CN103898078B - The heat-resisting gene TOG1 of paddy rice and application thereof - Google Patents
The heat-resisting gene TOG1 of paddy rice and application thereof Download PDFInfo
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Abstract
The invention discloses the heat-resisting gene TOG1 of paddy rice and proteins encoded thereof.Does is TOG1 gene one of following nucleotide sequences: 1) SEQ ID DNA sequence dna shown in No:1; 2) and SEQ ID DNA sequence dna shown in No:1 has more than 90% homology, and coding identical function protein DNA sequence.Does TOG1 have SEQ ID the protein of the aminoacid sequence shown in No:2, or by SEQ ID does aminoacid sequence shown in No:2 pass through one or several amino acid whose replacement, disappearance or interpolation and has and SEQ ID the activity that aminoacid sequence shown in No:2 is identical by SEQ ID the protein that No:2 is derivative.The invention also discloses and improve Heat Resistance of Plant proterties and the method for effective increase yield by improving TOG1 expression amount.
Description
Invention field
The invention belongs to plant genetic engineering field.Particularly, the present invention relates to a kind of yield enhancing gene TOG1 (ThermaltolerantGrowthl) giving 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 contain the carrier of described TOG1 gene nucleotide series and contain the host cell of this gene nucleotide series or this carrier; In addition, the invention still further relates to a kind of method that cultivation has the plant of the thermotolerance of raising.
Background technology
As the farm crop extensively planted in the torrid zone, subtropics and temperate zone, the Optimal 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], and the high temperature higher than Optimal growth temperature 5 DEG C can cause violent negative impact [1] to plant at cell and metaboilic level.But in rice breeding process, Holstein Cattle but for a long time ignore by people, thus it is very limited to cause the heat-resisting germ plasm resource of current high-quality.Due to heat-resisting often closely related with volume increase, the cultivation of heat resistant variety can increase per mu yield further undoubtedly on the increase adaptive basis of paddy rice.
And on the other hand, because the Heat-Resisting Mechanism of plant is very complicated, the research of this area receives much attention always.Except the adaptation of physiological level regulates, be that the heat shock protein protein family of representative can as the effective protected protein of molecular chaperones at high temperature normally folding [3,4,5] with HSP70.And in another aspect, histone H2A.Z also participates in the perception to high temperature, and the extensive response [6] of mediate transcription level.Therefore, plant obviously regulates and controls by multiple pathways the response of High Temperature Stress and tolerance.
DEAD-boxRNA helicase family is extensively present in [7] in nearly all life entity.This family has 9 extremely conservative functional zone [8] on aminoacid sequence, thus gives its very conservative basic de-rotation function [9].And on the other hand, this family differentiates the various functions participating in 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 the rna helicase ensureing false folding to be also correctly folded into specific conformation, by its heterogeneous activity that untwists, DEAD-boxRNA helicase ensure that various RNA's is correct folding just, therefore the investigators of this area more and more tend to be defined as RNA companion [10].Large quantifier elimination evidence has shown that RNA companion has played 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 the participation [10] of RNA companion.
The heat-resisting factor TOG1 of the paddy rice that the present invention identifies is the one in DEAD-boxRNA helicase, very high homology is had with the known Rrp3 [ref.11] having played key effect in the yeast rRNA precursor course of processing, the normal expression of this gene ensure that paddy rice is to high temperature tolerance to a certain degree, improve by genetic engineering modified appropriateness the thermotolerance that its expression amount can improve paddy rice further, thus 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 a kind of method that cultivation has the plant (such as, rice varieties) of the thermotolerance of raising.
The present 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-boxRNA unwindase gene.
To be found by the present inventor and the heat-resisting factor names of paddy rice that the heat-resisting gene TOG1 of the paddy rice of identifying encodes is abbreviated as TOG1 (called after DEAD-boxRNA helicase), be the protein with the aminoacid sequence shown in SEQIDNo:2, or the aminoacid sequence shown in SEQIDNo:2 passed through one or several amino acid whose replacement, disappearance or interpolation and there is the protein derived by SEQIDNo:2 of the activity identical with the aminoacid sequence shown in SEQIDNo:2.Preferably, wherein said " identical activity " refers to that DBPA is active, specifically refers to that identical DBPA active in the DBPA of the aminoacid sequence shown in SEQIDNo:2 is active.
The protein that aminoacid sequence shown in SEQIDNo:2 is made up of 472 amino acid.
The present inventor finds and the encoding gene TOG1 of the heat-resisting factor TOG1 of the paddy rice of identifying, is one of following nucleotide sequences:
1) DNA sequence dna shown in SEQIDNo:1;
2) with the DNA sequence dna shown in SEQIDNo:1, there is more than 90% homology, and the nucleotide sequence of the protein of coding identical function.
Preferably, above-mentioned " identical function " refers to that DBPA is active, specifically refers to that the DBPA identical with the DEAD-boxRNA helicase of the DNA sequence encoding shown in SEQIDNo:1 is active.
SEQ ID No: the DNA sequence dna shown in 1 is by 4746 based compositions.
Preferably, described paddy rice heat-resisting gene TOG1 is the gene of the following protein (a) of coding and (b):
A () has the protein of the aminoacid sequence shown in SEQIDNo:2;
B aminoacid sequence shown in SEQIDNo:2 is had the protein derived by SEQIDNo:2 of the activity identical with the aminoacid sequence shown in SEQIDNo:2 through one or more amino acid whose replacement, disappearance or interpolation by ().
Those skilled in the art should understand that, the carrier utilizing any one can guide foreign gene to express in plant, by TOG1 gene transfered plant cell provided by the present invention, or the gene leader sequence of TOG1 shown in SEQIDNo:3 district is transformed thus changes TOG1 expression amount, such as insert and strengthen element or delete straining element, clone and the plant of Holstein Cattle change can be obtained.Gene of the present invention, when being building up in plant expression vector, can add that before its transcription initiation Nucleotide any one strengthens promotor or inducible promoter.For the ease of identifying transgenic plant cells or plant and screening, can use carrier be processed, mark as added plant alternative or there is the antibiotic marker thing of resistance.The expression vector carrying TOG1 gene of the present invention imports vegetable cell by using the standard biologic technological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, the plant host be converted both can be monocotyledons, also can be dicotyledons, preferred grass, more preferably paddy rice.Gene pairs of the present invention cultivates thermophytes new variety, particularly cultivates heat-resisting new rice variety significant.When gene of the present invention is used to change paddy rice Holstein Cattle, following methods can be adopted: heat-resisting for paddy rice of the present invention gene TOG1 is cloned in plant conversion carrier by (1); (2) constructed plant conversion carrier is transformed renewable rice tissue (or organ) and the heat-resisting gene of paddy rice of the present invention is expressed in the tissue transformed; (3) tissue be converted (or organ) is trained plant.
More specifically, the invention provides the following:
1, a kind of DEAD-boxRNA helicase TOG1 giving paddy rice Holstein Cattle, it is for having the protein of the aminoacid sequence shown in SEQIDNo:2, or the aminoacid sequence shown in SEQIDNo:2 is passed through one or several amino acid whose replacement, disappearance or interpolation and has the protein derived by SEQIDNo:2 of the activity identical with the aminoacid sequence shown in SEQIDNo:2.
2, the DEAD-boxRNA helicase TOG1 according to the 1st, is characterized in that: it is the protein with the aminoacid sequence shown in SEQIDNo:2.
3, the encoding gene of TOG1, it is one of following nucleotide sequences:
1) DNA sequence dna shown in SEQIDNo:1;
2) with the nucleotide sequence shown in SEQIDNo:1, there is more than 90% homology, and the nucleotide sequence of the protein of coding identical function.
4, the gene according to the 3rd, is characterized in that: the encoding gene of described TOG1 is the nucleotide sequence shown in SEQIDNo:1.
5, a kind of carrier containing the 3rd or gene described in 4 or its fragment.
6, the carrier according to the 5th, it is plant expression vector, is preferably suitable for the carrier of expressing in paddy rice.
7, the carrier according to the 6th, it is pCAMBIA1300.
8, a host cell, this cell contains the 3rd or gene described in 4 or its fragment, or containing the carrier described in any one of 5-7 item.
9, the host cell according to the 8th, described cell is selected from Bacillus coli cells, agrobatcerium cell or vegetable cell.
10, cultivation has a method for the plant of the thermotolerance of raising, and described method comprises the cell or tissue with plant described in the vector described in any one of 5-7 item, and the vegetable cell of conversion or tissue cultivating are become plant.
11, the method according to the 10th, wherein said conversion is undertaken by agrobacterium-mediated transformation or particle bombardment.
12. methods according to the 10th or 11, wherein said plant is grass, preferred paddy rice.
13. methods according to claim 10, wherein said method also improves the output of transgenic plant.
Accompanying drawing explanation
Below in conjunction with in the detailed description of accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1, tog1 compare with the phenotype of Xian in wild-type 3037.
Rice material is planted respectively Beijing and May to the September in 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.
Location and the candidate gene of Fig. 3, TOG1 gene are determined.
The DNA sequence dna (SEQIDNo:1) of Fig. 4, TOG1 gene.
The aminoacid sequence (SEQIDNo:2) of Fig. 5, TOG1 genes encoding.
Fig. 6, comprise the leader sequence (SEQIDNo:3) of TOG1 gene promoter.
Fig. 7, TOG1 functional complementation and over-express vector collection of illustrative plates.
The experimental verification of Fig. 8, TOG1 functional complementation, left figure is wild-type (WT) rice plant, and right figure is complemented mutant body (tog1+TOG1).
Scale: 20 centimetres.
Fig. 9, TOG1RNAi plant is compared with wild-type.
Plant is planted Pekinese May to September.
Scale: 20 centimetres.
Figure 10, TOG1RNA unwinding activities Validation in vitro.
Swimming lane 1 is double-stranded RNA substrate, and swimming lane 2 is the RNA substrate after hatching with TOG1.
Figure 11, TOG1 process LAN compares with wild-type by plant.
A, process LAN type (TOG1-ox) and wild type seedlings (WT) growing state under 37 DEG C of high temperature.
Scale: 5 centimetres.
B, process LAN type and wild-type are in the self-sow situation in Beijing May to September.
Scale: 20 centimetres.
The process LAN type (TOG1-ox) of c, Beijing physical environment growth (May, average max. daily temperature was about 30 DEG C to September) is solid with the individual plant of wild-type (WT).
The process LAN type (TOG1-ox) of Figure 12, Beijing physical environment growth is added up with the individual plant solid (A) of wild-type (WT) and tillering number (B).
The process LAN type (TOG1-ox) of Figure 13, Beijing physical environment growth and the expression amount of TOG1 in wild-type (WT).
Embodiment
Come by the following examples to illustrate the present invention further.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
The sensitive carrying out map based cloning research in the present invention downgrades paddy rice tog1 from Xian in indica rice variety 3037 spontaneous mutation.When tog1 kind is planted in the day cool environment of the highest temperature lower than 30 DEG C, its morphological specificity and wild-type are as good as; And when being grown on the summer weather of the more sky a few days highest temperature higher than 30 DEG C, tog1 blade attenuates, obviously short in wild-type, and setting percentage significantly reduces; Be grown on more hot Yangzhou one when being with summer weather, tog1 more downgrades and loses ability (Fig. 1).Keep other condition identical, the seed germination under contrast differing temps and the experiment of growth of seedling 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 at the F2 of cross combination in segregating population, be typical 3: 1 to be separated (320: 96) normally with high temperature derived Dwarf Plants.This result shows that tog1 mutant character controls by single recessive gene.
Two, the heat-resisting gene TOG1 of map based cloning paddy rice
In order to clone TOG1 gene, the present inventor first by the sensitive mutant tog1 isozygotied and in spend 11 to hybridize, the F of acquisition
1f is obtained for selfing
2colony, carries out the Primary Location of TOG1 gene to wherein 645 F2 recessive individual (the F2 generation with sensitive phenotype is individual).As shown in Figure 3, application SequenceTaggedSite (STS, sequence tagged site) molecule marker, utilize the method for PCR, find that the STS be positioned on No. 3 karyomit(e) marks P1, P2, P3, P4, P5, P6, P7 (table 1) and has obvious linksystem in position with mutational site.Exchange individual plant between mutational site and P4, the overwhelming majority also exchanges in mutational site with between P1, P2, P3, and the exchange individual plant between mutational site and P5, the overwhelming majority is included in mutational site and the exchange individual plant between 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 that mutator gene may in mark P4 and the region marked between P5.On this basis, expand further mutant tog1 and middle spend 11 cross combination, obtain the F comprising 1272 plant mutant individual plants
2segregating population is used for TOG1 Fine Mapping.With reference to the Rice Genome Sequence (http://www.tigr.org/tdb/e2kl/osal/ and http://btn.genomics.org.cn) completed, the sequence of japonica rice and long-grained nonglutinous rice is compared, 3 the new STS molecule markers (table 1) that utilized sequence difference to develop.TOG1 Fine Mapping is cloned between BAC5:AC133930 mark P8 and P10 in BAC the most at last, and the physical distance between these two marks is about 25kb.Utilize rice genome annotation database RiceGAAS (http://ricegaas.dna.affrc.go.jp/rgadb) to analyze to show, the gene of 4 functional annotations is had in 25kb region, these 4 genes are carried out determined dna sequence by us, compare mutant and wild-type sequence, find consistent all with wild-type of wherein three genes sequence in mutant, the gene of an encoding D EAD-boxRNA helicase and Os03g46610 is only had to there occurs sudden change, this gene there occurs displacement and the G → T of a single base at the Nucleotide place of the 140th of first exon, the amino acid at this place is caused to be transformed into a α-amino-isovaleric acid by a glycine.Therefore, DEAD-boxRNA unwindase gene is defined as target gene by us, called after TOG1.Utilize rice genome annotation database RiceGAAS information to show, this full length gene is the total length of 4746bp, 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.Comprise the ORF sequence that this gene is complete, the aminoacid sequence of coding is shown in accompanying drawing 4,5,6 respectively with the 2kb leader sequence comprising this gene promoter sequence.
The molecule marker that table 1 is located for TOG1
Three, the qualification of TOG1 gene and functional analysis
PCAMBIA1300 plasmid (purchased from CAMBIA company) is utilized to build complementing vector (Fig. 7), pass through transgenic technology, carry out the transgenic research had complementary functions, result shows that the present invention identifies the transgenic paddy rice (Fig. 8) making mutant recover normal function, TOG1RNAi plant shows temperature sensitive Dwarfing phenotypes (Fig. 9), demonstrates the present invention and has correctly cloned TOG1 gene.Amino acid sequence analysis shows that TOG1 genes encoding contains the albumen of DEAD-box structural domain.Vitro enzyme experiment alive shows that TOG1 albumen has the short spiral unwinding activities (Figure 10) of obvious RNA double-strand, proves that this albumen is a DBPA having function, so this protein designations is DEAD-boxRNA helicase TOG1.
The present invention has cloned a heat-resisting gene of paddy rice by map based cloning method---and-TOG1, it also can be called the DEAD-boxRNA unwindase gene of paddy rice.Further, 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 (Fig. 4) of TOG1 gene is cut with HindIII from BAC:AC133930 (purchased from American Joint Genome Institute, NCBI discloses this sequence (AccessionNo.AC133930)) and builds the HindIII restriction enzyme site entering pCAMBIA1300 (purchased from CAMBIA company).
The recombinant vectors of above-mentioned structure is transformed in E.coliDH5 α competent cell (Invitrogen Products), by kalamycin resistance screening positive clone, (cloning and identification method is shown in that Molecular Cloning: A Laboratory guide (third edition) (in translate version) Huang Peitang etc. translates, Science Press, in September, 2002 publishes ").Extract plasmid and carry out sequencing analysis, after qualification connection is correct, by the plasmid electroporated Agrobacterium EHA105 competent cell of structure, (Agrobacterium EHA105 is purchased from Clontech again, Agrobacterium EHA105 competent cell is conventionally prepared by this laboratory, with reference to " plant genetic engineering ", Wang Guanlin, Fang Hongjun, Science Press, 2004, the 2nd edition etc.).Agrobacterium-medialed transformation method is adopted to import in japonica rice variety A024.Concrete steps are as follows:
(1) induction of Mature Embryos of Rice callus.
By the rice paddy seed that shells through 70% ethanol surface sterilization after 1 minute, with aseptic water washing 3 ~ 5 times.Sterilize 15 minutes by 30%NaClO (every 50mL adds a Tween20) solution stirring, aseptic water washing 5 times, blots with filter paper after soaked overnight.Seed after sterilizing is inoculated in N
6on D substratum (table 2), cultivate 10 ~ 14 days evoked callus in 32 DEG C of continuous lights, then remove plumule.
(2) cultivation of Agrobacterium.By the pCAMBIA1300-TOG1 recombinant plasmid 1800v voltage built, within 2 seconds, electroporated EHA105 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) containing 50mg/L kantlex upper 28 DEG C cultivate 48 hours.Picking Agrobacterium mono-clonal is inoculated in 20mLYEB liquid nutrient medium, and 28 DEG C are shaken bacterium and are cultured to logarithmic growth late period; Get the strain transfer after 0.5mL activation in 50mLYEB substratum (50mg/L kantlex), 28 DEG C are cultured to OD
600be about 0.5.
(3) Dual culture and conversion, screening, differentiation.First the Agrobacterium thalline proceeding to pCAMBIA1300-TOG1 recombinant plasmid is collected, 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 this AAM-AS bacterium liquid, rock 1.5 minutes gently; Aseptic filter paper sucks unnecessary bacterium liquid, dry up.Callus is connected to N
6d-As substratum, substratum spreads in advance the aseptic filter paper that is soaked with AAM-AS, 25 DEG C of light culture 3 days.The aseptic water washing of callus use after Dual culture containing 400mg/L carboxylic benzyl 5 times, and soak 1 hour.Aseptic filter paper sucks moisture and dries up.Then callus is transferred to containing 50mg/L Totomycin and 400mg/L carboxylic benzyl N
6on D substratum, at 32 DEG C, continuous light is cultivated two weeks.Eugonic resistant calli is transferred on the RE substratum containing 50mg/L Totomycin and 250mg/L carboxylic benzyl, differentiation-inducing and be cultured to generation regrowth under 32 DEG C of continuous light conditions.Treat that seedling grows to about 10cm, 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-Dichlorophenoxyaceticacid (2,4 dichloro benzene ethoxyacetic acid)
CH
*caseinacidhydrolysate (acid hydrolysis casein)
NAA
* *1-Naphthylaceticacid (1-naphthylacetic acid)
The phenotype analytical of embodiment 2, TOG1 process LAN transfer-gen plant
For inspection transgenosis is to the effect improving paddy rice Holstein Cattle, from the plant that transgenic experiments obtains, 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.
Positive strain seedling and contemporaneously wild type seedlings are divided into two batches:
A collection ofly being put in incubator, imposing a condition as continuing 37 DEG C, 12 little time: 12 hours dark.Cultivate the plant forms after 54 days as shown in fig. lla, the transfer-gen plant speed of growth is significantly higher than wild-type, illustrates that the process LAN of TOG1 gene significantly improves the high temperature resistant proterties of paddy rice;
A collection of self-sow (May ~ September, average max. daily temperature is about 30 DEG C) in the farm of During Summer In Beijing in addition.Ripe TOG1 process LAN plant is at single-strain fructification yield or on tillering number, is all significantly higher than WT lines about one times of (Figure 11 b, c; Figure 12).
The result shows that the process LAN of TOG1 in paddy rice also significantly can improve single plant yield on the basis of improving high temperature resistance.
In embodiment 3, TOG1 process LAN transfer-gen plant, TOG1 expresses quantitative analysis
In order to accurately grasp the above-mentioned expression amount carrying out this gene in the TOG1 process LAN transfer-gen plant of phenotype analytical, the relative quantity of TOG1mRNA with the methods analyst of qRT-PCR.Concrete grammar is, extracts rice leaf total serum IgE with TRIzol reagent (purchased from Invitrogen), detects the integrity of RNA, then use DNaseI (Takara) to digest through 1% agarose electrophoresis.With CDSIII (oligo (dT)) primer and M-MLV ThermoScript II (Invitrogen) synthesizing single-stranded cDNA, method detailed is shown in Invitrogen operational manual.CDNA dilutes the template as qRT-PCR after 10 times.Amplification reaction system used is PowerSYBRgreenPCRmastermix (purchased from AppliedBiosystems), and instrument is CFX96real-timePCRdetectionsystem (purchased from Bio-Rad).Primer TOGlQFl/TOGlQRl is used for detecting TOG1, primer OsACTINlF/OsACTINlR and is used for detecting interior source reference Actin.Primer sequence is as follows:
Table 3 Primer and sequence
QRT-PCR detected result shows, and the expression amount of TOG1 in process LAN transfer-gen plant is 1.7-3 times of level (Figure 13) of wild-type.
Conclusion:
Paddy rice is one of most important food crop in China and even the whole world, and investigators are devoted to improve its output and resistance always.Become today of prevalent variety cultivation important means gradually in genetically engineered and molecular breeding, the heat-resisting factor pair in discovery 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, gets a good chance of the object reaching rice genetic improvement and improve thermotolerance and output.
Should be appreciated that, although with reference to the embodiment that it is exemplary, the present invention shown particularly and describe, but will be understood by those skilled in the art that, do not deviating from by under the condition of the spirit and scope of the present invention as defined in the claims, the change of various forms and details can be carried out wherein, the arbitrary combination of various embodiment can be carried out.
Reference
1.Kondamudi，R.etal.HeatStressinRice-PhysiologicalMechanismsandAdaptationStrategies.InCropStressanditsManagement：PerspectivesandStrategies(edsVenkateswarlu，B.etal.)193-224(Springer，2012).
2.Morita，S.，Siratsuchi，H.，Takanashi，J.&Fujita，K.Effectofhightemperatureonripeninginriceplant.Analysisoftheeffectofhighnightandhighdaytemperatureappliedtothepanicleinotherpartsoftheplant.Jpn.J.CropSci.73，77-83(2004).
3.Glover，J.R.&Lindquist，S.Hsp104，Hsp70，andHsp40：anovelchaperonesystemthatrescuespreviouslyaggregatedproteins.Cell94，73-82(1998).
4.Montero-Barrientos，M.etal.TransgenicexpressionoftheTrichodermaharzianumHsp70geneincreasesArabidopsisresistancetoheatandotherabioticstresses.J.PlantPhysiol.167，659-665(2010).
5.Szabo，A.etal.TheATPhydrolysis-dependentreactioncycleoftheEscherichiacoliHsp70system-DnaK，DnaJ，andGrpE.Proc.Natl.Acad.Sci.USA91，10345-10349(1994).
6.Kumar，S.V.&Wigge，P.A.H2A.Z-containingnucleosomesmediatethethermosensoryresponseinArabidopsis.Cell140，136-147(2010).
7.Pyle，A.M.TranslocationandunwindingmechanismsofRNAandDNAhelicases.Ann.Rev.Biophys.37，317-336(2008).
8.Tanner，N.K.，Cordin，O.，Banroques，J.，Doere，M.&Linder，P.TheQmotif：anewlyidentifiedmotifinDEADboxhelicasesmayregulateATPbindingandhydrolysis.Mol.Cell11，127-138(2003).
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Claims (5)
1. a cultivation has the method for the rice varieties of the thermotolerance of raising, described method comprises the cell or tissue of paddy rice described in the vector with the nucleotide sequence comprising the DEAD-boxRNA helicase TOG1 shown in coding SEQIDNo:2, and the rice cell of conversion or tissue cultivating is become TOG1 process LAN type rice plant.
2. method according to claim 1, the nucleotide sequence of the DEAD-boxRNA helicase TOG1 shown in wherein said coding SEQIDNo:2 is as shown in SEQIDNo:1.
3. method according to claim 2, the wherein said carrier comprising the nucleotide sequence of the DEAD-boxRNA helicase TOG1 of coding shown in SEQIDNo:2 builds by the nucleotide sequence restructuring shown in SEQIDNo:1 being inserted in pCAMBIA1300 plasmid.
4. method according to claim 1, wherein said conversion is undertaken by agrobacterium-mediated transformation or particle bombardment.
5. method according to claim 1, wherein said rice tissue is Mature Embryos of Rice callus.
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