HU0303050A2 - Plant transcription factors - Google Patents

Plant transcription factors Download PDF

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HU0303050A2
HU0303050A2 HU0303050A HU0303050A HU0303050A2 HU 0303050 A2 HU0303050 A2 HU 0303050A2 HU 0303050 A HU0303050 A HU 0303050A HU 0303050 A HU0303050 A HU 0303050A HU 0303050 A2 HU0303050 A2 HU 0303050A2
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HU0303050A
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HU0303050A3 (en
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Rebecca E. Cahoon
Theodore M. Klein
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E. I. Du Pont De Nemours And Co.
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Priority to PCT/US2001/050908 priority patent/WO2002057439A2/en
Publication of HU0303050A2 publication Critical patent/HU0303050A2/en
Publication of HU0303050A3 publication Critical patent/HU0303050A3/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/11Specially adapted for crops
    • Y02A40/14Specially adapted for crops with increased yield
    • Y02A40/146Transgenic plants

Abstract

In particular, the present invention relates to nucleic acid fragments encoding transcription factors in plants and seeds, polypeptides encoded by fragments, and methods for their preparation. The present invention is suitable for the development of research model systems for plant embryogenesis as well as for genetic manipulation of plant growth. HE

Description

Extract

The present invention relates to the field of plant molecular biology. More particularly, the invention relates to nucleic acid fragments encoding transcription factors in plants and seeds, polypeptides encoded by fragments, and methods for their preparation.

The present invention is suitable for the development of research model systems for the study of plant embryogenesis and for the genetic manipulation of plant growth.

Plant Transcription Factors

The present invention relates to the field of plant molecular biology. More particularly, the invention relates to nucleic acid fragments encoding transcription factors in plants and seeds, polypeptides encoded by fragments, and methods for their preparation.

The present invention is suitable for the development of research model systems for the study of plant embryogenesis and for the genetic manipulation of plant growth.

In higher plants, embryogenesis is conceptually divided into two different phases: early morphogenetic processes that provide for the formation of embryonic cell types, tissues, and organ systems, and late maturation events that allow a fully adult embryo to enter a dehydrated and metabolically inactive state. state. After the appropriate signals arrive, the sleeping embryo germinates and the development of the seedling begins. Thus, the maturation of the nucleus and the metabolic rest period disrupt the morphogenetic processes that occur during embryogenesis and seed development. This unique form of development is based, in part, on the ability of the plant to produce seeds, which has a significant selection advantage for higher plants. Since lower plants do not produce seeds and do not go through the embryo maturation phase, it is assumed that this two-phase mode of embryogenesis comes from the incorporation of mutation events into the life cycle of higher plants. Little is known at the mechanism level about how the different processes that occur during the morphogenesis and seed ripening phases co-ordinate.

The leaf-like cotyledonl gene (leaf-like cotyledon 1 gene, LEC1) regulates many different aspects of embryogenesis. The lecl mutation is a pleiotropic, suggesting that LECl plays a role in late embryo development. For example, LEC1 is required for specific aspects of seed ripening, inhibiting premature germination and playing a role in the specification of embryonic identity. Finally, in the case of LEC1, it seems to work only during embryo development. For two other LEC classes, LEC2 and FUSCA3 (FUS3), we assume that they have functions similar to or overlapping with those of LEC1, including specification of cotyledon identity and retention of maturation. It is not known whether the GC class genes are acting at the molecular level, but their involvement in many different aspects of embryogenesis suggests that these genes are space · · ·

They encode products that serve as regulators in embryonic processes of higher plants. The LEC1 related transcription factors described below all exhibit homology to the B subunit of the DNA binding protein of the corn CAAT box and to the Arabidopsis LEC1 protein (WO 9837184-A) and as such can define a new family of LEC1 transcription factors.

Accordingly, the availability of nucleic acid factors encoding all or part of these LEC 1-related transcription factors may facilitate research into a better understanding of plant embryogenesis and may provide genetic tools for manipulating plant growth.

According to a first embodiment of the present invention, there is provided an isolated polynucleotide comprising: (a) a nucleotide sequence encoding a polypeptide comprising at least 50 or 100 amino acids, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8; Identity between any of the amino acid sequences of SEQ ID NO: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 is at least 90%, 91%, 92 %, 93%

94%, 95%, 96%, 97%, 98% or 99% by the Clustal fitting method, or (b) the complement of the nucleotide sequence, wherein the complementary and nucleotide sequences contain the same number of nucleotides and the complementarity is 100% . Preferably, the polypeptide is a

Any of the amino acid sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 included. The nucleotide sequence is preferably 1, 3, 5,

Any of the nucleotide sequences of SEQ ID NO: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29. Preferably, the polypeptide is a Lec1-related transcription factor.

According to a second embodiment of the invention, the present invention also provides a chimeric gene comprising any of the isolated polynucleotides of the invention linked to a functionally controlling sequence, and the invention further comprises a cell, plant, and nucleus comprising the chimeric gene.

In a third embodiment, the invention further provides a vector comprising any of the isolated polynucleotides of the invention.

According to a fourth embodiment of the invention, the invention also provides an isolated polynucleotide comprising a nucleotide sequence comprising any of the polynucleotides of the first point, wherein the nucleotide sequence comprises at least 30, 40 or 60 nucleotides, and the invention further provides a cell, plant. or a core containing the isolated polynucleotide.

In a fifth embodiment of the invention, the invention further relates to a method for transforming a cell, wherein the cell is transformed with any of the isolated polynucleotides of the invention, and the invention also relates to a cell transformed by this method. Preferably, the cell may be a eukaryotic cell, such as a yeast or plant cell, or may be prokaryotic, such as a bacterium.

According to a sixth embodiment of the present invention, there is also provided a method for producing a transgenic plant in which a plant cell is transformed with any of the isolated polynucleotides of the invention and the plant is regenerated from the transformed plant cell and the invention relates to a transgenic plant produced by this method. seed harvested from a transgenic plant.

According to a seventh embodiment of the present invention, there is also provided an isolated polypeptide having an amino acid sequence of at least 50 or 100 amino acids, wherein the amino acid sequence and the amino acid sequence is 2, 4, 6, 8, 10, 12, 14 ., 16, 18, 20, 22,

Identity between any one of amino acid sequences 24, 26, 28 or 30 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% based on the Clustal fitting method. Preferably, the amino acid sequence is 2, 4, 6, 8, 10, 12,

Contains any of the amino acid sequences of SEQ ID NO: 14, 16, 18, 20, 22, 24, 26, 28 or 30. Preferably, the polypeptide is a Lee 1-related transcription factor.

According to an eighth embodiment of the invention, the invention further relates to a method for isolating a polypeptide, wherein the polypeptide is encoded by any of the polynucleotides of the first point and the cell is transformed with the polynucleotide, resulting in the transformed cell producing the polypeptide and isolating the polypeptide from its transformations. cell.

According to a ninth embodiment of the invention, the invention further provides a virus, preferably a baculovirus, comprising any of the isolated polynucleotides of the invention or any chimeric gene of the invention.

According to a tenth embodiment of the present invention, there is also provided a method for selecting an isolated polynucleotide, which polynucleotide affects the expression level or activity of a gene encoding a Lecloc transcription factor protein in a host cell, preferably in a plant cell, wherein: an isolated polynucleotide or isolated chimeric gene is constructed; (b) introducing the isolated polynucleotide or isolated chimeric gene into a host cell; (c) measuring the level of protein or activity of Lee 1-related transcription factor in the host cell containing the isolated polynucleotide; and (d) ···

Comparison of the Lee 1-related transcription factor protein or activity level in the host cell containing the isolated polynucleotide with Lee 1-related transcription factor protein or activity level in a host cell that does not contain the isolated polynucleotide.

According to an eleventh embodiment of the present invention, there is also provided a method for producing a nucleic acid fragment encoding a substantial portion of Lee 1-related transcription factor protein, preferably a portion of a plant Lee 1-related transcription factor protein, wherein: an oligonucleotide prime is synthesized comprising a nucleotide sequence of at least 30 (preferably at least 40, most preferably at least 60) continuous nucleotides, the nucleotide sequence of which is 1, 3, 5, 7, 9, 11, 13, 15,

May be any of the nucleotide sequences of SEQ ID NOs: 17, 19, 21, 23, 25, 27 and 29, and may be complementary to such nucleotide sequences; and then amplifying the nucleic acid fragment (preferably a cDNA inserted into a cloning vector) using the oligonucleotide prime. Preferably, the amplified nucleic acid fragment encodes a substantial portion of the amino acid sequence of a protein of Lee 1-related transcription fragment.

( According to a twelfth embodiment of the present invention, there is also provided a method for producing a nucleic acid fragment, wherein said nucleic acid fragment encodes the entire amino acid sequence of a Lee 1-related transcription factor protein, or a substantial portion thereof, comprising: testing a cDNA or genomic clone in accordance with the present invention. an isolated polynucleotide; identifying a DNA clone that hybridizes to the isolated polynucleotide of the invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment containing the isolated DNA clone.

In a thirteenth embodiment of the invention, the invention further provides a method for positive selection of transformed cells, comprising: (a) transforming a host cell with the chimeric gene of the present invention or expression kit of the invention; and (b) the transformed host cell, preferably a plant cell, such as a monocot or dicotyledon, is grown under conditions that allow for sufficient expression of the Lecloc transcription factor polynucleotide to complement a null mutant to provide positive selection.

According to a fourteenth embodiment of the present invention, there is provided a method for altering the expression level of Lee 1-related transcription factor protein in a host cell, comprising: (a) transforming a host cell with the chimeric gene of the invention; and (b) the transformed host cell is grown under conditions suitable for the expression of the chimeric gene, wherein the expression of the chimeric gene is related to the Lecl.

- results in altered levels of 5 transcription factor proteins in the transformed host cell.

The invention will be more fully understood by reference to the following detailed description and accompanying drawings and sequence lists, and are to be considered as part of the teaching in its entirety.

Figure 1 shows a comparison of the amino acid sequence of Lee 1-related transcription factors from the following species: Momordica (SEQ ID NO: 2, SID2), eucalyptus (SID4), maize (SID6 and 8), rice (SITE 10). and 12), soy (SID14, 16, 18, 20 and 22), wheat (SID24, 26 and 28) and Canna (SID30); compared to the nearest previously known sequences from Arabidopsis (SID31, 33, 34 and 35) and maize (SID32). The EQDRXLPIAN and QECVSEFISFXTXE sequence elements, where X represents any amino acid, are found within the active site region of the polypeptide and are believed to be characteristic of Lecl-related transcription factors.

Table 1 shows the name of the cDNA clone containing the polypeptides of the present invention, the nucleic acid fragments encoding the polypeptides, which clone represents all or a substantial part of these polypeptides as well as the corresponding identifiers (sequence identification numbers) as shown in the attached Sequence Lists. is used. The sequence descriptions and the attached sequence lists correspond to the nucleotide and / or amino acid sequence communications required by patent applications as described in U.S. Pat.

CFR in §1.821-1.825.

Table 1

LEC 1-related transcription factors

Sequence Identification Number protein Name of clone nt amino acids LEC 1-related transcription factor fds.pkOOO3.h5 1 2 LEC 1-related transcription factor eeflc.pk004.c8 3 4 LEC 1-related transcription cbnl0.pk0005.e6 5 6

-6 »* · *

factor LEC 1-related transcription factor p0006.cbysa51r 7 8 LEC 1-related transcription factor rl0n.pk0061.c8 9 10 LEC 1-related transcription factor rslln.pk002.gl0 11 12 LEC 1-related transcription factor ses4d.pk0037.e3 13 14 LEC 1-related transcription factor src2c.pk003.il 3 15 16 LEC 1-related transcription factor src2c.pk011.ml2 17 18 LEC 1-related transcription factor src2c.pk025.b3 19 20 LEC 1-related transcription factor src3c.pk028.j21 21 22 LEC 1-related transcription factor wkmlc.pk0002.d7 23 24 LEC 1-related transcription factor wlk8.pk0001.el0 25 26 LEC 1-related transcription factor wlm96.pk037.k9 27 28 LEC 1-related transcription factor ectlc. pk007.pl8: fis 29 30

In the sequence lists, the one-letter code is assigned to the nucleotide sequence characters and the three-letter code is used for amino acids as defined by the IUPAC-IUBMB standards, which are published in Nucleic Acids. E3 3021 (1985) and Biochemical J. 219 (2) 345 (1984), these publications are incorporated herein by reference in their entirety. Symbols and formats used for nucleotide and amino acid sequence data correspond to 37 CFR §1,822

rules laid down in a grid.

Many terms are used herein. Polynucleotide, polynucleotide sequence, nucleic acid sequence, and nucleic acid fragment / isolated nucleic acid fragment expressions are used interchangeably. These terms include nucleotide sequences and the like. A polynucleotide RNA or DNA polymer may be single-stranded or double-stranded and optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a DNA polymer may comprise one or more cDNA segments, genomic DNA, synthetic DNA, or mixtures thereof. The isolated polynucleotide of the present invention may comprise at least 30 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 contiguous nucleotides which comprise 1, 3, 5, 7, 9, 11, 13, 15 ., 17, 19, 21,

May be derived from any of SEQ ID NOs: 23, 25, 27, and 29 and may be complementary to these sequences.

As used herein, the term "isolated" refers to substances such as nucleic acid molecules and / or proteins that are substantially free or otherwise free from components that normally associate with them or interact with them in the natural state of the materials. The isolated polynucleotides can be purified from the host cell in which they occur naturally. Conventional nucleic acid purification methods known to those skilled in the art may be used to produce isolated polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

As used herein, the term "recombinant" is understood to mean, for example, the production of a nucleic acid sequence by artificial combination of two otherwise distinct sequence segments, such as chemical synthesis or manipulation of isolated nucleic acids by genetic engineering means.

The term "contig" as used herein refers to a nucleotide sequence that is composed of two or more constituent nucleotide sequences comprising common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acid fragments can be compared and matched to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and hence their respective nucleic acid fragments) can be assembled into a single continuous matching nucleotide sequence.

-8 · ♦ · · ··· <

As used herein, a substantially similar term refers to nucleic acid fragments in which changes in one or more nucleotide bases result in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. A substantially similar term, however, includes nucleic acid fragments in which the changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression alteration by muting genes, such as antisense or co-suppression methods. The term "nucleotide fragments of the present invention", such as deletion or insertion of one or more nucleotides, which does not significantly affect the functional properties of the resulting transcript of vis-a-vis the ability of the gene to mute or suppress the gene is also substantially understood. results in altering the functional properties of the resulting protein molecule. We therefore draw attention to the fact that the scope of the invention includes more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. As used herein, substantially similar and substantially equivalent terms are used interchangeably.

Essentially similar nucleic acid fragments can be selected by screening nucleic acid fragments representing sub-fragments or modifications of the nucleic acid fragments of the present invention, where one or more nucleotides are substituted, excised and / or inserted into the screening for their ability to influence the level of the polypeptide encoded by the unmodified nucleic acid fragment by plant or plant cell. For example, a substantially similar nucleic acid fragment can be produced and introduced into a plant or plant cell which comprises at least 30 continuously matching nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 60 continuous matching nucleotides from the nucleic acid fragment of the invention. The level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell that is in contact with a substantially similar nucleic acid fragment can then be compared to the level of the polypeptide in a plant or plant cell that has not been contacted with a substantially similar nucleic acid fragment.

For example, it is well known in the art that anti-sense suppression and co-suppression of gene expression can be accomplished by the use of nucleic acid fragments that represent less than the entire portion of a coding region of a gene and nucleic acid fragments that do not show 100 % azo · ~ ———

-9 with the gene that we want to suppress. Furthermore, changes in a nucleic acid fragment that result in the formation of a chemically equivalent amino acid at a given site but which do not affect the functional properties of the encoded polypeptide are well known in the art. Thus, a codon of the alanine amino acid, a hydrophobic amino acid, can be replaced by a codon coding for a less hydrophobic amino acid, such as glycine, or a more hydrophobic amino acid such as valine, leucine, or isoleucine. Likewise, changes that result in the substitution of a negatively charged amino acid with another such as replacement of aspartic acid with glutamic acid or substitution of a positively charged amino acid with another such as lysine replacement with arginine are expected to result in a functionally equivalent product. In the case of nucleotide exchanges that result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule, it is also not expected that they will alter the activity of the polypeptide. All of the proposed modifications may be known to those skilled in the art, and likewise the determination of the retention of the biological activity of the encoded products. Consequently, the identification numbers 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29 An isolated polynucleotide comprising a nucleotide sequence comprising at least 30 (preferably at least 40, most preferably at least 60) nucleotide sequences of at least 30 nucleotide sequences or complementary to these nucleotide sequences may be used to influence expression and / or function of a LEC 1-related transcription factor in a host cell. In a method of influencing the expression level of an isolated polynucleotide in a host cell (eukaryotic, such as a plant or yeast, prokaryotic, such as bacterial), the following steps may be performed: an isolated polynucleotide of the invention or an isolated chimeric gene of the invention; isolating the isolated polynucleotide or isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide by the level of the polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.

Furthermore, substantially similar nucleic acid fragments can be characterized by their ability to hybridize. The estimation of such homologies can be performed either by DNA DNA or DNARNA hybridizations under different stringency conditions as is well known to those skilled in the art (Hames and Higgins, Nucleic Acid Hybridization, IRL Press, Oxford, United Kingdom, (1985)). Stringency conditions can be adjusted by screening moderately similar fragments, such as homologous sequences from distantly related living organisms, to substantially similar phage fragments, such as genes that duplicate functional enzymes from closely related living organisms. Stringency conditions are determined by washings after hybridization. A group of preferred conditions consists of the following wash sequence: first 6x SSC, 0.5% SDS at room temperature for 15 minutes, followed by repeated washing with 2x SSC, 0.5% SDS at 45 ° C for 30 minutes and then repeated twice. washing with 0.2x SSC, 0.5% SDS at 50 ° C for 30 minutes. A more preferred group of stringent conditions employ higher temperatures where the washings are the same as described above, except that the last two 30 minute washings (0.2x SSC, 0.5% SDS) are raised to 60 ° C. For another preferred group of highly stringent conditions, two final washes were used in 0.1x SSC, 0.1% SDS at 65 ° C.

At the same time, essentially similar nucleic acid fragments of the present invention can be characterized by the% identity between the amino acid sequences encoded by them and the amino acid sequences disclosed herein, as defined by those of ordinary skill in the art. Suitable nucleic acid fragments (isolated polynucleotides of the invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences disclosed herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences disclosed herein. Most preferred nucleic acid fragments encode amino acid sequences that are at least about 95% identical to the amino acid sequences disclosed herein. Suitable nucleic acid fragments have not only the properties listed above, but also typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and% identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, WI). Multiple alignment of sequences was performed using the Clustal fitting method (Higgins and Sharp: CABIOS 5 151 (1989)) with the basic parameters (gap failure = 10, gap length error point = 10). In the case of a pairwise alignment using the Clustal method, the basic parameters were: Ktuple = 1, gap error - 11 -

point = 3, window = 5 and saved diagonals = 5.

A substantial portion of an amino acid or nucleotide sequence is understood to mean an amino acid or nucleotide sequence sufficient to permit the presumed identification of the protein or gene contained in the amino acid or nucleotide sequence. Amino acid and nucleotide sequences can be evaluated manually by methods known to those skilled in the art, or by means of computer-based sequence comparison and identification tools, such as algorithms such as the Basic Local Alignment Search Tool (BLAST) [Altschul et al. .: J. Mol. Biol. 215, 403 (1993), see also explanation of the BLAST algorithm at the National Center forum

Biotechnology Information website at the National Institutes of Health National Library of Medicine. In general, the sequences of ten or more sequentially matching amino acids or thirty or more sequentially matching nucleotide sequences are required to identify a polypeptide or nucleic acid sequence as a homologous sequence with a known protein or gene. In addition, gene-specific oligonucleotide probes containing 30 or more continuous nucleotides in the nucleotide sequences may be used in sequence-dependent gene identification (such as Southern hybridization) and isolation (such as bacterial colony or bacteriophage plaque in siti hybridization). In addition, short oligonucleotides of 12 or more nucleotides can be used as amplification primers during PCR to produce a given nucleic acid fragment containing primers. Accordingly, a significant portion of a nucleotide sequence is understood to mean a nucleotide sequence that permits specific identification and / or isolation of a nucleic acid fragment containing the sequence. The present invention provides teaching of amino acid and nucleotide sequences encoding polypeptides comprising one or more specific plant proteins. One of ordinary skill in the art will be able to utilize all or a substantial part of the described sequences for the purposes known to the person skilled in the art. Accordingly, the entire sequence, as disclosed in the attached Sequence Lists, is part of the description, just as the major portions of these sequences as defined above.

As used herein, the term "codon degeneracy" refers to the di30-vergenesis found in the genetic code, which allows variations of the nucleotide sequence without altering the amino acid sequence of the encoded polypeptide. Accordingly, the present invention provides any nucleic acid fragment comprising a nucleotide sequence encoding all or a substantial portion of the amino acid sequences disclosed herein. It is known to those skilled in the art

·· · · r

-12 can be used to identify the codon differentiation that a specific host cell exhibits in the nucleotide codons used to determine the particular amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment in such a manner that its codon usage frequency approaches the use of the preferred codon of the host cell.

Synthetic nucleic acid fragments can be assembled from chemically synthesized oligonucleotide building blocks using methods known to those skilled in the art. These building blocks are ligated and annealed to produce larger nucleic acid fragments, which can then be enzymatically assembled to generate the entire desired nucleic acid fragment. As used herein, the term "chemically synthesized" refers to a nucleic acid fragment that is used to construct the component nucleotides in vitro. Manual chemical synthesis of nucleic acid fragments can be accomplished by well-known methods or automated chemical synthesis can be performed using any of the commercially available devices. Accordingly, nucleic acid fragments can be generated for optimal gene expression by optimizing the nucleotide sequence when considering host cell codon differentiation. One skilled in the art will appreciate the likelihood of successful gene expression by pushing the codon usage toward the codons preferred by the host cell. The determination of the preferred codons can be based on the analysis of genes from the host cell in which sequence information is available.

As used herein, the term gene refers to a nucleic acid fragment that expresses a specific protein, including precursor sequences (5 'non-coding sequences) and sequencing (3' non-coding sequences) preceding the coding sequence. As used herein, the term "gene of natural origin" refers to a gene that is found in nature along with its own regulatory sequences. As used herein, the term "chimeric gene" refers to any gene that is a non-natural gene and contains regulatory and coding sequences that are not found in nature. Accordingly, a chimeric gene may contain regulatory sequences and coding sequences that are derived from different sources or may contain regulatory sequences and coding sequences from the same source, but are arranged differently from those found in nature. As used herein, the term "endogenous gene" means a gene of natural origin in its natural site in the genome of a living organism. As used herein, the term "foreign gene" refers to a gene that is not naturally present in the host but which has been introduced into the host by gene transfer. The foreign genes may contain genes of natural origin introduced into a non-natural living organism or chimeric genes. A transgene is a gene introduced into the genome by a transformation process.

As used herein, the term "coding sequence" refers to a nucleotide sequence that encodes a specific amino acid sequence. As used herein, the term "regulatory sequences" refers to nucleotide sequences that are upstream (5 'non-coding sequences) or downstream (3' non-coding sequences) relative to a coding sequence that affect the transcription of the associated coding sequence. , RNA processing or RNA stability or translation. The regulatory sequences may include promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

As used herein, the term "promoter" refers to a nucleotide sequence capable of controlling expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'to the promoter sequence. The promoter sequence is comprised of adjacent and distal 5 'elements, the latter being often referred to as enhancers. Accordingly, an enhancer is a nucleotide sequence that is capable of stimulating promoter activity and may be a promoter element or may be a heterologous element introduced to increase expression level or promoter tissue specificity. The promoters may be derived entirely from a naturally occurring gene or may consist of different elements derived from different promoters found in nature, or may contain even synthetic nucleotide segments. It will be apparent to those skilled in the art that different promoters may direct expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that result in a nucleic acid fragment being expressed in most cell types are generally referred to as constitutive promoters. Various new types of promoters that can be used in plant cells are continuously discovered; there are many examples of these in Okamuro and Goldberg's summary work [Okamuro and Goldberg: Biochemistry of Plants 15 1 (1989)]. Furthermore, it has been recognized that, in most cases, the exact limits of the regulatory sequences have not been fully determined, nucleic acid fragments of different lengths may have the same promoter activity.

*

The term "translational leader" as used herein refers to a nucleotide sequence located between a promoter sequence of a gene and a coding sequence. The translational leader sequence is found in the fully processed mRNA in the 5 'direction of the translation start sequence. The translational leader sequence may affect the processing of the primary transcript into mRNA, may affect the stability of the mRNA or the translational efficiency. Examples of translational leader sequences [Turner and Foster: Mol. Biotechnol. 3,225 (1995)].

As used herein, the term 3 'non-coding sequences refers to nucleotide sequences that are located in the 3' direction relative to a coding sequence and include polyadenylation recognition sequences and further sequences that control regulatory signals capable of influencing mRNA processing or gene expression. encoded. The polyadenylation signal is generally characterized by influencing the addition of polyadenylic acid moieties to the 3 'end of the mRNA precursor. Examples of the use of the various 3 'non-coding sequences can be found in Ingelbrecht et al., Plant CellJ.671 (1989).

As used herein, the term RNA transcript refers to the product of catalyzed transcription of a DNA sequence RNA polymerase. When the RNA transcript is the perfect complementary copy of the DNA sequence, it is referred to as a primary transcript or may be an RNA sequence derived from post-transcription processing of the primary transcript and referred to as mature RNA. As used herein, the term messenger RNA (messenger RNA, mRNA) refers to an RNA that does not contain introns and can be translated into polypeptides by the cell. As used herein, the term "cDNA" refers to DNA derived from and complementary to an mRNA template. The cDNA can be converted into a single-stranded or double-stranded form, for example, using the Klenow fragment of DNA polymerase I. The term "sense RNA" refers to an RNA transcript that encompasses mRNA and can be translated into a polypeptide by the cell. An antisense RNA term is an RNA transcript complementary to or part of a target primary transcript or mRNA that blocks the expression of a target gene (see U.S. Patent No. 5,107,065, which is hereby incorporated by reference in its entirety). The complementarity of an antisense RNA may be accomplished with any part of the specific nucleotide sequence, i.e., may occur in the 5 'non-coding sequence, the 3' non-coding sequence, the introns, or the coding sequence. The term functional RNA as used herein includes sense RNA, antisense RNA, ribozyme ···· ····

- 15 RNAs or any other RNA that may not be translated but still have an effect on cellular processes.

As used herein, the term &quot; functionally-linked &quot; refers to the assembly of two or more nucleic acid fragments on a single polynucleotide such that one of its functions is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of influencing the expression of a particular coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequences can be functionally linked to regulatory sequences in sense or antisense orientation.

As used herein, the term expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA from a nucleic acid fragment of the invention. At the same time, expression of expression can mean the translation of mRNA into a polypeptide. The term antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. As used herein, the term overexpression refers to the production of a gene product at a level in a transgenic living organism where it exceeds the level of production in normal or non-transformed living organisms. As used herein, the term "co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes [U.S. Patent No. 5,231,020, the disclosure being incorporated by reference in its entirety],

The term "protein or polypeptide" refers to a chain of amino acids in a specific sequence defined by the coding sequence in the polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.

The term "altered levels" or "altered expression" as used herein refers to the production of a gene product (s) in transgenic living organisms in amounts or ratios that are different from those found in normal or non-transformed living organisms.

The mature protein or mature term, when a post-translationally processed polypeptide is used to describe a protein, i.e. a polypeptide from which each of the pre- or propeptides in the primary translation product has already been removed. As used herein, the term precursor protein or precursor, when used to describe a protein, refers to the primary product of mRNA translation, i.e. a product in which pre- and propeptides are still present. Pre- and propeptides may be, but are not limited to, intracellular localization signals.

V * -16- ............

A chloroplast transit peptide refers to an amino acid sequence that is translated for a protein and directs the protein to the chloroplast or other plastid type found in the cell in which the protein is produced. The term "chloroplast transit sequence" refers to a nucleotide sequence encoding a chloroplast transit peptide. As used herein, the term "signal peptide" refers to an amino acid sequence that is translated into a protein and directs its protein to the secretory system [Chrispeels: Ann. Port. Plant Phys. Plans for Mol. Biol. 42 21 (1991). If the peptide is to be directed to a vacuum, a vacuum target signal may be added (see above) or, if directed to the endoplasmic reticulum, an endoplasmic reticulum retention signal may be added (see above). If the protein is to be directed to the nucleus, the signal peptide present must be removed and replaced with a nuclear localization signal [Raikhel: Plant Phys. 100: 1627 (1992)].

As used herein, the term transformation refers to the introduction of a nucleic acid fragment into the genome of a host that results in genetically stable inheritance. Host hosts containing the transformed nucleic acid fragment are referred to as transgenic living organisms. Examples of suitable methods for plant transformation include: Agrobacterium-mediated transformation (De Blaere et al., Meth. Enzymol. 143 277 (1987)] and Particle Acceleration or Gene Transformation Technology [Klein et al., Natúré 32770 (1987) and U.S. Patent No. 4,945,050, which is incorporated by reference in its entirety], so that the isolated polynucleotides of the invention can be inserted into a recombinant form. constructs - typically DNA constructs - that are able to enter and replicate into a host cell. Such a construct may be a vector comprising a replication system and sequences capable of transcribing and translating a polypeptide coding sequence into a host cell. A vector suitable for stable transfection of many plant cells or for generating transgenic plants has been described, for example, in Pouwels et al. : Cloning Vectors: Laboratory Manual, (1985), Supplement 1987; Weissbach and Weissbach, Methods Forum Planning Molecular Biology, Academic Press, (1989); and Flevin et al., Plant

Molecular Biology Manual, Kluwer Academic Publishers, (1990). Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5 'and 3' regulatory sequences and a dominant selectable marker. Such plant expression vectors may, however, contain a promoter regulatory region (i.e., a regulatory region that can be induced or constitutively regulated, exhibits environmental or developmental control, or cell or tissue specific expression). ), may also include a transcription initiation site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

The standard recombinant DNA and molecular cloning methods used herein are well known in the art and are described in more detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, (1989). hereinafter referred to as Maniatis.

PCR or polymerase chain reaction is well known to those skilled in the art as a method for amplifying specific DNA segments [US Patents 4,683,195 and 4,800,159],

The present invention further provides an isolated polynucleotide comprising a nucleotide sequence encoding a LEC 1-related transcription factor polypeptide, and wherein the polypeptide 15 has at least 90% identity according to the Clustal fusion method. A polypeptide characterized by any of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30.

However, the present invention relates to an isolated complement of such polynucleotides, wherein the complementary and polynucleotides contain the same number of nucleotides, and the complementary and polynucleotides contain the same number of nucleotides and the complementary and polynucleotide nucleotide sequences exhibit 100% complementarity.

Previously, nucleic acid fragments encoding at least a portion of LEC 1-related transcription factors have been isolated and identified by comparing randomly selected plant cDNA sequences with public databases containing nucleotide and protein sequences using a well-known BLAST algorithm as is well known to those skilled in the art. The nucleic acid fragments of the present invention may be used to isolate genes encoding DNAs and homologous proteins from the same or other plant species. Isolation of homologous genes by sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, nucleic acid hybridization methods and DNA and RNA amplification methods, such as various applications of nucleic acid amplification methods (such as polymerase chain reaction, ligase chain reaction).

For example, genes encoding additional LEC 1-like transcription factors, either cDNA or genomic DNA, can be isolated directly from all or part of the nucleic acid fragments of the invention using DNA hybridization probes to screen for cloning from any desired plant using methods well known to those skilled in the art. . Specific oligonucleotide probes based on the nucleic acid sequences of the present invention can be designed and synthesized by methods known in the art [Maniatis]. Furthermore, a complete sequence can be used directly for the synthesis of DNA probes by methods known to those skilled in the art, such as random primer DNA tagging, nick translation, end marker techniques or RNA probes using available in vitro transcription systems. Furthermore, specific primers can be designed and used to amplify all or part of the sequences of the invention. The resulting amplification products may be labeled directly during amplification reactions or labeled after amplification reactions and used as probes to isolate full-length cDNA or genomic fragments under appropriate stringency conditions.

Furthermore, two short segments of the nucleic acid fragments of the invention can be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction can also be performed at the cloning site of cloned nucleic acid fragments, where one of the primary sequences is derived from the nucleic acid phage fragments of the invention and the other primer sequence utilizes the presence of the polyadenylic acid moiety at the 3 'end of the mRNA precursor encoding the plant genes. Alternatively, the second primer sequence may be based on sequences from the cloning vector. For example, one skilled in the art can follow the RACE protocol [Frohman et al., Proc. Natl. Acad. Sci. USA 85 8998 (1988)] to generate cDNAs by PCR by amplifying a single point on the transcript and copies of the region between the 3 'or 5' end. The primers according to the invention can be designed for the 3 'and 5' directions. Commercially available 3 'RACE or 5' RACE systems (BRL) can be used to isolate specific 3 'or 5' cDNA fragments [Ohara et al., Proc. Natl. Acad. Sci. USA 86, 5673 (1989); Loh et al., Science, 243: 217 (1989). The products produced by the 3 'and 5' RACE methods can be combined to create full-length cDNAs (Frohman and Martin, Techniques I 165 (1989)). As a consequence, a polynucleotide comprising at least 30 (preferably at least 40, most preferably at least 60) nucleotide sequences of a continuous matching nucleotide is provided, wherein the sequence is 1, 3, 5, 7, 9, 11, 13. SEQ ID NOs: 15, 17, 19, 21, 23, 25, 27 and 29

- a nucleotide sequence derived from any of the 19 and the nucleotide sequences that may be complementary may be employed in methods for producing a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.

The nucleotide and the deduced amino acid sequences of the present invention facilitate immunological screening of cDNA expression clones. Synthetic peptides representing portions of the amino acid sequences of the present invention can be synthesized synthetically. These peptides can be used to immunize animals to produce polyclonal and monoclonal antibodies that exhibit specificity for peptides or proteins containing amino acid sequences. These antibodies can then be used to screen cDNA expression clones to isolate desired full-length cDNA clones [Lerner: Adv. Immunol. 361 (1984); Maniatis].

According to another aspect of the present invention, there are provided viruses and host cells comprising either the chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein. Examples of host cells useful in the practice of the present invention include, but are not limited to, yeast, bacteria, and plant cells.

As noted above, the nucleic acid fragments of the invention may be used to generate transgenic plants in which the expressed polypeptides are found at a higher level when overexpressed - or at lower levels in co-suppression - than normal levels or in cell types or developmental stages in which normally not found. This has the effect of altering the level of gene expression in an LEC1-related transcription factor, which then leads to altered expression of the genes regulated by Lee 1-related transcription factor. This, in turn, leads to developmental and phenotypic variants, such as, but not limited to, increased accumulation of oils in plant tissues. For example, a core-specific promoter that directs overexpression of a Lecl-like transcription factor leads to overexpression of oil in the nucleus.

Overexpression of the proteins of the invention can be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing the expression of a gene in the desired tissue in the desired development phase. The chimeric gene may contain promoter sequences and translational leader sequences derived from the same genes. In addition, 3 'non-coding sequences encoding transcription termination signals may be provided. The chimeric gene of the invention may further comprise

-20 *. · : ··: ···.

··· *. ··· also one or more introns to promote gene expression.

Plasmid vectors containing the isolated polynucleotide (or chimeric gene) of the invention may be constructed. The selection of the plasmid vector depends on the method used to transform the host plants. Those skilled in the art will be aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells that contain the chimeric gene. However, it will be appreciated by those skilled in the art that different independent transformation events may result in different expression levels and patterns (Jones et al., EMBO J. 4: 2411 (1985); De Almeida et al., Mol. Gene. Genetics 218 78 (1989)] and thus it is also clear that more events need to be screened to produce lines with the desired expression level and pattern. Such screening can be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

In some applications, it may be useful to target the polypeptides of the invention to different cell compartments or to facilitate its secretion from the cell. Thus, it is also conceivable that the chimeric gene described above may be supplemented by providing a sequence encoding the polypeptides of the invention with appropriate intracellular targeting sequences, such as transit sequences [Keegstra: Cell 56, 247 (1989)], with signal sequences or endoplasmic reticulum localization. coding sequences [Chrispeels: Ann. Port. Plant Phys. Plans for Mol. Biol. 42 21 (1991)] or seed with localization signals [Raikhel: Plant Phys. 100 1627 (1992)] by removing or leaving the targeting sequences already present. While the references cited here serve as an example of each of these, the list is not exhaustive and further applicable targeting signals can be discovered in the future.

In some cases it may also be necessary to reduce or eliminate expression of genes encoding the polypeptides of the invention in plants for some applications. In order to accomplish this, a chimeric gene designed for the co-suppression of the polypeptide of the invention may be constructed by attaching the gene or gene fragment encoding the polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed for expression of an antisense RNA on the whole or part of a nucleic acid fragment of the invention may be constructed by linking the gene or gene fragment to reverse promoter sequences.

-21 Co-suppression or antisense chimeric genes can be introduced into plants by transformation, where the expression of the appropriate endogenous genes is reduced or absent.

Molecular genetic solutions for producing plants with altered gene expression have a definite advantage over conventional plant breeding solutions. Changes in plant phenotypes can be generated by specifically inhibiting the expression of one or more genes by antisense inhibition or co-suppression [US Patents 5,190,931, 5,107,065 and 5,283,323]. An antisense or cosuppression construct works as a dominant negative regulator of gene activity. Although conventional mutations may result in negative regulation of gene activity, these effects are likely to be recessive. The dominant negative regulation available with the transgenic approach may be beneficial from the point of view of breeding. Furthermore, the ability to limit the expression of a specific phenotype to the reproductive tissues of the plant by using tissue-specific promoters compared to conventional mutations in which the effect of the mutation can occur in all tissues in which the mutant gene is expressed under normal conditions may have agricultural advantages.

It will be appreciated by those skilled in the art that special considerations are used with the use of antisense or co-suppression technologies to reduce the expression of particular genes. For example, the appropriate expression level of sense or antisense genes may require the use of different chimeric genes that use various regulatory elements known to those skilled in the art. Once transgenic plants have been produced by any of the methods described above, it will be necessary to screen each transgenic one after those which show the desired phenotype most effectively. Accordingly, one skilled in the art can develop methods for screening a large number of transformants. The nature of this screening is generally selected on the basis of practical considerations. For example, screening for changes in gene expression can be screened using antibodies specific for the protein encoded by the gene, or assays that specifically measure enzyme activity can be constructed. A preferred method is to allow rapid processing of a large number of samples, as a large number of transformants are expected to be negative for the desired phenotype.

According to another aspect of the invention, there is provided a LEC 1-related transcription factor polypeptide having an amino acid sequence of at least 90% identical to that of the Clustal fusion method. ., 12, 14, 16, 18, 20, 22

Characterized by any of the polypeptides of SEQ ID NO: 2224, 26, 28 and 30.

The polypeptides of the invention (or a portion thereof) may be produced in heterologous host cells, more specifically, in cells of microbial hosts, and may be used to produce antibodies against these proteins using methods known to those skilled in the art. Antibodies are useful for detecting polypeptides of the invention in in situ cells or in vitro cell extracts. Preferred heterologous host cells for producing the polypeptides of the invention are microbial host cells. Expression vectors containing microbial expression systems and regulatory sequences that direct high-level expression of foreign proteins are well known to those skilled in the art. Any of these may be used to construct a chimeric gene for the production of polypeptides of the invention. This chimeric gene can then be introduced into appropriate microorganisms by transformation to provide high-level expression of the encoded LEC1-related transcription factor. For example, a high level expression of a polypeptide of the invention in a bacterial host may be provided by a vector as shown in Example 6 in the experimental part.

However, all or a substantial part of the polynucleotides of the present invention may be used as probes for the genetic and physical mapping of the genes that are part of, and may be used as markers for, the properties associated with these genes. Such information may be useful in plant breeding to produce lines with the desired phenotype. For example, the nucleic acid fragments of the invention may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots of the digested plant genomic DNA of the restriction enzyme [Maniatis] can be tested using the nucleic acid fragments of the invention as probes. The resulting band patterns can then be genetically analyzed using computer programs such as MapMaker [Lander et al., Genomics 1 174 (1987)] to construct a genetic map. Furthermore, the nucleic acid fragments of the invention may be used as probes for Southern blots, where the biotics contain restriction endonuclease-treated genomic DNA from a defined genetic crossing group of parent and descendants. The segregation of the DNA polymorphism is recorded and used to calculate the position of the nucleic acid sequence of the present invention on a genetic map previously obtained using this population (Botstein et al., Am. J. Him. Genet. 32, 314 (1980)].

-23 Production and application of plant gene probes during genetic mapping has been previously reported [Bernatzky and Tanksley: Plant Mol. Biol. Reporter 4: 37 (1986)]. Many publications describe the genetic mapping of specific cDNA clones using the methods outlined above or variations thereof. For example, F2 crossover populations, reciprocal populations, randomly paired populations, near isogenic lines, and other individuals may be used for mapping. Such methods are well known to those skilled in the art.

However, nucleic acid probes derived from nucleic acid sequences of the present invention may also be used for physical mapping (i.e., positioning of sequences on physical maps, see Hoheisel et al., Nonmammalian Genomic Analysis: Practical Guide, Academic Press, 319-346, (1996), and references cited therein).

Nucleic acid probes derived from nucleic acid sequences of the present invention can be used in direct fluorescence in situ hybridization (FISH) mapping [Trask: Trends Gene. 7, 149 (1991)]. Although current methods of FISH mapping favor the use of large clones (from a few to several hundred kilobases (kb); see Laan et al., Genome, Vol. 5, 13 (1995)), fixations made in sensitivity may allow the use of FISH mapping to be shorter. even in the case of rehearsals.

Various nucleic acid amplification based genetic and physical mapping methods can be performed using the nucleic acid sequences of the present invention. Examples include allele-specific amplification [Kazazian, J. Leg. Clin. Med. 95 (1989)], Polymorphism (CAPS) of PCR Amplified Fragments (Sheffield et al., Genomics 16: 325 (1993)), allele-specific ligation [Landegren et al., Science 241, 1077 (1988)] Extension Reactions [Sokolov: Nucleic Acid Slit. 18: 3671 (1990)], Radiation Hybrid Mapping [Walter et al., Nat. Genet. 7 22 (1997)] and Happy Mapping mapping [Dear and Cook: Nucleic Acid Slot. 17, 6795 (1989)]. In these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs that are used in the amplification reaction or primary extension reactions. The design of such primers is well known to those skilled in the art. In methods using PCR based genetic mapping, it may be necessary to identify differences in DNA sequence between parent of the mapping cross in the region corresponding to the nucleic acid sequence of the invention. However, this is generally not necessary for mapping methods.

Functional mutant phenotypes can be identified for the cDNA clones of the present invention using either targeted gene destruction protocols or identifying specific mutants in a maize population containing mutations for all possible genes [Ballinger and Benzer: Proc. Natl. Acad. Sci. USA 86 9402 (1989); Koes et al., Proc. Natl. Acad. Sci. USA 92 8149 (1995)]; Bensen et al., Plant Cell 7, 75 (1995)]. The latter approach can be implemented in two ways. First, the short segments of the nucleic acid fragments of the invention can be used in polymerase chain reaction protocols with DNA mutated in a mutation marker primer in a population of plants into which mutator transposons or any other mutant DNA element was previously introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation marker in or near the plant gene encoding the polypeptides of the invention. Alternatively, the nucleic acid fragment of the invention may be used as a hybridization probe against PCR amplification products produced from the mutation population, wherein the mutation marker primer is used in conjunction with an arbitrarily selected genomic site primer, such as a synthetic adapter anchored to a restriction enzyme cleavage site. For each method, a plant containing a mutation in the endogenous gene encoding the polypeptides of the invention can be identified and produced. This mutant plant can then be used to determine or confirm the natural function of the polypeptides of the invention described herein.

For a better understanding of the invention, the following examples are set forth in which ratios and percentages are given by weight, degrees degrees Celsius unless otherwise indicated. Note that while the examples illustrate preferred embodiments of the invention, these are merely illustrative. Based on the above description and these examples, one of ordinary skill in the art can determine the essential features of the invention and may make various changes and modifications to the invention to adapt it to different uses and conditions, but such modifications do not differ from the inventive idea and the attached.

Claims (1)

  1. are claimed within the scope of the claimed claims. Thus, various modifications of the invention, as described and described herein, will be readily apparent to those skilled in the art from the disclosure herein. Such modifications do not differ from the invention and are considered within the scope of the claimed claims.
    -25 • · · ····· · · · · · · ···· ·· ·· * ·· · * ··
    All references described in the description are to be considered in their entirety as part of the disclosure.
    Example I Composition of cDNA Cloning Libraries; Isolation and sequencing of cDNA clones 5 cDNA clones were created that represent mRNAs from Momordica charantia, Eucalyptus tereticornis, corn, rice, soya, wheat and Canna edulis. The characteristics of the cloning facilities are described below.
    Table 2 cDNA clones Momordica charantia, Eucalyptus tereticornis, maize, rice, soya, wheat and Canna edulis
    clone repository fabric clone fds Momordica charantia developing seed fds.pk0003.h5 ectlc Canna edulis tubers ectlc.pk007.pl 8 eeflc Eucalyptus tereticornis flower bud from adult tree eeflc.pk004.c8 cbnlO corn developing seed (embryo and endosperm); 10 days after pollination cbnlO.pkOOO5.e6 POOO corn young shoot p0006.cbysa51r rlOn rice 15 day letter * rl0n.pk0061.c8 rslln rice 15 days seedling * rslln.pk002.gl0 ses4d soy embryogenic suspension 4 days after subculture ses4d.pk0037.e3 src2c soy 8 day root Heterodera glycenis cyst infected with nematode src2c.pk003.il 3 src2c.pk011.ml2 src2c.pk025.b3 src3c soy 8 day root Heterodera glycenis cyst infected with nematode src3c.pk028.j21 wkmlc wheat seed for 55 hours at 22 ° C wkm 1 c. pk0002. d7 wlk8 wheat seedlings 8 hours after herbicide treatment ** wlk8.pk0001.el0 wlm96 wheat seedling 96 hours Erysiphe graminis f. sp. after tritic inoculation wlm96.pk037.k9
    • · · · · · · · · · · · · · · · · ·• · · · · · ··· ···
    -26 * These chlorine bases were normalized essentially as described in U.S. Patent No. 5,482,845, the disclosure being incorporated by reference in its entirety.
    ** Use of 6-iodo-2-propoxy-3-propyl-4 (3H) -quinazolinone; the synthesis and method of application of this compound are disclosed in U.S. Pat. No. 08 / 545,827; the reference is to be considered in its entirety as part of the teaching.
    cDNA cloning sites can be prepared by any of the many methods available. For example, cDNAs may be introduced into plasmid vectors by first constructing cDNA clones in Uni-ZAP ™ XR vectors according to the manufacturer's instructions (Stratagene Cloning Systems, La Jolla, CA). Uni-ZAP ™ XR Cloning Blocks are transformed into plasmid cloning libraries according to the protocol provided by Stratagene. After transformation, the cDNA inserts will be contained in the plasmid vector pBluescript. In addition, cDNA inserts can be directly introduced into the pre-cleaved Bluescript II SK (+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs) followed by transfection into DH10B cells according to the manufacturer's instructions (GIBCO BRL Products). After the cDNA inserts were inserted into plasmid vectors, plasmid DNAs were prepared from randomly selected bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences were amplified by polymerase chain reaction using primers specific for vector sequences bounding inserted cDNA sequences. The amplified insert DNAs or plasmid DNAs are sequenced by dye primer sequencing reactions to generate partial cDNA sequences (expressed sequence markers or ESTs; see Adams et al., Science 252, 1651 (1991)). The resulting ESTs were analyzed using a Perkin Elmer Model 377 fluorescence sequencer.
    Complete insert sequence (FIS) data is obtained using a modified transposition protocol. The clones identified for FIS were recovered from stored glycerine strains as individual colonies and plasmid cDNAs isolated by alkaline lysis. Isolated DNA templates are reacted with vector-initiated M13 pre and reverse oligonucleotides in a PCR-based sequencing reaction and applied to automated sequencers. Confirmation of clone identification is performed by sequence alignment with the original EST sequence from which FIS was required.
    Certified templates are transposed using Primer Island transposition kit (PE Applied Biosystems, Foster City, CA), which is based on the transposable element of Saccharomyces cerevisiae Tyl [Devine and Boeke: Nucleic Acids Slit. 22,365 • · · · (1994)]. The in vitro transposition system incorporates individual binding sites randomly into a large population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL / Life Technologies, Rockville, MD) by electroporation. The transposable element contains an additional selectable marker (called DHFR) [Fiing and Richards: Nucleic Acids Slit. JJL 5147 (1983)] which allows double selection on agar plates when only those subclones containing the integrated transposon are selected. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared by alkaline lysis, and the templates are sequenced (ABI Prism dye terminator ReadyReaction mixture) out of the transposition site using individual primers specific for the transposon site sites.
    Sequence data are collected (ABI Prism Collections) and matched using Phred / Phrap (P. Green, University of Washington, Seattle). Phred / Phrap is a public software program that re-reads ABI sequence data, recreates bases, assigns quality values, and writes base calls and quality values to editable output files. The Phrap Sequence Editor uses these quality values to increase the accuracy of contig sequences in the matched sequence. Conversions can be viewed using the Consed sequence editor (D. Gordon, University of Washington, Seattle).
    In some clones, the cDNA fragment corresponds to a portion of the 3'-terminus of the gene and does not cover the entire open reading phase. One or two different protocols are used to obtain information in the 5'-direction. The first of these methods results in the production of a DNA fragment containing a portion of the desired gene sequence, while the second method results in the production of a fragment containing the complete open reading phase. Both methods use two PCR amplification rounds to obtain fragments from one or more chlorine libraries. In some cases, chlorine libraries are selected based on prior knowledge, for example, knowing that the specific gene must be present in a particular tissue, while in other cases it is randomly selected. Reactions for selection of the same gene can be carried out in parallel at multiple cloning sites or by pooling clones. In general, pooled chlorine blends are formed by combining 3-5 different clones, normalized to uniform dilution. In the first round of amplification, both methods are used for a vector-specific (forward) prime, corresponding to a portion of the vector at the 5'-terminus of the clone, a gene-specific (reverse) pri · · · · · · · · · 9 ·
    9999 * «··· 99 9 999
    -28mer connected. The first method uses a sequence that is complementary to a portion of the known gene sequence, while the second method uses a gene-specific primer that is complementary to a portion of the 3 'untranslated region (also referred to as UTR). In the second amplification round, a nested primer set is used for both methods. The resulting DNA fragment was ligated into the pBluescript vector using a commercially available kit and following the manufacturer's instructions. This kit may be any of a number of available kits available from many vendors, including Invitrogen (Carlsbad, CA), Promega Biotech (Madison, WI), and Gibco-BRL (Gaithersburg, MD). The plasmid DNA was isolated by an alkaline lysis method and sequenced and assembled using the above-mentioned Phred / Phrap software.
    Example 2 Identification of cDNA clones
    We have identified cDNA clones encoding LEC 1-related transcription factors by performing BLAST searches (Basic Local Alignment Search Tool; [Altschul et al., J. Mol. Bioi. 215, 403 (1993)], see also BLAST algorithm. explanation on the website of the National Center for Biotechnology Information at the National Library of Medicine of the National Institutes of Health) similar to the sequences in the BLAST nr database. This database contains all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structures of Brookhaven Protein Data Bank, the latest major release of the SWISS-PROT protein sequence database, the EMBL and DDBJ databases. The DNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences found in the nr database, using the BLASTN algorithm provided by the National Center Forum Biotechnology Information (NCBI). The DNA sequences were translated into all the reading phases and compared to all publicly available protein sequences found in the nr database using the BLASTX algorithm provided by the NCBI [Gish and States: Nat. Genet. 3,266 (1993)]. From a comfort point of view, the P-value (probability) of a cDNA sequence and the sequence matching in the searched databases calculated using BLAST is given here as a pLog value representing the negative logarithm of the observed P-value. Accordingly, the higher the pLog value, the greater the likelihood that the relationship between the cDNA sequence and the BLAST hit represents homologous proteins.
    · ♦ · · · · ··· · ··· · · · · · · · · · · · · · · · · · · ·
    -29The ESTs for analysis were compared with the GenBank database as described above. ESTs containing multiple 5 or 3-prime sequences can be found in the BLASTn algorithm [Altschul et al., Nucleic Acids. 25, 3389 (1997)] compared to the DuPont protected database by comparing nucleotide sequences containing regions of common or overlapping sequence homology. Where common or overlapping sequences are found between two or more nucleic acid fragments, the sequences may be assembled into a single continuous matching nucleotide sequence to extend the original fragment in either the 5- or 3-prime direction. Once the most remote 5prime EST has been identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Different species homologous genes can be found by comparing the amino acid sequence of a known gene (either from a protected source or from a public database) with an EST database using the BLASTn algorithm. The BLASTn algorithm examines amino acid requests against a nucleotide database that is translated in all 6 possible reading phases. This search provides space for differences in nucleotide codon usage between different species and codon degeneration.
    Example 3
    Characterization of cDNA clone encoding LEC 1-related transcription factors
    The BLASTX search using the EST sequences from the clones listed in Table 3 revealed similarities between the polypeptides encoded in the cDNAs and the LCC-related transcription factors derived from the species listed below: Arabidopsis thaliana (NCBI General Identification Number: 6729485), Arabidopsis thaliana ( NCBI General Identification Number: gi 2398529), Arabidopsis thaliana (NCBI General Identification Number gi 3738293), Zea mays (NCBI General Identification Number 22380). Table 3 shows BLAST results for each EST (EST), sequences of complete cDNA inserts containing labeled cDNA clones (FIS), sequences of contigs constructed from two or more ESTs (Contig), an FIS, and sequences (contig *) of one or more EST-based contigs or full protein coding sequences (CGS) obtained by FIS, a contig, or a FIS and PCR.
    • 4 ···· ·· «· 4 · ♦« · · · · · · · · · · · · ··· · · · · · · · · · · «
    -303. table
    BLAST results for sequences encoding polypeptides homologous to Arabidopsis thaliana and Zea mays LEC 1-related transcription factors
    clone condition BLAST pLog score (NCBI General Identification Number) fds.pk0003.h5 CGS 57.70 (g 6729485) eeflc.pk004.c8 CGS 61.70 (g 22380) cbnl0.pk0005.e6 CGS 72.22 (g 22380) p0006.cbysa51r CGS 55.52 (g 2244810) rl0n.pk0061.c8 CGS 46.52 (g 22380) rslln.pk002.gl0 CGS 68.70 (g 22380) ses4d.pk0037.e3 CGS 49.00 (g 2398529) src2c.pk003.il 3 CGS 41.10 (g 3738293) src2c.pk011.ml2 CGS 62.00 (g 6729485) src2c.pk025.b3 CGS 45.52 (g 22380) src3c.pk028.j21 CGS 54.30 (g 22380) wkmlc.pk0002.d7 CGS 79.52 (g 22380) wlk8.pk0001.el0 CGS 52.70 (g 2398529) wlm96.pk037.k9 CGS 73.52 (g 22380) ectlc.pk007.pl 8 FIS 44.70 (g 22380)
    The total cDNA insert sequences from the clones listed in Table 3 were determined. Further sequencing and searching for the DuPont protected database enabled further identification of Momordica, Eucalyptus, Canna, corn, rice, soya and / or wheat clones encoding transcription factors on Leclr. Using the EST sequences from the clones listed in Table 4, the BLASTX search revealed similarities between polypeptides encoded in cDNAs and Lee 1-related transcription factors from the species listed below: Arabidopsis thaliana (NCBI general identification number: gi 6729485; Zea mays (NCBI General Identification Number: gi 22380; SEQ ID NO: 32); Arabidopsis thaliana (NCBI General Identification Number 2244810; 33.
    · · ···· ···· • · · «· *« · · · «· ··· · · · · ·« · · · · · · ·
    -31), Arabidopsis thaliana (NCBI General Identification Number: gi 2398529; SEQ ID NO: 34), Arabidopsis thaliana (NCBI General Identification Number gi 3738293; SEQ ID NO: 35). Table 4 shows the BLAST results for each EST (EST), the sequences of complete cDNA inserts containing the labeled cDNA clones (FIS), the sequences of contigs composed of two or more ESTs (Contig), an FIS and sequences (contig *) of one or more EST-based contigs or full protein coding sequences (CGS) obtained by FIS, a contig, or a FIS and PCR.
    Table 4
    % Identity of amino acid sequences based on the nucleotide sequences of the cDNA clone encoding polypeptides homologous to Arabidopsis thaliana and Zea mays LEC 1-related transcription factors.
    sequence identification number % identity (NCBI General Identification Number) 2 68% (g 6729485) 4 62% (g 22380) 6 80% (g 22380) 8 48% (gi 2244810) 10 45% (g 22380) 12 81% (g 22380) 14 47% (g 2398529) 16 52% (gi 3738293) 18 73% (gi 6729485) 20 64% (g 22380) 22 62% (g 22380) 24 86% (g 22380) 26 54% (g 2398529) 28 77% (g 22380) 30 70% (g 22380)
    · · · · · · · · · »· * ·» · · · »»
    -32it are summarized in Figure 1. A central region of about 90 amino acids is conserved in all polypeptide sequences. This region contains a required functional domain for the transcription factor. Sequence alignments and% identity calculations were performed using the Megalign program of the LASERGENE bioinformatics software package (DNASTAR Inc., Madison, WI). Multiple alignment of sequences was performed using the Clustal fitting method (Higgins and Sharp: CABIOS 5 151 (1989)) with the baseline values (gap point = 10, gap length error point = 10). In the case of pairwise alignments using the Clustal method, the default values were: Ktuple = 1, gap point = 3, window = 5, and saved diagonals =
    5. Sequence alignments and BLAST values as well as probabilities indicate that the nucleic acid fragments containing the cDNA clones of the present invention encode a substantial portion of an LEC 1-related transcription factor. These sequences are the first Morordica, Eucalyptus, rice, soy and wheat as well as new maize sequences which, according to the inventors' knowledge, encode transcription factors related to LEC 1.
    Example 4
    Expression of genes in monocotyledonous cells
    It is possible to construct a chimeric gene containing a cDNA encoding the polypeptides of the invention in a sense orientation, compared to a 27 kDain maize promoter located 5 'to the cDNA fragment and a 3' end of the 10 kDa in 3 'direction is located relative to the cDNA fragment. The cDNA fragment of this gene can be generated using oligonucleotide primers corresponding to the cDNA clone polymerase chain reaction (PCR). Cloning sites (VcoI and Smai) can be incorporated into the oligonucleotides to provide proper orientation to the DNA fragment when inserted into the digested vector pML103 as described below. The amplification was then performed by standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SMI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with the 7.9 kb NcoI-Sma ffagment of plasmid pML103. Plasmid pML103 was deposited at the ATCC (American Type Breeding Collection, 10801 University Blvd., Manassas, VA 20110-2209) and received ATCC 97366. The DNA segment from plasmid pML103 contains a 1.05 kb Salí-Ncol promoter fragment from the maize 27 kDa gene and contains a 0.96 kb Smal-Sal fragment from the 3 'end of the maize 10 kDa zein gene at pGem9Zf (+) in vector (Promega). The vector and insert DNA can be ligated at 15 ° C overnight, essentially prior to the «9» 4 «« *> · · 9 * · ♦ · «· · · t»
    - · * · -7 · »9 · *« »* * ·» «
    -33 as described in [Maniatis]. The ligated DNA can then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue ™; Stratagene) cells. Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase ™ DNA Sequencing Kit; US Biochemical). The resulting plasmid construct will contain a chimeric gene encoding the 5 'to 3' direction: the 27 kDain maize promoter, the cDNA fragment encoding the polypeptides of the invention, and the 10 kDein 3 'region.
    The chimeric gene described above can then be introduced into corn cells by the following procedure. Unripe corn embryos are excised from developing caries, which are the crosses of inbred maize lines H99 and LH132. The embryos were isolated 10-11 days after pollination when they were 1.0-1.5 mm long. The embryos are then placed on the shaft side down and in contact with agarose-solidified N6 medium (Chu et al., Sci. Sin. Beijing 18: 659 (1975). The embryos were kept in the dark at 27 ° C. The scotellum of these immature embryos proliferates a fragile embryogenic callus, which contains an undifferentiated mass of cells with embryoids on somatic proembroids and suspension structures. The embryogenic callus isolated from the primary explants can be cultured on N6 medium and subcultured on this medium every 2-3 weeks.
    Plasmid p35S / Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) can be used in transformation experiments to provide selectable markers. This plasmid contains the Pat gene (see European Patent Publication No. 0 242 236) which encodes phosphinotricin acetyl transferase (PAT). PAT provides enzyme resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The Pat gene in plasmid p35S / Ac is the cauliflower mosaic virus 35S promoter [Odell et al., Natúré 313,810 (1985)] and the Ti plasmid of Agrobacterium tumefaciens Ti under the control of the 3 'region of the nopaline synthase gene from TDNA.
    The particle bombardment method (Klein et al., Nat. 32770 (1987)) is well suited for the transfer of genes into cells of callus culture. In this method, gold particles (1 µm in diameter) are coated with DNA using the following procedure. Ten µg of plasmid DNA was added to 50 µl of gold particle suspension (60 mg / ml). Calcium chloride (50 µl of 2.5 M solution) and spermidine free base (20 µl of 1.0 M solution) were added to the particles. The suspension is vortexed while adding the solutions. After ten minutes, the tubes are briefly cent * * # #
    - vortex (5 seconds, 15,000 rpm) and remove the supernatant. The particles were resuspended in 200 μΐ anhydrous ethanol, centrifuged again and the supernatant removed. The ethanol rinse was repeated and the particles were resuspended in a final volume of 30 µl ethanol. Place 5 µΐ of the DNA coated gold particles in the center of a Kapton ™ disc (BioRad Labs). The particles are then accelerated into corn tissue by means of a Biolistic ™ PDS-1000 / He apparatus (BioRad Instruments, Hercules, CA) using a helium pressure of 6900 kPa (1000 psi) at a gap spacing of 0.5 cm and a clearance of 1.0 cm.
    For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The fabric is arranged in a thin layer and covered with a circular area of about 5 cm in diameter. The tissue-containing Petri dish is placed in the PDS-1000 / He chamber at a distance of about 8 cm from the stop mesh. The air is then pumped into the chamber to a pressure of 710 mmHg. The macro carrier is accelerated by a helium shock wave using a cleavage disk that breaks when the pressure of the helium in the pressure chamber reaches 6900 kPa.
    Seven days after the bombardment, the tissue was transferred to N6 medium containing bialophos (5 mg / l) without casein or proline. The tissue is slowly growing on this medium. After a further 2 weeks, the tissue was transferred to fresh N6 medium containing bialophost. After six weeks, approximately 1 cm diameter of the actively growing callus can be identified on some of the plates containing the medium supplemented with bialophos. These calluses continue to grow when subcultures are created on selective media.
    Plants from transgenic callus can be regenerated by first transferring tissue nodules to N6 medium supplemented with 0.2 mg of 2,4-D per liter. Two weeks later, the tissue can be transferred to a regenerating medium (Fromm et al., Bio / Technology 8,833 (1990)).
    Example 5
    Expression of chimeric genes in dicotyledonous cells
    A core-specific expression cassette from the gene encoding the β-subunit of the phaseol core storage protein from Phaseolus vulgar beans [Doyle et al., J. Bioi. Chem. 261, 9228 (1986)] contains the promoter and the transcriptional terminator, which may be used to express the polypeptides of the invention in transformed soy. The phaseol cassette encompasses about 500 nucleotides in a 5'-direction from the translation initiation codon and about 1650 nucleotides in the 3'-direction from the phasein translation translation codon. The 5'- and 3'-regions are stones. <* Ί »<·» · K »e * * · · <>
    • * ·· * * Ί * λ * »-» / *. · ».« <, ·
    -35 restriction sites are unique restriction endonuclease cleavage sites: Ncol (which includes the ATG translation initiation codon), Smal, Kpril and YMI. The Complete Cartridge Hindii! boundaries.
    The cDNA fragment of this gene can be generated by polymerase chain reaction (PCR) starting from the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to ensure that the DNA fragment is properly aligned when inserted into the expression vector. Subsequently, the amplification is performed as described above and the isolated fragment is inserted into a pUC18 vector carrying the core expression cassette.
    Subsequently, soy embryos can be transformed with the expression vector containing sequences encoding the polypeptides of the invention. For the induction of somatic embryos, the cotyledons of sterilized immature seeds of the A2872 surface were cut with 3-5 mm long cotyledons and grown in light or dark at 26 ° C on a suitable agar medium for 6-10 weeks. Somatic embryos that produce secondary embryos are then excised and placed in a suitable liquid medium. After repeated selection of the somatic embryo nodes, which were propagated as early globular phase embryos, the suspensions were maintained as described below.
    Soy embryogenic suspension cultures can be maintained in 35 ml of liquid medium on a rotary shaker at 150 rpm at 26 ° C with fluorescent illumination over a 16: 8 hour light / dark period. The cultures were subcultured every two weeks by inoculating about 35 mg of tissue onto 35 ml of liquid medium.
    Soybean embryogenic suspension cultures can then be transformed by a particle-bed bombardment method (Klein et al., Nat. U.S. Patent No. 4,945,050]. A DuPont Biolistic ™ PDS-1000 / He (helium retrofit) can be used for these transformations.
    A selectable marker gene for promoting soy transformation is a chimeric gene comprising the 35S promoter from the cauliflower mosaic virus (Odell et al., 1985, Nat. 313 810), the hygromycin phosphotransferase gene from plasmid pJR225 (E. coli-böY). [Gritz et al., Gene 25, 179 (1983)] and the 3 'region of the T1 DNA plasmid T-DNA of Agrobacterium tumefaciens Ti. The core expression cassette containing the phaseol 5 'region, the fragment encoding the polypeptides of the invention, and the phaseol 3' region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
    • · · ·
    -3650 μΐ To a 60 mg / ml concentration of 1 μιη gold particle suspension, add the following: 5 μ 5 DNA (1 µg / µl), 20 µΐ spermidine (0.1 M) and 50 µΐ CaCl 2 (2.5 M). . The particle composition was then shaken for three minutes, centrifuged for 10 seconds in a microcentrifuge and the supernatant removed. The DNA-coated particles were then washed once with 400 μΐ of 70% ethanol and resuspended in 40 μΐ of anhydrous ethanol. The DNA / particle suspension was sonicated three times over a period of one second. Subsequently, 5 µből of the DNA coated gold particles are applied to each macro carrier disk.
    Approximately 300-400 mg of a two-week suspension culture was placed in an empty 60 x 15-ram Petri dish and the remaining liquid was removed from the tissue by pipette. For each transformation experiment, tissue from 5 to 10 plates was generally bombarded. The membrane fission threshold was set at a pressure of 7590 kPa (1100 psi) and the chamber was aspirated to 710 mmHg. The tissue was placed about 9 cm from the retention grid and bombarded three times. After the bombardment, the tissue was divided into two and reinserted into the liquid and then cultured as described above.
    5-7 days after bombardment, the liquid medium was replaced with fresh medium and replaced with fresh medium containing 50 µg / ml hygromycin 11 to 12 days after bombardment. This selective medium was renewed weekly. Green, transformed tissue was observed 7-8 weeks after bombardment as it emerged from untransformed, necrotic embryogenic nodules. The isolated green tissue is removed and inoculated into individual bottles to produce new, cloned, transformed, embryogenic suspension cultures. Each new line can be treated as an independent transformation event. These suspensions can then be subcultured and maintained as immature embryos in lumps or regenerated as a result of the maturation and germination of individual somatic embryos.
    Example 6
    Expression of chimeric genes in microbial cells
    The cDNAs encoding the polypeptides of the invention may be inserted into the E. coli pBT430 expression vector T7. This vector is a derivative of pET-3a (Rosenberg et al., Gene 56 125 (1987)) which uses the bacteriophage T7 RNA polymerase / T7 promoter system. Plasmid pBT430 was constructed by first destroying EcoR1 and ffindlll sites in plasmid pET-3a at their original position. An oligonucleotide adapter containing EcoR1 and HindIII sites was then inserted at the site of pET-3α / wHI. Hereby, we created the ···· ···· pET-3aM vector with additional unique cloning sites to insert genes into the expression vector. The Ndel site was then converted to Ncol site using translational initiation using oligonucleotide directed mutagenesis. The DNA sequence of pET-3aM in this region - 5'-CATATGG - was converted to the 5'-CCCATGG sequence in plasmid pBT430.
    A cDNA-containing plasmid DNA can be appropriately digested to release the nucleic acid fragment encoding its protein. This fragment can then be purified on a 1% low melting agarose gel. Both the buffer and the agarose gel contain 10 pg / ml ethidium bromide to render the DNA fragment visible. The fragment can then be purified from the agarose gel by digestion with GELase ™ (Epicentre Technologies, Madison, WI) according to the manufacturer's instructions, then precipitated with ethanol, dried and resuspended in 20 μΐ of water. Appropriate oligonucleotide adapters can be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, MA). The fragment containing ligated adapters can be purified from excess adapters using low melting agarose as described above. The pBT430 vector was digested, dephosphorylated with alkaline phosphatase (NEB), and the protein / chloroform mixture was released as described above. The prepared pBT430 vector and fragment were then ligated at 16 ° C for 15 hours followed by transformation into DH5 electro-competent cells (GIBCO BRL). Transformants may be selected on LB medium and agar plates containing 100 pg / ml ampicillin. Transformants containing the gene encoding the polypeptides of the invention are then screened by restriction enzyme analysis for correct orientation to the T7 promoter.
    For high-level expression, a plasmid clone can be transformed into E. coli BL21 (DE3) in which the cDNA insert relative to the T7 promoter is located in the appropriate orientation [Studier et al., J. Mol. Biol. 189, 113 (1986). Cultures were grown on ampicillin (100 mg / l) LB medium at 25 ° C. When the optical density at 600 nm is about 1, IPTG (isopropylthio-β-galactoside, inducer) is added to a final concentration of 0.4 mM and incubation is continued for 3 hours at 25 ° C. The cells were then harvested by centrifugation and resuspended in 50 µl of buffer (50 mM Tris-HCl, pH 8.0 containing further 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride). Small amounts of 1 mm glass beads were added and the mixture was sonicated three times for 5-5 seconds with a microprojector sonicator. The mixture was centrifuged and the supernatant protein content was determined. A pg protein from the soluble fraction of culture was separated by SDS-polyacrylamide gel electrophoresis. The gels
    -38 examining protein bands that migrate with the expected molecular weight.
    • · «·
    -39FREE DETAILS
    An isolated polynucleotide comprising:
    (a) a nucleotide sequence encoding a polypeptide having LEC1-related transcriptional activity, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 16, 16
    Any of the amino acid sequences of SEQ ID NO: 18, 20, 22, 24, 26, 28 or 30, having at least 90% sequence identity based on the Clustal fitting method; or (b) a complement of the nucleotide sequence, wherein the complementary and nucleotide sequences comprise the same number of nucleotides and are 100% complementary to each other.
    The polynucleotide of claim 1, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 Any of the amino acid sequences of SEQ ID NO: 26, 28, or 30 will exhibit sequence identity of at least 92% based on the Clustal fitting method.
    3. The polynucleotide of claim 1, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 Any of the amino acid sequences of SEQ ID NO: 26, 28, 30 or 30 will have at least 94% sequence identity based on the Clustal fitting method.
    The polynucleotide of claim 1, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 Any of the amino acid sequences of SEQ ID NO: 26, 28, or 30 will exhibit sequence identity of at least 96% based on the Clustal fitting method.
    . The polynucleotide of claim 1, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, Any of the amino acid sequences of SEQ ID NO: 26, 28, or 30 will exhibit sequence identity of at least 98% based on the Clustal fitting method.
    6. The polynucleotide of claim 1, wherein the polypeptide has an amino acid sequence comprising 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 , Amino acid sequences of SEQ ID NO: 26, 28, or 30.
    The polynucleotide of claim 1, wherein said nucleotide sequence is
    Any of the nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27 or 29 .
    • · ·
    -408. A vector comprising a polynucleotide according to claim 1.
    A recombinant DNA construct comprising a polynucleotide according to claim 1 linked to a functional regulatory sequence.
    10. A method for transforming a cell comprising transforming the cell with a polynucleotide according to claim 1.
    A cell comprising a recombinant DNA construct according to claim 9.
    12. A method for preparing a plant comprising transforming a plant cell with a polynucleotide according to claim 1 and regenerating the plant from the transformed plant cell.
    13. A plant comprising a recombinant DNA construct according to claim 9.
    14. A core comprising a recombinant DNA construct according to claim 9.
    An isolated polynucleotide comprising a first nucleotide sequence comprising at least 30 nucleotides and wherein the first nucleotide sequence comprises another polynucleotide comprising the other polynucleotide comprising:
    (a) a second nucleotide sequence encoding a polypeptide having Lee 1-related transcription factor activity, wherein the polypeptide has the amino acid sequence and 2, 4, 6, 8, 10, 12, 14, 14 and 14; Any of the amino acid sequences of SEQ ID NOs: 16, 18, 20, 22, 24, 26, 28, or 30 exhibits sequence identity of at least 90% based on the Clustal fitting method; or (b) a complement of the second nucleotide sequence, wherein the complementary and second nucleotide sequences comprise the same number of nucleotides and are 100% complementary to each other.
    16. An isolated polypeptide having Lecl-like transcription factor activity, the amino acid sequence of the polypeptide and the sequences of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 22; Any of the amino acid sequences of SEQ ID NO: 24, 24, 26, 28, or 30 will exhibit sequence identity of at least 90% based on the Clustal fitting method.
    17. The polypeptide of claim 16, wherein the polypeptide is an amino acid sequence and a polypeptide of claim 2,
    Any of the amino acid sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 has similar sequence identity based on the Clustal fitting method.
    18. A polypeptide according to claim 16, wherein the polypeptide is an amino acid sequence and a polypeptide of claim 2, wherein:
    Any of the amino acid sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 has the same degree of sequence identity for the · · ·
    -41 ··· *
    Based on Clustal Matching Method.
    19. A polypeptide according to claim 16, wherein the polypeptide is an amino acid sequence and the polypeptide of claim 2 is selected from the group consisting of:
    Any of the amino acid sequences of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 has the same degree of sequence identity a
    Based on Clustal Matching Method.
    20. A polypeptide according to claim 16, wherein the polypeptide is an amino acid sequence and a polypeptide of claim 2, wherein:
    4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26.28. or amino acid sequences of SEQ ID NO: 30 or SEQ ID NO: 30 shows sequence identity of at least 98% based on the Clustal fitting method.
    21. The polypeptide of claim 16, wherein the amino acid sequence of the polypeptide is 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24. , SEQ ID NOs: 26, 28, 30.
    22. A method for isolating a polypeptide encoded by a polynucleotide according to claim 1, wherein the polypeptide is isolated from a cell carrying a recombinant DNA construct linked to a polynucleotide functionally regulatory sequence.
    Who
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    Fig. 1B »·« · · · · · · · · · · · · · · · · · · · ·
    PCT / US01 / 50 908
    3/4
    SLID 2: GGDESAK RDAVC-ALPGQNS ---- QQYMQPG AMTYINTQG ----------------- QHLIIPSMQNNE-
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    Figure 1C
    <3 · HORSE co He H (\ ri vr (f lm r t co «Η He CL co CL CM co CL CO He C0 rL CO CL ΓΟ ΓΟ ro 'L 1 co 1 í ' main Q Q G Q Q Q He Q Q Q G Q Q Q Q Q G G r-1 I-4 t-1 I-1 i-1 -1 FH · - · OJ EH • rL M hl H4 hl hl Ht co CO co 00 ω co co co CO CO CO CO CO ω co co CO 2 co
    «· * · ·· * · ·« ·
    PCT / US01 / 50908 ··· «* ·» ··
    4/4
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    Figure 1D
    ο Ο] <Ί ' X co ο X <0 C0 ο {Γ- Γ0 LP, C0 Η ~ ι ί-1 t-1 «-1 1-1 CN CN CM OJ C0 ΓΟ Γθ CO ΓΟ ΓΟ ο Q Ω ο X Q Q ο α G D Q X α α ο Q Μ rh Μ Μ > -ί Μ Μ 1-1 Μ ί-ΐ h l Η-1 κ-ι Μ Η4 > Η Μ C0 0 0 0 0 0 0 0 0 ο: ω 0 0 0 0 09 0
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