CN103172716B - Heat-resistant plant gene and application thereof - Google Patents

Abstract

The invention relates to a heat-resistant plant gene and the application thereof. The invention discloses the heat-resistant gene capable of obviously improving the heat resistance of plants; and the heat-resistant gene can be applied to heat-resistant molecular breeding, so that the heat resistance of the plants is improved, and the plants with breed improvement can be obtained.

Description

Genes For Plant Tolerance hot radical because of and application

Technical field

The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to a kind of Genes For Plant Tolerance hot radical because of and application.

Background technology

Temperature stress affects one of Vegetable produce and the Main Factors limiting its areal distribution.Because " Greenhouse effect " increasingly sharpen, hot weather occurs more and more frequent, especially in recent years the continuous high temperature in China Yangtze valley summer, and the g and D of serious restriction food crop and fruits and vegetables class plant, agriculture production faces a severe challenge.Nearly 50 years there are great paddy rice heat evil event 6 times in Yangtze River in China basin altogether.The last time occurs in 2003, and conservative estimation full basin injured area reaches 3 × 10 ⁷hectare, loss paddy reaches 5.18 × 10 ⁷ton ^[1].Late 1980s, south China citrus producing region once suffered 3 large-scale high temperature upsurges, produces cause huge loss to fruits and vegetables.Environment stress can cause a series of biochemical reactions in plant materials: cell leakage increases, and kytoplasm volumes of extravasation result increases; Lipid peroxidation metabolism degree is aggravated, and MDA accumulates; Active o content increases.Antioxidase and the reaction of non-enzymatic antioxidant generation responsiveness is started, to stop, to reduce or to repair the damage caused by high temperature in plant materials ^[2,3].As can be seen here, the plant under adverse circumstance is not that driven bearing is hurt, but adjustment initiatively adapts to.Just to the continuous adaptive process of this adverse circumstance, make plant define in long-term evolutionary process perfect with the degeneration-resistant system of complexity with maladjustment environment.Therefore, carrying out more deep research to Genes For Plant Tolerance heat engine reason, is one of understanding plant and environmental concerns and the important channel of cultivating new resistant variety, theory and application aspect all significant.

The research hot to Genes For Plant Tolerance has become a current important research topic.Be subject to the restriction of germ plasm resource, traditional breeding way is difficult to the heat impedance of the significantly nonrefractory crop of improvement in a short time, so the heat impedance realizing improving nonrefractory crop by the heat resistanceheat resistant gene of recombinant heat-proof species is the important directions of research now.Plant materials is subject to the poor environments such as high temperature when coercing, in body, the immunity system of self can make emergency reaction in time, produce a series of emergency reaction, wherein heat shock factor (Heat Shock factor, HsF) be central transcription factor in stress-inducing process, by the expression of its downstream target gene of transcriptional activation, produce various enzyme, add film fat saturation ratio, improve the content of soluble proteins and some protectiveness cellular components, protection body protein exempts from damage or repairs the protein of wound in damaged condition, plant is shielded, thus make plant obtain thermotolerance, improve the stress ability of organism and the survival rate in adverse circumstance ^[2,3,4,5].Together with being closely connected because heat shock factor and Genes For Plant Tolerance are hot, therefore the expression regulation of heat shock protein and downstream gene thereof is the physiological important research content of current molecular biology, protein biochemistry and plant stress-resistance.Regrettably, be how to start and the Mechanism Study of carrying out heat resistanceheat resistant physiological responses it be unclear that in plant materials for its target gene.

Rapidly, especially the application of biochip technology on crop molecular breeding is also more and more extensive in molecular biology research development in recent years ^[6,7].Its fast, responsive, efficiently, the expression change of analyzing gene quantitatively, meet Genes For Plant Tolerance hot radical because carrying out the screening requirements of large flux.Therefore, it is platform that this area is necessary with gene engineering, screens the gene in plant with heat resistance, carries out plant species improvement, improves plant heat resistance property, and horn of plenty and balance China farm crop market provide heat resistanceheat resistant germ plasm resource.

Summary of the invention

The object of the present invention is to provide a class Genes For Plant Tolerance hot radical because of and application.

In a first aspect of the present invention, provide a kind of HTT polypeptide ( heat induced ta-siR255 targets) or the purposes of the polynucleotide of this polypeptide of encoding, for improving plant heat resistance property.

In a preference, described HTT polypeptide is selected from:

A () has the polypeptide of aminoacid sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21; Or

(b) by aminoacid sequence shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21 through one or more (as 1-50; Preferably 1-40; Preferably 1-30; Preferably 1-20; Preferably 1-10; More preferably 1-5) replacement of amino-acid residue, disappearance or interpolation form, and have the polypeptide derivative by (a) improving Heat Resistance of Plant sexual function;

C aminoacid sequence shown in () and SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21 has 40% (preferably 50%; More preferably 60%; More preferably 70%; More preferably 80%; More preferably 90%; More preferably 95%; More preferably 98%) above homogeny, and there is the polypeptide derivative by (a) improving Heat Resistance of Plant sexual function.

In another preference, the polynucleotide of described coding HTT polypeptide are selected from: have SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:14, or SEQ ID NO:16, or the polynucleotide of nucleotide sequence shown in SEQ ID NO:18 or SEQ ID NO:20.

In another preference, described plant is selected from: cress; More preferably be selected from Brassica plants or mouse ear mustard; More preferably be selected from Chinese cabbage (Brassica.rapa) or Arabidopis thaliana (Arabidopsis thaliana).

In another aspect of this invention, provide a kind of polynucleotide, be selected from lower group:

A () has the polynucleotide of SEQ ID NO:23 or the nucleotide sequence shown in SEQ ID NO:24; Or

(b) by 11-14 position Nucleotide in the nucleotide sequence shown in SEQ ID NO:23 through one or more (as 1-8; Preferably 1-6, better 1-5, as 2,3,4) replacement of Nucleotide, disappearance or interpolation form, and have the polynucleotide of the function that antagonism ta-siR255 is combined with the mRNA of HTT polypeptide of encoding; Or

(c) by 236-239 position Nucleotide in the nucleotide sequence shown in SEQ ID NO:24 through one or more (as 1-8; Preferably 1-6, better 1-5, as 2,3,4) replacement of Nucleotide, disappearance or interpolation form, and have the polynucleotide of the function that antagonism ta-siR255 is combined with the mRNA of HTT polypeptide of encoding; Or

The nucleotide sequence of nucleotide complementary described in (d) and above-mentioned (a), (b), (c).

In another aspect of this invention, provide the purposes of described polynucleotide, for improving plant heat resistance property.

In a preference, described plant is selected from: cress; More preferably be selected from Brassica plants or mouse ear mustard; More preferably be selected from Chinese cabbage (Brassica.rapa) or Arabidopis thaliana (Arabidopsis thaliana).

In another aspect of this invention, provide a kind of method improving plant heat resistance property, described method comprises: the expression or the activity that improve HTT polypeptide in plant; Or

Reduce expression or the activity of ta-siR255 in plant; Or

Improve the expression of the target gene of ta-siR255 in plant.

In a preference, described method comprises: proceed in plant by the polynucleotide of coding HTT polypeptide; Or

Described polynucleotide are proceeded in plant.

In another preference, described method comprises step:

I () provides the Agrobacterium of carrying expression vector, described expression vector contains the polynucleotide of coding HTT polypeptide or described polynucleotide;

(ii) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (i), thus make the polynucleotide of described coding HTT polypeptide or described polynucleotide proceed to plant.

In another preference, described method also comprises:

(iii) select and proceeded to the described polynucleotide of coding HTT polypeptide or vegetable cell, tissue, the organ of described polynucleotide; And

(iv) by the vegetable cell in step (c), tissue, neomorph select transgenic plant.

In another preference, described plant is selected from: cress; More preferably be selected from Brassica plants or mouse ear mustard; More preferably be selected from Chinese cabbage (Brassica.rapa) or Arabidopis thaliana (Arabidopsis thaliana).

In another aspect of this invention, provide a kind of heat-resisting plant, it is the transgenic plant prepared by preceding method.

In another aspect of this invention, the purposes of the polynucleotide of a kind of HTT polypeptide or this polypeptide of encoding is provided, as the molecular marked compound of the thermotolerance of plant identification.

Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.

Accompanying drawing explanation

The expression analysis of Figure 1A-C, HTT1, HTT2, HTT3, At4G29760 and At1G51670.Wherein, ck represents WT lines.

The clustering relationships analysis of Fig. 1 D, HTT1, HTT2, HTT3, At4G29760 and At1G51670 and other candidate gene.

The sequence signature analysis of Fig. 2, HTT1, HTT2 and HTT3.

The mRNA of A, HTT1, HTT2 and HTT3 all exists the binding site of ta-siR255.

The structure schematic diagram of B, MIM255 over-express vector.

All there is nnGAAnnTTCnnGAAnn and AGGGG motif in C, HTT1.1, HTT2 and HTT3 promotor.

The transgenic line heat treatment experiment of Fig. 3, HTT1, HTT2 and HTT3 overexpression.

Plant strain growth situation after the transgenic line thermal treatment of A-C overexpression, respective left figure represents plant-growth situation, and right figure is the schematic diagram of the plant of each section post representative in left figure.

D-G, the quantitative RT PCR analysis mRNA accumulation volume in adjoining tree and process LAN plant.

Fig. 4 A, Ta-siR255 precursor TAS1a, TAS1b, TAS1c before heat treatment after response.

Fig. 4 B, Ta-siR255 before heat treatment after response.

Plant survival rate after the thermal treatment of Fig. 5 A, HTT1 and HTT2 process LAN strain.

Plant survival rate after the thermal treatment of Fig. 5 B, HTT3 process LAN strain.

Three strain survival rates after the thermal treatment of Fig. 5 C, MIM255 process LAN strain.

The homology Predicting and analysis of corresponding polypeptide in HTT polypeptide and Chinese cabbage in Fig. 6, Arabidopis thaliana.

Embodiment

The present inventor is devoted to the screening of heat resistanceheat resistant gene, finally determines the heat resistanceheat resistant gene that a class can significantly improve plant heat resistance.These heat resistanceheat resistant genes can be applicable to heat resistanceheat resistant molecular breeding, improve plant heat resistance property, obtain the plant of breed improvement.

In the present invention, have no particular limits for being applicable to plant of the present invention, as long as it is applicable to the conversion operation of carrying out gene, as various farm crop, flower plant or forestry plant etc.Described plant can be such as (being not limited to): dicotyledons, monocotyledons or gymnosperm.More specifically, described plant includes, but is not limited to: wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olea, Sunflower Receptacle, coconut, castor oil plant, cocoa beans, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, natsudaidai, spinach, piemarker lettuce, asparagus, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, onion, potato, tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, nut, coffee, eggplant, sugarcane, tealeaves, pepper, grapevine, oyster fiber crops grass, banana, natural rubber tree and ornamental plant etc.

As a kind of optimal way, described " plant " includes but not limited to: Cruciferae, Gramineae, rosaceous plant.Such as, described " plant " includes but not limited to: the Chinese cabbage of Cruciferae Brassica genus, Plantula Brassicae chinensis; Cruciferae mouse ear mustard; Paddy rice gramineous, wheat, Chinese sorghum, corn; Comprise tobacco, melon and fruit, vegetables, rape etc. in addition.More preferably, described " plant " is the plant that Cruciferae Brassica genus or mouse ear mustard belong to.

As used herein, " separation " refers to that material is separated from its primal environment (if natural substance, namely primal environment is natural surroundings).As the polynucleotide under the native state in active somatic cell and polypeptide do not have separation and purification, but same polynucleotide or polypeptide as from native state with in other materials existed separately, then for separation and purification.

As used herein, " HTT polypeptide " refer to derive from Arabidopis thaliana or other plant there is higher homology (if homology is higher than 40% with sequence SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19 or SEQ ID NO:21; Preferably higher than 50%; Preferably higher than 60%; More preferably higher than 70%; More preferably higher than 80%; More preferably higher than 90%; More preferably higher than 95%; More preferably higher than 98%) the general name of a class polypeptide.

As used herein, described " containing ", " having " or " comprising " include " comprising ", " primarily of ... form ", " substantially by ... form " and " by ... form "; " primarily of ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".

Polypeptide of the present invention (albumen) can be recombinant polypeptide, natural polypeptides, improvement on synthesis.Polypeptide of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to recombinant production scheme, polypeptide of the present invention can be glycosylated, can be maybe nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises the fragment of HTT polypeptide, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " refer to the polypeptide substantially keeping biological function that polypeptide of the present invention is identical or activity.Polypeptide fragment of the present invention, derivative or analogue can be the polypeptide that (i) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) and be substituted, and the amino-acid residue of such replacement can may not be and encoded by genetic code, or (ii) has the polypeptide of substituted radical in one or more amino-acid residue, or (iii) mature polypeptide and another compound (such as extend the compound of polypeptide transformation period, such as polyoxyethylene glycol) merge the polypeptide formed, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide formed (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying or proprotein sequence, or fusion rotein).The known scope of those skilled in the art is belonged to according to these fragments of definition herein, derivative and analogue.

The bioactive fragment of any one HTT polypeptide can be applied in the present invention.Here, the implication of described bioactive fragment refers to as a peptide species, and it still can keep all or part of function of corresponding full-length polypeptide.Under normal circumstances, described bioactive fragment at least keeps the activity of the full-length polypeptide of 40%.Under still more preferential conditions, described active fragments can keep the activity of 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of full-length polypeptide.

In the present invention, described " HTT polypeptide " refer to have improve plant heat resistance property functionally active SEQID NO:4, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:11 sequence polypeptide.This term also comprise have with described polypeptide identical function, the variant form of above-mentioned sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best, also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally within 20, is preferably within 10, within being more preferably 5) amino acid at C-terminal and/or N-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of polypeptide can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change polypeptide usually.This term also comprises active fragments and the reactive derivative of described polypeptide.

The variant form of polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, the albumen coded by DNA can hybridized with above-mentioned protein D NA under high or low stringency conditions and the polypeptide utilizing the antiserum(antisera) of anti-described albumen to obtain or albumen.

Any with described peptides homologous high and there is the polypeptide improving Heat Resistance of Plant sexual function be also included within the present invention.

Invention also provides the analogue of described albumen or polypeptide.The difference of these analogues and native protein can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplified.

(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.

In the present invention, " conservative variation's polypeptide of polypeptide of the present invention " refers to compared with the aminoacid sequence of SEQ ID NO:4, SEQID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19 or SEQ ID NO:21, there are 50 at the most, preferably at the most 40, more preferably at the most 30, more preferably at the most 20, more preferably at the most 10, more preferably at the most 5, best at the most 3 amino acid replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out amino acid replacement according to table 1 and produce.

Table 1

Amino-acid residue	Representational replacement	Preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention still further relates to the polynucleotide sequence of code book invention polypeptide or its conservative variation's polypeptide, i.e. HTT gene or its homologous gene.Described polynucleotide can be DNA form or rna form.DNA form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-strand.DNA can be coding strand or noncoding strand.Described polynucleotide can with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 or the sequence shown in SEQ ID NO:20 is identical or the varient of degeneracy.

Term " polynucleotide of coded polypeptide " can be the polynucleotide comprising coding said polypeptide, also can be the polynucleotide also comprising additional code and/or non-coding sequence.The polynucleotide of encoding mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, the sum analogous to general Dedekind sum of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function of polypeptide changing in fact its coding.

The invention still further relates to and above-mentioned sequence hybridization and have at least 50% between two sequences, preferably at least 70%, the more preferably polynucleotide of at least 80% homogeny.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.In the present invention, " stringent condition " refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.I%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences, at least more than 90%, is just hybridized when being more preferably more than 95%.Further, the polypeptide of interfertile polynucleotide encoding also has the function improving plant heat resistance property.

A.lyrata (Arabidopsis.lyrata) and Arabidopis thaliana belong to for being all Cruciferae mouse ear mustard, and Chinese cabbage (Brassica.rapa) belongs to Cruciferae Btassica.In Arabidopis thaliana, in HTT polypeptide and Chinese cabbage, the homology Predicting and analysis of corresponding polypeptide is as Fig. 6, and the polypeptide in visible two kind of plant sources is very high homology, and have same structural domain, the function played in plant is also roughly the same.

Should understand, although plant heat resistanceheat resistant (heat-resisting) gene of the present invention is preferably available from cress, but (as have more than 40% available from the HTT gene very high homology of originating with Arabidopis thaliana of other plant, preferably more than 50%, more than 60%, more preferably more than 70%, more preferably more than 80%, as 85%, 90%, 95%, even 98% sequence thereto) the scope also considered in the present invention of other gene within.The Method and kit for of aligned sequences homogeny is also that this area is known, such as BLAST.

The Nucleotide full length sequence of polynucleotide of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

The present invention also relates to the carrier comprising described polynucleotide, and with the host cell that described carrier or polypeptid coding sequence produce through genetically engineered.

Polynucleotide sequence of the present invention can be inserted in recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually containing replication orgin, promotor, marker gene and translation controlling elements.

Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.

When described polynucleotide are expressed in higher eucaryotic cells, if will make to transcribe to be enhanced when inserting enhancer sequence in the carrier.Enhanser is the cis-acting factors of DNA, and nearly 10 to 300 base pairs, act on promotor transcribing with enhancing gene usually.

Persons skilled in the art all know how to select suitable carrier, promotor, enhanser and host cell.

Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Conversion of plant can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as spraying, leaf disk method, Rice Young Embryo conversion method etc.Can ordinary method regeneration plant be used for the vegetable cell transformed, tissue or organ, thus the plant that acquisition Holstein Cattle changes.

The invention provides described polypeptide or the purposes of its encoding gene, for improving the thermotolerance of plant.

The invention still further relates to a kind of method improveing plant, the method comprises the expression improving HTT polypeptide in described plant.

After the purposes obtaining the polypeptide described in cicada, multiple method well known in the art can be adopted to improve the expression of described polypeptide.Such as by the approach that those skilled in the art are known, the ceneme (such as expression vector or virus etc.) carrying polynucleotide is delivered on target spot, and makes it the polypeptide of expression activity.

As one embodiment of the present invention, polynucleotide are cloned in suitable carrier by conventional method, the described recombinant vectors with foreign gene is imported in vegetable cell, make the described polypeptide described in vegetable cell expression.By described Plant cell regeneration is become plant, obtain the plant of polypeptide described in overexpression.

Preferably, provide a kind of method preparing transgenic plant, comprising:

(1) the HTT gene of external source or its homologous gene are proceeded to vegetable cell, tissue, organ or tissue, obtain and be transformed into HTT gene or its homogenic vegetable cell, tissue, organ or seed; With

(2) what step (1) obtained has proceeded to external source HTT gene or its homogenic vegetable cell, tissue, organ or seed regeneration plant.

As the preferred example of one, described method comprises step:

(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains HTT gene or its homologous gene;

(s2) vegetable cell, tissue, organ are contacted with the Agrobacterium in step (s1), thus make HTT gene or its homologous gene proceed to vegetable cell, and be incorporated on the karyomit(e) of vegetable cell;

(s3) select and proceed to HTT gene or its homogenic vegetable cell, tissue, organ or seed; And

(s4) by the vegetable cell in step (s3), tissue, organ or seed regeneration plant.

Other method increasing HTT gene or the expression of its homologous gene is that this area is known.Such as, by driving with strong promoter thus strengthening HTT gene or its homogenic expression.Or strengthen HTT gene or its homogenic expression by enhanser (as paddy rice waxy gene First Intron, Actin gene First Intron etc.).The strong promoter being applicable to the inventive method includes but not limited to: 35s promotor, the Ubi promotor etc. of paddy rice, corn.

Any suitable conventional means can be adopted, comprise reagent, temperature, pressure condition etc. and implement described method.

In addition, the invention still further relates to and utilize HTT gene or its homologous gene to mark as a kind of tracking of gene transformation progeny of plants.The invention still further relates to and utilize HTT gene or its homologous gene as a kind of molecule marker, by detecting HTT gene or its homogenic expression in plant, the thermotolerance of plant identification, and the cue mark that can be used as true hybrid in hybrid seeding process.

The present inventor also finds under study for action, the mRNA of HTT gene exists the binding site of ta-siR255 (this siRNA is see document [10]), and in plant, HTT gene or its homogenic expression are also subject to the regulation and control of ta-siR255.The combination of the mRNA of ta-siR255 and HTT gene causes the reduction of HTT genetic expression, affects the temperature capacity of plant.Therefore, the material suppressing the mRNA of the expression of ta-siR255 or the material of activity or minimizing ta-siR255 and HTT gene to combine is useful for the thermotolerance of raising plant.

Expression specificity and a kind of blocking-up, minimizing or antagonism ta-siR255 or the polynucleotide that combine complementary with the mRNA of coding HTT polypeptide in plant, the thermotolerance for raising plant is also useful.Process LAN blocks, reduce or antagonism ta-siR255 is complementary with the mRNA of coding HTT polypeptide or the polynucleotide that combine, and endogenous ta-siR255 can be made to be combined with these polynucleotide competitively, thus decreases the interference that endogenous ta-siR255 expresses HTT.Those skilled in the art should be understood that based on known general knowledge and anyly or can combine and can block, to reduce or polynucleotide that antagonism ta-siR255 is combined with the mRNA of HTT polypeptide of encoding are all useful for the present invention with ta-siR255 complementation (partial complementarity or all complementary).

As optimal way of the present invention, described blocking-up, minimizing or antagonism ta-siR255 are MIM255 with the mRNA of coding HTT polypeptide polynucleotide that are complementary or that combine, and it has the nucleotide sequence shown in SEQ ID NO:23.MIM255 can be combined with endogenous ta-siR255 competitively, makes ta-siR255 not play shearing action, thus decreases the interference that ta-siR255 expresses HTT.Or described blocking-up, minimizing or antagonism ta-siR255 have the nucleotide sequence (wherein including the nucleotide sequence of MIM255) shown in SEQ ID NO:24 with the mRNA of coding HTT polypeptide polynucleotide that are complementary or that combine.

On the polynucleotide basis with the nucleotide sequence shown in SEQ ID NO:23 or SEQ ID NO:24, through one or more (as 1-8; Preferably 1-6, better 1-5, as 2,3,4) replacement of Nucleotide, disappearance or interpolation and the polynucleotide that formed are also included within the present invention, as long as it also has blocking-up, to reduce or antagonism ta-siR255 is complementary with the mRNA of coding HTT polypeptide or the activity that combines.When designing this kind of polynucleotide, suitable Nucleotide replaces, lack or interpolation is technology well known in the art, and described technology is easy to be implemented and guarantees not change the biological activity (blocking-up, minimizing or antagonism ta-siR255 are combined with the mRNA of coding HTT polypeptide) of gained molecule.

Therefore, present invention also offers a kind of method improving plant heat resistance property, described method comprises step:

I () provides the Agrobacterium of carrying expression vector, described expression vector contains can the polynucleotide that are combined with the mRNA of coding HTT polypeptide of antagonism ta-siR255;

(ii) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (i), thus make described to proceed to plant by the polynucleotide that are combined with the mRNA of HTT polypeptide of encoding of antagonism ta-siR255.

(iii) vegetable cell, tissue, the organ that have proceeded to described construction is selected; And

(iv) by the vegetable cell in step (iii), tissue, neomorph select transgenic plant.

Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.

Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.

I. materials and methods

The sequence explanation of some genes

In Arabidopis thaliana:

HTT1 (At4G29770) genome sequence is as SEQ ID NO:1;

HTT1.1 (At4G29770.1) open reading frame sequence is as SEQ ID NO:2;

HTT1.2 (At4G29770.2) open reading frame sequence is as SEQ ID NO:3;

HTT1.1 (AT4G29770) coding protein sequence is as SEQ ID NO:4;

HTT1.2 (AT4G29770) coding protein sequence is as SEQ ID NO:5;

HTT2 (At5G18040) genome sequence is as SEQ ID NO:6;

HTT2 (At5G18040) open reading frame sequence is as SEQ ID NO:7;

HTT2 (At5G18040) coding protein sequence is as SEQ ID NO:8;

HTT3 (At5G18065) genome sequence is as SEQ ID NO:9;

HTT3 (At5G18065) open reading frame sequence is as SEQ ID NO:10;

HTT3 (At5G18065) coding protein sequence is as SEQ ID NO:11;

AT4G29760 open reading frame sequence is as SEQ ID NO:12;

AT4G29760 coding protein sequence is as SEQ ID NO:13.

In Chinese cabbage, HTT homologous gene has four, is respectively Bra039897, Bra011210, Bra010277 and Bra010276:

Bra039897 open reading frame sequence is as SEQ ID NO:14;

Bra039897 protein sequence is as SEQ ID NO:15;

Bra011210 open reading frame sequence is as SEQ ID NO:16;

Bra011210 protein sequence is as SEQ ID NO:17;

Bra010277 open reading frame sequence is as SEQ ID NO:18;

Bra010277 protein sequence is as SEQ ID NO:19;

Bra010276 open reading frame sequence is as SEQ ID NO:20;

Bra010276 protein sequence is as SEQ ID NO:21.

RNA interfering:

Ta-siR255 sequence is as SEQ ID NO:22.

The sequence of MIM255 is as SEQ ID NO:23.This sequence is as the complementary sequence of ta-siR255, and more than ta-siR255 3 bases, this can reach the effect of competitive binding effectively.

Plant tissue Total RNAs extraction

TaKaRa RNAiso Reagent extraction agent box is adopted to extract plant tissue total serum IgE.Step:

A) fully ground in liquid nitrogen by material, add in sample, fully mix with the amount of 100mg material 1ml extraction buffer, room temperature leaves standstill 10 minutes.

B) centrifugal 5 minutes of 13000rpm, proceeds to supernatant in new centrifuge tube, adds 200 μ l chloroforms, fully mixes, and room temperature leaves standstill and makes its layering in 10 minutes.

C) centrifugal 5 minutes of 13000rpm, careful supernatant of drawing is in new centrifuge tube.

D) add equal-volume Virahol, after mixing, room temperature leaves standstill 10 minutes.

E) centrifugal 5 minutes of 13000rpm, washes once with 1ml 75% ethanol after abandoning supernatant.

F) centrifugal 5 minutes of 7800rpm, abandon low-speed centrifugal after supernatant, suck residual liquid with rifle head, room temperature is dried, and adds the appropriate water without RNase after RNA just drying, is stored in-70 DEG C after within 65 DEG C, 10 minutes, fully dissolving.

Quantitation RT-PCR primer

HTT1：

HTT1-RT-S：5’＞GAAGCGTTGAAGCTGGGTATG＜3’；(SEQ ID NO：25)

HTT1-RT-A：5’＞GTCGTCACCATTAAGCGGGAG＜3’(SEQ ID NO：26)；

HTT1.1：

HTT1.1-S：5’＞AACTACGCTGAGGGGGAAATAAG＜3’(SEQ ID NO：27)；

HTT1.1-A：5’＞ACTAAGGAGGTTTCCGACAGTGT＜3’(SEQ ID NO：28)；

HTT1.2：

HTT1.2-S：5’＞GCAACGAAAATTGACAAAAACGG＜3’(SEQ ID NO：29)；

HTT1.2-A：5’＞ACTAAGGAGGTTTCCGACAGTGT＜3’(SEQ ID NO：30)；

HTT2：

HTT2-RT-S：5’＞TGGATAATGCTGGGAAGGAAG＜3’(SEQ ID NO：31)；

HTT2-RT-A：5’＞CTGGACAAACATACGGCTCAA＜3’(SEQ ID NO：32)；

HTT3：

HTT3-RT-S：5’＞GACTAAGAGTTTGGATGAAGCGT＜3’(SEQ ID NO：33)；

HTT3-RT-A：5’＞TTAGTTGACAGATAAGCGTGGG＜3’(SEQ ID NO：34)；

ACTIN：

Forward: 5 ' > TGGCATCAYACTTTCTACAA < 3 ' (SEQ ID NO:35);

Reverse: 5 ' > CCACCACTDAGCACAATGTT < 3 ' (SEQ ID NO:36).

TAS1a：

Forward: 5 ' > AGTAAACATGAGCGCCGTCAA < 3 ' (SEQ ID NO:37);

Reverse: 5 ' > TGGCTGGTGACAAATAGACAGG < 3 ' (SEQ ID NO:38).

TAS1b：

Forward: 5 ' > AGTAAACATGAGCGCCGTCAA < 3 ' (SEQ ID NO:39);

Reverse: 5 ' > CAAAGTTGTCTCCTCCTGAAA < 3 ' (SEQ ID NO:40).

TAS1c：

Forward: 5 ' > TCCTTCCCGTCCTCTTCTTTG < 3 ' (SEQ ID NO:41);

Reverse: 5 ' > GATGCTTCTTCGCTACACCTC < 3 ' (SEQ ID NO:42).

Northern hybridization probe

Ta-siR255：5’＞TACGCTATGTTGGACTTAGAA(biotin)＜3’(SEQ ID NO：43)。

U6：5’＞TCATCCTTGCGCAGGGGCCA(biotin)＜3’(SEQ ID NO：44)。

Real Time RT-PCR

Reagent:

AMV ThermoScript II (TAKARA); RNase inhibitor (TAKARA); DNase I (RNase free) (TAKARA); SYBR green Kit (TAKARA).

Step:

A) extract the total serum IgE of the Arabidopsis leaf of different heat treatment respectively, phenol chloroform after processing 30 minutes with DNase I (RNase free), precipitation, dries up, without the water dissolution of RNase.

B) survey OD260 and electrophoresis quantitatively after, get 1 μ g total serum IgE, by specification operates, 42 DEG C of reactions 1 hour, 94 DEG C 5 minutes to make ThermoScript II inactivation.

C) reaction uses the SYBR green Kit of TAKARA.Program is 94 DEG C of 2min, 94 DEG C of 15sec, annealing 15sec, 72 DEG C of 30sec, 40 circulations.CDNA adds 10 μ l after diluting 10 times, using the primer of ACTIN as internal reference.

PCR system (25 μ l):

SYBR green Kit 12.5μl；

5 ' primer 0.5 μ l;

3 ' primer 0.5 μ l;

cDNA 10μl；

ddH ₂O 1.5μl。

Build over-express vector PCR primer

HTT1.1：

HTT1.1-S：5’＞C TCTAGAGATGGAGTCATTGTTAGAATGTT＜3’(SEO IDNO：45)；

HTT1.1-A：

5’＞CGCCATGGC GTCGACGTTGATAGAAAAACGTGGGATACAG＜3’(SEQ ID NO：46)；

HTT1.2：

HTT1.2-S：5’＞CGG GGTACCCGATGTTGTTGTTTAAGCCTTCA＜3’(SEQ ID NO：47)；

HTT1.2-A：5’＞CGC GGATCCTTAGTTGATAGAAAAACGTGGGATAC＜3’(SEQ ID NO：48)；

HTT2：

HTT2-S：5’＞CGG GGTACCATGAATATGATTCAGCGATTC＜3’(SEO IDNO：49)；

HTT2-A：5’＞AA CTGCAGTTAGTTGATAGATAAGCGTGGGAT＜3’(SEOID NO：50)；

HTT3：

HTT3-S：5’＞CG GGATCCTATGGATATGAATCAGCTATTCATGC＜3’(SEQID NO：51)；

HTT3-A：

5’＞CATGCCATGGT GTCGACCTTGAAAGCACATTCAAGGC＜3’(SEQ IDNO：52)；

Mutant IPS1：

MIM255-S：

5’＞AATACGCTATGTGCTAGGACTTAGAAAGCTTCGGTTCCCCTCG＜3’(SEQ ID NO：53)；

MIM255-A：

5’＞CTTTCTAAGTCCTAGCACATAGCGTATTTCTAGAGGGAGATAA＜3’(SEQ ID NO：54)；

IPS1-S：5’＞GT GGATCCAAGAAAAATGGCCATCCCCTAGC＜3’(SEQ IDNO：55)；

IPS1-A：5’＞ACGC GTCGACGAGGAATTCACTATAAAGAGAATCG＜3’(SEQ ID NO：56)。

35S over-express vector builds

Heat resistanceheat resistant transgenic line uses the method for genomic fragment PCR to clone from arabidopsis gene group STb gene herein.Concrete construction process is as follows:

Adopt HTT1.1 primer: added Xba1, Sal1 restriction endonuclease sites respectively at the two ends of primer, after being cloned into the gDNA fragment of HTT1.1, after using Xba1, Sal1 double digestion, be connected in expression vector pCAMBIA1301 (purchased from CAMBIA).

Adopt HTT1.2 respectively, HTT2, HTT3 primer, respectively by Kpn1/BamH1; Kpn1/Pst1; BamH1/Sal1 double digestion is connected in expression vector pCAMBIA1301.

Mutant IPS1 (SEQ ID NO:24, containing MIM255 sequence, in plant materials after process LAN, can be combined with endogenous ta-siR255, thus decrease the interference that ta-siR255 expresses HTT, improve the expression amount of HTT in plant materials) build: utilize IPS1-S, MIM255-A and MIM255-S, IPS1-A two pairs of primers clone respectively MIM255 precursor 5 ' and 3 ' end after, then by IPS1-S, IPS1-A with 5 ' and 3 ' end fragment be template Cloning of full length fragment.Because the two ends of IPS1-S, IPS1-A primer add BamH1/Sal1 restriction endonuclease sites respectively, so after using BamH1/Sal1 double digestion, be connected in expression vector pCAMBIA1301.

C) freeze thawing method for transformation is utilized to be imported by transgene carrier in Agrobacterium GV3101 (purchased from Invitrogen), and PCR qualification.

The preparation of freeze-thaw method Agrobacterium competent cell and conversion

A) cultivate the mono-bacterium colony of picking Agrobacterium GV3101 the fresh plate of 48 hours from 28 DEG C, forward in 20ml LB liquid nutrient medium (rif 50mg/l, GM 50mg/l), spend the night (can not be too dense) in 28 DEG C of 250rpm shaking culture.

B) ice bath is after 20 minutes, by the centrifuge tube (often pipe 4ml) of bacterium liquid packing 5ml, and ice bath 10 minutes.

C) centrifugal 10 minutes of 4000rpm (5-10 DEG C), abandons bacterium liquid.

D) often pipe adds the 20mM CaCl of the abundant precooling of 1ml ₂with resuspended thalline.Ice bath 10 minutes.

E) centrifugal 10 minutes of 4000rpm (5-10 DEG C), abandons supernatant.

F) often pipe adds 300 μ l 20mM CaCl ₂(depending on cell concentration), is in charge of in the centrifuge tube of 1.5ml after merging.

G) often pipe adds 1 μ l plasmid or all connection products, ice bath 5 minutes, then puts into liquid nitrogen 4-5 minute.

H) put 5 minutes for 37 DEG C, often pipe adds 400 μ l LB and cultivates and within 2 hours, make bacteria resuscitation based on 28 DEG C of incubations, and expresses corresponding antibiotics resistance gene.

I) respectively get 200 μ l volume coated plates, room temperature is placed a little while, in 28 DEG C of cultivations.

Flower-dipping method arabidopsis thaliana transformation and screening

Reagent:

Transfer buffer (1L): macroelement (50 ×): 10ml; Trace element (1000 ×): 0.5ml; CaCl ₂(100 ×): 5ml; Molysite (200 ×): 2.5ml; Organic (100 ×): 10ml; Sucrose: 50g; 6-BA (1mg/ml): 10 μ l; Silwet L-77:400 μ l (vacuum filtration then uses 200 μ l); PH5.8 is adjusted to, constant volume 1L with KOH.

Screening culture medium is dull and stereotyped: 3% sucrose MS ₀solid medium (pH5.8) adds kantlex (Kan) to 50mg/l (screening for Nossen background Arabidopis thaliana).

Step:

A) can transform when stem height after Arabidopis thaliana bolting about 5 centimetres, if plant setting percentage to be transformed is low, will carry out after plant is pinched 4 days.

B), before transforming, the flower of having pollinated and angle fruit are got rid of.And soil water sorption is spent the night.

C) Agrobacterium 1: 100 of overnight incubation is diluted in large bottle substratum, cultivate after 24 hours for 28 DEG C, centrifugal 20 minutes of 4 DEG C of 5000rpm, abandon supernatant, Agrobacterium precipitation is suspended in the transfer buffer of the two volumes of original bacteria liquid, makes OD600 about 0.8.

D) over-ground part of Arabidopis thaliana to immerse in bacterium liquid 30 seconds completely, and taking-up keeps flat, and covers preservative film and newspaper, spends the night under dark, moves into phytotron normal upright next day and cultivates.Drying 2 weeks after sowing.

E) seed is laid in the Ms0 solid plate containing 50mg/l Kan after Aseptic sterilisation, moves on to group training room two days later, block that resistance seedling and move on to continued growth in soil through 4 DEG C of vernalization.

F) get blade extraction genomic dna and obtain positive seedling through PCR detection, then screening obtains genetically modified pure lines, for further analysis through two generations.

Chinese cabbage transgenic method

Vacuum filtration method transforms Chinese cabbage: the Agrobacterium of inoculation containing destination carrier is in liquid YEP medium (peptone 10g/L, yeast extract 10g/L, beef extract 5g/L adjust ph is 7), and 28 DEG C, light culture is to proper concn.4 DEG C, 5000rpm, collects thalline in centrifugal 15 minutes.Transfer buffer (MS is added in bacterial sediment ₀add appropriate silwet77), with glass stick, precipitation is broken up, shake up that to be placed on vacuum tank to be transformed.The Chinese cabbage inflorescence of bolting is immersed bacterium liquid, and build vacuum container cover, vacuum pump is bled 5 minutes, exits 2 minutes.Plant lucifuge after suction filtration is spent the night.Every other day overcover is taken away, the backsight degree of blooming pollinate.The seed of results is containing corresponding antibiotic MS ₀substratum carries out resistance screening.Positive plant can be used for further phenotype analytical.

Transgenosis heat resistanceheat resistant phenotype analytical

Select respectively and turn HTT1, HTT2, HTT3 gene overexpression is sowed at MS for strain and wild-type respectively with the Arabidopis thaliana T3 turning MIM255 ₀on substratum, cultivate 7 days under 22 DEG C of illumination conditions.Under the long consistent condition of wild-type and transgenic line, under culture dish being placed on 44 DEG C of conditions, heat shock is after 1 hour, take out under being placed on 22 DEG C of illumination conditions and cultivate 7 days again, now can be observed transgenic line comparatively wild-type energy survive after heat shock, show it and there is remarkable heat impedance.The transgenosis heat resistanceheat resistant phenotype of Chinese cabbage also adopts this survival test to analyze.

II. embodiment

Embodiment 1, quantitative RT-PCR detect the expression after heat treatment of candidate's heat resistanceheat resistant gene

Quantitative RT-PCR experiment result is consistent with chip data, and after thermal treatment, HTT1, HTT2 and HTT3 are all increased significantly (Fig. 1) before expressing comparatively process.Wherein, the expression amount of HTT2 (At5G18040) rises about about 10 times in thermal treatment after 0.5 hour, then continue afterwards to rise slowly, within this test treatment time, its highest multiple finally reached is about 18 times (Figure 1A) before treatment; The expression amount of HTT3 (At5G18065) improves about about 23 times for 0.5 hour in process, and keeps slowly rising, and the highest multiple that can reach in the treatment time is about 40 times (Figure 1A); What is interesting is most HTT1, in Arabidopis thaliana, the present inventor detects two transcripts altogether, respectively called after HTT1.1 (At4G29770.1) and HTT1.2 (At4G29770.2).Generally speaking, the expression amount of HTT1 shows as in thermal treatment and sharply raises for 0.5 hour, reaches about 80 times before treatment, still continues afterwards to raise.In treatment time, its highest multiple that can reach is about 280 times (Figure 1B).Wherein there is similar change in the expression of HTT1.1 and HTT1.2, and just, under point of same treatment time, the expression amount of HTT1.1 comparatively HTT1.2 exceeds 20-80 doubly left and right; In addition, the present inventor also have detected At4G29760 and At1G51670 before heat treatment after expression pattern (Fig. 1 C), result shows that At4G29760 shows as the trend raised gradually after treatment, and in the treatment time, its highest multiple that can reach is about 8 times.Then there is downward trend in 0.5-3 hour in the expression of At1G51670, raises until process first about 5 times gradually again after heat treatment afterwards.

To sum up, the response that this five gene mRNA levels show thermal treatment, especially HTT1, HTT2 and HTT3, discloses them invariably and plant heat resistanceheat resistant also exists close relationship.

The present inventor has cloned HTT1, HTT2 and HTT3 respectively, and analyzes its sequence.Result shows:

(1) HTT1, HTT2 and HTT3 in sibship closely (Fig. 1 D), redundancy functionally may be there is between prompting three.

(2) their mRNA all exists the binding site (Fig. 2 A) of ta-siR255, this discovery shows that the genetic expression of HTT1, HTT2 and HTT3 exists post-transcriptional level regulation and control.

(3) promoter sequence analysis shows, HTT1.1, HTT2 and HTT3 promotor all exists nnGAAnnTTCnnGAAnn and AGGGG motif (Fig. 2 C), and this two classes motif is considered to HSF binding motif in plant materials ^[8,9].

All these sequence signatures are the present inventor's better utilised genetic engineering means undoubtedly, by the expression of various aspect regulation and control HTT1, HTT2 and HTT3, thus improve that Genes For Plant Tolerance is hot provides possibility.

The heat resistanceheat resistant gene transgenic plant phenotype of embodiment 2, Arabidopis thaliana

The present inventor constructs multiple transgene carrier and arabidopsis thaliana transformation respectively, obtains the transgenic line of multiple HTT1, HTT2 and HTT3 overexpression, and has carried out heat treatment experiment to these transgenic lines.Result is as follows:

HTT1 and HTT2 process LAN strain comparatively wild type control and hsfA1a/1b double-mutant (see reference [11]) (HsfA1A/1B is heat shock factor, they can activate the expression of HTT1, HTT2 and HTT3) all there is heat resistanceheat resistant phenotype (Fig. 3 A), quantitative RT PCR analysis shows that the accumulation volume of its mRNA in process LAN Arabidopsis plant is apparently higher than contrast (Fig. 3 D and 3E).

In Arabidopis thaliana, the heat resistanceheat resistant of HTT3 process LAN strain is analyzed and is shown, in all 7 process LAN strains, have 2 strains to have obvious heat resistanceheat resistant phenotype (Fig. 3 B), similarly, in these two strains, the present inventor also detects a large amount accumulation (Fig. 3 F) of mRNA.

The pCAMBIA1301 recombinant vectors containing HTT1.2, HTT2, HTT3 of aforementioned structure is imported Agrobacterium GV3101 by the present inventor, transform Chinese cabbage, obtain the transgenic line of multiple HTT1, HTT2 and HTT3 overexpression, and experiment is heat-treated to these transgenic lines.

Transgenosis Chinese cabbage heat resistanceheat resistant experimental result: 44 DEG C of heat shocks are carried out after 1 hour for positive seedling to transgenosis Chinese cabbage T2, cultivate 7 days under 22 DEG C of illumination conditions, turn HTT1 as seen, HTT2, the Chinese cabbage survival rate of seedling of HTT3 overexpression is far away higher than Wild type control plants, high by more than 50%.

To improve Genes For Plant Tolerance hot in the expression of embodiment 3, regulation and control HTT1, HTT2 and HTT3

The present inventor intends the expression being regulated and controled HTT1, HTT2 and HTT3 by post-transcriptional level, and then it is hot to improve Genes For Plant Tolerance.

For this reason, the present inventor constructs MIM255 over-express vector (Fig. 2 B) and proceeds to Arabidopis thaliana.In 4 strains obtained, 3 strains are had to show obvious heat resistanceheat resistant phenotype (Fig. 3 C).What correspond is the excess accumulation (Fig. 3 G) of its mRNA.

The present inventor also measured were Ta-siR255 precursor TAS1a, TAS1b, TAS1c before heat treatment after response, result is as Fig. 4 A; And determine Ta-siR255 before heat treatment after response, result is as Fig. 4 B.After the heat treatment, there is remarkable up-regulated in visible Ta-siR255 precursor; But, the time of Ta-siR255 response thermal treatment rise obviously lags behind the time of its target gene thermal response, this phenomenon hint target gene expresses that sharply to increase may be caused by the shearing effect of Ta-siR255 is temporarily removed after heat treatment, along with the prolongation of heat treatment time, Ta-siR255 also can raise, and the expression that this guarantees HTT gene can not be out of control.

Embodiment 4, be heat-treated to the quantitative statistics result of motility rate

The T3 of all acquisitions adds up as Fig. 5 for the surviving rate after the thermal treatment of homozygous transgenic Arabidopis thaliana strain.After the thermal treatment of Fig. 5 A, HTT1 and HTT2 process LAN strain, plant survival rate is all more than 90%.90% is about from plant survival rate after the thermal treatment of Fig. 5 B, HTT3 process LAN strain.From after the thermal treatment of Fig. 5 C, MIM255 process LAN strain, three strain survival rates are more than 85%, and indivedual strain survival rate is about 12%, correspondingly be that the expression amount of MIM255 in this strain is also relatively low.In addition, the survival rate of thermal treatment WT lines (CK) is only about 5%.

To sum up, Arabidopis thaliana HTT1, HTT2 and HTT3 is very effective heat resistanceheat resistant gene.Reasonably utilize their diversified control methods, improved the heat impedance of plant by genetically engineered, improving the viability of plant under heat stress adverse environmental factor has boundless application prospect.

The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.

Reference:

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[2] Zeng Qingping, Guo Yong. plant Stress response and system resistant are induced. the chemical .1997 of life, 17 (3): 31-33.

[3] Song Hongyuan, thunder is built up the Army, Li Chengqiong. vegetable hot Stress responses and heat tolerance and evaluation. and China's Vegetable .1998, (1): 48-50.

[4] Ma Dehua, Pang Jinan, Huo Zhenrong etc. cucumber is to the Resistence research of differing temps adverse circumstance. Scientia Agricultura Sinica 1999,32 (5): 28-35.

[5] Yao Yuangan, Shi Xuehui, Yang Jianguo. the relation of capsicum thermotolerance and leaf membrane permeability and several biochemical substances content. Agricultural University Of Hunan journal .2000,26 (2): 97-99.

[6] Li Tongxiang, Wang Jinke. gene chip and plant gene Differential expression analysis. plant research .2002.22 (3): 310-313.

[7]Seki M.Narusaka M.Abe H.Monitoring the expression pattern of 1300Arabidopsis genes under drought and cold stresses by using a full-length cDNA microarray.Plant cell.2001.13(1)：61-72.

[8]Ayako N，Ryota N，Hideki H，Hitoshi T，Takanori M，Masahiro T，Miho I，Masaru OT，Kazuya Y，Yukinori Y and Shigeru S.HsfA1d and HsfA1e Involved in the Transcriptional Regulation of HsfA2 Function as Key Regulators for the Hsf Signaling Network in Response to Environmental Stress.Plant Cell Physiol.2011.52(5)：933-945.

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Claims (10)

1. a purposes for the polynucleotide of HTT polypeptide or this polypeptide of encoding, for improving plant heat resistance property; Described HTT polypeptide is selected from:

The polypeptide of (a) aminoacid sequence as shown in SEQ ID NO:4 or SEQ ID NO:5; Or

B aminoacid sequence shown in SEQ ID NO:4 or SEQ ID NO:5 is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation by (), and have the polypeptide derivative by (a) improving Heat Resistance of Plant sexual function.

2. purposes as claimed in claim 1, it is characterized in that, the polynucleotide of described coding HTT polypeptide are the polynucleotide of nucleotide sequence as shown in SEQ ID NO:1.

3. purposes as claimed in claim 1, it is characterized in that, described plant is cress.

4. purposes as claimed in claim 3, it is characterized in that, described cress is selected from Brassica plants or mouse ear mustard.

5. purposes as claimed in claim 4, it is characterized in that, described cress is selected from Chinese cabbage (Brassica.rapa) or Arabidopis thaliana (Arabidopsis thaliana).

6. improve a method for plant heat resistance property, described method comprises: the expression or the activity that improve HTT polypeptide in plant; Or

Reduce expression or the activity of ta-siR255 in plant; Or

Improve the expression of the target gene of ta-siR255 in plant;

Described HTT polypeptide is selected from:

The polypeptide of (a) aminoacid sequence as shown in SEQ ID NO:4, SEQ ID NO:5; Or

B aminoacid sequence shown in SEQ ID NO:4, SEQ ID NO:5 is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation by (), and have the polypeptide derivative by (a) improving Heat Resistance of Plant sexual function.

7. method as claimed in claim 6, it is characterized in that, described method comprises: proceed in plant by the polynucleotide of coding HTT polypeptide; Or

The polynucleotide of nucleotide sequence as shown in SEQ ID NO:23 or SEQ ID NO:24 are proceeded in plant.

8. method as claimed in claim 7, it is characterized in that, described method comprises step:

I () provides the Agrobacterium of carrying expression vector, described expression vector contains polynucleotide or the polynucleotide of nucleotide sequence as shown in SEQ ID NO:23 or SEQ ID NO:24 of coding HTT polypeptide;

(ii) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (i), thus make the polynucleotide of described coding HTT polypeptide or the polynucleotide of nucleotide sequence as shown in SEQ ID NO:23 or SEQ ID NO:24 proceed to plant.

9. method as claimed in claim 6, it is characterized in that, described plant is cress.

10. a purposes for the polynucleotide of HTT polypeptide or this polypeptide of encoding, as the molecular marked compound of the thermotolerance of plant identification; Described HTT polypeptide is selected from:

The polypeptide of (a) aminoacid sequence as shown in SEQ ID NO:4, SEQ ID NO:5; Or

B aminoacid sequence shown in SEQ ID NO:4, SEQ ID NO:5 is formed through the replacement of 1-5 amino-acid residue, disappearance or interpolation by (), and have the polypeptide derivative by (a) improving Heat Resistance of Plant sexual function.