WO2012005589A1 - Gène végétal de résistance à la chaleur hsf1 et son utilisation - Google Patents

Gène végétal de résistance à la chaleur hsf1 et son utilisation Download PDF

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WO2012005589A1
WO2012005589A1 PCT/NL2011/050497 NL2011050497W WO2012005589A1 WO 2012005589 A1 WO2012005589 A1 WO 2012005589A1 NL 2011050497 W NL2011050497 W NL 2011050497W WO 2012005589 A1 WO2012005589 A1 WO 2012005589A1
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plant
protein
polynucleotide
seq
sequence
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PCT/NL2011/050497
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English (en)
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Chuan-bao SUN
Yu-ke HE
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
De Lang, R.J.
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Publication of WO2012005589A1 publication Critical patent/WO2012005589A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • the present invention belongs to the fields of biotechnology and botany.
  • the present invention relates to a new method for improving heat resistance of a plant.
  • the invention involves the use of a protein in said plant for improving heat resistance.
  • the present invention relates to the enhancement of the expression or activity of the protein, thereby providing improved heat resistance to a plant in comparison to a plant not modified to enhance expression of the protein.
  • Cabbages mainly include Brassica campestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensis.
  • Brassica campestris L. ssp. chinensis is also named as green cabbage, and baby Brassica campestris L. ssp. chinensis in the north of China.
  • Brassica campestris L. ssp. chinensis exhibits high adaptability, growth, productivity and nutrition. It is the most consumed vegetable among various vegetables and widely grown in the provinces in the regions of Changjiang valley in China.
  • Cabbages have a short growth period, wide adaptability, and high productivity. They are also easy to plant, which allows for a sustained perennial supply.
  • the products of Brassica campestris L. ssp. chinensis are fresh and tender, have rich nutrition and win favor of consumers.
  • Brassica campestris L. ssp. chinensis comprises about 30-40% of the total domestic vegetable productivity a year, and also makes a significant contribution in supplementing vegetables in slack seasons and balancing the vegetable supply over a whole year. Both the Brassica campestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensis favor cool whether and can be planted perennially. The most suitable growth temperature is 15-20°C.
  • Brassica campestris L. ssp. chinensis In recent years, to meet the market demand, Brassica campestris L. ssp. chinensis generally needs to be planted in different manners in different seasons. In the past, Brassica campestris L. ssp. chinensis was mainly planted in spring and winter. Now people begin to plant Brassica campestris L. ssp. chinensis in torrid summer and autumn by various culture manners. This will undoubtedly make Brassica campestris L. ssp. chinensis subject to the stress from high temperatures during its growth, especially in late spring, summer and early autumn. The Brassica campestris L. ssp.
  • chinensis cultured in the seasons of high temperature can go to the market in bulk after a 20-day culture.
  • the high temperatures usually lead to an elongated internode, slowed growth, bitter taste and undesirably increased fiber, etc. This will result in low productivity and poor quality.
  • the consumer demand cannot be met.
  • Brassica campestris L. ssp. Pekinensis has poor tolerance to high temperature. It is highly temperature sensitive in the rosette stage and the heading stage. If the average temperature is too high, the heart leaf can not amplexate to built a tight bulb, or can not bulb up at all. Even if it constrainedly bulbs up, the heading is loose.
  • chinensis were originally planted in China. In foreign countries, there is few studies on breeding of cabbages. Varieties of Japanese, Korean and Formosan origins are poor in heat resistance, and unsuitable for planting in China. Domestically dominant are mainly the disease resistant varieties planted in autumn. Vegetables of cabbages have a narrow gene library for heat-resistance. Breeding of heat-resistance cabbage variety is limited to the screening among the cabbage materials, whereby only some varieties with poor heat resistance and low stress resistance have been obtained.
  • the domestic breeding experts have utilized the traditional breeding methods to widely screen and culture heat-resistance varieties of vegetables of cabbages, to introduce heat- resistance genes, and broaden the sources of exploitation, which improved the heat- resistance of vegetables of cabbages to a certain degree and have produced effect in actual production.
  • the current methods are limited to the assessment of heat resistance under the local climate and the morphological changes under a high temperature stress. These methods are not suitable for the temperate areas, which can not provide the field conditions with suitable selection stresses. Even if a single heat-resistance plant was selected, a series of complicated methods and means would be required to maintain the heat-resistance in the seeds collected until the next spring.
  • the screening requires a long period, and is geographically limited, which can not provide a heat resistant variety universally adaptable. Therefore, it is an urgent task in breeding of heat-resistance vegetables of cabbages to intensively study the occurrence and development of the heat damages during the seedling stage, and to develop a method and technique for screening heat resistance in seedling stage, which provides improved operability, stability, efficiency and adaptability.
  • the traits closely associated with the heat resistance in cabbages are of a quantitative nature, which poses great difficulties in genotyping. Particularly for molecular breeding, the difficulties include not only the limited number of DNA markers useful in the auxiliary selection, but also the inconsistence of the number and the significance of the quantitative traits loci (QTL).
  • Gene chip comprises a support on which a lot of specific oligonucleotide fragments or gene fragments as probes are arranged and fixed, which forms a DNA microarray.
  • the DNA or RNA in a sample is fluorescently labeled via various techniques such as PCR amplification and in vitro transcription. After the probes hybridize to the labeled molecules in the sample, the chip is scanned by a fluorescence detection system and the fluorescent signals of all the probes are compared and measured by using a computer system.
  • gene chip technique has been widely used in various fields, such as drug screening, agriculture, diagnosis and treatment of disease, identification of species of traditional Chinese medicine, judicial expertise, supervise on food and sanitation, environment detection, national defense and the like.
  • gene chips There are not many reports about using gene chips in plants. The reports mainly focus on Arabidopsis thaliana, strawberry, morning glory and the like.
  • analysis and detection of gene expression level may be the most popular and established. Since thousands of probes can be fixed onto a chip, it is possible to simultaneously detect a lot of genes.
  • an isolated protein which is:
  • (c) a protein derived from the protein of (a), having at least 60% identity to the amino acid sequence of SEQ ID NO:3 and having an equivalent function as the amino acid sequence represented by SEQ ID NO:3.
  • an isolated plant heat resistance protein has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence represented by SEQ ID NO:3.
  • 1 -20, preferably 1 -10, more preferable 1 -5, most preferably 1 -3 residues are substituted, deleted or added in the amino acid sequence of SEQ ID No:3.
  • the plant is a plant of Cruciferae.
  • the Cruciferae plant is selected from the group consisting of Brassica spp. plant and Abrabidopsis spp. plant.
  • the Brassica spp. plant is Brassica campestris.
  • the Abrabidopsis spp. plant is Arabidopsis thaliana (L.) Heynh.
  • the protein is derived from the Brassica spp. plant, preferably, it is derived from Brassica campestris L. ssp. Pekinensis.
  • an isolated polynucleotide is provided, which is selected from the group consisting of:
  • nucleotide sequence of said polynucleotide is set forth in SEQ ID NO: 1 or 2.
  • a vector which contains said polynucleotide.
  • a genetically engineered host cell which comprises said vector or the genome of which is integrated with said polynucleotide.
  • a plant which contains any of the aforementioned polynucleotides.
  • a method for preparing the aforementioned protein which comprises:
  • use of the aforementioned protein or its coding gene for improving heat- resistanceof a plant is provided, or for enhancing expression of HSP70 in a plant.
  • a method for improving the heat-resistance of a plant is provided, which comprises enhancing the expression or activity of the aforementioned protein in said plant.
  • said method comprises transforming the polynucleotide encoding the aforementioned protein into the genome of the plant.
  • said method comprises:
  • step (1 ) (2) contacting plant cell, organ or tissue with the agrobacterium of step (1 ), such that the coding sequence of the protein is introduced into the plant cell ;
  • the introduced coding sequence is integrated into the genome of the plant cell.
  • a transgenic plant obtained or obtainable by the aforementioned method is provided.
  • a molecular marker for identifying heat- resistance or improved heat-resistance in a plant wherein said molecular marker comprises at least 50 nucleotides of the sequence of SEQ ID. No 1 or 2.
  • a method is provided wherein said molecular marker is identified in a plant by sequencing the DNA of a plant cell. In one embodiment, a method is provided wherein said molecular marker is identified by amplifying the said sequence of SEQ ID No. 1 or 2 and detecting the amplicon. In one embodiment, a pair of primers is provided capable of amplifying the said sequence of SEQ ID No. 1 or 2. In one embodiment, a pair of primers is provided represented by the nucleotide sequences SEQ ID NO: 4 and 5.
  • Figure 1 shows the expression patterns of the BcpHSFI and the gene AtHSP70
  • WT refers to wild type Arabidopsis.
  • OE-line1 and OE-line2 refer to transgenic plants, with over-expressed BcpHSFI. After a heat treatment of the Arabidopsis plants, which have been cultured for 14 days, at 42°C for
  • ACTIN is the marker.
  • gDNA refers to the genomic DNA.
  • the "28c” indicates a PCR of 28 cycles, “26c” indicates a PCR of 26 cycles, while “20c” means 20 cycles.
  • Figure 2 shows that the transgenic plants 35S::BcpHSF1 , which over-express BcpHSFI, exhibit improved heat-resistance.
  • Panel A indicates the growth status of the transgenic plants 35S::BcpHSF1 over-expressing BcpHSFI (OE-line1 and OE-line2) under the conditions of heat treatment.
  • the 7-day old seedlings of the wild type and transgenic plants were treated at 44°C for 1 hour and then switched back to 22°C for another 7 days before photos were taken.
  • Panel B indicates that the survival rate of the two transgenic plants (OE-line1 and OE-line2) was significantly increased as compared with the wild type plant.
  • the conditions for heat treatment are identical to panel A.
  • the data are the average values of five experiments.
  • Figure 3 shows the analysis on the domains in the BcpHSFI protein (SEQ ID NO:3).
  • a method for isolating "a" DNA molecule includes isolating a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).
  • heat stress refers to a sub-optimal environmental condition associated with temperature.
  • heat refers to an environmental condition wherein the temperature of the atmosphere and/or soil is higher than optimal for growth and/or development.
  • the optimal temperature of the atmosphere for growing cabbages is in the range of 15-20°C.
  • the cabbages are subjected to "heat stress".
  • the effect of subjecting plants to "heat stress” may be that plants do not have optimal growth and/or development. For example, subjecting Brassica campestris L. ssp.
  • chinensis to heat stress may have the effect of elongating internode, slowing growth, providing bitter taste, increasing fiber content etc.
  • Subjecting Brassica campestris L. ssp. Pekinsis to heat stress during the rosette stage and the heading stage may have the effect that the heart leaf can not amplexate to built a tight bulb, or it can not bulb up at all. Even if the heart leaf constrainedly bulbs up, the heading may be loose.
  • heat resistant or “heat resistance” refers to plants which, when provided with heat resistance (or being heat resistant), when subjected to heat stress do not show effects or show alleviated effects as observed in plants not provided with heat resistance
  • heat resistance is a relative term determined by comparing plants with another plant, whereby the plant most capable of sustaining (normal) growth may be a "heat resistant” plant, whereas the plant less capable may be termed a "heat sensitive” plant.
  • Aligning and alignment With the term “aligning” and “alignment” is meant the comparison of two or more nucleotide sequences based on the presence of short or long stretches of identical or similar nucleotides. Several methods for alignment of nucleotide sequences are known in the art, as will be further explained below.
  • “Expression of a gene” refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, e.g. which is capable of being translated into a biologically active protein or peptide or active peptide fragment.
  • An active protein in certain embodiments refers to a protein being constitutively active.
  • the coding sequence is preferably in sense- orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.
  • modifying the expression level of the gene e.g. by enhancing expression or reducing expression
  • the quantitative and/or qualitative feature of a plant is affected.
  • enhancing gene expression may lead to heat resistance.
  • the skilled person will have no difficulties in testing functionality with regard to abiotic stresses such as heat.
  • gene means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter).
  • a gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3' non- translated sequence comprising e.g. transcription termination sequence sites.
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using published techniques. See, e.g.: (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTER ANALYSIS OF
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J.
  • Methods to determine identity and similarity are codified in computer programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1 ):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of a certain sequence it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference polypeptide sequence.
  • nucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted and/or substituted with another nucleotide, and/or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: 1 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 1 .
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a nucleic acid according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800
  • the present invention contemplates any deoxyribonucleotide, ribonucleotide or peptide nucleic acid component, and any chemical variants thereof, such as methylated,
  • the polymers or oligomers may be heterogenous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex,
  • heteroduplex and hybrid states.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein".
  • a “chimeric protein” or “hybrid protein” is a protein composed of various protein "domains” (or motifs) which is not found as such in nature but which a joined to form a functional protein, which displays the functionality of the joined domains.
  • a chimeric protein may also be a fusion protein of two or more proteins occurring in nature.
  • domain as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain.
  • Plant refers to either the whole plant or to parts of a plant, such as cells, tissue or organs (e.g. pollen, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing or crossing.
  • Plant cell(s) include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.
  • promoter includes herein also the 5' UTR region (5' Untranslated Region) (e.g.
  • the promoter may herein include one or more parts upstream (5') of the translation initiation codon of a gene, as this region may have a role in regulating transcription and/or translation.
  • a "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions.
  • inducible promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmental ⁇ regulated.
  • tissue specific promoter is only active in specific types of tissues or cells.
  • promoter active in plants or plant cells refers to the general capability of the promoter to drive transcription within a plant or plant cell. It does not make any implications about the spatio-temporal activity of the promoter.
  • protein or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein”.
  • isolated protein is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.
  • a "genetically modified plant” refers herein to a plant or plant cell having been transformed, e.g. by the introduction of a mutation in an endogenous gene or part there of such that expression is enhanced, or by the introduction of an exogenous gene or additional copy or copies of an endogenous gene, said exogenous gene or additional endogenous gene may be integrated into the genome.
  • transgenic plant cell may refer to a plant cell in isolation or in tissue culture, or to a plant cell contained in a plant or in a differentiated organ or tissue, and both possibilities are specifically included herein.
  • a reference to a plant cell in the description or claims is not meant to refer only to isolated cells or protoplasts in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present.
  • Methods for obtaining transgenic plant cells and plants are well known in the art and include but are not limited to ⁇ grobacfer/i/m-mediated transformation of plant explants, particle bombardment of plant explants, transformation of plant explants using whiskers technology, transformation using viral vectors, electroporation of plant protoplasts, direct uptake of DNA by protoplasts using polyethylene glycol, microinjection of plant explants and/or protoplasts./ ⁇ grot»acier/ ' t/m- mediated transformation is a preferred method to introduce the nucleic acid molecule of the invention into plant explants.
  • Agrobacterium tumefaciens harbors a natural vector called Ti plasmid which was engineered to make it suitable for introduction of exogenous nucleic acid molecules into plant genomes.
  • plant-derived explants are incubated with suspension of Agrobacterium cells followed by cultivation of the explants on the medium containing a selective agent that promotes growth and regeneration of the transformed cells only.
  • the present inventors by using the chip technique in developing plant heat-resistance genes, have isolated a new plant heat-resistance gene from Brassica spp., which can be used to improve the heat-resistance in a plant.
  • the isolated gene is named as "HSF1", based on which, transgenic plants with improved heat-resistancecan be produced.
  • the plants include various crops, flower plants or plants of forestry, etc. Specifically, the plants include, but are not limited to, dicotyledon, monocotyledon or gymnosperm.
  • the plants include, but is not limited to, wheat, barley, rye, rice, corn, sorghum, beet, apple, pear, plum, peach, apricot, cherry, strawberry, Rubus swinhoei Hance, blackberry, bean, lentil, pea, soy, rape, mustard, opium poppy, olea europea, helianthus, coconut, plant producing castor oil, cacao, peanut, calabash, cucumber, watermelon, cotton, flax, cannabis, jute, citrus, lemon, grapefruit, spinach, lettuce, asparagus, cabbage, Brassica campestris L. ssp. Pekinensis, Brassica campestris L. ssp.
  • chinensis carrot, onion, murphy, tomato, green pepper, avocado, cassia, camphor , tobacco, nut, coffee, aubergine, sugar cane, tea, pepper, grapevine, nettle grass, banana, natural rubber tree and ornamental plant, etc.
  • plant(s) includes, but is not limited to, plants of Cruciferae, Gramineae and Rosaceae.
  • the "plant” includes but is not limited to Brassica campestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensis of Brassica spp. of the Cruciferae; Abrabidopsis spp. plant of the Cruciferae; rice of Gramineae; and tobacco, melon and fruit, vegetable, rape and the like. More preferably, the "plant” is a plant of the Brassica spp. or Abrabidopsis spp. of the Cruciferae.
  • isolated means that a substance has been separated from the original or native environment where it is initially found.
  • a polynucleotide and a polypeptide in a natural state in the living cell is not isolated or purified.
  • the same polynucleotide or polypeptide is separated from the other substances that coexist in the said natural state, it is called “isolated” and/or “purified”.
  • isolated plant heat-resistance protein polypeptide
  • isolated polypeptide that improves the plant heat-resistance isolated BcpHSFI protein
  • isolated BcpHSFI polypeptide refers to the BcpHSFI protein substantially free of the other proteins, lipids, saccharides and other substances that may be naturally associated with said protein.
  • a person skilled in the art can utilize standard protein purification techniques to purify the BcpHSFI protein.
  • the substantially pure polypeptide may form a single major band on a non-reduced polyacrylamide gel.
  • the term “comprising”, “having” or “containing” includes “comprising”, “consisting substantively of”, “consisting essentially of”, and “consisting of”.
  • the “consisting substantively of”, “consisting essentially of” and “consisting of” are specific concepts of the generic terms “comprising", “having” and “containing”.
  • the polypeptide of the present invention can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. Preferably, it is a recombinant polypeptide.
  • the polypeptide of the current invention can be a product purified from a natural source, chemically synthesized, or recombinantly produced by prokaryotic or eukaryotic hosts (such as, bacterium, yeast, higher plant, insect and mammalian cell). Depending onthe host used in recombinant production, the polypeptide of the present invention can be glycosylated or non-glycosylated.
  • the polypeptide of the current invention can further include or not include the first native methionine residue.
  • the present invention further includes fragments, derivatives and analogs of the BcpHSFI protein.
  • fragments, derivatives and analogs of the BcpHSFI protein As used herein, the terms “fragment”, “derivative” and “analog” refer to the polypeptide that have substantially the same biological function and/or activity of the
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or several conservative (preferred) or non-conservative amino acid residues are substituted by one or more amino acid residues that are genetically encoded or not, or (ii) a polypeptide with one or more amino acid residues bearing a substituent, or (iii) a fusion polypeptide of the mature polypeptide and another compound (such as a compound for extending the half life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by an additional amino acid sequence (such as a leader sequence or a secretion sequence, or a sequence facilitating purification, or a proteinogen sequence, or a fusion protein) fusing to the polypeptide sequence.
  • these fragments, derivatives and analogs are understood by a person skilled in the art.
  • BcpHSFI protein refers to a polypeptideproviding improved heat- resistance based on the sequence of SEQ ID NO:3. This also includes the variants of SEQ ID NO:3 that exhibit improved plant heat-resistance. Mutations include but are not limited to deletion, insertion and/or substitution of one or more (generally 1 -50, preferably 1 -30, more preferably 1 -20, most preferably 1 -10, further more preferably 1 -8 or 1 -5) amino acids, and addition or deletion of one or more (generally within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • substitution with an amino acid residue having close or similar property will generally not affect the function of the protein.
  • addition or deletion of one or more amino acids from the C-terminus and/or N-terminus will generally not affect the function of the protein.
  • the term also includes the active fragments and active derivatives of the BcpHSFI protein.
  • Variants of the polypeptide includes its homologous sequence, conservative mutants, allelic mutant, natural mutant, induced mutant, protein encoded by a DNA that could hybridize to the DNA of BcpHSFI protein under a high or low stringent condition, and polypeptide or protein obtained by utilizing an anti-serum against the HSF1 protein.
  • the present invention also provides more related polypeptides, such as fusion proteins containing BcpHSFI protein or fragments thereof.
  • the present invention also includes the soluble fragments of the BcpHSFI protein.
  • the fragment contains at least about 20, generally at least about 30, preferably at least about 50, more preferably at least about 80, most preferably at least about 100 continuous amino acid of the BcpHSFI protein.
  • the present invention also provides analogs of the BcpHSFI protein or polypeptide. These analogs may be different from the native BcpHSFI protein in the primary sequence or in modification patters along the same primary sequence, or both. These polypeptides include the natural or induced genetic mutants. The induced mutants may be obtained via various techniques, for example, by radiation or by exposure to a mutagen so as to produce a random mutagenesis. They may also be obtained by site-directed mutagenesis or some other known biological technologies. The analogs also include those having residues different from the natural L-amino acid (such as D-amino acid), and those having un-natural or synthetic amino acid(s), such as ⁇ - and ⁇ - amino acids.
  • L-amino acid such as D-amino acid
  • un-natural or synthetic amino acid(s) such as ⁇ - and ⁇ - amino acids.
  • polypeptide of the subject invention is not limited to the above representative examples.
  • Modification patterns which will not change the primary structure, include in vivo or in vitro chemical derivation, such as acetylation or carboxylation. Modification may also be glycosylation. Modification may also be phosphorylation of the amino acid residues (such as, phosphorylated tyrosine, phosphorylated serine, and phosphorylated threonine) in the sequence. Also included are polypeptides which are modified to have an improved anti- proteolysis property or optimize the solubility property.
  • a conservative mutant of BcpHSFI protein refers to a polypeptide having up to 20, preferably up to 10, more preferably up to 5, most preferably up to 3 amino acids in the amino acid sequence of SEQ ID NO:3 being replaced by amino acids with a similar properties. These mutant polypeptides may be produced according to the amino acid replacement shown below in Table 1 .
  • the present invention further provides polynucleotide sequences encoding the BcpHSFI protein of the current invention or conservative variant polypeptides thereof.
  • the polynucleotides of the present invention may be DNA or RNA molecules.
  • the DNA molecules include cDNA, genomic DNA and synthetic DNA.
  • the DNA molecules may be in the form of a single strand or of double strands.
  • the DNA molecule may be the coding strand or the non-coding strand.
  • the coding sequence encoding the mature polypeptide may be identical to the coding sequence of SEQ ID NO: 1 or 2, or may be their degeneration variants.
  • a degeneration variant refers to a nucleic acid molecule that encodes a protein having the sequence of SEQ ID NO: 3 with a nucleotide sequence different from the coding sequence as set forth in SEQ ID NO: 1 or 2.
  • the polynucleotides encoding the polypeptide of SEQ ID NO:3 may comprise a coding sequence only encoding the polypeptide; a coding sequence of the polypeptide and an additional coding sequence; the coding sequence of the polypeptide and a non-coding sequence, optionally as well as an additional coding sequence.
  • polynucleotide encoding a polypeptide may optionally include, in addition to the polynucleotide encoding said polypeptide, an additional coding and/or a non-coding polynucleotide.
  • the present invention further relates to variants of the above polynucleotides, which encode the same amino acid sequence of the polypeptide of the present invention, and fragments, analogs and derivatives thereof.
  • the variants of the polynucleotides may be the naturally occurring allelic mutants or non-naturally occurring mutants.
  • the nucleotide variants include substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, wherein the mutation may be substitution, deletion or insertion of one or more nucleotides, but the function of the polypeptide encoded by the allelic variant is substantively un-altered.
  • the present invention also relates to a polynucleotide hybridizing to any of the above sequences and having at least 50%, preferably at least 70%, more preferably at least 80% sequence identity between the two sequences.
  • the present invention specifically relates to a polynucleotide hybridizing to the polynucleotides of the present invention under stringent conditions.
  • the "stringent condition” refers to: (1 ) hybridization and elution at a relatively lower ionic strength and relatively higher temperature, such as 0.2xSSC, 0.1 %SDS, 60°C; or (2) presence of denaturation agent during hybridization, such s 50%(v/v) formamide, 0.1 % calf serum/0.1 %Ficoll, 42°C, and the like; or (3) conditions only allowing hybridization between two sequences that have at least 80%, preferably at least 90%, more preferably at least 95% identity.
  • the polypeptide encoded by the hybridizing polynucleotide exhibits the same biological function and activity as those of the mature polypeptide as shown in SEQ ID NO: 3.
  • nucleic acid fragments that can hybridize to the any of the above sequences.
  • a "nucleic acid fragment” contains at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, most preferably at least 100 nucleotides.
  • the fragment of nucleic acid may be used in the amplification technique of nucleic acid (such as PCR) to determine and/or isolate the polynucleotide encoding the BcpHSFI protein.
  • the full-length nucleotide sequence of the BcpHSFI protein of the present invention or fragment thereof can typically be prepared via PCR amplification method, recombinant method or artificial synthesis.
  • the sequences of interests can be amplified by designing primers according to the related nucleotide sequence disclosed in the present invention, e.g. the open-reading frame, and using a commercially available cDNA library or a cDNA library prepared according to conventional methods known in the art as a template.
  • a commercially available cDNA library or a cDNA library prepared according to conventional methods known in the art as a template For large sequence, two or more PCR amplications may be needed, the fragments thus obtained in each amplification may be fused together, e.g. by ligation, in a correct orientation.
  • the sequence may cloned into a vector.
  • the vector may be transformed into a cell, and then the sequence can be isolated from the proliferated host cells using conventional means.
  • the related sequence can be synthesized by artificial synthesis, especially when the fragment is relatively short. Generally, several small fragments are first
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be prepared completely via chemical synthesis.
  • the obtained DNA sequence can be incorporated into various known DNA molecules (such as vectors) and then into cells. Further, mutations may be introduced into the protein sequence of the present invention through the chemical synthesis.
  • the present invention also relates to a vector containing the polynucleotide of the present invention, a host cell genetically engineered to contain the vector or the coding sequence of the BcpHSFI protein of the present invention, and a method for recombinantly producing the polypeptide of the present invention.
  • the polynucleotide of the present invention can be used to express or produce a
  • the polynucleotide sequence of the BcpHSFI protein can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus and any other vectors known in the art. Any plasmids and vectors can be used as long as they can replicate and retain stably in the host. Expression vectors may compriset a replication origin, promoter, markers and/or translation control element.
  • an expression vector containing a DNA sequence encoding the BcpHSFI protein and suitable transcription/translation regulatory signals. These methods include in vitro recombinant techniques, DNA synthesis, in vivo recombinant techniques, etc.
  • the DNA sequence may be operably linked under a suitable promoter for directing mRNA synthesis in the expression vector.
  • the expression vector can further include a ribosome binding site for initiating the translation and a transcription terminator.
  • the expression vector may containone or more selectively labeled genes to provide phenotypic traits for selecting the transformed host cells.
  • the labeled genes may encode, for example, dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for culture of eukaryotic cells, and kanamycin or ampicillin resistance for E. coli.
  • GFP green fluorescent protein
  • the vector containing the above suitable DNA sequence and suitable promoter or regulatory sequence can be used to transform suitable host cells for protein expression.
  • the host cell may be a prokaryotic cell, such as bacterial cell; or lower eukaryotic cell, such as yeast cell; or higher eukaryotic cell, such as plant cell. Examples include E. coli,
  • the transcription may be enhanced when an enhancer sequence is inserted into the vector.
  • the enhancer is a cis-acting factor of DNA, which may contain about 10 to 300 bp and can act on a promoter to enhance the transcription of the gene.
  • a person skilled in the art knows how to select a suitable vector, promoter, enhancer and host cell.
  • Transformation of a host cell with the recombinant DNA can be carried out using
  • the host cells are prokaryotic cells, such as E. coli
  • competent cells that can uptake the DNA can be harvested after the exponential growth phase and then treated by CaCI 2 method, towel described in the art.
  • Another method is to use MgCI 2 .
  • the transformation could be conducted using electroporation.
  • the host cell is of an eukaryotic origin, one or more of the following DNA transfecting methods may be used: calcium phosphate precipitation, conventional mechanical method such as micro-injection, electroporation, liposome packing, etc. Transformation of plant may also be achieved by using agrobacterium or gene gun transformation, and the like, such as leaf discs transformation, rice immature embryo transformation, etc.
  • the transformed plant cell, tissue or organ can be regenerated into a plant via conventional methods, so as to obtain a plant having altered traits.
  • the transformant may be cultured in conventional ways to express the polypeptide encoded by the gene of the present invention.
  • the culture medium used in the culture may be selected from various conventional culture mediums. Culturing is carried out under conditions suitable for growth of the host cell. When the host cell grows to a suitable density, the selected promoter may be induced by a suitable method (such as temperature change or chemical induction), after which the cell may be further cultured for a period of time.
  • the recombinant polypeptide can be expressed in the cell, or on the cell membrane, or be secreted outside the cell. If desired, the recombinant protein could be isolated and purified via various isolation methods by utilizing the physical, chemical or other properties of the protein. These methods are well known in the art. Examples include but are not limited to the conventional renaturation treatment, treatment with protein precipitant (such as salting out), centrifugation, osmosis (for disrupting the bacterium), ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption
  • the recombinant BcpHSFI can be used in many applications. For example, it can be used to screen for the antibody, polypeptide or the other ligands agonistic or antagonistic to the function of the BcpHSFI protein. Screening a polypeptide library with the expressed recombinant BcpHSFI protein may help finding valuable polypeptide molecules that could inhibit or stimulate the function of the BcpHSFI protein.
  • the whole polynucleotide of the present invention or a portion thereof can be used as a probe, which may be fixed onto a microarray or a DNA chip (also termed as "gene chip") to perform an analysis of gene differential expression.
  • Primers specific for the BcpHSFI protein to perform RNA reverse transcription polymerase chain reaction (RT-PCR) for in vitro amplification can also be used to detect the transcription products of the BcpHSFI protein.
  • the present invention also relates to a method for modifying a plant (to improve the heat- resistance of the plant), comprising enhancing the expression of the BcpHSFI gene and/or the activity of encoded protein in the plant. Methods for enhancing the expression of the BcpHSFI gene are well known in the art.
  • plants can be transformed with an expression construct carrying the BcpHSFI coding gene to over-express the BcpHSFI gene.
  • a promoter can be used to enhance the expression of the BcpHSFI gene.
  • An enhancer (such as the first intron of the rice waxy gene or the first intron of the Actin gene, and the like) can be used to enhance the expression of the BcpHSFI gene. Promoters include but are not limited to the 35S promoter, and the Ubi promoter in rice and corn.
  • a method for obtaining a plant with high expression of BcpHSFI protein includes:
  • step (1 ) (2) contacting a plant cell, tissue or organ with the agrobacterium of step (1 ) such that the DNA coding sequence of the BcpHSFI protein is transformed into the plant cell and integrated into the chromosome;
  • step (3) selecting the plant cell or tissue transformed with the DNA coding sequence of the BcpHSFI protein; and (4) regenerating the plant cell or tissue of step (3) into a plant.
  • Any suitable conventional means including reagents, temperature and pressure controls, can be used in this process.
  • the present invention also includes agonists to the BcpHSFI protein or its coding gene. Since the agonists of the BcpHSFI protein can regulate the activity or expression of the BcpHSFI protein, the said agonists can also enhance the heat-resistance of a plant through the influences on the BcpHSFI protein, so that to achieve the improvement on traits.
  • the agonists of the BcpHSFI protein refer to any substance that can enhance the activity of BcpHSFI , maintain the stability of BcpHSFI , promote the expression of BcpHSFI , prolong effect duration of BcpHSFI , or promote transcription and translation of BcpHSFI. These substances can be used in the present invention as agents for enhancing the heat- resistance of plant.
  • a BcpHSFI gene is provided, the genomic sequence of which is listed in SEQ ID NO: 1 , and the CDS sequence of which is indicated in SEQ ID NO: 2.
  • Said gene encodes a protein containing 435 amino acids (SEQ ID NO:3).
  • Said BcpHSFI gene provides a new route for modification of tolerance in a plant..
  • Primers used in the RT-PCT include:
  • AMV reverse transcriptase (TAKARA)
  • TAKARA RNase inhibitor
  • the PCR reaction conditions are as follows: 94°C 3 min; 94°C 30sec, 55°C 30 sec, 72°C 30sec, 25-28 cycles; 72°C 5 min.
  • the primers of Actin are used as an internal control in a parallel PCR reaction.
  • 2 x CTAB buffer (100ml): 10 ml 1 M Tris pH 8.0; 4 ml 0.5 M EDTA pH8.0; 8.19 g NaCI; 2 g CTAB; 1 g PVP K30; qs to 100ml.
  • 1 xCTAB buffer (100ml): 5 ml 1 M Tris pH 8.0; 2 ml 0.5 M EDTA pH8.0; 1 g CTAB; qs to 100ml.
  • High-salt TE 100ml: 1 ml 1 M Tris pH 8.0; 200 ⁇ 0.5 M EDTA pH8.0; 5.844 g NaCI; qs to 100ml.
  • step e) Removing the supernatant, repeating step d) for 2-3 times, and then transferring the supernatant to a new centrifuge tube, adding more than 2 volumes of precipitation buffer (1 xCTAB), gently mixing to form a uniform solution, standing at room temperature for 30 min.
  • Primers used for amplifying BcpHSFI genomic DNA from the genomic DNA are as follows: Forward: 5' C G AC C C AC AC AC AAGTAGTATATAA 3' (SEQ ID NO: 10);
  • step b) The strain solution of step a) was placed in an ice-bath for 20 min and then separated into aliquots in 5ml centrifuge tubes (4ml per tube). The tubes were placed on an ice-bath for 10 min.
  • Transformation was conducted when the stem of Arabidopsis thaliana (L.) Heynh has reached 5cm in height after bolting. For plants with a low fruition rate, transformation is to be conducted 4 days after topping.
  • the seeds were spread on a MS0 solid plate containing 50mg/l Kan. After jarovization at 4°C for two days, the plate was moved into a tissue culture chamber. The seedlings having Kan resistance were selected and transferred to grow in soil.
  • Genomic DNA was extracted from leaves. After PCR identification, the positive seedlings were obtained. A pure transgenic linage was obtained via two further passages, which were used for further analysis.
  • the transformation solution containing agrobacterium was prepared according to the methods for transforming Arabidopsis.
  • Brassica campestris L. ssp. chinensis was transformed by vacuum leaching.
  • the transformation method and conditions are identical to those used for Brassica campestris L. ssp. Pekinensis.
  • Gene expression, especially functional genes' expression, in plant is temporally and/or spatially specific.
  • the inventors detected the expression of functional genes in cabbage specimens under different heat treatment conditions by hybridizing mRNAs extracted from specimens having been subjected to different heat treatments with a chip presenting all of the functional genes in cabbages.
  • Conventional methods for detecting gene expression require a large scale of sequencing, which can only detect a few gene expressions in one time with low detection sensitivity.
  • Using gene chip technique allows for not only quantitatively and qualitatively determining gene expression level in a high sensitivity, but also studying expression of thousands of genes in one sample
  • Gene chip technology enables not only to shorten the screening time, but also to obtain more stable and more pinpointed results. It is recommendable for its high adaptability and utility value.
  • AFLP Aminified Fragment Length Polymorphism
  • This method has been widely used in various fields, including genetic mapping in vegetables, analysis on genetic diversity and relationship, location of important genes, study on regulation of gene expression, genetic fingerprinting in vegetables and identification of purity of lineage, and molecular marker-assisted selection.
  • the inventors of the present invention screened for and obtained a heat-resistance gene in cabbages using gene chip technology in combination with cDNA-AFLP technology.
  • the inventors have also developed transgenic lines that expresses said gene.
  • Brassica campestris L. ssp. Pekinensis Its genomic sequence is indicated in SEQ I D NO: 1 , its CDS sequence is indicated in SEQ I D NO:2. It encodes a protein "BspHSFV having 435aa (SEQ I D NO:3).
  • Example 2 Detection of the candidate heat-resistance genes' expression by RT- PCR after heat treatment
  • expression vector 35::BrpHSF1 (pCAMBIA1300-HSF1 ) that comprised the 35S promoter. This vector was used to transform Arabidopsis, which gave the transgenic plants OE Iine1 and OE Iine2.
  • the inventors used a heat treatment system to verify the phenotype the transgenic Arabidopsis plants. 7-day old seedlings were treated at 44°C for 1 hour, 45°C for 3 hours and 46°C for 2.5 hours.
  • the transgenic plants 35S::BcpHSF1 exhibited a higher tolerance to the heat stress as compared with the wild type plants, as showed in Fig. 2. In other words, the transgenic plants were provided with heat resistance (or heat resistance was enhanced).
  • Example 4 Phenotypes of the plants of transgenic Brassica campestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensis after heat treatment
  • the inventors of the subject invention used a heat treatment system to verify the phenotypes of the plants of transgenic Brassica campestris L. ssp. Pekinensis and Brassica campestris L. ssp. chinensis after heat treatment.
  • the seeds of the transgenic plants were accelerated to sprout, and then planted in a plastic culture bowl. The seedlings were cultured at 25°C.
  • the seedlings having consistent growth status were selected and placed in a culture box for heat treatment at an increased temperature.
  • the temperature was set to 32°C, and the treatment lasted for 10 days. Then the temperature was switched back to 25°C for 2 days.
  • Heat damage indexes were calculated and analyzed. The representative symptoms of heat damage, including leaf crimple and warp, chlorosis of leaf, growth tardiness, wilting and death of the plants, were determined and scored.
  • leaf crimple and warp lightly, A; moderately, A+; seriously, A++;
  • chlorosis of leaf lightly, B; moderately, B+; seriously, B++;
  • Brassica campestris L. ssp. Pekinensis, B-hot cabbage) were all scored as serious, which was expressed as A++B++C++D++.
  • the inventors of the subject application has identified the domains in the BcpHSFI protein (SEQ ID NO: 3), as shown in Fig. 3.
  • the results showed that positions 38-132 constitute a conservative HSF DNA-binding domain. This domain may be important for the protein's heat-resistance function.
  • the inventors constructed several variants of the BcpHSFI protein as specified below: In the sequence of the BcpHSFI protein (SEQ ID NO: 3), amino acid 7 was changed from A to V, so as to obtain BcpHSFI -M1 variant.
  • amino acid 428 was changed from L to I, so as to obtain BcpHSF1 -M2 variant.
  • amino acid 292 was changed from S to T, so as to obtain BcpHSFI -M3 variant.
  • the CDS sequence of the BcpHSFI gene shown in SEQ ID NO: 2 was first cloned into the pCAMBIA1300 vector at the Kpn I site to obtain a recombinant vector containing said CDS. Then, site-directed mutagenesis was conducted to introduce the corresponding substitution, deletion and addition to obtain the recombinant vectors containing the above-said variants respectively.
  • the recombinant vectors thus constructed were transformed into strains of agrobacterium, and then the agrobacterium strains were used to transform Arabidopsis, so that the following transgenic Arabidopsis plants were obtained: M1 -Line1 , M1 -Line2; M2-Line1 , M2-Line2; M3- Linel , M3-Line2; M4-Line1 , M4-Line2; M5-Line1 , M5-Line2; M6-Line1 , M6-Line2.
  • a heat treatment system was used to verify the phenotype of these transgenic Arabidopsis plants. 7-day old seedlings were treated at 44°C for 1 hour, 45°C for 3 hours and 46°C for 2.5 hours. The transgenic plants could better tolerate heat stress as compared the wild type plants.
  • BcpHSFI of Brassica campestris L. ssp. Pekinensis and its variants are effective heat-resistance gene which can be used to improve the heat-resistance in crops.

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Abstract

Cette invention concerne un gène végétal de résistance à la chaleur HSF1 et son utilisation. Les inventeurs de la présente ont isolé pour la première fois un nouveau gène de résistance à la chaleur à partir d'une plante de la famille des Brassica spp., qui peut considérablement améliorer la capacité de résistance à la chaleur d'une plante. Cette invention concerne, en outre, une protéine codée par ledit gène et son procédé de préparation, des vecteurs et des cellules hôtes contenant ledit gène, et un procédé de préparation d'une plante transgénique contenant ledit gène. Le procédé concerne également une plante transgénique ou comprenant ledit gène.
PCT/NL2011/050497 2010-07-08 2011-07-07 Gène végétal de résistance à la chaleur hsf1 et son utilisation WO2012005589A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109722441A (zh) * 2019-01-22 2019-05-07 广东省农业科学院蔬菜研究所 一种黄瓜小热激蛋白Cu-sHSP基因及其应用
CN114480432A (zh) * 2022-03-31 2022-05-13 四川农业大学 一种芹菜耐热基因AgHSFA6a-1及其应用
CN116814647A (zh) * 2023-05-24 2023-09-29 安徽农业大学 一种提升不结球白菜种子产量的基因cuc2及其载体、宿主细胞和应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531717B (zh) * 2014-12-18 2017-02-22 武汉大学 水稻稻瘟病抗性基因Pike及其应用
CN109609544B (zh) * 2019-01-14 2021-01-29 安徽农业大学 一种提高乌菜外植体瞬时表达率的定向遗传转化方法
CN110922460B (zh) * 2019-11-15 2022-06-17 湖南省蔬菜研究所 一种热激蛋白基因CaHSP90-1及其辅助选育耐热辣椒品种的方法
CN112981000B (zh) * 2021-04-30 2022-12-06 新疆农业大学 与棉花抗旱性相关的InDel分子标记及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102965A2 (fr) * 2008-02-15 2009-08-20 Ceres, Inc. Tolérance à la sécheresse et à la chaleur chez les plantes
WO2010075143A1 (fr) * 2008-12-22 2010-07-01 Monsanto Technology Llc Gènes et leurs utilisations en amélioration des plantes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101585870B (zh) * 2009-06-25 2011-11-16 中国农业大学 与植物耐热性相关的蛋白及其编码基因与应用
CN101585871B (zh) * 2009-06-25 2012-05-23 中国农业大学 耐热性相关蛋白及其编码基因与应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009102965A2 (fr) * 2008-02-15 2009-08-20 Ceres, Inc. Tolérance à la sécheresse et à la chaleur chez les plantes
WO2010075143A1 (fr) * 2008-12-22 2010-07-01 Monsanto Technology Llc Gènes et leurs utilisations en amélioration des plantes

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
"BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS", 1993, ACADEMIC PRESS
"COMPUTATIONAL MOLECULAR BIOLOGY", 1988, OXFORD UNIVERSITY PRESS
"COMPUTER ANALYSIS OF SEQUENCE DATA, PART", 1994, HUMANA PRESS
"GUIDE TO HUGE COMPUTERS", 1994, ACADEMIC PRESS
"Methods in Enzymology", ACADEMIC PRESS
"SEQUENCE ANALYSIS PRIMER", 1991, M STOCKTON PRESS
ALBERT L. LEHNINGER, PRINCIPLES OF BIOCHEMISTRY, 1982, pages 793 - 800
AMANJOT SINGH ET AL: "Genetic engineering for heat tolerance in plants", REVIEW ARTICLE/PHYSIOL. MOL. BIOL. PLANTS, SPRINGER, vol. 14, no. 1, 2, 1 January 2008 (2008-01-01), pages 155 - 166, XP007919321 *
ATSCHUL, S. F. ET AL., J. MOLEC. BIOL., vol. 215, 1990, pages 403
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1987, JOHN WILEY & SONS
CARILLO, H., LIPTON, D., SIAM J. APPLIED MATH, vol. 48, 1988, pages 1073
DATABASE EMBL [Online] 21 April 2008 (2008-04-21), "Brassica rapa subsp. pekinensis clone KBrH090J04, complete sequence.", XP002660569, retrieved from EBI accession no. EM_HTG:CU695316 Database accession no. CU695316 *
DATABASE Geneseq [online] 15 October 2009 (2009-10-15), "Brassica oleracea drought-resistance related protein, SEQ ID 335.", XP002660574, retrieved from EBI accession no. GSP:AXQ37086 Database accession no. AXQ37086 *
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, no. 1, 1984, pages 387
SAMBROOK ET AL.: "Molecular Cloning. A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2002, COLD SPRING HARBOR LABORATORYPRESS
VON HEINJE, G.: "SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY", 1987, ACADEMIC PRESS
YAN ZHU ET AL: "Ectopic over-expression of BhHsf1, a heat shock factor from the resurrection plant Boea hygrometrica, leads to increased thermotolerance and retarded growth in transgenic Arabidopsis and tobacco", PLANT MOLECULAR BIOLOGY, KLUWER ACADEMIC PUBLISHERS, DORDRECHT, NL, vol. 71, no. 4-5, 23 August 2009 (2009-08-23), pages 451 - 467, XP019752765, ISSN: 1573-5028, DOI: 10.1007/S11103-009-9538-2 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109722441A (zh) * 2019-01-22 2019-05-07 广东省农业科学院蔬菜研究所 一种黄瓜小热激蛋白Cu-sHSP基因及其应用
CN114480432A (zh) * 2022-03-31 2022-05-13 四川农业大学 一种芹菜耐热基因AgHSFA6a-1及其应用
CN114480432B (zh) * 2022-03-31 2022-08-30 四川农业大学 一种芹菜耐热基因AgHSFA6a-1及其应用
CN116814647A (zh) * 2023-05-24 2023-09-29 安徽农业大学 一种提升不结球白菜种子产量的基因cuc2及其载体、宿主细胞和应用
CN116814647B (zh) * 2023-05-24 2024-03-22 安徽农业大学 一种提升不结球白菜种子产量的基因cuc2及其载体、宿主细胞和应用

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