WO2012005590A1 - Gène végétal de résistance à la chaleur, bccdreb2a, et son utilisation - Google Patents

Gène végétal de résistance à la chaleur, bccdreb2a, et son utilisation Download PDF

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WO2012005590A1
WO2012005590A1 PCT/NL2011/050498 NL2011050498W WO2012005590A1 WO 2012005590 A1 WO2012005590 A1 WO 2012005590A1 NL 2011050498 W NL2011050498 W NL 2011050498W WO 2012005590 A1 WO2012005590 A1 WO 2012005590A1
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plant
protein
polynucleotide
seq
sequence
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PCT/NL2011/050498
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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 WO2012005590A1 publication Critical patent/WO2012005590A1/fr

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    • 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
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

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.
  • plants and plant products that can be obtained by the method according to the invention. . Background Art
  • 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, and 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.
  • plants provided with heat resistance it is e.g. possible to obtain higher yields of crop and/or plant product when the plant is subjected to a period or periods of heat when compared to plants not provided with heat resistance. It was found a plant can be provided with heat resistance when the expression in said plant of a DREB2A gene is enhanced.
  • the present invention thus provides for an isolated plant heat-resistance protein , which may be :
  • 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 plant heat-resistance protein is derived from the Brassica spp. plant.
  • an isolated polynucleotide is provided, which is selected from the group consisting of:
  • the nucleotide sequence of said polynucleotide is set forth in SEQ ID NO: 1 or 2.
  • a vector is provided, which contains said polynucleotide.
  • a genetically engineered host cell is provided, which comprises said vector or the genome of which is integrated with said polynucleotide.
  • a plant is provided, which comprises any of the above mentioned polynucleotides.
  • a method for preparing the aforementioned protein which comprises:
  • any of the above mentioned proteins and/or encoding genes for the above mentioned proteins is provided for providing a plant with heat-resistance.
  • a method for providing a plant with heat-resistance which comprises enhancing the expression or activity of the aforementioned protein in said plant.
  • said method comprises transforming the polynucleotide encoding any of the above mentioned proteins into the genome of the plant.
  • said method comprises:
  • step (1 ) (3) contacting the 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 and integrated into the chromosome of the plant cell.
  • said method comprises further:
  • a transgenic plant obtainable by the above method, comprising a polynucleotide encoding a heat resisitance protein according to the invention is provided.
  • said transgenic plant is obtainable or obtained by the the aforementioned method.
  • a molecular marker for identifying heat- resistance in a plant is provided, 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.
  • a method is provided wherein said molecular marker is identified by amplifying the said sequence of SEQ ID No.
  • a pair of primers is provided capable of amplifying the said sequence of SEQ ID No. 1 or 2.
  • a pair of primers is provided represented by the nucleotide sequences SEQ ID NO: 4 and 5.
  • Figure 1 shows expression patterns of the Brassica campestris L. ssp. chinensis
  • BccDREB2A homologous genes in heat-resistance and heat-sensitive green cabbage, as detected by RT-PCT. After a heat treatment at 46°C for one hour, total RNA was extracted and detected by electrophoresis. ACTIN is the marker. The “26c” indicates a PCR of 26 cycles, while “20c” means 20 cycles. “gDNA” refers to the genomic DNA.
  • Figure 2 shows that over-expression of BccDREB2A improved the heat-resistance in Arabidopsis thaliana (L.) Heynh.
  • Panel A indicates that 3 transgenic plants which over- express BccDREB2A have improved heat-resistance.
  • the upper right figure shows the result of NOS as a control, which was transformed with a vector without the BccDREB2A gene.
  • Panel B shows the results obtained from the other 3 transgenic plants expressing BccDREB2A. The seedlings were cultivated at 22°C for 7 days, transferred to 44°C for 60 minutes, and then switched back to 22°C for another 7 days before photos were taken.
  • Panel C shows the identities of the plants grown in each of the areas as shown in panels A and B.
  • Figure 3 shows the domains in the BccDREB2A protein (SEQ ID NO:3). Definitions
  • 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.
  • 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.
  • 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.
  • These 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 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes).
  • 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, has isolated for the first time 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 "BccDREB2A", based on which, transgenic plants with improved heat resistance can 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 BccDREB2A protein
  • isolated BccDREB2A polypeptide refers to the BccDREB2A protein substantially free of other proteins, lipids, saccharides and other substances that may be naturally associated with said protein.
  • a skilled person in the art can utilizea standard protein purification techniques to purify the BccDREB2A protein.
  • the substantially pure polypeptide forms a single major band on a non-reduced polyacrylamide gel.
  • the term “comprising", “having” or “containing” includes “comprising",
  • 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 present 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). According to the 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
  • the terms “fragment”, “derivative” and “analog” refer to the polypeptide that have substantiallythe same biological function and/or activity of the BccDREB2A protein of the present invention.
  • 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 proteinogenic sequence, or a fusion protein) fusing
  • BccDREB2A protein refers to a polypeptide providing improved heat resistance based on the sequence of SEQ ID NO:3. This term also includes the variants of SEQ ID NO:3 that can provide 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. For example, it is understood that substitution with an amino acid residue having close or similar property will generally not affect the function of the protein.
  • the term also includes the active fragments and active derivatives of the BccDREB2A 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 BccDREB2A protein under a high or low stringent condition, and polypeptide or protein obtained by utilizing an anti-serum against the BccDREB2A protein.
  • the present invention also provides more related polypeptides, such as fusion proteins containing BccDREB2A protein or fragments thereof.
  • the present invention also includes the soluble fragments of the BccDREB2A 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 BccDREB2A protein.
  • the present invention also provides analogs of the BccDREB2A protein or polypeptide. These analogs may be different from the native BccDREB2A 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. It should be understood that the 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 BccDREB2A 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 the amino acids with similar or close property. These mutant polypeptides preferably are produced according to the amino acid replacement shown below in Table 1 .
  • the present invention further provides polynucleotide sequences encoding the BccDREB2A protein of the current invention or 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.
  • 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
  • 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 BccDREB2A protein.
  • the full-length nucleotide sequence of the BccDREB2A protein of the present invention or fragment thereof can typically be prepared via PCR amplification method, recombinant method or artificial synthesis.
  • PCR amplification 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 any of the conventional methods known in the art as a template.
  • two or more PCR amplifications may be needed, and, the fragments thus obtained in each amplification may be fused together in the correct orientation.
  • the related sequence can be synthesized by artificial synthesis, e.g. when the fragment is relatively short. Several small fragments may be first synthesized and then fused, e.g. by ligation or fusion PCR, into a large fragment.
  • 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 vectorcomprising the polynucleotide of the present invention, a host cell genetically engineered to comprise the vector or the coding sequence of the BccDREB2A 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
  • an expression vector containing a DNA sequence encoding the BccDREB2A protein and 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 contain one 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.
  • the vector containing the above DNA sequence and promoter and/or regulatory sequence can be used to transform 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,
  • Streptomyces Streptomyces, agrobacterium, fungi cell such as yeast, and plant cell, etc.
  • the transcription may be enhanced when an enhancer sequence is inserted into the vector.
  • the enhancer may be a cis-acting factor of DNA, which may contains about 10 to 300 bp and can act on a promoter to enhance the transcription of the gene.
  • Transformation of a host cell with the recombinant DNA can be carried out using
  • the competent cells that can uptake the DNA may be harvested after the exponential growth phase and then treated by CaCI 2 method, as well described in the prior art. Another method is to use MgCI 2 . If desired, 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
  • the transformant can 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.
  • the selected promoter may be induced by a suitable method (such as temperature change or chemical induction), after which the cells 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.
  • the recombinant BccDREB2A 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 BccDREB2A protein. Screening a polypeptide library with the expressed recombinant BccDREB2A protein may help finding valuable polypeptide molecules that could inhibit or stimulate the function of the BccDREB2A 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 BccDREB2A 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 BccDREB2A 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 BccDREB2A gene or the activity of encoded protein in the plant.
  • BccDREB2A coding gene to express the BccDREB2A gene A a promoter may be used to enhance the expression of the BccDREB2A gene.
  • An enhancer e.g. the first intron of the rice waxy gene or the first intron of the Actin gene, and the like
  • Promoters useful in the subject invention include but are not limited to the 35S promoter, and the Ubi promoter in rice and corn.
  • a method for obtaining a plant with enhanced expression of BccDREB2A 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 BccDREB2A protein is transformed into the plant cell and integrated into the chromosome;
  • the present invention also includes agonists to the BccDREB2A protein or its coding gene. Since the agonists of the BccDREB2A protein can regulate the activity or expression of the BccDREB2A protein, the said agonists can also enhance the heat resistance of a plant through iaffecting the BccDREB2A protein, to achieve improvement on traits.
  • the agonists of the BccDREB2A protein refer to any substance that can enhance the activity of BccDREB2A, maintain the stability of BccDREB2A, promote the expression of BccDREB2A, prolong effect duration of BccDREB2A, or promote transcription and translation of BccDREB2A. These substances can be used in the present invention as agents for enhancing the heat resistance of plant.
  • Primers used in the RT-PCT include:
  • AMV reverse transcriptase (TAKARA)
  • TAKARA RNase inhibitor
  • chinensis after different heat treatments; treating with DNase I (RNase free) for 30 min, and then extracting by phenol-chloroform; precipitating, blow-drying, dissolving in water free of RNase.
  • DNase I RNase free
  • 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.
  • 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.
  • 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.
  • 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 a transgenic line that over- express said gene.
  • the gene "BccDREB2A” obtained in the present invention is initially obtained from Brassica campestris L. ssp. chinensis. 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
  • Example 2 Detection of the candidate heat-resistance genes' expression by RT- PCR after heat treatment
  • the transgenic plants 35S::BccDREB2A all exhibited strong expression after heat treatment.
  • the inventors of the present invention used a heat treatment system to verify the phenotype of these transgenic 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::BccDREB2A exhibited a higher tolerance to the heat stress as compared with the wild type plants, as showed in Fig. 2.
  • 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.
  • 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 experimental results showed that the symptoms of heat damage in the transgenic plants of Brassica campestris L. ssp. chinensis plant were all scored as light, which was expressed as ABCD.
  • the symptoms of heat damage in the control plants wild type Brassica campestris L. ssp. chinensis, heat-sensitive Brassica campestris L. ssp. chinensis HS 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 BccDREB2A protein (SEQ ID NO:3), as shown in Fig. 3.
  • the results showed that positions 78-141 constitute a conservative AP2 functional domain (DNA-binding domain). This domain is the critical active site for the protein's heat-resistance function.
  • amino acid 15 was changed from L to I, so as to obtain BccDREB2A-M1 variant.
  • amino acid 307 was changed from A to V, so as to obtain BccDREB2A-M2 variant.
  • the CDS sequence of the BccDREB2A 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 as previously taught 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 a strain of agrobacterium, and then each transformed agrobacterium strain was re used to transform Arabidopsis, so that the following transgenic Arabidopsis plants were obtained: M1 -Line1 , M1 -Line2; M2- Linel , M2-Line2; M3-Line1 , M3-Line2; M4-Line1 , M4-Line2; M5-Line1 , M5-Line2; M6-Line1 , M6-Line2.

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Abstract

La présente invention concerne un gène végétal de résistance à la chaleur, BccDREB2A, et son utilisation. Les inventeurs de la présente invention ont isolé pour la première fois, chez une plante du genre Brassica spp., un nouveau gène de résistance à la chaleur qui améliore considérablement la capacité de résistance à la chaleur de la plante. La présente invention concerne en outre une protéine codée par ledit gène et sa méthode de préparation, les vecteurs et les cellules hôtes contenant ledit gène, et une méthode de préparation d'une plante transgénique contenant ledit gène.
PCT/NL2011/050498 2010-07-08 2011-07-07 Gène végétal de résistance à la chaleur, bccdreb2a, et son utilisation WO2012005590A1 (fr)

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CN114391518B (zh) * 2021-12-29 2022-11-25 广东省农业科学院蚕业与农产品加工研究所 一种基于温水浸渍的蚕种孵化方法
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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