CN103898078A

CN103898078A - Paddy rice heat-resistant gene TOG1 and application thereof

Info

Publication number: CN103898078A
Application number: CN201210574391.9A
Authority: CN
Inventors: 薛勇彪; 王冬; 程祝宽; 覃宝祥; 张玉娥
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2012-12-26
Filing date: 2012-12-26
Publication date: 2014-07-02
Anticipated expiration: 2032-12-26
Also published as: CN103898078B

Abstract

The invention discloses a paddy rice heat-resistant gene TOG1 and encoding protein thereof. TOG1 gene has one of the following nucleotide sequences: 1) a DNA sequence shown as SEQ ID No: 1; and 2) a DNA sequence which has 90% or more of homology with the DNA sequence shown by SEQ ID No: 1 and is capable of encoding protein with same functions. TOG1 is protein possessing the amino acid sequence shown as SEQ ID No: 2, or protein which is derived from protein of SEQ ID No: 2 by performing substitution, deletion or addition of one or multiple amino acids on the amino acid sequence shown by SEQ ID No: 2 and has the activity same to that of the amino acid sequence shown by SEQ ID No: 2. The invention also discloses a method for improving plant thermotolerance and effectively increasing output by improving TOG1 expression level.

Description

The heat-resisting gene TOG1 of paddy rice and application thereof

Invention field

The invention belongs to plant genetic engineering field.Particularly, the present invention relates to a kind of volume increase gene TOG1 (Thermaltolerant Growthl) that gives paddy rice high temperature tolerance proterties, the function fragment of this gene, the protein of this coded by said gene and functional analogue thereof, and the carrier that contains described TOG1 gene nucleotide series and the host cell that contains this gene nucleotide series or this carrier; In addition, the invention still further relates to a kind of cultivation and have the method for the stable on heating plant of raising.

Background technology

Plant in the farm crop in the torrid zone, subtropics and temperate zone as extensive, the desirable growth temperature of paddy rice is 33 degrees Celsius [1].The high temperature of 34 DEG C can cause the obvious paddy rice underproduction [2], can cause violent negative impact [1] at cell and metaboilic level to plant higher than the high temperature of 5 DEG C of desirable growth temperatures.But in rice breeding process, heat-resisting proterties is but ignored by people for a long time, thereby cause the heat-resisting germ plasm resource of current high-quality very limited.Due to heat-resisting often closely related with volume increase, the cultivation of heat resistant variety can increase the further per mu yield that increases on the adaptive basis of paddy rice undoubtedly.

And on the other hand, very complicated due to the Heat-Resisting Mechanism of plant, the research of this area receives much attention always.Except the adaptation of physiological level regulates, the heat shock protein protein family using HSP70 as representative can at high temperature normally fold [3,4,5] as the effective protected protein of molecular chaperones.And in another aspect, histone H2A.Z also participates in the perception to high temperature, and mediate extensively the replying of transcriptional level [6].Therefore, plant replying and tolerating and regulated and controled by multiple approach High Temperature Stress.

DEAD-box DBPA family is extensively present in [7] in nearly all life entity.This family has 9 extremely conservative functional zone [8] on aminoacid sequence, thereby gives its very conservative basic de-rotation function [9].And on the other hand, this family differentiates and participates in the various functions of multiple life process in long-term evolution process, comprise the processing etc. [7] of the transcribing of mRNA, montage, transport and translation and rRNA precursor.All above-mentioned processes all need to ensure the rna helicase of false folding and are correctly folded into specific conformation, DEAD-box DBPA has been guaranteed the correct folding of various RNA by its heterogeneous activity that untwists just, and therefore the investigators of this area more and more tend to be defined as RNA companion [10].A large amount of research evidences has shown that RNA companion has brought into play keying action [10] in the degeneration-resistant mechanism of bacterium.Equally, the correct fold height of plant RNA under hot and cold, salt and oxidative stress depends on RNA companion's participation [10].

The heat-resisting factor TOG1 of paddy rice that the present invention identifies is the one in DEAD-box DBPA, with the known Rrp3[ref.11 that has brought into play key effect in the yeast rRNA precursor course of processing] there is a very high homology, the normal expression of this gene has ensured that paddy rice is to high temperature tolerance to a certain degree, improve its expression amount by genetic engineering modified appropriateness and can further improve the thermotolerance of paddy rice, thereby reach the object of volume increase.

Summary of the invention

Therefore, an object of the present invention is to provide the heat-resisting factor of paddy rice of the heat-resisting gene of a kind of paddy rice and coding thereof.Another object of the present invention is to provide the method for the stable on heating plant (for example, rice varieties) that a kind of cultivation has raising.

The inventor has cloned the heat-resisting gene TOG1 of paddy rice, because this genes encoding is containing the DBPA of DEAD-box structural domain, so it is named as DEAD-box DBPA gene.

Found and the heat-resisting factor names of paddy rice of the heat-resisting gene TOG1 coding of the paddy rice of identifying is abbreviated as TOG1 (called after DEAD-box DBPA) by the inventor, the protein with the aminoacid sequence shown in SEQ IDNo:2, or by aminoacid sequence shown in SEQ ID No:2 through one or several amino acid whose replacement, disappearance or interpolation and have identical with the aminoacid sequence shown in SEQ ID No:2 active in the derivative protein of SEQ ID No:2.Preferably, wherein said " identical activity " refers to DBPA activity, specifically refers to the active identical DBPA activity of DBPA with the aminoacid sequence shown in SEQ ID No:2.

The protein that aminoacid sequence shown in SEQ ID No:2 is made up of 472 amino acid.

The inventor finds and the encoding gene TOG1 of the heat-resisting factor TOG1 of paddy rice that identifies, is one of following nucleotide sequences:

1) DNA sequence dna shown in SEQ ID No:1;

2) there is 90% above homology with the DNA sequence dna shown in SEQ ID No:1, and the nucleotide sequence of the protein of coding identical function.

Preferably, above-mentioned " identical function " refers to DBPA activity, specifically refers to the DBPA activity identical with the DEAD-box DBPA of the DNA sequence encoding shown in SEQID No:1.

In sequence table, the DNA sequence dna shown in SEQ ID No:1 is by 4746 based compositions.

Preferably, the heat-resisting gene TOG1 of described paddy rice is the following protein of coding (a) and gene (b):

(a) there is the protein of the aminoacid sequence shown in SEQ ID No:2;

(b) by aminoacid sequence shown in SEQ ID No:2 through one or more amino acid whose replacements, disappearance or interpolation and have identical with the aminoacid sequence shown in SEQ ID No:2 active in the derivative protein of SEQ ID No:2.

Those skilled in the art should understand that, utilize any carrier that can guide foreign gene to express in plant, by TOG1 gene transfered plant cell provided by the present invention, or thereby TOG1 gene leader sequence district shown in SEQ ID No:3 is transformed and changes TOG1 expression amount, strengthen element or delete straining element such as inserting, can obtain clone and plant that heat-resisting proterties changes.Gene of the present invention, in the time being building up in plant expression vector, can add any enhancing promotor or inducible promoter before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant are identified and screened, can process used carrier, as add the alternative mark of plant or there is the antibiotic marker thing of resistance.The expression vector that carries TOG1 gene of the present invention can import vegetable cell by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity the conventional biotechnological means such as to lead, the plant host being converted can be both monocotyledons, also can be dicotyledons, preferably grass, more preferably paddy rice.Gene pairs of the present invention is cultivated thermophytes new variety, particularly cultivates heat-resisting new rice variety significant.In the time that gene of the present invention is used to change the heat-resisting proterties of paddy rice, can adopt following methods: (1) is cloned into heat-resisting paddy rice of the present invention gene TOG1 in plant conversion carrier; (2) constructed plant conversion carrier is transformed to renewable rice tissue (or organ) and the heat-resisting gene of paddy rice of the present invention is expressed in the tissue transforming; (3) tissue being converted (or organ) is cultivated into plant.

More specifically, the invention provides the following:

1, a kind of DEAD-box DBPA TOG1 that gives the heat-resisting proterties of paddy rice, it is the protein with the aminoacid sequence shown in SEQ ID No:2, or by aminoacid sequence shown in SEQ ID No:2 through one or several amino acid whose replacement, disappearance or interpolation and have identical with the aminoacid sequence shown in SEQ ID No:2 active in the derivative protein of SEQ ID No:2.

2, according to the DEAD-box DBPA TOG1 described in the 1st, it is characterized in that: it is the protein with the aminoacid sequence shown in SEQ ID No:2.

3, the encoding gene of TOG1, it is one of following nucleotide sequences:

1) DNA sequence dna shown in SEQ ID No:1;

2) there is 90% above homology with the nucleotide sequence shown in SEQ ID No:1, and the nucleotide sequence of the protein of coding identical function.

4, according to the gene described in the 3rd, it is characterized in that: the encoding gene of described TOG1 is the nucleotide sequence shown in SEQ ID No:1.

5, a kind of contain the 3rd or 4 described in gene or the carrier of its fragment.

6, according to the carrier described in the 5th, it is plant expression vector, is preferably the carrier that is suitable for expressing in paddy rice.

7, according to the carrier described in the 6th, it is pCAMBIA1300.

8, a host cell, this cell contain the 3rd or 4 described in gene or its fragment, or contain the carrier described in 5-7 item any one.

9, according to the host cell described in the 8th, described cell is selected from Bacillus coli cells, agrobatcerium cell or vegetable cell.

10, cultivation has a method for the stable on heating plant of raising, and described method comprises the cell or tissue that transforms described plant with the carrier described in 5-7 item any one, and the vegetable cell of conversion or tissue cultivating are become to plant.

11, according to the method described in the 10th, wherein said conversion is undertaken by agrobacterium-mediated transformation or particle bombardment.

12. according to the 10th or 11 described in method, wherein said plant is grass, preferably paddy rice.

13. methods according to claim 10, wherein said method also improves the output of transgenic plant.

Brief description of the drawings

In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:

The phenotype comparison of Xian 3037 in Fig. 1, tog1 and wild-type.

Rice material is planted respectively May to the September in Beijing and Yangzhou and the December in Hainan to April.

Scale: 20 centimetres.

The seed germination of Xian 3037 and growth of seedling in tog1 and wild-type under Fig. 2, differing temps.

Scale: 3 centimetres.

The location of Fig. 3, TOG1 gene and candidate gene are determined.

The DNA sequence dna (SEQ ID No:1) of Fig. 4, TOG1 gene.

The aminoacid sequence (SEQ ID No:2) of Fig. 5, TOG1 genes encoding.

Fig. 6, the leader sequence (SEQ ID No:3) that comprises TOG1 gene promoter.

Fig. 7, TOG1 functional complementation and over-express vector collection of illustrative plates.

Fig. 8, the experimental verification of TOG1 functional complementation, left figure is wild-type (WT) rice plant, right figure is complementary mutant (tog1+TOG1).

Scale: 20 centimetres.

Fig. 9, TOG1RNAi plant and wild-type comparison.

Plant is planted Pekinese May to September.

Scale: 20 centimetres.

Figure 10, the TOG1RNA active Validation in vitro that untwists.

Swimming lane 1 is double-stranded RNA substrate, and swimming lane 2 is the RNA substrate after hatching with TOG1.

Figure 11, TOG1 cross expression plant and wild-type comparison.

A, mistake expression type (TOG1-ox) and the growing state of wild-type seedling (WT) under 37 DEG C of high temperature.

Scale: 5 centimetres.

B, excessively expression type and wild-type are in Beijing self-sow situation in May to September.

Scale: 20 centimetres.

The individual plant of crossing expression type (TOG1-ox) and wild-type (WT) of c, Beijing physical environment growth (May, average day top temperature was about 30 DEG C to September) is solid.

The expression type (TOG1-ox) of crossing of Figure 12, the growth of Beijing physical environment is added up with individual plant solid (A) and the tillering number (B) of wild-type (WT).

The expression amount of crossing TOG1 in expression type (TOG1-ox) and wild-type (WT) of Figure 13, the growth of Beijing physical environment.

Embodiment

Carry out by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.

One, the separation of the heat-resisting gene of paddy rice and genetic analysis

In the present invention, carry out the sensitive of map based cloning research and downgrade paddy rice tog1 from 3037 spontaneous mutations of Xian in indica rice variety.Plant in the time that day, the highest temperature was lower than the nice and cool environment of 30 DEG C when tog1 kind, its morphological specificity and wild-type are as good as; And in the time growing in the more day a few days highest temperature higher than the summer weather of 30 DEG C, tog1 blade attenuates, obviously short in wild-type, setting percentage significantly reduces; When growing in more hot Yangzhou one and being with summer weather, tog1 downgrades and loses ability (Fig. 1) more.Keep other condition identical, the seed germination under contrast differing temps and the experiment of growth of seedling have further determined that tog1 is temperature-sensitive mutant (Fig. 2).Tog1 and normal water rice varieties carry out positive crossing and negative crossing, and it is of short stem that all F1 plant all do not show high temperature.And the F2 of cross combination for segregating population in, be typical 3: 1 with high temperature derived Dwarf Plants normally and separate (320: 96).This result shows that tog1 mutant character is subject to single recessive gene control.

Two, the heat-resisting gene TOG1 of map based cloning paddy rice

In order to clone TOG1 gene, the inventor first by the sensitive mutant tog1 isozygotying and in spend 11 to hybridize, the F of acquisition ₁obtain F for selfing ₂colony, carries out the Primary Location of TOG1 gene to 645 F2 recessive individual (having the F2 of sensitive phenotype for individuality) wherein.As shown in Figure 3, application Sequence Tagged Site (STS, sequence tagged site) molecule marker, utilize the method for PCR, find to be positioned at No. 3 STS mark P1, P2, P3, P4, P5, P6, the P7 (table 1) on karyomit(e) and there is in position obvious linksystem with mutational site.Exchange individual plant between mutational site and P4 most also exchanges between mutational site and P1, P2, P3, and exchange individual plant between mutational site and P5, the overwhelming majority is included in the exchange individual plant between mutational site and P6, P7.Exchange individual plant between simultaneous mutation site and P4 is different from the exchange individual plant between mutational site and P5, therefore infers the region that mutator gene may be between mark P4 and mark P5.On this basis, further expand mutant tog1 and middlely spend 11 cross combination, having obtained the F that comprises 1272 plant mutant individual plants ₂segregating population is for TOG1 Fine Mapping.With reference to the rice genome sequence (http://www.tigr.org/tdb/e2kl/osal/ and http://btn.genomics.org.cn) having completed, sequence to japonica rice and long-grained nonglutinous rice compares, and utilizes sequence difference to develop 3 new STS molecule markers (table 1).TOG1 Fine Mapping is between BAC clone BAC5:AC133930 mark P8 and P10 the most at last, and the physical distance between these two marks is about 25kb.Utilizing rice genome annotation database RiceGAAS (http://ricegaas.dna.affrc.go.jp/rgadb) to analyze shows, in 25kb region, there is the gene of 4 functional annotations, these 4 genes are carried out determined dna sequence by we, mutant and wild-type sequence are compared, find all consistent with wild-type of three genes sequence in mutant wherein, the gene that only has an encoding D EAD-box DBPA is that sudden change has occurred Os03g46610, the displacement that this gene, at the Nucleotide place of the 140th of first exon, a single base has occurred is G → T, cause the amino acid at this place to be transformed into a α-amino-isovaleric acid by a glycine.Therefore, DEAD-box DBPA gene is defined as target gene by we, called after TOG1.Utilize rice genome annotation database RiceGAAS information to show, this full length gene is 4746bp, and the total length of mRNA is 1817bp, has 13 exons, 12 introns, and CDS section length is 1419bp, 472 amino acid of encoding altogether.This gene has complete cDNA sequence at the fine cDNA database of KOME (http://cdna01.dna.affrc.go.jp/cDNA) paddy rice Japan, and accession number is AK067769.The ORF sequence that comprises this gene complete, the aminoacid sequence of coding and the 2kb leader sequence that comprises this gene promoter sequence are shown in respectively accompanying drawing 4,5,6.

Table 1 is for the molecule marker of TOG1 location

Three, the qualification of TOG1 gene and functional analysis

Utilize pCAMBIA1300 plasmid (purchased from CAMBIA company) to build complementary carrier (Fig. 7), pass through transgenic technology, the transgenic research having complementary functions, result shows that the present invention has identified the transgenic paddy rice (Fig. 8) that makes mutant recover normal function, TOG1 RNAi plant shows temperature sensitive and downgrades phenotype (Fig. 9), has proved that the present invention has correctly cloned TOG1 gene.Amino acid sequence analysis shows the albumen that TOG1 genes encoding contains DEAD-box structural domain.The vitro enzyme experiment of living shows that TOG1 albumen has the double-stranded short spiral of the obvious RNA activity (Figure 10) of untwisting, and proves that this albumen is a DBPA that has function, so this albumen called after DEAD-box DBPA TOG1.

The present invention has cloned a heat-resisting gene of paddy rice----TOG1 by map based cloning method, and it also can be called the DEAD-box DBPA gene of paddy rice.And, utilize the functional performance of this gene, in paddy rice, carry out Molecular design breeding, the high-yield variety of seed selection high temperature resistance has very strong operability.

Embodiment 1, TOG1 over-express vector build and rice transformation

The ORF of TOG1 gene (Fig. 4) is above cut and builds with HindIII the HindIII restriction enzyme site that enters pCAMBIA1300 (purchased from CAMBIA company) from BAC:AC133930 (purchased from Joint Genome Institute of the U.S., NCBI discloses this sequence (Accession No.AC133930)).

The recombinant vectors of above-mentioned structure is transformed in E.coli DH5 α competent cell (Invitrogen company product), by kalamycin resistance screening positive clone, (cloning and identification method is shown in that molecular cloning experiment guide (third edition) (in translate version) Huang Peitang etc. translates, Science Press, publish in September, 2002 ").Extract plasmid and carry out sequencing analysis, qualification transforms Agrobacterium EHA105 competent cell by the plasmid electric shock building after connecting correctly again, and (Agrobacterium EHA105 is purchased from Clontech, Agrobacterium EHA105 competent cell is prepared according to ordinary method by this laboratory, with reference to " plant genetic engineering ", Wang Guanlin, Fang Hongjun, Science Press, 2004, the 2nd edition etc.).Adopt agriculture bacillus mediated method for transformation to import in japonica rice variety A024.Concrete steps are as follows:

(1) induction of Mature Embryos of Rice callus.

The rice paddy seed shelling after 1 minute, is used to aseptic water washing 3～5 times through 70% ethanol surface sterilization.With 30%NaClO (every 50mL adds a Tween 20) solution stirring sterilization 15 minutes, aseptic water washing 5 times, blotted with filter paper after soaked overnight.Seed after sterilizing is inoculated in to N ₆d substratum (table 2) is upper, cultivates 10～14 days evoked callus in 32 DEG C of continuous lights, then removes plumule.

(2) cultivation of Agrobacterium.By the pCAMBIA1300-TOG1 recombinant plasmid 1800v voltage building, within 2 seconds, electric shock Transformed E HA105 Agrobacterium competent cell (is prepared according to a conventional method by this laboratory, with reference to " plant genetic engineering ", Wang Guanlin, Fang Hongjun, Science Press, 2004 the 2nd edition), the YEB plate culture medium (table 2) that contains 50mg/L kantlex upper 28 DEG C cultivate 48 hours.Picking Agrobacterium mono-clonal is inoculated in 20mL YEB liquid nutrient medium, and 28 DEG C are shaken bacterium and are cultured to logarithmic growth late period; Get strain transfer after 0.5mL activation to 50mLYEB substratum (50mg/L kantlex), 28 DEG C are cultured to OD ₆₀₀it is 0.5 left and right.

(3) cultivate altogether and transform, screen, break up.First collect the Agrobacterium thalline that proceeds to pCAMBIA1300-TOG1 recombinant plasmid, 4 DEG C, 4000g, 10min is centrifugal, resuspended in equal-volume AAM-AS substratum (table 2), the preculture embryo callus of 4 days is immersed to this AAM-AS bacterium liquid, rock gently 1.5 minutes; On aseptic filter paper, suck unnecessary bacterium liquid, dry up.Callus is connected to N ₆d-As substratum, spreads an aseptic filter paper that is soaked with AAM-AS in advance on substratum, 25 DEG C of dark cultivations 3 days.The aseptic water washing that callus use after common cultivation contains 400mg/L carboxylic benzyl 5 times, and soak 1 hour.On aseptic filter paper, suck moisture and dry up.Then callus is transferred to containing 50mg/L Totomycin and 400mg/L carboxylic benzyl N ₆on D substratum, continuous light is cultivated two weeks at 32 DEG C.Eugonic resistant calli is transferred on the RE substratum that contains 50mg/L Totomycin and 250mg/L carboxylic benzyl, under 32 DEG C of continuous light conditions, induction is broken up and is cultured to generation regrowth.Treat that seedling grows to 10cm left and right, open sealed membrane, after hardening 2-3 days, seedling is moved into experimental plot, carry out phenotypic evaluation.

The substratum that table 2 agrobacterium mediation converted paddy rice is used

2,4-D ^*2,4-Dichlorophenoxyacetic acid (2,4 dichloro benzene ethoxyacetic acid)

CH ^*casein acid hydrolysate (acid hydrolysis casein)

NAA ^{* *}1-Naphthylacetic acid (1-naphthylacetic acid)

Embodiment 2, TOG1 cross the phenotype analytical of express transgenic plant

For inspection transgenosis is to improving the effect of the heat-resisting proterties of paddy rice, the plant obtaining from transgenic experiments, with PCR, (primer is 35SF:5 '-AGAGATAGATTTGTAGAGAGAG-3 '; 35SR:5 '-ATGGTGGAGCACGACACTC-3 ') evaluation and screening, positive strain selfing obtains F1 generation, again carries out PCR and screens positive strain.

By positive strain seedling and the contemporaneously wild-type seedling be divided into two batches:

A collection of being put in incubator, imposes a condition as continuing 37 DEG C, 12 little time: 12 hours dark.Cultivate plant forms after 54 days as shown in Figure 11 a, the transfer-gen plant speed of growth is significantly higher than wild-type, illustrates that crossing of TOG1 gene expressed to have significantly improved the high temperature resistant proterties of paddy rice;

A collection of self-sow (in May～September, an average day top temperature is about 30 DEG C) in the farm of During Summer In Beijing in addition.It is to be all significantly higher than wild-type plant approximately one times of (Figure 11 b, c at single-strain fructification yield or on tillering number that ripe TOG1 crosses expression plant; Figure 12).

The result shows that the excessively expression of TOG1 in paddy rice also can significantly improve single plant yield on the basis of improving high temperature tolerance.

Embodiment 3, TOG1 cross TOG1 in express transgenic plant and express quantitative analysis

Cross the expression amount of this gene in express transgenic plant in order accurately to grasp the above-mentioned TOG1 that carries out phenotype analytical, with the methods analyst of qRT-PCR the relative quantity of TOG1mRNA.Concrete grammar is, with the total RNA of TRIzol reagent (purchased from Invitrogen) extraction rice leaf, through the integrity of 1% agarose electrophoresis detection RNA, then to use DNaseI (Takara) digestion.With CDSIII (oligo (dT)) primer and the synthesizing single-stranded cDNA of M-MLV ThermoScript II (Invitrogen), method detailed is shown in Invitrogen operational manual.After 10 times of cDNA dilutions as the template of qRT-PCR.Amplification reaction system used is Power SYBR green PCRmaster mix (purchased from Applied Biosystems), and instrument is CFX96real-time PCRdetection system (purchased from Bio-Rad).Primer TOGlQFl/TOGlQRl is used for detecting TOG1, and primer OsACTINlF/OsACTINlR is used for detecting interior source reference Actin.Primer sequence is as follows:

Table 3 primer title and sequence

QRT-PCR detected result shows, the 1.7-3 times of level (Figure 13) that TOG1 is wild-type at the expression amount of crossing in express transgenic plant.

Conclusion:

Paddy rice is one of most important food crop of China and even the whole world, and investigators are devoted to improve its output and resistance always.Become gradually today of prevalent variety cultivation important means at genetically engineered and molecular breeding, find that heat-resisting factor pair in paddy rice improves rice yield and tolerance tool is of great significance.The application of TOG1 in paddy gene engineering and molecular breeding has very large feasibility, the object that gets a good chance of reaching rice genetic improvement and improve thermotolerance and output.

Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and described, but will be understood by those skilled in the art that, under the condition not deviating from by the spirit and scope of the present invention as defined in the claims, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.

Reference

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Claims

1. give the DEAD-box DBPA TOG1 of the heat-resisting proterties of paddy rice for one kind, it is the protein with the aminoacid sequence shown in SEQ ID No:2, or by aminoacid sequence shown in SEQ ID No:2 through one or several amino acid whose replacement, disappearance or interpolation and have identical with the aminoacid sequence shown in SEQID No:2 active in the derivative protein of SEQ ID No:2.

2. DEAD-box DBPA TOG1 according to claim 1, is characterized in that: it is the protein with the aminoacid sequence shown in SEQ ID No:2.

The encoding gene of 3.TOG1, it is one of following nucleotide sequences:

1) DNA sequence dna shown in SEQ ID No:1;

4. gene according to claim 3, is characterized in that: the encoding gene of described TOG1 is the nucleotide sequence shown in SEQ ID No:1.

5. one kind contains gene described in claim 3 or 4 or the carrier of its fragment.

6. carrier claimed in claim 5, it is plant expression vector, is preferably the carrier that is suitable for expressing in paddy rice.

7. carrier according to claim 6, it is pCAMBIA1300.

8. a host cell, described cell contains gene or its fragment described in claim 3 or 4, or contains the carrier described in claim 5-7 any one.

9. host cell according to claim 8, described cell is selected from Bacillus coli cells, agrobatcerium cell or vegetable cell.

10. cultivation has a method for the stable on heating plant of raising, and described method comprises the cell or tissue that transforms described plant with the carrier described in claim 5-7 any one, and the vegetable cell of conversion or tissue cultivating are become to plant.

11. methods according to claim 10, wherein said conversion is undertaken by agrobacterium-mediated transformation or particle bombardment.

12. according to the method described in claim 10 or 11, and wherein said plant is grass, preferably paddy rice.