CN103304652A - Plant stress tolerance related protein TaHSF3 and coding gene as well as application thereof - Google Patents
Plant stress tolerance related protein TaHSF3 and coding gene as well as application thereof Download PDFInfo
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Abstract
The invention discloses a plant stress tolerance related protein TaHSF3 and a coding gene as well as application thereof. The protein provided by the invention comes from triticum aestivum cv., is named TaHSF3 and is the following (a) or (b), wherein (a) is a protein formed by amino acid sequences shown in a sequence 1 in a sequence table; and (b) is a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequences in the sequence 1, is related to plant stress tolerance and is derived from the sequence 1. The TaHSF3 gene is expressed under induction of high temperature, drought, salt, low temperature and abscisic acid (ABS) but has different stress response time and strength, wherein the gene TaHSF3 responds to high and low temperatures more strongly. The protein TaHSF3 and the coding gene thereof provide a foundation for expression of genes related to artificial control of stress resistance and stress tolerance and can play an important role in breeding for culturing plants with enhanced stress resistance and stress tolerance.
Description
Technical field
The present invention relates to a kind of plant stress tolerance correlative protein TaHSF3 and encoding gene and application.
Background technology
Environment stresses such as high temperature are the obstruction factors that influences wheat growth, growth.Therefore, the understanding wheat is replied and signal transduction mechanism adverse environmental factor, improves the resistance of wheat breed, becomes one of vital task of wheat genetic research and wheat breed improvement.
Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Clear and definite plant is to the reaction mechanism of adverse circumstance, will provide the science argument for adversity gene engineering research and application.At present, the plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, explores and improves plant growth characteristics with biotechnology, its objective is and improves plant to the adaptive faculty of adverse circumstance.
Summary of the invention
The purpose of this invention is to provide a kind of plant stress tolerance correlative protein TaHSF3 and encoding gene and application.
Protein provided by the invention, available from the Xiao Bai wheat, called after TaHSF3 albumen is following (a) or (b):
(a) protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance protein of being derived by sequence 1.
In order to make the protein in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| |
8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned (b) but in the protein synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of the protein in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene of encoding said proteins also belongs to protection scope of the present invention.
Described gene can be following 1) to 4) in arbitrary described dna molecular:
1) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 133rd to 885 Nucleotide;
2) dna molecular shown in the sequence 2 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant stress tolerance correlative protein;
4) with 1) or 2) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant stress tolerance correlative protein.
Described stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain described gene all belong to protection scope of the present invention.
Available existing expression vector establishment contains the recombinant expression vector of described gene.Described expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of micropellet bombardment.Described expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of identifying and screening, can process used expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding to encode.Also can not add any selected marker, directly according to phenotypic screen.
Described recombinant expression vector specifically can be described gene is inserted the recombinant plasmid that the multiple clone site of pBI121 obtains.
The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.
Described gene specifically can import in the described purpose biology by described recombinant expression vector.Carry the recombinant expression vector of described gene can be by using conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated, particle gun transform the cell or tissue of described biology.
Described purpose plant can be monocotyledons or dicotyledons.Described dicotyledons can be Arabidopis thaliana, as the environmental Arabidopis thaliana of Colombia.
Described resistance of reverse specifically can be high temperature resistant and/or low temperature resistant.
The present invention also protects the application of described albumen in the regulation and control plant stress tolerance.Described plant can be monocotyledons or dicotyledons.Described dicotyledons can be Arabidopis thaliana, as the environmental Arabidopis thaliana of Colombia.
Protein provided by the invention, from the information biology structural analysis, (Heat shock protein HSP), is positioned at nucleus and part vacuole to belong to heat shock protein.
The TaHSF3 gene is at the abduction delivering of high temperature, arid, salt, low temperature and dormin (ABA), but to various time and intensity differences of coercing response, and is wherein, stronger to high temperature, low temperature response ratio.TaHSF3 albumen provided by the invention and encoding gene thereof will play an important role in the plant breeding of cultivating the enhancing of resistance and resistance of reverse for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.
Description of drawings
Fig. 1 is the result of TaHSF3 protein biology information science structural analysis.
Fig. 2 is the structural representation of recombinant plasmid pBI121-TaHSF3.
Fig. 3 is the photo after normal 1 week of cultivation of pyroprocessing and recovery.
Fig. 4 changes relatively for the chlorophyll content in the Arabidopsis leaf before and after the pyroprocessing.
Fig. 5 is the photo after normal 1 week of cultivation of subzero treatment and recovery.
Fig. 6 is that the relative conductivity of Arabidopsis leaf before and after the subzero treatment changes.
Fig. 7 coerces for difference and handles TaHSF3 expression of gene down.
Fig. 8 is the structural representation of recombinant plasmid 16318hGFP-TaHSF3.
Fig. 9 is the Subcellular Localization of TaHSF3 albumen.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Xiao Bai wheat (Triticum aestivum cv.Xiaobaimai) is the kind of common wheat (Triticum aestivum L.), the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, and the public also can obtain (being numbered ZM242) from national germplasm resource bank; The document of mentioning the Xiao Bai wheat is: Sun Haitao etc., the screening of wheat TaDREB6 transcription factor interact protein, Scientia Agricultura Sinica, 2011,44 (22): 4740-4747.; The document of mentioning the Xiao Bai wheat is: Isolation and molecular characterization of the Triticum aestivum L.ethylene-responsive factor 1 (TaERF1) that increases multiple stress tolerance, Plant Mol Biol (2007) 65:719-732, Zhao Shi Xu, Lan Qin Xia, Ming Chen, Xian Guo Cheng, Rui Yue Zhang, Lian Cheng Li, Yun Xing Zhao, Yah Lu, Zhi Yong Ni, Li Liu, Zhi Gang Qiu, You Zhi Ma).
The discovery of embodiment 1, TaHSF3 albumen and encoding gene thereof
The sowing of Xiao Bai wheat on the seedbed, about 20～24 ℃ of growth 10d, is taken out from soil, clean with distilled water flushing, be placed on arid processing 2h on the filter paper, get blade and put into liquid nitrogen immediately, under-80 ℃ of conditions, preserve.
Adopt Trizol method (TianGen) to extract total RNA of wheat leaf blade, the first chain cDNA is synthetic with ThermoScript II XL (AMV) (TaKaRa), adopts SMART method (BD) to synthesize ds cDNA.Pcr amplification product carries out 1.0% agarose gel electrophoresis.New gene shown in the sequence 2 of a sequence table of discovery, the albumen shown in the sequence 1 of this gene coded sequence table.
With the protein called after TaHSF3 albumen shown in the sequence 1 of sequence table.TaHSF3 albumen is carried out the information biology structural analysis, the results are shown in Figure 1.TaHSF3 albumen belongs to heat shock factor, mainly comprise following structural domain: conservative N end DNA is in conjunction with territory (DNA-binding domain, DBD), the oligomerization territory (HR-A/B) formed by two 7 hydrophobic peptide repeat region A and B, nuclear localization signal (Nuclear localization signal, NLS), AHA (the activation structure territory of acid C end).With the encoding gene called after TaHSF3 gene of TaHSF3 albumen, its open reading frame as the sequence 2 of sequence table from shown in 5 ' terminal the 133rd to 885 Nucleotide.
The acquisition of embodiment 2, transgenic plant and resistance of reverse thereof are identified
One, the structure of recombinant expression vector
1, the clone of TaHSF3 gene
To (TaHSF3-1-121F and TaHSF3-1-121R), the primer end introduces BamHI respectively and the SpeI enzyme is cut recognition site according to the sequences Design primer of TaHSF3 gene.
TaHSF3-1-121F：5’-CG
GGATCCGTCTCTAACTTCCTCGCTTCTC-3’；
TaHSF3-1-121R：5’-GG
ACTAGTTCACCAGAGCAATTCCCGAC-3’。
Total RNA and the reverse transcription of extracting the Xiao Bai wheat are cDNA, and the primer that to be template with cDNA form with TaHSF3-1-121F and TaHSF3-1-121R is to carrying out pcr amplification, the pcr amplification product of the about 885bp of recovery.
2, the structure of recombinant expression vector
1. use the pcr amplification product of restriction enzyme BamHI and SpeI double digestion step 1, reclaim enzyme and cut product.
2. use restriction enzyme BamHI and SpeI double digestion pBI121 plasmid (Clontech company), reclaim carrier framework (about 12.7kb).
3. step enzyme 1. being cut product is connected with step carrier framework 2..
4. step connection product electric shock is 3. transformed TOP10 bacterial strain (TIANGEN Biotech (Beijing) Co., Ltd.), 37 ℃ of incubated overnight, the picking positive colony checks order.Sequencing result shows, has obtained recombinant plasmid pBI121-TaHSF3.According to sequencing result, it is as follows that recombinant plasmid pBI121-TaHSF3 is carried out structrual description: inserted the dna fragmentation shown in the sequence 2 of sequence table (open reading frame for from 5 ' terminal the 133rd to 885 Nucleotide) between the BamHI of pBI121 plasmid and SpeI restriction enzyme site.The structural representation of recombinant plasmid pBI121-TaHSF3 is seen Fig. 2.
Two, the acquisition of transgenic plant
1, transforms Agrobacterium C58 (Beijing Baeyer enlightening biotech company) with recombinant plasmid pBI121-TaHSF3, obtain the Agrobacterium of recombinating.
2, the Agrobacterium of will recombinating is inoculated in the YEP liquid nutrient medium, and 28 ℃, 3000rpm were cultivated about 30 hours.
3, the bacterium liquid with step 2 goes in the YEP liquid nutrient medium (containing 50 μ g/ml kantlex and 50 μ g/ml Rifampins), and 28 ℃, 300rpm are cultivated about 14 hours (bacterium liquid OD600 reaches 1.5-3.0).
4, with 4 ℃ of bacterium liquid, the centrifugal 10min of 4000g of step 3, also being diluted to OD600 with 10% sucrose solution (containing 0.02% silwet-L77) is about 0.8-1.0 to collect thalline.
5, will put in order the environmental Arabidopis thaliana of strain Colombia (Col-0, SALK company) and tip upside down in the container of the bacterium liquid that fills step 4 with flowerpot, flower is soaked about 50s, after immersion finishes, take out flowerpot, be sidelong in pallet, cover black plastic cloth; Open plastic cloth after 24 hours, uprightly place flowerpot, carry out normal illumination cultivation, results T
1For seed.
T
1For seed results back screening resistant plant in MS substratum (kantlex that contains 50 μ g/L), resistant plant is transplanted in the soil results T
2Be plant (T for seed and with its cultivation
2For plant).
Extract T respectively
1For plant and T
2Total RNA and reverse transcription for the blade of plant are cDNA, identify that to the cDNA from each sample being carried out PCR it is transfer-gen plant that PCR is accredited as positive plant with the primer that TaHSF3-1-121F and TaHSF3-1-121R form.For a certain T
1For plant, if its T
2Equal PCR is accredited as the positive for plant, and then this plant is the transfer-gen plant that isozygotys, and this plant and offspring thereof are 1 transgenic line.
T
2Produce T for the transfer-gen plant selfing
3For seed.
Three, change the acquisition of empty carrier plant
Replace pBI121-TaHSF3 with the pBI121 plasmid, other same step 2 obtains changeing the T of empty carrier plant
3For seed, as transfer-gen plant T
3Contrast for seed.
Four, the high temperature resistant evaluation of transgenic plant
With three transgenic lines (strain is 1, strain be 2 and strain be 3) T
3For plant, a T who changes empty carrier strain system
3Carry out high temperature resistant evaluation (each strain system adopts parallel processing, repeated experiments is set three times, results averaged) respectively for plant and the environmental Arabidopis thaliana of Colombia (each strain is 60 strains), concrete steps are as follows:
22 ℃ of plant are normal to be cultivated, and the seedling that will sprout then after 4 weeks was placed 3 hours in 42 ℃ of high temperature, recovered normal 22 ℃ of cultivations then, takes pictures after normally cultivating for 1 week, and adds up survival rate.Photo is seen Fig. 3.The environmental Arabidopis thaliana of Colombia is most of dead, and survival rate only is 25%.And the survival rate of three transgenic line plant is all up to more than 75%.It is all consistent with Colombia ecotype Arabidopis thaliana to change the phenotype of empty carrier plant and survival rate.
Pyroprocessing is preceding and recover normal cultivation after 2 days, and the blade of getting plant respectively carries out measuring chlorophyll content (adopting acetone-ethanol mixed determining method, Yang Minwen, 2002).The results are shown in Figure 4.Preceding each strain of pyroprocessing is that the chlorophyll content in the plant leaf does not have significant difference, all between 1.3-1.5mg/g.After the pyroprocessing, the chlorophyll content in the environmental Arabidopsis leaf of Colombia is about 0.35mg/g, and the chlorophyll content in three transgenic line plant leafs is between 0.52-0.67mg/g.Before and after the pyroprocessing, the chlorophyll content that changes the empty carrier plant leaf is all consistent with the chlorophyll content of Colombia ecotype Arabidopsis leaf.
Five, the low temperature resistant evaluation of transgenic plant
With three transgenic lines (strain is 1, strain be 2 and strain be 3) T
3For plant, a T who changes empty carrier strain system
3Carry out low temperature resistant evaluation (each strain system adopts parallel processing, repeated experiments is set three times, results averaged) respectively for plant and the environmental Arabidopis thaliana of Colombia (each strain is 60 strains), concrete steps are as follows:
22 ℃ of plant are normal to be cultivated, and the seedling that will sprout then after 4 weeks was placed 2 hours in-10 ℃ of low temperature, recover normally 1 week of cultivation then after, take pictures and add up survival rate.Photo is seen Fig. 5.The environmental Arabidopis thaliana of Colombia is most of dead, and survival rate only is 10%.And the survival rate of three transgenic line plant is all up to more than 85%.It is all consistent with Colombia ecotype Arabidopis thaliana to change the phenotype of empty carrier plant and survival rate.
Subzero treatment is preceding and recover normal cultivation after 2 days, and the blade of getting plant respectively carries out relative conductivity and measures (referring to modern plants experiment guide (1999, Beijing: scientific publication is learned)).The results are shown in Figure 6.The relative conductivity that preceding each strain of subzero treatment is the transgenic line plant leaf does not have significant difference, all about 9.0%.After the subzero treatment, the relative conductivity of the environmental Arabidopsis leaf of Colombia is about 37.5%, and the relative conductivity of three transgenic line plant leafs is between 17.56-18.6%.Before and after the subzero treatment, the relative conductivity that changes the empty carrier plant leaf is all consistent with the relative conductivity of Colombia ecotype Arabidopsis leaf.
The structure of embodiment 3, bait carrier and self activation detect
According to the sequence of TaHSF3 gene and the cloning site of pGBKT7 (Clontech), introduce EcoR I and BamH I (Promaga) restriction enzyme site respectively at the two ends of gene.(T4 ligase enzyme is cut, connected to enzyme; Promaga) to plasmid pGBKT7.Sequence verification fusion plasmid pGBKT7-TaHSF3 clone is correct.
PGBKT7-TaHSF3, empty pGBKT7 and negative control are changed among the yeast strain AH109 jointly with pGADT7 respectively, and SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade plate screening are cultivated, and checking pGBKT7-TaHSF3 has the self activation activity.
One, carries out the various processing of coercing
Seedling age is 10 days Xiao Bai wheat seedling, carries out following processing respectively:
(1) arid is handled: the wheat seedling of water planting is taken out the moisture that blots on the root, place on the dry filter paper, arid is cultivated after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours and is taken out material, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby;
(2) salt marsh is handled: with wheat seedling place 2% by NaCl and Na
2SO
4(NaCl and Na in the sodium salt solution of forming
2SO
4Mass percent be 3: 2) in, illumination cultivation is taken out material respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby;
(3) damage to plants caused by sudden drop in temperature processing: wheat seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby;
(4) ABA handles: wheat seedling is placed the ABA solution of 100 μ M, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby;
(5) pyroprocessing: wheat seedling is placed under 42 ℃, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby.
Two, the separation of mRNA
Adopt Quikprep Micro mRNA Purification Kit (Pharmacia) to carry out the separation of mRNA.
Three. reverse transcription is cDNA
Adopting R103-Quant_Reverse_Transcriptase (TIANGEN) is cDNA with the mRNA reverse transcription of purifying.
Four, real-time fluorescence quantitative PCR
According to the TaHSF3 gene order, at its variable region design special primer HSF3F and HSF3R.Be internal control gene with actin, primer is actin-2F and actin-2R.
HSF3F：5’-CAAGCACAATAACTTCTCCAGC-3’；
HSF3R：5’-GCCGATGTATGTCACAAAGC-3’。
actin-2F：5’-CTCCCTCACAACAACCGC-3’；
actin-2R：5’-TACCAGGAACTTCCATACCAAC-3’。
Each coerces the TaHSF3 gene pairs and hormone shows response, sees Fig. 7.Among Fig. 7, A is the result of pyroprocessing, and B is the result who damages to plants caused by sudden drop in temperature processing, and C is the result that ABA handles, and D is the result that arid is handled, and E is the result that salt marsh is handled.The various time and intensity differences of coercing response of TaHSF3 gene pairs.
One, vector construction
Pcr amplification TaHSF3 full length gene.The purpose fragment is connected on the Subcellular Localization carrier 163hGFP of same double digestion heat shock transformed into escherichia coli Top10.Recombinant plasmid is carried out PCR to be detected.Positive colony is checked order.Extract plasmid, obtain recombinant plasmid 16318hGFP-TaHSF3 (see figure 8).
Two, material is prepared
It is the bronze suspension 6 μ l (50mg/ml) of 1.0 μ M that 3 μ g recombinant plasmid dnas add diameter, 0.1M spermidine (spermidine) 4 μ l, 2.5M CaCl
26 μ l with the first mixing that vibrates respectively of bronze, DNA, spermidine and calcium chloride, behind the mixing vibration mixing 3min, leave standstill 15min on ice then.The centrifugal 10s of 12000rpm (rotating speed reach 12000rpm after 10s) abandons supernatant.Add 140 μ l dehydrated alcohols, the centrifugal 10s of thick vibration back (breaing up bronze) 12000rpm collects the bronze precipitation.20 μ l dehydrated alcohols suspend and precipitate, the some film.
Three, particle gun bombardment receptor material
1. select the split film (this experiment 1100psi) of certain pressure for use, with the bombardment film, soak 1～2h in 70% alcohol, taking-up is dried.
2. metal baffle is sterilized at spirit lamp with alcohol-pickled, the Bechtop ultraviolet sterilization of particle gun.
3. get the above-mentioned bronze for preparing of 20 μ l-plasmid complex body, evenly coat on the mid-way of bombardment film, be not applied on the whole film, size is consistent with the pore diameter range on the carrier fixed ring, dries, and is fixed on then on the carrier fixed ring.
4. above-mentioned carrier fixed ring is installed on the launching device.
5. can split film and be installed to gas acceleration tube lower end.
6. the onion epidermis culture dish is put into vacuum chamber, take off the culture dish lid.
7. vacuumize pointer to 26In/Hg.
8. put helium in the gas acceleration tube, pressure reaches in the time of can splitting the pressure that film can bear in pipe, can split film and break.
9. gas is flushed on the bombardment film, and carrier moves downward, and blocked by metal baffle, and following bronze-plasmid complex body sees through the mesh directive target cell of metal baffle.
10. will bombard good onion epidermis cell and put into 25 ℃ of incubators, under laser confocal microscope, observe after secretly cultivating 16～24h.
Four, onion epidermis cell microscopy
Particle gun is bombarded, secretly cultivates 16-24h onion epidermis compressing tablet afterwards, then at laser scanning co-focusing microscope (Bio-Rad MicroRadiance) (Laser scanning confocal microscopy, LSMC) observe GFP (green fluorescent protein) fluorescence, the line scanning of going forward side by side is taken pictures.The result shows that TaHSF3-1 navigates in the tenuigenin.The working parameter of LSCM is: Ex=488nm, Em=525 ± 15nm, Power=10%, Zoom7, medium sweep, Frame512 * 512.Software is TIME-COURSE and PHOTOSHOP5.0.See Fig. 9.TaHSF3-GFP fusion rotein excited fluorescent mainly is distributed in tenuigenin.
Claims (10)
1. protein is following (a) or (b):
(a) protein of being formed by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance protein of being derived by sequence 1.
2. the gene of coding claim 1 described albumen.
3. gene as claimed in claim 2, it is characterized in that: described gene is following 1) to 4) in arbitrary described dna molecular:
1) sequence 2 of sequence table is from the dna molecular shown in 5 ' terminal the 133rd to 885 Nucleotide;
2) dna molecular shown in the sequence 2 of sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coded plant stress tolerance correlative protein;
4) with 1) or 2) dna sequence dna that limits has 90% above homology, and the dna molecular of coded plant stress tolerance correlative protein.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.
5. recombinant vectors as claimed in claim 3 is characterized in that: the recombinant plasmid that described recombinant expression vector obtains for the multiple clone site with claim 2 or 3 described genes insertion pBI121.
6. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.
7. method as claimed in claim 6, it is characterized in that: claim 2 or 3 described genes import in the described purpose plant by claim 4 or 5 described recombinant vectorss.
8. as claim 6 or 7 described methods, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.
9. as arbitrary described method in the claim 6 to 8, it is characterized in that: described resistance of reverse is high temperature resistant and/or low temperature resistant.
10. the application of the described albumen of claim 1 in the regulation and control plant stress tolerance.
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