (McElroy et al., 1990), which contains the promoter of the constitutively expressed Actinl gene from rice (Figure 4D), both the number and the size of the spots obtained with the Ltp2-GUS construct is dramatically smaller (Figure 4E).
I
I II WO 95/15389 PCT/N094/0019 In order to quantify Ltp2 gene promoter activity in particle bombardment experiments, another control plasmid containing the LUC gene under the control of the 35S-promoter was co-bombarded with the Ltp2-GUS constructs. In this way, after particle bombardment and incubation on tissue culture medium, protein was extracted from the seeds in a buffer that allowed measurement of both LUC and GUS activity (for details, see Materials and Methods section). In such experiments, calculating GUS expression standardized on the base of the LUC-activity, the Ltp2-GUS activity was not significantly higher than background, corresponding approximately to 1.5% of the Actinlf promoter activity in parallel experiments.
For the Ltp2-Shl intron-GUS construct, however, the activity was significantly higher than background, corresponding to 5% of that of the Actinl promoter. Blue spots from the activity of the Ltp2-promoter were never observed in other tissues than the aleurone layer of developing seeds. From these experiments it is concluded that the -807 bp promoter of the Ltp2 gene is capable of directing transient gene expression in a fashion similar to that of the endogenous Ltp2 gene, in the cells of the aleurone layer of immature barley seeds.
4. The Ltp2 gene promoter directs aleurone sp-cific expression of the GUS-reporter gene in transgenic rice seeds The gene was transformed into rice by electroporation of embryogenetic protoplasts following the teachings of Shimamoto et al. 1989.
Four fertile transgenic rice plants were obtained and integration of the Ltp2-GUS gene was examined by Southern blot analysis. The results demonstrated that a 2.9 b fragment containing the Ltp2-GUS gene is integrated in all the transgenic lines. Histochemical GUS analysis was carried out with developing rice seeds of 20 DAP and 5 day old seedings derived from transgenic seeds (Figure In developing seeds the GUS expression is strictly limited to the aleurone layer, with no staining observed in the maternal tissues, starchy endosperm or in the embryo of the transgenic seeds (Figure 6 nor in untransformed control seeds (Figure 6 No GUS staining was observed in leaves or roots of seedlings transformed with the Ltp2 GUS gene (Figure 6 E).
WO 95/15389 PCT/N94100190 26 In contrast, seedlings transformed with the CaMV35S GUS gene GUS expression is detected in the coleptile, shoots and roots (see Figure 6 F; Terada and Shimamoto 1990).
T’ese results clearly demonstrate the aleurone-specific expression of the Ltp2 GUS gene in transgenic rice plants.
The Ltp2 gene promoter contains sequence elements implicated in the transcriptional control of cereal endosperm specific genes The studies with the deletion spanning the myb and myc sites in the Ltp2 gene promoter showed that levels of expression were about 10% of that of the wild-type gene promoter.
These studies indicated that both the myb and myc sites are important for expression.
In addition, the Ltp2 gene promoter may even contain another sequence element that has been implicated in regulation of gene expression in maize aleurone cells, namely the octanucleotide CATGCATG (Figure This sequence, previously referred to as the SphI element, has been shown to mediate the transcriptional activation of maize Cl by interaction Slith VP1 (Hattori et al., 1992). As in the maize Cl promoter (Paz-Ares et al., 1987), the putative SphI element of the barley Ltp2 gene promoter is located between the TATA-box and the myb binding site.
In addition, the Ltp2 gene promoter may contain two further sites that could play an important role in transcription. The first site is an «AL» site and has the sequence
CATGGAAA
This AL sequence ends at position -366 in the sequence shown in Figure 1.
The second site is a «DS» site that has a high degree of similarity or identity with the binding site for 5′ transcriptional factors from other eucaryotic organisms. This DS site, which a dyad-symmetry, has the sequence
TCGTCACCGACGA
This DS sequence ends at position -121 in the sequence shown in Figure 1.
i r WO 95/15389 PCT/N094/00190 27 D. DISCUSSION The above examples of the present invention concern the barley gene Ltp2, which encodes an aleurone specific 7 kDa nsLTP.
The identification of the Ltp2 protein as a nsLTP is based on the high identity between the predicted Ltp2 amino acid sequence and the 7 kDa protein isolated from wheat seeds using the in vitro lipid transfer assay (Monnet, 1990). The high degree of sequence identity between the two barley aleurone Ltp gene products and the homologous proteins and transcripts from wheat seeds strongly suggests that the aleurone layer of these two cereals contain two related classes of nsLTPs with molecular masses of 10 and 7 kDa, respectively.
While the sequence identity is more than 70% within the two classes, it is only around between them. However, several conserved features are apparent in the cereal seed nsLtps, including similar N-terminal signal peptides, an internal stretch of 20 amino acids with similarity, and 8 cysteine residues that are believed to be important for the activity of plant Ltps (Tchang et al., 1988). Studies also showed that the Ltp2 gene lacks an intron.
Hybridization experiments using Ltp2 probes to barley genomic Southern blots indicate that the barley haploid genome contains only one copy of each gene (Jakobsen et al., 1989; Skriver et al., 1992).
According to a suggestion by Sterk et al. (1991) plant nsLTPs may be involved in the extracellular transport of cutin or other lipid monomers from the endoplasmic reticulum to the site of synthesis of extracellular matrix components, such as the cuticle. Therefore, one possible role for the aleurone specific nsLTPs in barley and wheat could be in the formation of the earlier described amorphous layer on the outside of the aleurone cells in wheat seeds (Evers and Reed, 1988). The function of this layer is unknown, but it may be involved in the regulation of the osmotic pressure in the endosperm during seed development and germination. If this holds true, the absence of the Ltp2 transcript in the modified aleurone cells in the ventral crease area is functionally significant, since an impermeable layer on the outside of these cells would prevent the influx of soluble synthates from the vegetative plant parts.
‘I WO 95/15389 PCT/N094/00190 28 Of the nine cDNAs isolated in the differential screening experiment design to identify clones representing transcripts differentially expressed in the aleurone layer of developing barley seeds, Ltp2 hybridizes to transcripts present exclusively in the aleurone layer. Thus, the Ltp2 gene represents a suitable gene for the search for promoter sequences responsible for the control of gene transcription in the aleurone layer.
Due to the lack of a routine protocol for stable barley transformation, demonstration of Ltppromoter specificity in barley has to rely on transient assays using the particle bombardment method. Using this method, it was demonstrated that the -807 bp Ltp2 gene promoter carried on the BglI restriction fragment is capable of driving the expression of the GUS reporter gene in immature barley aleurone layers. From this it is concluded that the promoter fragment carries sequences that are responsible for barley aleurone specific gene transcription.
The Ltp2 gene promoter can be weaker than constitutive cereal promoters like that of the Actinlfgene even after the introduction of the Shl-intron (see Maas et al. (1991) and their work on tobacco protoplasts) into the Ltp2-GUS construct which increases the expression levels by around three-fold. However, this lower expression does not result in any damage to the developing seedling unlike the constitutive cereal promoters. Moreover, and again unlike the constitutive cereal promoters, the Ltp2 gene promoter directs desirable tissue and stage specifc expression.
As demonstrated by the histochemical assays shown in Figure 6, the Ltp2 BglI promoter fragment shows the same aleurone specific expression in developing rice seeds as in barley.
Thus, the conclusion from the transient assays in barley that this promoter fragment contains sequences responsible for aleurone specific gene transcription is confirmed. Furthermore, the data from rice provide support to the view that the molecular mechanisms underlying aleurone specific gene transcription in developing seeds are conserved among the cereal species.
i ll- i WO 95/15389 PCT/NO94/00190 29 E. SUMMATION The Examples describe the isolation of the promoter for the barley gene Ltp2, which encodes a novel class of cereal 7 kDa nsLTPs. The gene was isolated by the use of a cDNA from a differential screening experiment in which the positive probe was constructed from aleurone cell poly rich RNA, and the negative probe from the starchy endosperm of immature seeds.
In situ hybridization analysis demonstrates that the Ltp2 transcript is expressed exclusively in the aleurone layer from the beginning of the differentiation stage and half way into the maturation stage. Similar to previously identified 10 kDa plant nsLTPs, the Ltp2 protein contains the eight conserved cysteine residues.
The results indicate that the Ltp2 protein is involved in the synthesis of a lipid layer covering the outside of the cereal aleurone cells.
Using particle bombardments it was shown that the -807 bp Ltp2 gene promoter fused to the GUS-reporter gene is active in the aleurone layer of developing barley seeds, giving 5% of the activity of the strong constitutive actinlf-promoter from rice. Transformed into rice, the barley Ltp2-promoter directs strong expression of the GUS-reporter gene exclusively in the aleurone layer of developing rice seeds. Analysis of the Ltp2 gene promoter reveals the presence of sequence motives implicated in endosperm specific gene expression in maize, i.e.
the myb and myc protein binding sites. In short, the Ltp2 gene promoter represents a valuable tool for the expression of GOIs in the aleurone layers of cereal seeds.
Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.
I M i WO 95/15389) PCT/NO94/00190
REFERENCES
Baker, R.E, Gabrielsen, and Hall, B.D. (1986) Effects of tRNATyr point mutations on the binding of the yeast RNA polymerase III transcription factor C. J. Biol. Chem. 261, 5275 5282.
Barkardottir, Jensen, Kreiberg, Nielsen, P.S. and Gausing, K. (1987) Expression of selected nuclear genes during leaf development in barley. Dev. Genet. 8, 495 511.
Bosnes, Weideman, F. and Olsen, (1992) Endosperm differentiation in barley wild-type and sex mutants. Plant J. 2, 661-674.
Dellaporta, Greenblatt, Kermicle, Hick, J.B. and Wessler, S. (1988) Molecular cloning of the R-nj allelel by transposon tagging with Ac. In Chromosome structure and function: Impact ofnew concepts, 18th Stadler Genetics Symposium (Gustafson, J.P. and Appels, eds.), New York: Plenum Press, pp. 263-282.
Dieryck, Gautier, Lullien, V. and Joudrier, P. (1992) Nucleotide sequence of a cDNA encoding a lipid transfer protein from wheat (Triticum durum Desf.). Plant Mol.
Biol. 19, 707-709.
Dooner, H.K. (1983) Coordinate genetic regulation of flavonoid biosynthetic enzymes in maize. Mol. Gen. Genet. 198, 136-14.
Dooner, H.K. (1985) Viviparous -1 mutation in maize conditions pleiotropic enzyme deficiencies in the aleurone. Plant Physiol. 77, 486-488.
Evers, A.D. and Reed, M. (1988) Some novel observations by scanning electron microscopy on the seed coat and nucellus of the mature wheat grain. Cereal Chem. 65, 81- Fincher, G.B. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 305-346.
Fleming, AJ, Mandel, Hofmann, Sterk, de Vries, and Kuhlemeier, C.
(1992) Expression pattern of a tobacco lipid transfer protein gene within the shoot apex.
Plant J. 2, 855-862.
Franken, Niesbach-Klesgen, Weydeman, Marechal-Drouard, Saedler,H., and Wienand, U. (1991). The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for translational control of Whp expression by the anthocyanin intensifying gene EMBO J. 10, 2605-2612.
Friedman, W.E. (1992) Evidence of a pre-angiosperm origin of endosperm: implication for the evolution of flowering plants. Science 255, 336-339.
I; WO 95/15389 PCT/NO94/00190 31 Gabrielsen, Sentenac, A. and Fromageot, P. (1991) Specific DNA binding by c- MYB: Evidence for a double helix-tun-helix-related motif. Science, 253 1140-1143.
Goff, Cone, K.C. and Chandler, V.L. (1992) Fuctional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between to classes of regulatory proteins. Genes Dev. 6, 864-875.
Gordon-Kamm, WJ., Spencer, Mangano, Adams, Daines, R.J., Start, O’Brien, Chambers, Adams, Willetts, Rice, T.B., Mackey, Krueger, Kausch, A.P. and Lemaux, P.G. (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2, 603-618.
Hammond-Kosack, Holdsworth, M.J. and Bevan, M. (1993) In vivo footprinting of a low molecular weigth glutenin gene (LMWG-1D1) in wheat endosperm. EMBO J. 12, 545-554.
Hattori, Vasil, Rosenkrans, Hannah, McCarty, D.L. and Vasil, I.K.
(1992) The Viviparous-1 gene and abscisic acid activate the Cl regulatory gene for anthocyanin biosynthesis during seed maturation in maize. Genes Dev. 6, 609-618.
Jacobsen, Knox, R.B. and Pyliotis, N.A. (1971) The structure and composition of aleurone grains in the barley aleurone layer. Planta 101, 189-209.
Jakobsen, Klemsdal, Aalen, Bosnes, Alexander, D. and Olsen, O.-A.
(1989) Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains. Plant Mol. Biol. 12, 285-293.
Jefferson, R.A. (1987) Assaying chimeric genes in plants: the GUS gene fusion system.
Plant Mol. Biol. Rep. 5, 387-405.
Joshi, C.P. (1987) Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucleic Acid Res. 15, 9627-9640.
Kalla, Lonneborg, Linnestad, Potter, Aalen, Nielsen, P.S. and Olsen, (1993) Characterisation of promoter elements of aleurone specific genes from barley. In Pre-harvest sprouting in cereals 1992. Eds. M.K.Walker-Simmons and J.J.Ried.
The American Association of Cereal Chemists, Inc. ISBN 0-913250-81-3. pp 236-245.
Karrer, Litts, J.C and Rodriguez, R.L. (1991) Differential expression of a-amylase genes in germinating rice and barley seeds. Plant Mol. Biol. 16, 797-805.
Kader, Julienne, M. and Vergnolle, C. (1984) Purification and characterisation of a spinach-leaf protein capable of transferring phospholipids from liposomes to mitochondria or chloroplasts. Eur. J. Biochem. 139, 411-416.
I WO 95/15389 PCT/NO94/00190 32 Koltunow, Truettner, Cox, Wallroth, M. and Goldberg, R.B. (1990) Different temporal and spatial expression patterns occur during anther development. Plant Cell 2, 1201-1224.
Kosugi, Ohashi, Nakajima, K. and Arai, Y. (1990) An improved assay for bglucuronidase in transformed cells: methanol almost completly suppresses a putative endogenous b-glucuronidase activity. Plant Sci. 70, 133-140.
Kvaale, A. and Olsen, (1986) Rates of cell division in developing barley endosperms.
Ann. Bot. 57, 829-833.
Kyozuka, J. and Shimamoto, K. 1991) Transformation and regeneration of rice protoplasts. In Plant Tissue Culture Manual Bl (Lindsey, ed.) Dordrecht: Kluwer Academic Publishers, pp. 1-6.
Kyozuka,J., Fujimoto, Izawa,T. and Shimamoto, K. (1991). Anaerobic induction and tissue-specific expression of maize Adhl promoter in transgenic rice plants and their progeny.
Mol. Gen. Genet. 228, 40-48.
Lanahan, Ho, Rogers, S. and Rogers, J. (1992) A Gibberellin response complex in cereal a-amylase gene promoters. Plant Cell 4, 203-211.
Lea, Tommerup, Svendsen, I. and Mundy, J. (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J. Biol. Chem. 266, 1564-73.
Linnestad, Linueborg, Kalla, R. and Olsen, (1991) The promoter of a lipid tranfer protein gene expressed in barley aleurone cells contains similar Myb and Myc recognition sites as the maize Bz-McC allele. Plant Physiol. 97, 841-843.
Logemann, Schell, J. and Willmitzer, L. (1987) Improved method for isolation of RNA from plant tissues. Anal. Biochem. 163, 16-20.
Ludwig, Habera, Dellaporta, S.L. and Wessler, S.R. (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc.
Natl. Acad. Sci. USA 86, 7092-7096.
Maas, Laufs, Grant, Korfhage, C. and Werr, W. (1991) The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intronl enhances reporter gene expression up to 1000 fold. Plant. Mol. Biol. 16, 199-207.
Madrid, S.M. (1991) The barley lipid transfer protein is targeted into the lumen of the endoplasmic reticulum. Plant Physiol.Biochem. 29, 695-703.
I I WO 95/15389 PCT/NO94/00190 33 Marocco, Wissenbach, Becker, Paz-Ares, Saedler, H. and Salamini, F.
(1989) Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins. Mol. Gen. Genet. 216, 183-187.
McCarty, Carson, Stinard, P.S. and Robertson, D.S. (1989) Molecular analysis of Viviparous-1: An abscisic acid-insensitive mutant of maize. Plant Cell 1, 523-532.
McCarty, Hattori, Carson, Vasil, Lazar, M. and Vasil, I.K. (1991) The Viviparous-1 developmental gene of maize encodes a novel transcriptional activator. Cell 66, 895-905.
McClintock, B. (1978) Development of the maize endosperm as revealed by clones. In The clonal basis of development (Subtelny, S. and Sussex, eds.), New York: Academic Press, pp.217-237.
McElroy, Zhang, Cao, J. and Wu, R. (1990) Isolation of an efficient actin promoter for use in rice transformation. Plant Cell 2, 163-171.
Monnet, (1990) Ph.D thesis. Universite des Sciences et Techniques du Languedoc, Montpellier, France, pp. 121.
Mundy, J. and Rogers, J. (1986) Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: comparison to the barley amylase/subtilisin inhibitor.
Planta 169, 51-62.
Murashige, and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.
Olsen, Potter, R.H. and Kalla, R. (1992) Histo-differentiation and molecular biology of developing cereal endosperm. Seed Sci. Res. 2, 117-131.
Paz-Ares, Ghosal, Wienand, Peterson, P.A. and Saedler, H. (1987) The regulatory cl locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 6, 3553-3558.
Qu, Wang, Lin, Vance, V.B. and Huang, A.H.C. (1986) Characteristics and biosynthesis of membrane proteins of lipid bodies in the scuttela of maize. Biochem J. 235, 57-65.
Rohde, D6rr, Salamini, F. and Becker, B. (1991) Structure of a chalcone synthase gene from Hordeum vulgare. Plant Mol. Biol. 16, 1103-1106.
Roth, Goff, Klein, T.M. and Fromm, M.E. (1991) Cl- and R-dependent expression of the maize Bzl gene requires sequences with homologies to mammalian myb and myc binding sites. Plant Cell 3, 317-325.
WO 95/15389 WO 9515389PCTIN094100190 34 Samibrook, Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Schmelzer, Jahnen, W. and Hahibrock, K. (1988) In situ localization of light-induced chalcone synthetase mRNA, chalcone synthetase, and flavonoid end products in epidernmal cells of parsley leaves. Proc. Natl. Acad. Sd. U.S.A 85, 2989-2993.
Selden, R.F. (1987) Analysis of RNA by Northern Hybridization. In Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds.). New York: Green Publishing Associates and Wiley-Interscience, pp. 4.9.1-4.9.8.
Shimamoto, Terada, Izawa, T. and Fujimoto, H. (1989) Fertile transgenic rice plants regenerated from transformed protoplasts. Nature 338, 274-276.
Skriver, Leahi, Mfilier-IUri, Olsen, and Mundy, J. (1992) Structure and expression.
Slakeski, N. and Fincher, G.B. (1992) Developmental regulation of l-4)-b-glucanase gene expression in barley. Plant Physiol. 99, 1226-123 1.
Smith, Handley, J1., Li, Martin, Donovan, L. and Bowles, D.J. (1992) Temporal and spatial regulation of a novel gene in barley embryos. Plant Mol. Biol. 20, 255- 266.
Sonissich, Scinneizer, Kawalieck, P. and Halilbrock, K. (1988) Gene structure and in situ transcript localization of the pathogenesis-related protein 1 in parsley. Mol. Gen.
Genet. 213, 93-98.
Sossountzov, Riuz-Avila, Vignois, Jolliot, Arondel, Tchang, F., Grosbois, Guerbette, Miginiac, Deisney, Puigdomenech, P. and Kader, (1991) Spatial and temporal expression pattern of a maize lipid transfer protein gene.
Plant Cell 3, 923-933.
Sterk, BooUj, Schellekens, Van Kamnien, A. and De Vries, S.C. (1991) Cell-specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell 3, 907-921.
Terada, R. and Shimamoto,K. (1990) Expression of CaMV35S-GUS gene in transgenic rice plants. Mol. Gen. Genet. 220, 3 89-392.
Terada,R., Nakayama,T., Iwabuchi,M., Shiniamoto, K. (1993) A wheat histone H3 promoter confers cell division-dependent and -independent expression of the GUSA gene in transgenic rice plants. Plant J 3: 241-252.
Tchang, This, Stiefel, ArrondeI, Morcb, Pages, Puigdomenech, Greliet, Deisney, Bouillon, Huet, Guerbette, Beauvais-Cante, Duranton, Pernoliet, J.C. and Kader, (1988) Phospholipid transfer protein: Full-length cDNA and amino acid sequence in maize. J. Biol. Chem. 263, 16849-16855.
WO 95/15389 PCTNO94/00190 Thoma,S., Kaneko,Y. and Soinmervile,C. (1993) A non-specificlipid transfer protein from Arabidopsis is a cell wall protein. The Plant Journal 427-436.
von Heijne, G. (1988) Transcending the impenetrable: How proteins come to term with the membranes. Biochim.Biophys. Acta. 947, 307-333.
Watanabe, S. and Yamada, M. (1986) Purification and characterization of a nonspecific lipid transfer protein from germinated castor bean endosperms which transfers phospholipids and galactolipids. Biochim. Biophys. Acta. 876, 116-123.
Weston, K. (1992) Extension of the DNA binding consensus of the chicken c-Myb and v- Myb proteins. Nucleic Acids Res. 20, 3043-3049.
WO 95/15389 WO 9515389PCINO94/00190 36 SEQUENCE LISTING GENERAL INFORMATION BUSINESS ADDRESS: Ti 1’LE OF INVENTION: OLSEN AND R. KALLA PLANT MOLECULAR BIOLOGY LABORATORY DEPARTMENT OF BIOTECHNICAL SCIENCE-S AGRJCTU(RAL UNIVERSITY OF NORWAY AND AGRICULTURAL BIOTECHNOLOGY PROGRAM NRC NORWAY N-1432
PROMOTER
INFORMATION FOR SEQUENCE I.D. 1 SEQUENCE TYPE: MOLECULE TYPE: ORIGINAL SOURCE: SEQUENCE LENGTH:
STRANDEDNESS:
TOPOLOGY:
SEQUENCE:
-807 -780 GTTAACCGTC
AAGCCGATGA
CAAGAGGTT
-660 TCTAGTAGTA AAATATTTfG
ATGTCACTCT
-540 AGGTITTGAC ACAATT1TAT
GTATCACAAA
-420 GAATGTGAAA
‘TTCATGGCAT
TAAGAAAAAA
-300 TCATGAGACA
GGATGATGCG
CGCCTACCGC
-180 CGAACGACCC
GCTACCTTCG
GTGCCCCCGC
GTGCGTGGCT
AGCTAGAAAC
NUCLEIC ACID DNA (GENOMIC)
BARLEY
-807
DOUBLE
LINEAR
GATCTCG
TCTITCGTGAG
GGATAAATAA
ACTCATCAAG
CATCGGACCT
TGCTCAT1TA AGG’TrTGAC
AAATAATITC
TACTTTTA
TGCCACTCTA
AAAAACACTC
GGAAATGTGA
TTGTACTCCT
ATCGCGTITG
CATGAATGGA
CCACTGAGTC
AGCTGACCTC
TCAGCGACGA
ATGCATGGCG
GGCTACAAAT
1TACACCTGC
ATGTGTAGTC
AATAACCGTG
AATGTGGTGG
AGGATGCTT
CACATACCTC
GTGATGGGTA
ATTTCAGTITT
CAITCCGCGG
CCACTCTTAG
GAAATI’CTGT
ACTTATITGA
CATAAAGTAA
CGTAACAAGA
GAAGGCTITG
GTCGTCTGCT
CGGGCGGCAA
TACCGACCGG
TGGCCGCGTA
GCACATGGCG
ACGTACCCCG
TACGAGAAGG
GCCTAAAAAT
TACAGTACTT
TCCGATGAGC
CATrGTGGTG
AA’TTTGTT
TGCCACTCTT
CAAAAGCAAA
CTTTCACAAT
TTATGCCACA
AGCCAAGGTG
CGTTCGTGTA
GACGGAAACA
CATCACCI11r
TGCTAGCCTI’
CTACCATCGG
AQTGAATGC
CGCTGGCGAC
AGCTCAGACC
TGAGTGCCCT
NOTE: ABOVE SEQUENCE IS A RETYPED VERSION OF FIGURE 2A WHICH IS TO BE TAKEN AS THE CORRECT SEQUENCE 1 3 WO 95/15389 PCT/N094/00190 37 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 11 ,line 6 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited (NCIMB) Address of depositary institution (including postal code and country) 23 St. Machar Drive Aberdeen Scotland AB2 1RY United Kingdom Date of deposit Accession Number 22 November 1993 NCIMB 40598 C. ADDITIONAL INDICATIONS (leave blank ifnot applicable) This information is continued on an additional sheet In respect of those designacions in which a European patent is sought, and any other designated state having equivalent legislation, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4)
EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (ifthe indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later (spccifytithgeneralnatureoftheindications ‘Accession Number ofDeposit’) For receiving Office use only For International Bureau use only This sheet was received with the international application This sheet was received by the International Bureau on: Authorized officer Form PCT/RO/134 (July 1992) Authorized officer I r: 1 II I WO 95/15389 PCT/NO94/00190 38 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page 1 line 12 B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [7 Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited (NCIMB) Address of depositary institution (including postal code and country) 23 St. Machar Drive Aberdeen Scotland AB2 1RY United Kingdom Date of deposit Accession Number 22 November 1993 NCIMB 40599 C. ADDITIONAL INDICATIONS (leave blank ifno applicable) This information is continued or an additional sheet In respect of those designations in which a European patent is sought, and any other designated state having equivalent legislation, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4)
EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if lle indicatons are not for all designated Stales) E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) The indications listed belowwill be submitted to the International Bureau later (specify thegencralnature ofthe indications ‘Accession Number of Deposit) S For receiving Office use only For International Bureau use only This sheet was received with the international application This sheet was received by the International Bureau on: .ol Authorized officer Form PCT/RO/134 (July 1992) Authorized officer i r -I I WO 95/15389 PCT/N04/0019 39 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganis.;, referred to in tLe description on page 11 j ,line B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet Name of depositary institution The National Collections or Industrial and Marine Bacteria Limited (NCIMB) Address of depositary institution (including postal code and country) 23 St. Machar Drive Aberdeen Scotland AB2 1RY United Kingdom Date of deposit Accession Number 22 November 1993 NCIMB 40601 C. ADDITIONAL INDICATIONS (leave blank ifnoc applicable) This informition is continued on an additional sheet In respect of those designations in which a European patent is sought, and any other designated state having equivalent legislation, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4)
EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if th indications are nod for all designated States) E. SEPARATE FURNISHING OF INDICATIONS (leave blank ifnot applicable) The indications listed belowwill be submitted to the International Bureau la ter .;cify te generalnature ofthe indications ‘Accession Number of Deposit) 7 For receiving Office use onl’ For International Bureau use only This sheet was eceived with the international application This sheet was received by the International Bureau on: Authorized officer Form PCT/RO/134 (J(t!y 1992) Authorized officer I WO 95/15389 PCT/N094/100190 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM (PCT Rule 13bis) A. The indications made below relate to the microorganism referred to in the description on page «1 ,line 2 B. IDENTIFICATION OF DEt’OS.T Further deposits are identified on an additional sheet Name of depositary institution The National Collections of Industrial and Marine Bacteria Limited (NCIMB) Address of depositary institution (including postal code and country) 23 St. Machar Drive Aberdeen Scotland AB2 1RY United Kingdom Date of deposit Accession Number 22 November 1993 NCIMB 40C00 C. ADDITIONAL INDICATIONS (leave blank ifnot applicable) This information is continued on an additional sheet In respect of those designations in which a European patent is sought, and any other designated state having equivalent legislation, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4)
EPC).
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (iftlle indications are not for all designated Sates) E. SEPARATE FURNISHING OF INDICATIONS (cave blank ifnot applicable) The indications listed belowwillbesubmitted to the International Bureau later (specify itegeneralnatt, ofthe indications ‘Accession Number ofDposit’) For receiving Office use only For International Bureau use only SThis sheet was received with the international application This sheet was received by the International Bureau on: Authorized officer Form PCT/RO/134 (July 1992) Authorized officer

Claims (12)

1. An in vivo expression system comprising a conjugate comprising a GOI and a Ltp2 gene promoter comprising the sequence shown in SEQ. I.D. 1, or a sequence that has substantial homology with that of SEQ. I.D. 1 as hereinbefore defined, or a variant thereof as hereinbefore defined; wherein the conjugate is integrated, preferably stably integrated, within a monocotyledon’s genomic DNA; and wherein use of the Ltp2 gene promoter results in specific aleurone expression of the GOI.

2. A transgenic cereal comprising a conjugate comprising a GOI and a Ltp2 gene promoter as defined in claim 1 wherein the conjugate is integrated, preferably stably integrated, within a cereal’s genomic DNA, and wherein use of the Ltp2 gene promoter results in specific aleurone expression of the GOI.

3. The in vivo expression in the aleurone cells of a monocotyledon of a conjugate comprising a GOI and a Ltp2 gene promoter as defined in claim 1; wherein the conjugate is integrated, preferably stably integrated, within the monocotyledon’s genomic DNA. g oo*

4. A method of enhancing in vivo expression of a GOI in just the aleurone cells of a monocotyledon which comprises stably inserting into the genome of those cells a DNA conjugate comprising a Ltp2 gene promoter as defined in claim 1 and a GOI; wherein in the formation of the conjugate the Ltp2 gene promoter is ligated to the GOI in such a manner that each of the myb site and the myc site in the Ltp2 gene promoter is maintained substantially intact. The invention of any one of claims 1 to 4 wherein the promoter is a barley aleurone specific promoter.

S

6. The invention of claim 5 wherein the promoter is for a 7 kDa lipid transfer q t a~ protein.

7. The invention of any one of claims 1 to 6 wherein the promoter is used for expression of a GOI in a cereal seed.

8. The invention of any one of claims 1 to 7 wherein the promoter is used for expression of a GOI in a transgenic cereal seed.

9. The invention of any one of claims 1 to 8 wherein the cereal seed is any one of a rice, maize, wheat, or barley seed, preferably maize.

10. The invention of any one of claims 1 to 9 wherein the promoter is the promoter for Ltp2 of Hordeum vulgare.

S11. The invention according to any one of the preceding claims wherein the conjugate further comprises at least one additional sequence to increase expression of a GOI or the GOI.

12. The invention according to any one of the preceding claims wherein the conjugate is stably integrated within the genome of a developing grain. DATED this 28th day of October 1997. ODD-ARNE OLSEN and ROGER KALLA WATERMARK PATENT TRADEMARK ATTORNEYS 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA KJS:CJH:JL VAX- doc 017 AU1250695.WPC

AU12506/95A
1993-12-02
1994-11-24
Promoter

Ceased

AU686291B2
(en)

Applications Claiming Priority (3)

Application Number
Priority Date
Filing Date
Title

GB9324707

1993-12-02

GB939324707A

GB9324707D0
(en)

1993-12-02
1993-12-02
Promoter

PCT/NO1994/000190

WO1995015389A2
(en)

1993-12-02
1994-11-24
Promoter

Publications (2)

Publication Number
Publication Date

AU1250695A

AU1250695A
(en)

1995-06-19

AU686291B2
true

AU686291B2
(en)

1998-02-05

Family
ID=10745996
Family Applications (1)

Application Number
Title
Priority Date
Filing Date

AU12506/95A
Ceased

AU686291B2
(en)

1993-12-02
1994-11-24
Promoter

Country Status (8)

Country
Link

US
(1)

US5525716A
(en)

EP
(1)

EP0731844B1
(en)

AT
(1)

ATE302281T1
(en)

AU
(1)

AU686291B2
(en)

CA
(1)

CA2110772A1
(en)

DE
(1)

DE69434458T2
(en)

GB
(1)

GB9324707D0
(en)

WO
(1)

WO1995015389A2
(en)

Families Citing this family (170)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

GB9403512D0
(en)

*

1994-02-24
1994-04-13
Olsen Odd Arne
Promoter

JPH10504706A
(en)

*

1994-06-06
1998-05-12
プラント・ジェネティック・システムズ・エヌ・ブイ

Use of anthocyanin genes for maintenance of male sterile plants

US6008437A
(en)

*

1995-06-06
1999-12-28
Plant Genetic Systems
Use of anthocyanin genes to maintain male sterile plants

WO1998008961A2
(en)

*

1996-08-30
1998-03-05
Olsen Odd Arne
Endosperm and nucellus specific genes, promoters and uses thereof

US6939958B1
(en)

1998-05-07
2005-09-06
Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture And Agri-Food
Regulatory region of a lipid transfer protein (LtpW1) from aleuron tissue of wheat

US6326528B1
(en)

*

1998-05-07
2001-12-04
Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of Agriculture
Wheat aleurone regulatory region

CA2230975C
(en)

*

1998-05-07
2007-02-20
Sharon Allard
Wheat aleurone promoter

US6307123B1
(en)

*

1998-05-18
2001-10-23
Dekalb Genetics Corporation
Methods and compositions for transgene identification

US6462257B1
(en)

1999-06-01
2002-10-08
Institute Of Paper Science And Technology, Inc.
Vicilin-like seed storage protein gene promoter and methods of using the same

JP3645522B2
(en)

1999-06-07
2005-05-11
ビーエーエスエフ プラント サイエンス ゲーエムベーハー

Δ6-acetylenase and Δ6-desaturase of Ceratodon purpures

DE10030976A1
(en)

2000-06-30
2002-01-10
Basf Ag
Production of unsaturated fatty acids, useful e.g. in nutrition, cosmetics or pharmaceuticals, in organisms transformed with Physcomitrella patens delta-6-desaturase nucleic acid

PL207026B1
(en)

2000-02-09
2010-10-29
Basf Ag
Novel elongase gene and method for producing multiple-unsaturated fatty acids

CA2789254A1
(en)

2000-04-07
2001-10-18
Basf Plant Science Gmbh
Signal transduction stress-related proteins and methods of use in plants

RU2286387C2
(en)

*

2000-07-07
2006-10-27
Эйсай Ко., Лтд.
Gene of fungus cell wall synthesis

AU2002246585A1
(en)

*

2000-11-09
2002-07-30
Washington State University Research Foundation
Plant promoters, and methods of use

CA2441265A1
(en)

2001-03-16
2002-09-26
Basf Plant Science Gmbh
Sugar and lipid metabolism regulators in plants

GB0107510D0
(en)

2001-03-26
2001-05-16
Univ Bristol
New elongase gene and a process for the production of -9-polyunsaturated fatty acids

EP1392104B1
(en)

2001-06-04
2012-08-15
BASF Plant Science GmbH
Sugar and lipid metabolism regulators in plants ii

US7294759B2
(en)

2001-06-29
2007-11-13
E. I. Du Pont De Nemours And Company
Alteration of oil traits in plants

AU2002355746A1
(en)

*

2001-08-02
2003-02-17
Pioneer Hi-Bred International, Inc.
Methods for improving seed and grain characteristics

MXPA04001012A
(en)

2001-08-09
2005-06-06
Univ Saskatchewan
Wheat plants having increased resistance to imidazolinone herbicides.

EP2604107B1
(en)

2001-08-09
2018-10-10
Northwest Plant Breeding Company
Wheat plants having increased resistance to imidazolinone herbicides

CA2456311C
(en)

2001-08-09
2014-04-08
University Of Saskatchewan
Wheat plants having increased resistance to imidazolinone herbicides

EP1414288B1
(en)

2001-08-10
2009-03-25
BASF Plant Science GmbH
Sugar and lipid metabolism regulators in plants iii

US7456335B2
(en)

2001-09-03
2008-11-25
Basf Plant Science Gmbh
Nucleic acid sequences and their use in methods for achieving pathogen resistance in plants

US7091402B2
(en)

2001-09-05
2006-08-15
Basf Plant Science Gmbh
Protein phosphates stress-related polypeptides and methods of use in plants

EP1451325B1
(en)

2001-11-09
2011-05-18
BASF Plant Science GmbH
Transcription factor stress-related polypeptides and methods of use in plants

GB2385852A
(en)

2002-02-27
2003-09-03
Rothamsted Ex Station
Delta 6-desaturases from Primulaceae

DE10212892A1
(en)

2002-03-20
2003-10-09
Basf Plant Science Gmbh

Constructs and methods for regulating gene expression

WO2003095655A2
(en)

2002-05-08
2003-11-20
Basf Plant Science Gmbh
Methods for increasing oil content in plants

EP2329708B1
(en)

2002-07-10
2016-10-19
The Department of Agriculture, Western Australia
Wheat plants having increased resistance to imidazolinone herbicides

EP2036984B1
(en)

2002-07-26
2012-02-22
BASF Plant Science GmbH
Reversion of the negative selective effect of negative marker proteins as selection procedure

EP2272966A3
(en)

2002-08-02
2011-07-06
BASF Plant Science GmbH
Sugar and lipid metabolism regulators in plants IV

AU2003246349B2
(en)

2002-08-07
2011-02-03
Basf Plant Science Gmbh
Nucleic acid sequences encoding proteins associated with abiotic stress response

AU2003294924A1
(en)

2002-12-20
2004-07-14
Metanomics Gmbh & Co. Kgaa
Method for producing aminoacids

EP2322633A3
(en)

2003-02-17
2011-08-17
Metanomics GmbH
Preparation of organisms with faster growth and/or higher yield

WO2004076617A2
(en)

2003-02-27
2004-09-10
Basf Plant Science Gmbh
Method for the production of polyunsaturated fatty acids

EP2365063B1
(en)

2003-03-31
2015-10-14
University Of Bristol
New vegetable acyltransferases specifically for long-chain polyunsaturated fatty acids

WO2004092398A2
(en)

2003-04-15
2004-10-28
Basf Plant Science Gmbh
Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stress

ES2389767T3
(en)

2003-05-28
2012-10-31
Basf Se

Wheat plants that have higher tolerance to imidazolinone herbicides

AU2004265250C1
(en)

2003-06-23
2008-05-01
Pioneer Hi-Bred International, Inc.
Engineering single-gene-controlled staygreen potential into plants

EP2434019A1
(en)

2003-08-01
2012-03-28
BASF Plant Science GmbH
Process for the production of fine chemicals

EP2166068B1
(en)

2003-08-01
2016-01-06
BASF Plant Science GmbH
Method for producing polyunsaturated fatty acids in transgenic organisms

WO2008015263A2
(en)

2006-08-02
2008-02-07
Cropdesign N.V.
Plants having improved characteristics and a method for making the same

CN1832961B
(en)

2003-08-11
2011-04-06
植物育种农业研究所有限公司
Fungus resistant plants and their uses

AR047107A1
(en)

2003-08-29
2006-01-11
Inst Nac De Tecnologia Agropec

RICE PLANTS THAT HAVE A GREATER TOLERANCE TO IMIDAZOLINONA HERBICIDES

BRPI0418048A
(en)

2003-12-23
2007-04-17
Basf Plant Science Gmbh

isolated lmp nucleic acid, expression vector, and methods for producing a transgenic plant and modulating the level of a seed storage compound in a plant

US9458436B2
(en)

2004-02-27
2016-10-04
Basf Plant Science Gmbh
Method for producing polyunsaturated fatty acids in transgenic plants

WO2005083053A2
(en)

2004-02-27
2005-09-09
Basf Plant Science Gmbh
Method for producing unsaturated omega-3 fatty acids in transgenic organisms

BR122019013200B1
(en)

2004-04-22
2022-04-12
Commonwealth Scientific And Industrial Research Organisation

ISOLATED POLYNUCLEOTIDE, VECTOR, RECOMBINANT MICROBIAL CELL CAPABLE OF SYNTHESISTING LONG-CHAIN POLYUNSATURATED FATTY ACID(S), METHOD FOR PRODUCTION OF SUCH RECOMBINANT MICROBIAL CELL, METHOD FOR PRODUCTION OF POLYUNSATURATED FATTY ACID(S) S) LONG CHAIN, METHOD FOR PRODUCTION OF OIL, METHOD FOR PRODUCTION OF OIL SEED, TRANSGENIC SEED EXTRACT, COMPOSITION, RATION AND USE OF SUCH RECOMBINANT CELL, VECTOR OR EXTRACT

EP2527362A1
(en)

2004-06-16
2012-11-28
BASF Plant Science GmbH
Nucleic acid molecules encoding wrinkled1-like polypeptides and methods of use in plants

WO2006069610A2
(en)

2004-07-02
2006-07-06
Metanomics Gmbh
Process for the production of fine chemicals

BRPI0514003A
(en)

2004-07-31
2008-05-20
Metanomics Gmbh

methods for preparing a non-human organism, preparing a vector, a transgenic plant, a plant cell, a plant tissue, a useful animal’s cell, a useful animal or a transgenic microorganism, and fine chemicals, and for identifying a gene product, polynucleotide , vector, host cell, antibody, antisense nucleic acid, non-human organism, transgenic microorganism, seed, tuber or propagation material of a plant, biomass of a microorganism, plant cell, plant cell organelle, plant tissue, plant or part thereof , and, uses of a polypeptide, and nucleic acid molecule

CN101076594A
(en)

2004-09-20
2007-11-21
巴斯福植物科学有限公司
Arabidopsis genes encoding proteins involved in sugar and lipid metabolism and methods of use

EP1794184A2
(en)

2004-09-24
2007-06-13
BASF Plant Science GmbH
Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stress

EP1794304B1
(en)

2004-09-24
2013-06-19
BASF Plant Science GmbH
Plant cells and plants with increased tolerance to environmental stress

EP1974049A2
(en)

2004-12-17
2008-10-01
Metanomics GmbH
Process for the control of production of fine chemicals

US20140199313A1
(en)

2005-03-02
2014-07-17
Metanomics Gmbh
Process for the Production of Fine Chemicals

CA2598792A1
(en)

2005-03-02
2006-09-08
Metanomics Gmbh
Process for the production of fine chemicals

EP1859037A2
(en)

2005-03-08
2007-11-28
BASF Plant Science GmbH
Expression enhancing intron sequences

DE102005013779A1
(en)

2005-03-22
2006-09-28
Basf Plant Science Gmbh

Process for the preparation of polyunsaturated C20 and C22 fatty acids having at least four double bonds in transgenic plants

EP1874936B1
(en)

2005-04-19
2013-10-30
BASF Plant Science GmbH
Improved methods controlling gene expression

US7550579B2
(en)

*

2005-04-29
2009-06-23
Pioneer Hi-Bred International, Inc.
Pericarp-preferred regulatory element

WO2006134162A2
(en)

2005-06-17
2006-12-21
Basf Plant Science Gmbh
Lecitin-like protein kinase stress-related polypeptides and methods of use in plants

EP1896594B1
(en)

2005-06-23
2011-01-12
BASF Plant Science GmbH
Improved methods for the production of stably transformed, fertile zea mays plants

AU2006264937B2
(en)

2005-07-06
2011-01-27
Cropdesign N.V.
Plant yield improvement by Ste20-like gene expression

AU2006270321B2
(en)

2005-07-18
2012-03-08
Basf Plant Science Gmbh
Yield increase in plants overexpressing the ACCDP genes

CN101228278A
(en)

2005-07-18
2008-07-23
巴斯福植物科学有限公司
Yield increase in plants overexpressing the SHSRP genes

EP1915452A2
(en)

2005-08-12
2008-04-30
BASF Plant Science GmbH
Nucleic acid sequences encoding proteins associated with abiotic stress response and plant cells and plants with increased tolerance to environmental stress

WO2007031581A2
(en)

2005-09-15
2007-03-22
Cropdesign N.V.
Plant yield improvement by group 3 lea expression

CN101268194A
(en)

2005-09-20
2008-09-17
巴斯福植物科学有限公司
Methods for controlling gene expression using ta-siRNA

GB2431158A
(en)

2005-10-13
2007-04-18
Rothamsted Res Ltd
Process for the production of arachidonic and/or eicosapentaenoic acid

DE102005052551A1
(en)

2005-11-02
2007-05-16
Rothamsted Res Harpenden

Process for the preparation of γ-linolenic acid and / or stearidonic acid in transgenic Brassicaceae and Linaceae

WO2007054441A2
(en)

2005-11-08
2007-05-18
Basf Plant Science Gmbh
Use of armadillo repeat (arm1) polynucleotides for obtaining pathogen resistance in plants

DK1965658T3
(en)

2005-11-18
2019-01-21
Commw Scient Ind Res Org

FEATURES FOR AQUACULTURE INCLUDING STEARIDONIC ACID

US7723574B2
(en)

2005-11-24
2010-05-25
Basf Plant Science Gmbh
Process for the production of Δ5-unsaturated fatty acids in transgenic organisms

US20070130644A1
(en)

*

2005-12-07
2007-06-07
Peter Hajdukiewicz
Oryza sativa LTP promoters useful for modulating gene expression in plants

EP2314606A1
(en)

2005-12-09
2011-04-27
BASF Plant Science GmbH
Nucleic acid molecules encoding polypeptides involved in regulation of sugar and lipid metabolism and methods of use VIII

US8178751B2
(en)

2006-01-12
2012-05-15
Basf Plant Science Gmbh
Use of stomatin (STM1) polynucleotides for achieving a pathogen resistance in plants

GB0603160D0
(en)

2006-02-16
2006-03-29
Rothamsted Res Ltd
Nucleic acid

EP2343376A3
(en)

2006-03-24
2011-11-23
BASF Plant Science GmbH
Proteins associated with abiotic stress response and homologs

US8642838B2
(en)

2006-03-31
2014-02-04
Basf Plant Science Gmbh
Plants having enhanced yield-related traits and a method for making the same

EP2090662A3
(en)

2006-04-05
2012-10-31
Metanomics GmbH
Process for the production of a fine chemical

EP2441839A1
(en)

2006-05-30
2012-04-18
CropDesign N.V.
Plants with reduced expression of REVOLUTA (REV) having enhanced yield-related traits and a method for making the same

ES2402309T3
(en)

2006-05-31
2013-04-30
Metanomics Gmbh

Nitrogen metabolism manipulation using ammonium transporter or glucose 6-phosphate dehydrogenases or farnesyl phosphate synthetases (FPP)

CN103215288A
(en)

2006-06-08
2013-07-24
巴斯福植物科学有限公司
Plants having improved growth characteristics and method for making the same

EP2500420B1
(en)

2006-08-24
2016-06-22
BASF Plant Science GmbH
Pythium omega 3 desaturase with specificity to all omega 6 fatty acids longer than 18 carbon chains

WO2008025711A2
(en)

2006-08-30
2008-03-06
Basf Plant Science Gmbh
Method for increasing resistance to pathogens in transgenic plants

US8710299B2
(en)

2006-10-06
2014-04-29
Basf Plant Science Gmbh
Processes for producing polyunsaturated fatty acids in transgenic organisms

EP2078087A1
(en)

2006-10-12
2009-07-15
BASF Plant Science GmbH
Method for increasing pathogen resistance in transgenic plants

CN101589148B
(en)

2006-10-13
2014-07-02
巴斯福植物科学有限公司
Plants with increased yield

CA2673361A1
(en)

2007-01-15
2008-07-24
Basf Plant Science Gmbh
Use of subtilisin (rnr9) polynucleotides for achieving a pathogen resistance in plants

MX2009007811A
(en)

2007-02-06
2009-07-31
Basf Plant Science Gmbh
Use of alanine racemase genes to confer nematode resistance to plants.

US20100011463A1
(en)

2007-02-06
2010-01-14
Basf Plant Science Gmbh
Compositions and Methods Using RNA Interference for Control of Nematodes

MX2009007766A
(en)

2007-02-09
2009-07-30
Basf Plant Science Gmbh
Compositions and methods using rna interference of cdpk-like for control of nematodes.

WO2008110522A1
(en)

2007-03-15
2008-09-18
Basf Plant Science Gmbh
Use of nematode chitinase genes to control plant parasitic nematodes

AU2008231785A1
(en)

2007-03-23
2008-10-02
Basf Plant Science Gmbh
Transgenic plant with increased stress tolerance and yield

EP2492345A3
(en)

2007-05-04
2012-12-12
BASF Plant Science GmbH
Seed enhancement by combinations of pyruvate kinases

CN102770542A
(en)

2007-05-22
2012-11-07
巴斯夫植物科学有限公司
Plants with increased tolerance and/or resistance to environmental stress and increased biomass production

WO2008142036A2
(en)

2007-05-22
2008-11-27
Basf Plant Science Gmbh
Plant cells and plants with increased tolerance and/or resistance to environmental stress and increased biomass production-ko

EP2152886A2
(en)

2007-05-29
2010-02-17
BASF Plant Science GmbH
Transgenic plants with increased stress tolerance and yield

MX2009013648A
(en)

2007-07-13
2010-01-27
Basf Plant Science Gmbh
Transgenic plants with increased stress tolerance and yield.

AR067795A1
(en)

2007-08-02
2009-10-21
Basf Plant Science Gmbh

TRANSGENIC PLANTS WITH HIGHER STRESS TOLERANCE AND HIGHER PERFORMANCE

WO2009037279A1
(en)

2007-09-18
2009-03-26
Basf Plant Science Gmbh
Plants with increased yield

EP2594647A3
(en)

2007-09-21
2013-07-24
BASF Plant Science GmbH
Plants with increased yield

AU2008328818A1
(en)

2007-11-27
2009-06-04
Basf Plant Science Gmbh
Transgenic plants with increased stress tolerance and yield

EP2231861B1
(en)

2007-12-13
2014-10-29
Philip Morris Products S.A.
Transgenic plants modified for reduced cadmium transport, derivative products, and related methods

DE112008003237T5
(en)

2007-12-14
2011-03-17
Basf Plant Science Gmbh

Promoters of Brassica napus for seed-specific gene expression

US20110035841A1
(en)

2007-12-17
2011-02-10
Basf Plant Science Gmbh
Lipid Metabolism Proteins, Combinations of Lipid Metabolism Proteins and Uses Thereof

US20110041215A1
(en)

*

2007-12-17
2011-02-17
Basf Plant Science Gmbh
Lipid Metabolism Protein And Uses Thereof I (BZIP Transcription Factor)

CN101952305B
(en)

2007-12-19
2014-12-24
巴斯夫植物科学有限公司
Plants with increased yield and/or increased tolerance to environmental stress (IV-BM)

AR069894A1
(en)

2007-12-21
2010-02-24
Basf Plant Science Gmbh

PLANTS WITH GREATER PERFORMANCE PRODUCING A KNOCK OUT (SILENCING) IN GENES RELATED TO EFFICIENCY IN THE USE OF NITROGEN (KO NUE) »

WO2009106596A2
(en)

2008-02-27
2009-09-03
Basf Plant Science Gmbh
Plants with increased yield

WO2009129583A1
(en)

2008-04-25
2009-10-29
Commonwealth Scientific Industrial Research Organisation
Recombinant cells and methods for hydroxylating fatty acids

WO2009147127A1
(en)

2008-06-03
2009-12-10
Basf Plant Science Gmbh
Fatty acid dehydratases and uses thereof

US8853383B2
(en)

2008-07-01
2014-10-07
Basf Plant Science Gmbh
Promoters from Brassica napus for seed specific gene expression

EP2331695B1
(en)

2008-08-26
2014-07-23
BASF Plant Science GmbH
Nucleic acids encoding desaturases and modified plant oil

CN102124025A
(en)

2008-08-27
2011-07-13
巴斯夫植物科学有限公司
Nematode-resistant transgenic plants

CA2736473A1
(en)

2008-09-23
2010-04-01
Basf Plant Science Gmbh
Plants with increased yield (lt)

CN102264907A
(en)

2008-10-23
2011-11-30
巴斯夫植物科学有限公司
Plants with increased yield (NUE)

CN102203262A
(en)

2008-10-23
2011-09-28
巴斯夫植物科学有限公司
A method for producing a transgenic cell with increased gamma-aminobutyric acid (gaba) content

CA2743880C
(en)

2008-11-18
2020-08-18
James Robertson Petrie
Recombinant cells comprising exogenous a5 elongase and methods for producing docosapentaenoic acid

ES2435572T3
(en)

2008-12-12
2013-12-20
Basf Plant Science Gmbh

Desaturases and process for the production of polyunsaturated fatty acids in transgenic organisms

EP2430166A1
(en)

2009-05-13
2012-03-21
BASF Plant Science Company GmbH
Acyltransferases and uses thereof in fatty acid production

JP2012529277A
(en)

2009-06-08
2012-11-22
ロザムステッド リサーチ リミテッド

Novel fatty acid elongation components and uses thereof

CA3037072C
(en)

2009-07-17
2022-07-26
Basf Plant Science Company Gmbh
Novel fatty acid desaturases and elongases and uses thereof

EP2456873A1
(en)

2009-07-23
2012-05-30
BASF Plant Science Company GmbH
Plants with increased yield

DE112010003389T5
(en)

2009-08-25
2012-06-14
Basf Plant Science Company Gmbh

Nematode resistant transgenic plants

CA2772267A1
(en)

2009-08-31
2011-03-03
Basf Plant Science Company Gmbh
Regulatory nucleic acid molecules for enhancing seed-specific gene expression in plants promoting enhanced polyunsaturated fatty acid synthesis

MX2012005719A
(en)

2009-11-17
2012-07-30
Basf Plant Science Co Gmbh
Plants with increased yield.

US9347049B2
(en)

2009-11-24
2016-05-24
Basf Plant Science Company Gmbh
Fatty acid elongase and uses thereof

EP2504427B1
(en)

2009-11-24
2018-06-27
BASF Plant Science Company GmbH
Novel fatty acid desaturase and uses thereof

AU2010330050A1
(en)

2009-12-09
2012-07-19
Basf Plant Science Company Gmbh
Methods for increasing the resistance of plants to fungi by silencing the fungal SMT1-gene

US20130091598A1
(en)

2010-02-23
2013-04-11
Basf Plant Science Company Gmbh
Nematode-Resistant Transgenic Plants

CN102884185A
(en)

2010-05-06
2013-01-16
先锋国际良种公司
Maize ACC Synthase 3 gene and protein and uses thereof

WO2011161093A1
(en)

2010-06-25
2011-12-29
Basf Plant Science Company Gmbh
Acyltransferases and uses therof in fatty acid production

RU2636344C2
(en)

2010-06-28
2017-11-22
Коммонуэлт Сайентифик энд Индастриал Рисерч Организейшн
Methods for lipids obtaining

EP2695936B1
(en)

2010-10-21
2016-04-20
BASF Plant Science Company GmbH
Novel fatty acid desaturases and uses thereof

WO2012084756A1
(en)

2010-12-20
2012-06-28
Basf Plant Science Company Gmbh
Nematode-resistant transgenic plants

WO2013024121A2
(en)

2011-08-18
2013-02-21
Basf Plant Science Company Gmbh
Increase of sucrose transporter activity in the seeds of plants

US8783588B2
(en)

2011-11-10
2014-07-22
Applied Milling Systems, Inc.
Recovery of aleurone-rich flour from bran

CA2860434C
(en)

2011-12-27
2023-08-08
Commonwealth Scientific And Industrial Research Organisation
Processes for producing lipids

US9347067B2
(en)

2011-12-27
2016-05-24
Commonwealth Scientific And Industrial Research Organisation
Production of dihydrosterculic acid and derivatives thereof

ES2744240T3
(en)

2011-12-27
2020-02-24
Commw Scient Ind Res Org

Gene silencing and suppression of simultaneous gene silencing in the same cell

EP2612918A1
(en)

2012-01-06
2013-07-10
BASF Plant Science Company GmbH
In planta recombination

EP2861059B1
(en)

2012-06-15
2017-05-03
Commonwealth Scientific and Industrial Research Organisation
Production of long chain polyunsaturated fatty acids in plant cells

WO2014020533A2
(en)

2012-08-03
2014-02-06
Basf Plant Science Company Gmbh
Novel enzymes, enzyme components and uses thereof

WO2014117990A1
(en)

2013-01-29
2014-08-07
Basf Plant Science Company Gmbh
Fungal resistant plants expressing hcp6

WO2014118018A1
(en)

2013-01-29
2014-08-07
Basf Plant Science Company Gmbh
Fungal resistant plants expressing ein2

WO2014117988A1
(en)

2013-01-29
2014-08-07
Basf Plant Science Company Gmbh
Fungal resistant plants expressing hcp7

CN105008542B
(en)

2013-03-08
2020-02-07
巴斯夫植物科学有限公司
Antifungal plants expressing MybTF

US9803214B2
(en)

2013-03-12
2017-10-31
Pioneer Hi-Bred International, Inc.
Breeding pair of wheat plants comprising an MS45 promoter inverted repeat that confers male sterility and a construct that restores fertility

EP3083949B1
(en)

2013-12-17
2020-02-12
BASF Plant Science Company GmbH
Methods for conversion of the substrate specificity of desaturases

KR102535223B1
(en)

2013-12-18
2023-05-30
커먼웰쓰 사이언티픽 앤 인더스트리알 리서치 오거니제이션
Lipid comprising long chain polyunsaturated fatty acids

SG11201610596PA
(en)

2014-06-27
2017-01-27
Commw Scient Ind Res Org
Lipid comprising docosapentaenoic acid

WO2016000237A1
(en)

2014-07-03
2016-01-07
Pioneer Overseas Corporation
Plants having enhanced tolerance to insect pests and related constructs and methods involving insect tolerance genes

US10472587B2
(en)

2014-07-07
2019-11-12
Commonwealth Scientific And Industrial Research Organisation
Processes for producing industrial products from plant lipids

CN107257630A
(en)

2014-11-14
2017-10-17
巴斯夫植物科学有限公司
The modification of vegetable lipid comprising PUFA

CA2971425A1
(en)

2014-12-16
2016-06-23
Pioneer Hi-Bred International, Inc.
Restoration of male fertility in wheat

AR103649A1
(en)

2015-02-11
2017-05-24
Basf Se

HYDROXYPHENYL PYRUVATE DIOXYGENASES RESISTANT TO HERBICIDES

WO2017074547A1
(en)

2015-10-30
2017-05-04
Pioneer Hi-Bred International, Inc.
Methods and compositions for rapid plant transformation

AU2016355920B2
(en)

2015-11-18
2020-11-12
Commonwealth Scientific And Industrial Research Organisation
Rice grain with thickened aleurone

JP2020536515A
(en)

2017-09-25
2020-12-17
パイオニア ハイ−ブレッド インターナショナル， インコーポレイテッド

Tissue-preferred promoters and how to use them

US11447786B2
(en)

2017-10-13
2022-09-20
Pioneer Hi-Bred International, Inc.
Systems and methods for cellular reprogramming of a plant cell

CA3089883A1
(en)

2018-02-15
2019-08-22
Monsanto Technology Llc
Compositions and methods for improving crop yields through trait stacking

CN113423837A
(en)

2019-02-14
2021-09-21
嘉吉公司
Brassica plants producing increased levels of polyunsaturated fatty acids

WO2020198408A1
(en)

2019-03-27
2020-10-01
Pioneer Hi-Bred International, Inc.
Plant explant transformation

BR112021020770A2
(en)

2019-04-18
2022-01-04
Pioneer Hi Bred Int

Embryogenesis factors for cellular reprogramming of a plant cell

AU2021355365A1
(en)

2020-09-30
2023-04-06
Corteva Agriscience Llc
Rapid transformation of monocot leaf explants

WO2022256695A1
(en)

2021-06-03
2022-12-08
Mazen Animal Health Inc.
Oral administration of coronavirus spike protein for altering cytokine levels and providing passive immunity to newborn pigs

Family Cites Families (1)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

EP0428572A1
(en)

*

1988-07-29
1991-05-29
Washington University School Of Medicine
Producing commercially valuable polypeptides with genetically transformed endosperm tissue

1993

1993-12-02
GB
GB939324707A
patent/GB9324707D0/en
active
Pending

1993-12-06
CA
CA002110772A
patent/CA2110772A1/en
not_active
Abandoned

1993-12-10
US
US08/165,315
patent/US5525716A/en
not_active
Expired – Lifetime

1994

1994-11-24
AT
AT95903463T
patent/ATE302281T1/en
not_active
IP Right Cessation

1994-11-24
AU
AU12506/95A
patent/AU686291B2/en
not_active
Ceased

1994-11-24
WO
PCT/NO1994/000190
patent/WO1995015389A2/en
active
IP Right Grant

1994-11-24
DE
DE69434458T
patent/DE69434458T2/en
not_active
Expired – Fee Related

1994-11-24
EP
EP95903463A
patent/EP0731844B1/en
not_active
Expired – Lifetime

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party

Title

AMERICAN ASSOC. CEREAL CHEMISTS, 1992, PP 236-245
*

KEYSTONE SYMPOSIUM ON EVOLUTION AND PLANT DEVELOPMENT, 1993, 17 PT B, PP 33
*

Also Published As

Publication number
Publication date

WO1995015389A2
(en)

1995-06-08

EP0731844B1
(en)

2005-08-17

DE69434458T2
(en)

2006-06-08

EP0731844A1
(en)

1996-09-18

AU1250695A
(en)

1995-06-19

WO1995015389A3
(en)

1995-06-29

DE69434458D1
(en)

2005-09-22

ATE302281T1
(en)

2005-09-15

CA2110772A1
(en)

1995-06-03

US5525716A
(en)

1996-06-11

GB9324707D0
(en)

1994-01-19

Similar Documents

Publication
Publication Date
Title

AU686291B2
(en)

1998-02-05

Promoter

WO1995015389A9
(en)

1995-07-06

Promoter

AU695526B2
(en)

1998-08-13

Promoter from a lipid transfer protein gene

Kalla et al.

1994

The promoter of the barley aleurone‐specific gene encoding a putative 7 kDa lipid transfer protein confers aleurone cell‐specific expression in transgenic rice

Hattori et al.

1995

Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis‐acting promoter elements required for regulation by abscisic acid and VP1

Hatfield et al.

1997

The ubiquitin‐activating enzyme (E1) gene family in Arabidopsis thaliana

US5712112A
(en)

1998-01-27

Gene expression system comprising the promoter region of the alpha-amylase genes

US5460952A
(en)

1995-10-24

Gene expression system comprising the promoter region of the α-amylase genes

JPH10504968A
(en)

1998-05-19

Acetohydroxy acid synthase promoter for expression of genes introduced into plants

US5767379A
(en)

1998-06-16

Commercial production of avidin in plants

MXPA02003098A
(en)

2002-09-30

Seed preferred promoter from maize.

WO1997017455A9
(en)

1998-05-22

Commercial production of avidin in plants

US20040244072A1
(en)

2004-12-02

Leaf specific gene promoter of coffee

Graham et al.

1990

Developmental regulation of expression of the malate synthase gene in transgenic plants

Klemsdal et al.

1991

Primary structure of a novel barley gene differentially expressed in immature aleurone layers

AU2009284691B2
(en)

2015-07-09

Seed active transcriptional control sequences

JP2001524315A
(en)

2001-12-04

Methods and compositions useful for silent transgene activation

Kojima et al.

2000

Analysis of a promoter for the NADH–glutamate synthase gene in rice (Oryza sativa): cell type-specific expression in developing organs of transgenic rice plants

AU713340B2
(en)

1999-12-02

Promoter from tobacco

US8648229B2
(en)

2014-02-11

Plant seed active transcriptional control sequences

CZ20021139A3
(en)

2002-06-12

Specific albumen promoters of plant grains

US7557264B2
(en)

2009-07-07

Gossypium hirsutum tissue-specific promoters and their use

Voronin et al.

2001

Temporal and tissue-specific expression of the tobacco ntf4 MAP kinase

US6794559B2
(en)

2004-09-21

Promoters for gene expression in caryopses of plants

AU2006308436B2
(en)

2012-08-16

Specific expression using transcriptional control sequences in plants

Download PDF in English