CN101921774A - Application of Dnaj-like protein and encoded gene thereof - Google Patents
Application of Dnaj-like protein and encoded gene thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene engineering and particularly relates to the application of a Dnaj-like protein and an encoded gene thereof in the resistance of plants to abiotic stress processes such as drought, low temperatures, exogenous abscisic acid and the like. A Dnaj-like protein gene is one of the following nucleotide sequences: 1) a deoxyribonucleic acid (DNA) sequence of SEQ ID No:1 in a sequence table, 2) polynucleotide of an SEQ ID No:2 protein in an encoded sequence table and 3) a DNA sequence which has over 80 percent of homology with the DNA sequence defined by the SEQ ID No:1 in the sequence table and encodes proteins with the same function. The Dnaj-like protein of the invention is a protein having an SEQ ID No:2 amino acid residue sequence in the sequence table or a protein which is obtained by performing replacement, deletion or addition of one or more amino acid residues on SEQ ID No:2 amino acid, has the same activity as an amino acid sequence of SEQ ID No:2 and is derived from SEQ ID No:2. The gene of the invention plays an important role in improving the stress tolerances such as drought tolerance, abscisic acid resistance and the like as well as relative characters of plants.
Description
One, technical field
The invention belongs to gene engineering technology field, be specifically related to the application in abiotic stress processes such as plant opposing arid, low temperature and Exogenous Abscisic Acid of a kind of Arabidopis thaliana DnaJ-like albumen and encoding gene thereof.
Two, background technology
Plant-growth can run into the influence of many poor environments such as hot and cold, non-irrigated, waterlogging, saline and alkaline, topsoil inevitably in physical environment.When poor environment acts on plant, can cause a series of physiological metabolism reaction takes place in the plant materials that the reversibility that shows as metabolism and growth suppresses, when serious even can cause and the irreversible injury of each tissue cause whole plant death.In various environment-stress, abiotic stress such as arid, low temperature and high salt are particularly outstanding to the influence of plant, these environment-stress factors are by the influence in various degree to water regime in the plant materials, thereby restrict growth of plant and the output of farm crop, become the principal element of the global agriculture underproduction.
Yet plant has formed a series of physiology, metabolism and systems of defense of replying environment stress gradually in evolution of long period of time.When plant when being subjected to coercing, many expression of gene can change, be the common processes of coordinating of a plurality of gene interactions, produced a series of responsiveness reaction and accommodation from the cell to the physiological level thus, change as membrane component, the increase of soluble proteins, the accumulation of sugar and proline(Pro) etc., thereby reduce or eliminate the harm that these poor environments bring to plant, make the resistance of reverse of plant strengthen (Bray E.A.et al. (2002) Response to Abiotic Stress In B Buchanan, WGruissem, R Jones, eds, Biochemistry and Molecular Biology of Plants.Academic Press, pp 1158-1204).Part adverse circumstance response gene often is exactly the gene of anti-the reverse function, the anti-counter-adaptation process of involved in plant, thus reach the purpose of resisting adverse circumstance, protection cell normal activity.Help the regulatory mechanism of people by analysis and research, thereby the help people transform reasonably with molecular biology method and optimize the stress-tolerance proterties to improve Crop Productivity from molecular level understanding plant stress tolerant to these genes and coded product thereof.
The molecule mechanism that discloses the plant stress answering is the long-term key subjects of exploring of people always; discover; external environment coerce can the inducing plant body in the proteic expression of DnaJ-like; these J albumen can prevent the protein aggegation with the protein bound of sex change or error state (ERST); protect cyto-architectural integrity, thereby complying with of aspects such as high temperature stress, salt stress and heavy metal stress brought into play in the process and will be acted on plant.And the domain C RR (Cysteine-rich region) that is rich in halfcystine in the DnaJ-like albumen has participated in the cell processes of a lot of high conservatives, comprise Protein Folding, the assembling of protein complex and decomposition etc. have crucial physiological function (Lu S et al. (2006) TheCauliflower Or Gene Encodes a DnaJ Cysteine-Rich Domain-ContainingProtein That Mediates High-Levels of b-Carotene Accumulation.Plant Cell18:3594-3605) in plant growth and development process.
Three, summary of the invention
The problem that the present invention need solve provides a kind of intend southern DnaJ-like albumen and the application of encoding gene in abiotic stress processes such as plant opposing arid, low temperature and Exogenous Abscisic Acid thereof.
The DnaJ-like protein gene relevant with resistance provided by the present invention, feature are to derive from Arabidopis thaliana (Arabidopsis thaliana), and name is called CRR1, is one of following nucleotide sequences;
(1) polynucleotide sequence is:
1?atgagtccga?tcgtcataac?ccagttagct?acaggaatca?gcgttttagc?50
51 cggtgcggtc?tttatcaaat?cagtcatgga?ccaaaagcct?atggctggtc?100
101?aattccctcg?ttgtccgact?tgtaacggta?ctggacgagt?cacgtgtttc?150
151?tgttctcgat?ggtccgatgg?tgacgttgga?tgtcgtaggt?gttctggttc?200
201?aggtcgtgcg?gcttgtagca?attgtggtgg?ttctggaact?ggtagacctt?250
251?taccggctca?gattacggtt?cagccaccga?atcgtcctta?ttga 294
(2) polynucleotide sequence of described protein amino acid sequence below the coding:
Met?Ser?Pro?Ile?Val?Ile?Thr?Gln?Leu?Ala?Thr?Gly?Ile?Ser?Val?Leu
1 5 10 15
Ala?Gly?Ala?Val?Phe?Ile?Lys?Ser?Val?Met?Asp?Gln?Lys?Pro?Met?Ala
20 25 30
Gly?Gln?Phe?Pro?Arg?Cys?Pro?Thr?Cys?Asn?Gly?Thr?Gly?Arg?Val?Thr
35 40 45
Cys?Phe?Cys?Ser?Arg?Trp?Ser?Asp?Gly?Asp?Val?Gly?Cys?Arg?Arg?Cys
50 55 60
Ser?Gly?Ser?Gly?Arg?Ala?Ala?Cys?Ser?Asn?Cys?Gly?Gly?Ser?Gly?Thr
65 70 75 80
Gly?Arg?Pro?Leu?Pro?Ala?Gln?Ile?Thr?Val?Gln?Pro?Pro?Asn?Arg?Pro
85 90 95
Tyr
97
(3) have 80% above homology with (1) described polynucleotide sequence, and the proteinic polynucleotide sequence of coding identical function.
The DnaJ-like albumen relevant with resistance provided by the present invention is characterized in that deriving from Arabidopis thaliana (Arabidopsis thaliana), and name is called CRR1, is one of following amino acid residue sequence:
(1) aminoacid sequence is:
Met?Ser?Pro?Ile?Val?Ile?Thr?Gln?Leu?Ala?Thr?Gly?Ile?Ser?Val?Leu
1 5 10 15
Ala?Gly?Ala?Val?Phe?Ile?Lys?Ser?Val?Met?Asp?Gln?Lys?Pro?Met?Ala
20 25 30
Gly?Gln?Phe?Pro?Arg?Cys?Pro?Thr?Cys?Asn?Gly?Thr?Gly?Arg?Val?Thr
35 40 45
Cys?Phe?Cys?Ser?Arg?Trp?Ser?Asp?Gly?Asp?Val?Gly?Cys?Arg?Arg?Cys
50 55 60
Ser?Gly?Ser?Gly?Arg?Ala?Ala?Cys?Ser?Asn?Cys?Gly?Gly?Ser?Gly?Thr
65 70 75 80
Gly?Arg?Pro?Leu?Pro?Ala?Gln?Ile?Thr?Val?Gln?Pro?Pro?Asn?Arg?Pro
85 90 95
Tyr
97
(2) with replacement and/or disappearance and/or interpolation and with (1) the described proteic amino acid residue sequence identical active deutero-protein amino acid sequence of (1) described proteic aminoacid sequence through one or several amino-acid residue.
The promotor (pCRR1) of the proteic gene C RR1 of DnaJ-like that Arabidopis thaliana of the present invention is relevant with resistance is characterized in that deriving from Arabidopis thaliana (Arabidopsis thaliana), is one of following nucleotide sequences:
(1) nucleotides sequence is classified as:
1 ccaactctct?cctacaactt?gacacatcag?cacatgagag?cttcccacct 50
51 aaacttcata?aaataaataa?tacataaata?gtaattaatt?caacataata?100
101 actaaaatag?gaggatataa?ttaatcagaa?cttgaaccat?cactctttct?150
151 acatttttta?attcaatata?tcctttttaa?ttttgagatc?caaacatgtt?200
201 acacatattc?cttgcctaaa?aattacacga?ttattatgaa?agaaactaaa?250
251 tgaatatgca?gattaaatat?atctaagctt?ttcaagattt?atgctaagaa?300
301 aacaagtaat?ttgaaagatg?tttgaggagt?tatccttaaa?actaatacgt?350
351 aagatagaag?attatgacaa?gaacttaact?aaactttcca?agaacatcaa?400
401 gttgaatatg?attagattgg?ttatgcagag?acatggttaa?taaaaacatg?450
451 ttatatttta?aatgtaagaa?agaaattttc?taaaatttca?ccgtttcaca?500
501 aactaaatat?ataaccaaaa?gaacaaggat?atataaatta?aaaaaaaaaa?550
551 ctgagaatct?agaacaacaa?taaaaaataa?ctcaagcatt?ttcgttttca?600
601 gatgaactaa?tatagttgga?ctttttgaaa?acaattacaa?atcatattta 650
651 tgtttcaaaa?ccgatgcatt?taactttact?ttatatgaac?agacattata 700
701 taaaacaatt?tgtaaatagt?aatacataaa?cccgcacgta?gtgcgggtac 750
751 tcatctagta?tacaataata?aaaacttcaa?aatgatgcaa?caatagctca 800
801 gaatcttcat?atatgggcca?ctcggcccac?gccgaatttg?aaaagcccga 850
851 tattggaagt?aagcacgtgc?ggttgtgaga?attgcttctt?cgtccaccat 900
901 tggacatgtt?tatggctgcg?ttcgacgcgt?gatttgtttg?tggagaagag 950
951 tgcatcgcct?gataattcaa?gcaacctagt?tatagtacaa?acgtgaaaga 1000
1001?aacaaaatta?acccaaaaaa?tgaaatggaa?ggttacaatt?gcaacccttg 1050
1051?caagatagta?caacaatata?atagtaacaa?tagaacaata?aaccctaacc 1100
1101?aaacaaagta?agaattacat?tcctctaatt?ttagtgcatg?taagttcggt 1150
1151?tatcactgca?aacttaatca?tttccctatt?gactaattcg?taacatagtt 1200
1201?tatactttcg?attaatcaat?taattaattt?acagtgaatt?tgtgaaaaac 1250
1251?ttctaacgag?aaacttatta?tgttataaaa?gtcaacaaaa?gaactgtatc 1300
1301?acacagaaca?tatatattgg?gtctgattgg?taaccaaaaa?ctatacaagt 1350
1351?tatttagctg?ttagctttat?cggaatagtg?taaaactgtt?aaaatgagct 1400
1401?gtacaaaaaa?actgagagta?aatttttttg?taacgctttt?gataaagttt 1450
1451?ttgtgattag?taactgtcaa?aattgtattg?ttattaaaat?ggttaccatt 1500
1501?caaatctatt?aggtaaaata?cagagccgtc?cataagtaca?agcgggtcaa 1550
1551?acacaagctt?gtggcctcaa?ttactataaa?taaaattttg?atttttttta 1600
1601?gtagaaaaat?tagttgcttg?cttctttgat?gacaaaaaaa?aaaaagcaag 1650
1651?cagtcagata?aatcaatgat?aatctaaaaa?aaagaaaaat?ctgacgaaaa 1700
1701?aaaaaaagaa?aaaaaagaaa?aatctgaagg?ttgtacactt?gtagaagata 1750
1751?ttttacacac?acagcccaat?aaaaacccaa?atccaaaaac?tggagtatcc 1800
1801?aaaaaaaaca?cggctgtttc?attcatctcc?tcacacaaca?acttaaccct 1850
1851?cactagccca?cgtgtttact?ttctcaacca?ttcccttatc?tcccaaaatt 1900
1901?gtatttactt?caaaaataaa?ccgaaccgta?tcaaattaaa?acaagaccac 1950
1951?acttttgtct?gaacacaaaa?aaaaaaaaaa?cttttatttt?ttcctcgaga 2000
2001?cgtctctctt?cttcaatttc?ttcacgattc?tacttttgcg?ttatccccaa 2050
2051?aattggcaac?caaaaccaaa?cctctctctc?ttattgaaac?caaca 2095
(2) have 80% above homology with (1) described dna sequence dna and have the nucleotide sequence that starts the described coding Arabidopis thaliana DnaJ-like proteic genetic transcription function relevant with resistance of reverse.
The recombinant expression vector, transgenic cell line and the host bacterium that contain gene of the present invention and/or promotor all belong to protection scope of the present invention.
Arbitrary segmental primer is to also within protection scope of the present invention in gene of the present invention and/or the promotor of increasing.
Technical scheme of the present invention is:
1) extraction of total RNA
Select for use RNAiso reagent (Takara company) to extract the total RNA of Arabidopis thaliana, through integrity, purity and the concentration of electrophoresis detection and UV spectrophotometer measuring affirmation RNA, in-80 ℃ of preservations;
2) clone of Arabidopis thaliana DnaJ-like protein gene CRR1
According to the complete encoding sequence of DnaJ-like protein gene CRR1, design the two ends primer and introduce EcoRI and XhoI restriction endonuclease sites respectively at upstream and downstream:
Upstream primer: CCATCCATGAGTCCGATCGTCATAACCCAG
Downstream primer: CTCGAGTCAATAAGGACGATTCGGTGGC
The total RNA that obtains with 1 μ g step 1) behind synthetic cDNA first chain of reverse transcription, carries out pcr amplification as the template of reverse transcription, and the PCR program is as follows: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 58 ℃ of annealing 1min, 72 ℃ are extended 30s, after 35 circulations, 72 ℃ of 10min are connected to pGEM-T (Promega company) carrier with the PCR product, after the order-checking acquisition are had the cDNA sequence SEQ ID N of the Arabidopis thaliana DnaJ-like protein gene CRR1 of complete coding region
o: 1.
3) structure of plant expression vector
With step 2) the pGEM-T recombinant vectors that contains the CRR1 complete coding region that obtains carries out enzyme with restriction enzyme EcoRI and XhoI (Takara company) and cuts, and after being connected to the CaMV35S promotor of intermediate carrier pRTL2, then utilize restriction endonuclease HindIII (Takara company) further to be cloned among the double base conversion carrier pCAMBIA1300;
4) acquisition of transfer-gen plant
The expression vector that step 3) is obtained changes Agrobacterium over to, further change the wild-type Arabidopis thaliana over to, the transfer-gen plant that obtains is carried out carrying out after resistance screening, PCR and RT-PCR verify the resistance of reverse evaluation of plant, and transfer-gen plant and wild-type adjoining tree carry out the resistance of reverse analysis.
The low temperature that CRR1 gene function provided by the invention is an involved in plant, arid and the reaction of dormin stress response, sxemiquantitative inverse transcription polymerase chain reaction (RT-PCR) analysis revealed CRR1 expresses under low temperature (4 ℃), arid (not watering for two weeks) and dormin (4uM) inductive condition and strengthens.
CRR1 protein function provided by the invention is the responsing reaction of low temperature, arid and dormin being coerced by the involved in plant that combines with myb class transcription factor, and the storehouse experiment of yeast two-hybrid sieve shows that a myb family transcription factor has interaction in CRR1 and the Arabidopis thaliana.
CRR1 gene source of the present invention is in Arabidopis thaliana, optimizing codon with dicotyledons expression such as suitable Arabidopis thalianas, its genetically engineered recipient plant is more suitable in dicotyledonss such as tobacco, soybean, cottons except monocotyledonss such as paddy rice, corn, wheats.
Utilize the CRR1 gene of this law invention to make up plant expression vector, can utilize cauliflower mosaic virus CAMV35S promotor, alcohol induced promotor etc., can comprise enhanser in case of necessity as goal gene.In order to simplify the evaluation of transformed plant, can use selected marker (as antibiotic enzyme).Used expression vector can use Ti-plasmids, Ri plasmid and plant viral vector etc.Method for transformation can transform plant with agrobacterium-mediated transformation or additive method.
The present invention compared with prior art its beneficial effect is:
Gene of the present invention not only can be used for improveing the resistance of reverse of farm crop, and the Study on Molecular Mechanism of the responsing reaction of plant opposing adverse circumstance is had crucial value.After cross expressing, can improve this gene anti-reverse functions such as the drought tolerance of plant and anti-Exogenous Abscisic Acid in plant, and significant to the improvement of plant variety.
Four, description of drawings
Fig. 1: the influence that different environment stresses are expressed gene C RR1, RD22, RD29A and KIN1
A: be each expression of gene situation behind the NaCl solution-treated wild-type Arabidopis thaliana of 250mM with concentration.3h, 6h, 9h represent the time of NaCl solution to plant treatment, and c represents control group, the control group water treatment.
B: the wild-type Arabidopis thaliana is each expression of gene situation in 4 ℃ of environment.1d, 2d, 3d represent the time that plant is handled in 4 ℃ of environment, c represents control group, and control group is 22 ℃ of following growing plants.
C: be each expression of gene situation behind the dormin solution-treated wild-type Arabidopis thaliana of 4uM with concentration.2h, 4h, 6h represent the time of dormin solution to plant treatment, and c represents control group, the control group water treatment.
D: the wild-type Arabidopis thaliana is carried out arid handle each expression of gene situation of back.12d represents that plant do not water in continuous 12 days, and c represents control group, and control group normally waters.
Fig. 2 CRR1 crosses each expression of gene situation and drought tolerance experiment in the expression mutant
A, C: before the drought-enduring experiment, CRR1 crosses the upgrowth situation of expressing No. 9 strains of mutant and wild-type
B, D: arid finishes, and the back CRR1 that recovers to water crosses the upgrowth situation of expressing No. 9 strains of mutant and wild-type
Before the arid: the upgrowth situation the when seedling in three weeks of growing is watered before the arid experiment for the last time
Arid back: drought-enduring experiment (calculatings of watering for the last time before the arid, continuous two weeks do not water) the recovery afterwards growth of seedlings situation the 3rd day time of watering.
E:CRR1 and stress-inducing gene are expressed expression in mutant and the wild-type in the mistake of CRR1
Test the Arabidopis thaliana seedling leaf of used plant for growing about 3 weeks, wherein, 1: wild-type Arabidopis thaliana, 2:CRR1 are crossed No. 9 strains expressing mutant, and 3:CRR1 crosses No. 11 strains expressing mutant.RD22, RD29A, DREB2A and KIN1 are the stress-inducing gene, and ACT8 is an internal control gene.
Fig. 3 CRR1 crosses the dormin tolerance of expressing mutant and wild-type Arabidopis thaliana and compares
Mutant: CRR1 crosses No. 9 strains of expression mutant
Substratum is the MS solid medium that contains the 0.5uM dormin, and the growth of seedling time is about two weeks
The tissue expression specificity of Fig. 4 CRR1
Experiment material is the transgenic arabidopsis (Pcrr1-GUS) that CRR1 promoters driven GUS expresses
A: two cotyledon seedling in period B: the petal of bolting plant
C: bolting plant D just: begin to tie the seed plant in period
The Subcellular Localization of Fig. 5 CRR1
A, laser scanning blade epidermis under B:20 * object lens, green fluorescent protein concentrate in the Stomacal guard cell of blade and express
Laser scanning blade epidermis under C:40 * object lens, green fluorescent protein is expressed in the plastid of blade cell
The experiment material therefor is transgenic arabidopsis (Pcrr1-GFP) seedling leaves of the CRR1 promoters driven GFP expression in three weeks of growth, and excitation wavelength is 405nm, and emission wavelength is 513nm
Fig. 6 yeast two-hybrid checking CRR1 and other proteic interaction
PGBKT7 empty carrier and CRR-pGBKT7 heat shock respectively change in the yeast AH109 bacterial strain;
AT1G12050-pGADT7，AT1G32100-pGADT7，AT1G70760-pGADT7，
AT5G29000-pGADT7 heat shock respectively changes in the yeast Y187 bacterial strain.With yeast crossbreeding method checking empty carrier and CRR1 albumen and other proteic interactions.
A: empty carrier and CRR1 and other proteic hybridization products are at the growing state that lacks on Serine and the leucic SD flat board
B: empty carrier and CRR1 and other protein hybridization products are lacking Serine, leucine and Histidine and are adding growing state on the SD flat board of VITAMIN B4
AT1G12050, AT1G32100, AT1G70760 and AT5G29000 represent recombinant vectors AT1G12050-pGADT7, AT1G32100-pGADT7, AT1G70760-pGADT7 and AT5G29000-pGADT7 respectively among the figure.
Five, embodiment
1) clone of Arabidopis thaliana CRR1 gene coded sequence
Get some Arabidopis thaliana wild type seeds in the 1.5mL centrifuge tube, at first use 75% ethanol surface disinfection 2～3 times, remove residual ethanol 2～3 times through sterile water wash again, the centrifuge tube lucifuge that the tinfoil paper parcel is equipped with seed places 4 ℃ of synchronizations 3 days, be seeded in (volume ratio of vermiculite and soil is 1: 4) in the soil that contains vermiculite, the photoperiod of plant-growth is 14h illumination/10h dark, and envrionment temperature is 22 ℃.
Select for use RNAiso reagent (Takara company) to extract the total RNA of wild-type Arabidopsis leaf, through integrity, purity and the concentration of electrophoresis detection and UV spectrophotometer measuring affirmation RNA, in-80 ℃ of preservations.As the reverse transcription template, use " the PrimeScript of Takara company with the total RNA of 1 μ g
TM1stStrand cDNA SynthesisKit " synthetic cDNA first chain of test kit counter-rotating.
According to the complete encoding sequence of DnaJ-like protein gene CRR1, design the two ends primer and introduce EcoRI and XhoI restriction endonuclease sites (primer is synthetic by Genscript company) respectively at upstream and downstream:
Upstream primer: CCATCCATGAGTCCGATCGTCATAACCCAG
Downstream primer: CTCGAGTCAATAAGGACGATTCGGTGGC
The total RNA that obtains with 1 μ g step 1) behind synthetic cDNA first chain of reverse transcription, carries out pcr amplification as the template of reverse transcription, and the PCR program is as follows: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 30s, 58 ℃ of annealing 1min, 72 ℃ are extended 30s, after 35 circulations, 72 ℃ of 10min.Reaction is connected to pGEM-T carrier (Promega company) with the PCR product after finishing, screening positive clone behind the connection product transformed into escherichia coli DH5 α competent cell, obtain carrying the recombinant vectors of CRR1, called after pGEM-T-CRR1, to its check order (Genscript company finishes), order-checking shows SEQ ID N in the acquisition sequence table
o: 1 nucleotide sequence, by 294 based compositions, SEQ ID N in the code sequence tabulation
o: 2 amino acid residue sequence, this sequence contain three placed in-line CxxCxGxG zinc fingerses.
Utilization Genevestigator database is analyzed at the expression of different growth phases them, finds that CRR1 expression amount in whole growth process is all lower, and begins maturation when the seed of plant, and promptly whole plant is taken place to express when old and feeble and increases suddenly; The expression of CRR1 in different tissues analyzed, and it is very high to find that CRR1 expresses in the sepal of flower, and expression amount is all lower in its hetero-organization of plant, expresses hardly in root; The expression of CRR1 under the different stimulated condition analyzed, and the result shows, CRR1 expresses under the water stress condition that abiotic factors such as arid, cold, dormin cause and increases.At last, promoter sequence to CRR1 upstream 1000bp is analyzed, find to exist on the sequence a plurality of as cis-acting elements such as TATA box, CAAT box, ABRE-like sequence, MYB/MYC recognition site, Binding sitefor MYB/MYC, DPBF-1/2binding core sequence, ABRE-related sequence and ARR1-bindingelement, these elements have participated in plant and have resisted the transcriptional control that drought stress or dormin are coerced.By above result, we infer that CRR1 has participated in the plant opposing and comprised the old and feeble physiological process relevant with water stress.
2) environment-stress is induced the expression of CRR1
In order to detect the response relation of CRR1 gene and environment stress, we choose NaCl, dormin, low temperature and these four kinds of adverse circumstances of arid the expression characterization of Arabidopis thaliana CRR1 gene on transcriptional level are studied.
The vegetable material of coercing processing is the wild-type Arabidopis thaliana about growing all around.When carrying out dormin and NaCl processing, selecting directly to be sprayed onto on the Arabidopis thaliana seedling that to handle the control group spray water under the normal growth condition with the dormin solution of 4uM or the NaCl solution of 250mM.When carrying out deepfreeze, seedling is positioned in 4 ℃ of environment, control group places normal growth temperature (22 ℃) environment.Needs are carried out plant that arid handles place under the normal growth condition and will not water about two weeks, control group normally waters.Analyze the differential expression (Fig. 1) of CRR1 and dehydration responsive genes RD29A, RD22 and KIN1 with RT-PCR.Experimental result shows that CRR1 is the same with KIN1 with RD29A, RD22 can be by arid, and cold and dormin stress-inducing is expressed, but salt stress can induce the expression of RD29A, RD22 and KIN1 but CRR1 to be expressed basic not influence.RT-PCR result shows simultaneously, CRR1 coerces later stage with drought stress by abduction delivering (Fig. 1-B cold, Fig. 1-D), and coerce 2 hours just by a large amount of abduction deliverings at dormin, the level before expression amount returns to substantially and induces after 4 hours and 6 hours (Fig. 1-C).The physiological process that above presentation of results CRR1 has participated in that the plant opposing is cold, arid and dormin are coerced, and, the abduction delivering of CRR1 needs the accumulation of dormin in the plant, infers that therefore CRR1 is the responsing reaction by dormin signal pathway involved in plant opposing water stress.In the experiment under the condition of salt stress expression of CRR1 unaffected substantially, this is because CRR1 may be positioned at the downstream of regulatory pathway, under slight stress conditions hardly by abduction delivering.
3) CRR1 crosses and expresses the drought tolerance that mutant strengthens plant
When plant transpiration speed surpassed moisture absorption speed or the available moisture of soil shortage plant, plant was subjected to drought stress.Drought stress is the most general form of plant adverse circumstance, is bottlenecks of agricultural development in many areas.Plant is in order to adapt to drought stress, and protective substance that some are relevant with drought stress and gene thereof are induced to increase and expressed.We can express other relevant with adverse circumstance in mutant expression of gene situation by the mistake of analyzing CRR1, thereby determine the direct or indirect signal transduction process that influences each gene that changes over to of CRR1.RT-PCR result shows, crosses in two strains expressing mutant at CRR1, and arid genes involved RD29A, DREB2A compare expression with KIN1 with wild-type all have rising, that RD22 changes is not obvious (Fig. 2-E).
For further research CRR1 gene is in the effect of plant reply drought stress process, CRR1 is crossed the expression mutant for we and the wild-type Arabidopis thaliana carries out drought-enduring reality.The Arabidopis thaliana wild-type and the mutant that carry out drought-enduring experiment are the growth seedling of size all around, growth conditions does not change, calculating does not water in continuous two weeks from watering for the last time carries out drought stress, observe the growth of seedlings situation, then observed growth of seedlings situation, statistics survival rate (Fig. 2 A-D) once more at the 3rd day that recovers to water.
Experiment shows under normal operation the growth conditions basically identical of mutant and wild-type.Under drought stress, the mistake expression mutant of CRR1 gene is than the more late wilting of wild-type, and survival rate also is higher than wild-type.Crossing the survival rate of the arid of expressing mutant after watering after two weeks is about 40%, and the survival rate of wild-type is about 2%.The drought tolerance of above presentation of results CRR1 gene pairs plant has enhancement.
4) CRR1 crosses and expresses the anti-acidity that comes off that mutant strengthens plant
According to the experimental result of RT-PCR, wild-type Arabidopis thaliana CRR1, RD22 and KIN1 expression of gene when being subjected to Exogenous Abscisic Acid and coercing all raise, and illustrate that CRR1 has participated in the answering that plant reply dormin is coerced (Fig. 1-C).In order further to study the coerce effect in process brought into play of CRR1 at plant reply dormin, we have carried out the experiment of dormin patience to expression mutant excessively and the wild-type Arabidopis thaliana of CRR1, when carrying out the experiment of dormin tolerance, configuration contains the MS solid medium of 0.5uM dormin, be seeded in media surface after sterilization of seed process and the synchronization process, be positioned in the normal habitat, regularly observe (Fig. 3).Experimental result shows: containing on the MS substratum of 0.5uM dormin, CRR1 crosses the growth of expressing mutant and wild-type and compare slowly, but mutant than more premature eruption of wild-type, and root growth is significantly better than wild-type, plant strain growth is health more.Above presentation of results, CRR1 gene can strengthen the tolerance of plant to dormin.
5) tissue expression specificity of CRR1
Whether have tissue expression specificity in order to investigate the CRR1 promotor, we have made up CRR1 promotor (CRR1 upstream 2095bp, SEQ ID N in the sequence table
o: 3) together with the recombinant vectors of CRR1 that removes terminator codon and gus reporter gene amalgamation and expression, and it is transferred in the wild-type Arabidopis thaliana by the mediation of Agrobacterium, utilize Totomycin (Hygromycin B purchases in BBI) screening to obtain positive plant (Pcrr1-GUS).Select the transgenic arabidopsis plant (Pcrr1-GUS) of the CRR1 promoters driven GUS expression of different growing stage to place 1.5mL or 10mL centrifuge tube (deciding) on the plant size, handle 20min with the decolouring of 90% acetone under the room temperature, be immersed in staining fluid (10mL prescription: 0.5mL 1mol/L sodium phosphate buffer then, pH7.2,0.2mL10%Triton X-100,0.4mL the 50mmol/L Tripotassium iron hexacyanide, 0.4mL 50mmol/L yellow prussiate of potash, 1mL 20mmol/L X-Gluc, 7.5mL ice bath 10min, then vacuum take-off 15～20min H2O).Sample is in the dyeing of 37 ℃ of lucifuges spend the night (about 20h).Washing after the decolouring with 70% ethanol can direct viewing or observe (Fig. 4) with microscope.
Experimental result shows: CRR1 promoter-driven GUS gene is expressed in root hardly, and its hetero-organization all has expression; The various piece of two leaf to six leaf leaves in period all has the expression of GUS, and more (Fig. 4-A) is expressed at the vein place; After the plant maturation, the various piece of plant all has the expression of GUS, but the expression ratio in the blade concentrates on the edge of blade, and the expression in the stem concentrates on petal and the part of fruit pod is arranged, and the expression of petal concentrates on (Fig. 4 B-D) on the sepal.Above result shows that CRR1 changes the The Plant Senescence process that participated in by impression by the ripe and old and feeble plant moisture that causes.
6) Subcellular Localization of CRR1
In order to investigate the Subcellular Localization of CRR1 in vegetable cell, we have made up CRR1 promotor (CRR1 upstream 2095bp, SEQ ID N equally
o: 3) together with the recombinant vectors of CRR1 that removes terminator codon and green fluorescent protein GFP reporter gene amalgamation and expression, mediation by Agrobacterium is transferred to it in wild-type Arabidopis thaliana, utilizes Totomycin (Hygromycin B) screening to obtain positive plant (Pcrr1-GFP).Transgenic arabidopsis plant (Pcrr1-GFP) different tissues that different strains about choosing around the growth contain CRR1 promoters driven GFP is observed (Fig. 5) under Laser Scanning Confocal Microscope.During laser scanning, excitation wavelength is 405nm, and emission wavelength is 513nm.Experimental result shows: GFP expresses (Fig. 5-A under the low power lens in the guard cell of blade pore, Fig. 5-B), find after improving the object lens multiple, there is GFP to express (Fig. 5-C) around cell walls, show that GFP expresses on plastid, this explanation CRR1 is by regulating pore involved in plant replying water stress
7) chip results analysis
Select for use wild-type and CRR1 about growth all around to cross expression mutant (No. 9 and No. 11 strains) Arabidopis thaliana seedling lotus throne leaf as chip material.Carry out experiments such as chip hybridization by Shanghai Biochip Co., Ltd (SBC).
Experimental result shows: CRR1 crosses No. 9 strains of expression mutant, and compare up-regulated gene with wild-type be 1038, and the gene of downward modulation is 758; CRR1 crosses No. 11 strains of expression mutant, and compare up-regulated gene with wild-type be 583, and the gene of downward modulation is 423; The wherein common gene that raises is 67, and the gene of common downward modulation is 49.
Common 67 genes that raise comprise:
AT1G73330 AT1G02340 AT2G18050 AT3G28270 AT5G43260 AT3G48360
AT5G04950 AT5G42900 AT3G16450 AT1G01720 AT4G14020 AT1G80130
AT5G54080 AT2G33380 AT3G59350 AT1G08230 AT5G01600 AT1G23040
AT3G19030 AT4G37610 AT5G39610 AT2G30360 AT1G56600 AT3G57520
AT4G15530 AT1G32170 AT2G39310 AT1G22190 AT1G21910 AT4G37410
AT5G09440 AT3G45730 AT1G31820 AT5G65870 AT5G57560 AT4G08950
AT4G11310 AT5G58650 AT5G44260 AT3G11410 AT2G27830 AT5G59820
AT1G61890 AT1G28330 AT5G64260 AT4G10960 AT4G27280 AT4G35750
AT3G50910 AT1G32920 AT3G62260 AT5G11650 AT2G40000 AT3G57450
AT2G37130 AT1G27200 AT1G44100 AT5G62020 AT3G61990 AT1G21000
AT5G49450 AT3G19580 AT5G01520 AT4G01950 AT1G66180 AT5G25350
AT2G30040
49 genes of common downward modulation comprise:
AT2G02810 AT3G57460 AT3G18000 AT2G03260 AT1G13470 AT1G10990
AT3G13950 AT2G19400 AT2G04450 AT1G69720 AT3G17990 AT4G04190
AT3G15140 AT3G04220 AT1G13750 AT1G23790 AT4G24190 AT2G35980
AT5G59680 AT2G30770 AT3G49620 AT3G49320 AT5G48470 AT3G48500
AT5G62550 AT2G44040 AT3G28540 AT4G23310 AT5G55450 AT2G02850
AT1G59980 AT4G25110 AT2G04530 AT4G10500 AT2G18660 AT3G57240
AT5G24530 AT5G60950 AT1G18370 AT3G20440 AT5G17160 AT1G15520
AT2G43570 AT4G23800 AT2G26400 AT2G29350 AT3G28510 AT2G14610
AT2G44240
Experimental result is carried out cluster analysis, show that the affiliated classification of common up-regulated gene and down-regulated gene is very similar, comprise the genes involved of stimulation responses gene, transferring enzyme, receptor binding protein transcription factor and formation cell walls and ECM etc.The gene that raises is jointly compared with the gene of common downward modulation and the expression of these genes in different stimulated condition and mutant in the genevestigator database, the result shows: the common gene up-regulated in osmotic pressure, salt, low carbon dioxide and drought stress that raises, carry out arid processing expression to mutant Aox1A and also raise; Gene down-regulated expression under drought stress of common downward modulation carries out arid processing expression to mutant Aox1A and also reduces.Mutant Aox1A is that oxidase gene AT3G22370 inserts mutant, responsing reaction, cellular respiration and plastosome signal pathway etc. that this gene involved in plant cold-peace pressure is coerced.In sum, gene C RR1 has mainly participated in the responsing reaction of the water stress that plant causes abiotic factor.
8) interaction of CRR1 and myb transcription factor
The test kit that provides according to clontch, make up CRR1 as the support C RR1-PGBKT7 of bait protein (bait protein) with Gal4DNA-binding domain (DNA-BD) amalgamation and expression, use the heat shock method to change among the yeast strain AH109, lacking Serine (screening positive clone on SD flat board Trp).Sieve positive colony enlarged culturing in 50mlYPDA, thalline and the Y187 yeast thalline that has transformed arabis protein carrier storehouse (prey-pGADT7) are joined among 50ml 2 * YPDA after centrifugal, hybridization is spent the night.The centrifugal back of bacterium liquid after the hybridization is resuspended with 10ml 0.5 * YPDA.Getting that wherein 100ul dilutes respectively is 1/10,1/100,1/1000 and 1/10000, is coated with to lack leucine or/and the SD flat board of Serine calculates yeast crossbreeding efficient; It is dull and stereotyped and lack leucine, Serine, Histidine and add the SD plate screening positive colony of VITAMIN B4 that residue bacterium liquid is coated with the SD that lacks leucine and Serine.The hybridization efficiency that is obtained twice experiment by the yeast crossbreeding formulae of efficiency is about 5%.In the crossover process, if bait protein (bait) has interaction with prey albumen (prey), DNA-BD and AD are close mutually so, thereby start GAL promotor 4 different reporter gene (HIS3 in back, ADE2, AUB1-C and MEL1) transcribe, thereby yeast can be grown on the defective flat board.After lacking leucine, Serine, Histidine and adding the positive colony enlarged culturing that the dull and stereotyped top sieve of SD of VITAMIN B4 gets, extract plasmid, (primer is that T7 and 3 '-DNA-AD) check order to product in the back to utilize pcr amplification, thereby determine interactional albumen to take place with CRR1, to from yeast, change yeast strain Y187 over to by extractive plasmid simultaneously, lack screening positive clone on the leucic SD flat board.These positive colonies are extracted the plasmid and the evaluation of checking order, obtain four with CRR1 interactional albumen is arranged, be respectively AT1G12050, AT1G32100, AT1G70760 and AT5G29000, wherein, AT1G32100 is rosin spirit reductase enzyme (pinoresinol reductase), AT1G70760 is a subunit forming chloroplast(id) NAD (P) H dehydrogenase complex, the transcription factor of a MYB family of AT5G2900 coding.The positive colony that obtains is hybridized with the AH109 positive colony that has transformed CRR1-pGBKT7 or pGBKT7 empty carrier, and whether the checking interaction between protein stable existence.Discovery when the yeast two-hybrid experiment (Fig. 6) of checking CRR1 and these protein-interactings, AT1G12050 and empty carrier pGBKT7 also have interaction, illustrate that the interaction of it and CRR1 is a false positive.The AT1G32100 rosin spirit reductase enzyme (pinoresinol reductase) of encoding, the biosynthesizing of lignan (lignan) in this enzyme involved in plant.Lignan is a kind of phytoestrogen, also is a kind of antioxidant, has certain anti-oxidant activity.AT1G70760 is a subunit forming chloroplast(id) NAD (P) H dehydrogenase complex, participates in the circulating electron transmittance process of PSI.The transcription factor of a MYB family of AT5G2900 coding.Myb transcription factor is one of maximum plant transcription factor family member, has participated in cytodifferentiation, regulation of Cell Cycle, and hormone and environmental factor are replied, and organ morphologies such as Secondary Metabolism of Plant and blade are built up has important regulatory role.In the transcriptional control process of environment stress, myb transcription factor is realized accuracy controlling to target gene by the conservative DNA of N end in conjunction with territory and target sequence specific combination, and the accumulation of the endogenous dormin of needs usually of the abduction delivering of MYB class transcription factor.In conjunction with CRR1 plant to the experimental result in stress response such as low temperature, dormin and the arid reactions, show that CRR1 relies on the stress response reactions such as arid, low temperature of dormin signal pathway by the involved in plant that combines with myb transcription factor.
Sequence table
＜110〉Nanjing University
＜120〉application of a kind of Arabidopis thaliana DnaJ-like albumen and encoding gene thereof
<160>3
<210>1
<211>294
<212>DNA
＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis Thaliana)
<400>1
1 atgagtccga?tcgtcataac?ccagttagct?acaggaatca?gcgttttagc?50
51?cggtgcggtc?tttatcaaat?cagtcatgga?ccaaaagcct?atggctggtc?100
101aattccctcg?ttgtccgact?tgtaacggta?ctggacgagt?cacgtgtttc?150
151tgttctcgat?ggtccgatgg?tgacgttgga?tgtcgtaggt?gttctggttc?200
201aggtcgtgcg?gcttgtagca?attgtggtgg?ttctggaact?ggtagacctt?250
251taccggctca?gattacggtt?cagccaccga?atcgtcctta?ttga 294
<210>2
<211>97
<212>PRT
＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis Thaliana)
<400>2
Met?Ser?Pro?Ile?Val?Ile?Thr?Gln?Leu?Ala?Thr?Gly?Ile?Ser?Val?Leu
1 5 10 15
Ala?Gly?Ala?Val?Phe?Ile?Lys?Ser?Val?Met?Asp?Gln?Lys?Pro?Met?Ala
20 25 30
Gly?Gln?Phe?Pro?Arg?Cys?Pro?Thr?Cys?Asn?Gly?Thr?Gly?Arg?Val?Thr
35 40 45
Cys?Phe?Cys?Ser?Arg?Trp?Ser?Asp?Gly?Asp?Val?Gly?Cys?Arg?Arg?Cys
50 55 60
Ser?Gly?Ser?Gly?Arg?Ala?Ala?Cys?Ser?Asn?Cys?Gly?Gly?Ser?Gly?Thr
65 70 75 80
Gly?Arg?Pro?Leu?Pro?Ala?Gln?Ile?Thr?Val?Gln?Pro?Pro?Asn?Arg?Pro
85 90 95
Tyr
97
<210>3
<211>2095
<212>DNA
＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis Thaliana)
<400>3
1 ccaactctct?cctacaactt?gacacatcag?cacatgagag?cttcccacct 50
51 aaacttcata?aaataaataa?tacataaata?gtaattaatt?caacataata?100
101?actaaaatag?gaggatataa?ttaatcagaa?cttgaaccat?cactctttct?150
151?acatttttta?attcaatata?tcctttttaa?ttttgagatc?caaacatgtt?200
201?acacatattc?cttgcctaaa?aattacacga?ttattatgaa?agaaactaaa?250
251?tgaatatgca?gattaaatat?atctaagctt?ttcaagattt?atgctaagaa?300
301?aacaagtaat?ttgaaagatg?tttgaggagt?tatccttaaa?actaatacgt?350
351?aagatagaag?attatgacaa?gaacttaact?aaactttcca?agaacatcaa?400
401?gttgaatatg?attagattgg?ttatgcagag?acatggttaa?taaaaacatg?450
451?ttatatttta?aatgtaagaa?agaaattttc?taaaatttca?ccgtttcaca?500
501?aactaaatat?ataaccaaaa?gaacaaggat?atataaatta?aaaaaaaaaa?550
551?ctgagaatct?agaacaacaa?taaaaaataa?ctcaagcatt?ttcgttttca?600
601?gatgaactaa?tatagttgga?ctttttgaaa?acaattacaa?atcatattta?650
651?tgtttcaaaa?ccgatgcatt?taactttact?ttatatgaac?agacattata?700
701?taaaacaatt?tgtaaatagt?aatacataaa?cccgcacgta?gtgcgggtac?750
751?tcatctagta?tacaataata?aaaacttcaa?aatgatgcaa?caatagctca?800
801?gaatcttcat?atatgggcca?ctcggcccac?gccgaatttg?aaaagcccga?850
851?tattggaagt?aagcacgtgc?ggttgtgaga?attgcttctt?cgtccaccat?900
901?tggacatgtt?tatggctgcg?ttcgacgcgt?gatttgtttg?tggagaagag?950
951?tgcatcgcct?gataattcaa?gcaacctagt?tatagtacaa?acgtgaaaga?1000
1001aacaaaatta?acccaaaaaa?tgaaatggaa?ggttacaatt?gcaacccttg?1050
1051caagatagta?caacaatata?atagtaacaa?tagaacaata?aaccctaacc?1100
1101aaacaaagta?agaattacat?tcctctaatt?ttagtgcatg?taagttcggt?1150
1151tatcactgca?aacttaatca?tttccctatt?gactaattcg?taacatagtt?1200
1201tatactttcg?attaatcaat?taattaattt?acagtgaatt?tgtgaaaaac?1250
1251?ttctaacgag?aaacttatta?tgttataaaa?gtcaacaaaa?gaactgtatc?1300
1301?acacagaaca?tatatattgg?gtctgattgg?taaccaaaaa?ctatacaagt?1350
1351?tatttagctg?ttagctttat?cggaatagtg?taaaactgtt?aaaatgagct?1400
1401?gtacaaaaaa?actgagagta?aatttttttg?taacgctttt?gataaagttt?1450
1451?ttgtgattag?taactgtcaa?aattgtattg?ttattaaaat?ggttaccatt?1500
1501?caaatctatt?aggtaaaata?cagagccgtc?cataagtaca?agcgggtcaa?1550
1551?acacaagctt?gtggcctcaa?ttactataaa?taaaattttg?atttttttta?1600
1601?gtagaaaaat?tagttgcttg?cttctttgat?gacaaaaaaa?aaaaagcaag?1650
1651?cagtcagata?aatcaatgat?aatctaaaaa?aaagaaaaat?ctgacgaaaa?1700
1701?aaaaaaagaa?aaaaaagaaa?aatctgaagg?ttgtacactt?gtagaagata?1750
1751?ttttacacac?acagcccaat?aaaaacccaa?atccaaaaac?tggagtatcc?1800
1801?aaaaaaaaca?cggctgtttc?attcatctcc?tcacacaaca?acttaaccct?1850
1851?cactagccca?cgtgtttact?ttctcaacca?ttcccttatc?tcccaaaatt?1900
1901?gtatttactt?caaaaataaa?ccgaaccgta?tcaaattaaa?acaagaccac?1950
1951?acttttgtct?gaacacaaaa?aaaaaaaaaa?cttttatttt?ttcctcgaga?2000
2001?cgtctctctt?cttcaatttc?ttcacgattc?tacttttgcg?ttatccccaa?2050
2051?aattggcaac?caaaaccaaa?cctctctctc?ttattgaaac?caaca 2095
Claims (8)
1. an Arabidopis thaliana DnaJ-like protein gene is characterized in that deriving from Arabidopis thaliana (Arabidopsis thaliana), and name is called CRR1, and nucleotides sequence is classified as:
1 atgagtccga?tcgtcataac?ccagttagct?acaggaatca?gcgttttagc 50
51 cggtgcggtc?tttatcaaat?cagtcatgga?ccaaaagcct?atggctggtc?100
101?aattccctcg?ttgtccgact?tgtaacggta?ctggacgagt?cacgtgtttc?150
151?tgttctcgat?ggtccgatgg?tgacgttgga?tgtcgtaggt?gttctggttc?200
201?aggtcgtgcg?gcttgtagca?attgtggtgg?ttctggaact?ggtagacctt?250
251?taccggctca?gattacggtt?cagccaccga?atcgtcctta?ttga 294?。
2. according to the described Arabidopis thaliana DnaJ-like of claim 1 albumen, it is characterized in that deriving from Arabidopis thaliana (Arabidopsis thaliana), name is called CRR1, and aminoacid sequence is:
Met?Ser?Pro?Ile?Val?Ile?Thr?Gln?Leu?Ala?Thr?Gly?Ile?Ser?Val?Leu
1 5 10 15
Ala?Gly?Ala?Val?Phe?Ile?Lys?Ser?Val?Met?Asp?Gln?Lys?Pro?Met?Ala
20 25 30
Gly?Gln?Phe?Pro?Arg?Cys?Pro?Thr?Cys?Asn?Gly?Thr?Gly?Arg?Val?Thr
35 40 45
Cys?Phe?Cys?Ser?Arg?Trp?Ser?Asp?Gly?Asp?Val?Gly?Cys?Arg?Arg?Cys
50 55 60
Ser?Gly?Ser?Gly?Arg?Ala?Ala?Cys?Ser?Asn?Cys?Gly?Gly?Ser?Gly?Thr
65 70 75 80
Gly?Arg?Pro?Leu?Pro?Ala?Gln?Ile?Thr?Val?Gln?Pro?Pro?Asn?Arg?Pro
85 90 95
Tyr
97。
3. according to the promotor of the described Arabidopis thaliana DnaJ-like of claim 1 protein gene, it is characterized in that nucleotides sequence classifies as:
1 ccaactctct?cctacaactt?gacacatcag?cacatgagag?cttcccacct 50
51 aaacttcata?aaataaataa?tacataaata?gtaattaatt?caacataata?100
101?actaaaatag?gaggatataa?ttaatcagaa?cttgaaccat?cactctttct?150
151?acatttttta?attcaatata?tcctttttaa?ttttgagatc?caaacatgtt?200
201?acacatattc?cttgcctaaa?aattacacga?ttattatgaa?agaaactaaa?250
251?tgaatatgca?gattaaatat?atctaagctt?ttcaagattt?atgctaagaa?300
301?aacaagtaat?ttgaaagatg?tttgaggagt?tatccttaaa?actaatacgt?350
351?aagatagaag?attatgacaa?gaacttaact?aaactttcca?agaacatcaa?400
401?gttgaatatg?attagattgg?ttatgcagag?acatggttaa?taaaaacatg?450
451?ttatatttta?aatgtaagaa?agaaattttc?taaaatttca?ccgtttcaca?500
501?aactaaatat?ataaccaaaa?gaacaaggat?atataaatta?aaaaaaaaaa?550
551?ctgagaatct?agaacaacaa?taaaaaataa?ctcaagcatt?ttcgttttca?600
601?gatgaactaa?tatagttgga?ctttttgaaa?acaattacaa?atcatattta?650
651?tgtttcaaaa?ccgatgcatt?taactttact?ttatatgaac?agacattata?700
701?taaaacaatt?tgtaaatagt?aatacataaa?cccgcacgta?gtgcgggtac?750
751?tcatctagta?tacaataata?aaaacttcaa?aatgatgcaa?caatagctca?800
801?gaatcttcat?atatgggcca?ctcggcccac?gccgaatttg?aaaagcccga?850
851 tattggaagt?aagcacgtgc?ggttgtgaga?attgcttctt?cgtccaccat?900
901 tggacatgtt?tatggctgcg?ttcgacgcgt?gatttgtttg?tggagaagag?950
951 tgcatcgcct?gataattcaa?gcaacctagt?tatagtacaa?acgtgaaaga?1000
1001?aacaaaatta?acccaaaaaa?tgaaatggaa?ggttacaatt?gcaacccttg?1050
1051?caagatagta?caacaatata?atagtaacaa?tagaacaata?aaccctaacc?1100
1101?aaacaaagta?agaattacat?tcctctaatt?ttagtgcatg?taagttcggt?1150
1151?tatcactgca?aacttaatca?tttccctatt?gactaattcg?taacatagtt?1200
1201?tatactttcg?attaatcaat?taattaattt?acagtgaatt?tgtgaaaaac?1250
1251?ttctaacgag?aaacttatta?tgttataaaa?gtcaacaaaa?gaactgtatc?1300
1301?acacagaaca?tatatattgg?gtctgattgg?taaccaaaaa?ctatacaagt?1350
1351?tatttagctg?ttagctttat?cggaatagtg?taaaactgtt?aaaatgagct?1400
1401?gtacaaaaaa?actgagagta?aatttttttg?taacgctttt?gataaagttt?1450
1451?ttgtgattag?taactgtcaa?aattgtattg?ttattaaaat?ggttaccatt?1500
1501?caaatctatt?aggtaaaata?cagagccgtc?cataagtaca?agcgggtcaa?1550
1551?acacaagctt?gtggcctcaa?ttactataaa?taaaattttg?atttttttta?1600
1601?gtagaaaaat?tagttgcttg?cttctttgat?gacaaaaaaa?aaaaagcaag?1650
1651?cagtcagata?aatcaatgat?aatctaaaaa?aaagaaaaat?ctgacgaaaa?1700
1701?aaaaaaagaa?aaaaaagaaa?aatctgaagg?ttgtacactt?gtagaagata?1750
1751?ttttacacac?acagcccaat?aaaaacccaa?atccaaaaac?tggagtatcc?1800
1801?aaaaaaaaca?cggctgtttc?attcatctcc?tcacacaaca?acttaaccct?1850
1851?cactagccca?cgtgtttact?ttctcaacca?ttcccttatc?tcccaaaatt?1900
1901?gtatttactt?caaaaataaa?ccgaaccgta?tcaaattaaa?acaagaccac?1950
1951?acttttgtct?gaacacaaaa?aaaaaaaaaa?cttttatttt?ttcctcgaga?2000
2001?cgtctctctt?cttcaatttc?ttcacgattc?tacttttgcg?ttatccccaa?2050
2051?aattggcaac?caaaaccaaa?cctctctctc?ttattgaaac?caaca 2095。
4. according to the described Arabidopis thaliana DnaJ-like of claim 1 protein gene, it is characterized in that having 80% above homology and the proteinic polynucleotide sequence of coding identical function with the polynucleotide sequence of the described gene of claim 1.
5. according to the described Arabidopis thaliana DnaJ-like of claim 2 albumen, the polynucleotide sequence of the described protein sequence of claim 2 that it is characterized in that encoding.
6. according to the promotor of the described Arabidopis thaliana DnaJ-like of claim 3 protein gene, it is characterized in that 90% above homology being arranged and have the nucleotide sequence of the functional transcription of relevant DnaJ-like protein coding gene that starts described Arabidopis thaliana with resistance of reverse with the polynucleotide sequence of the described promotor of claim 3.
7. according to the described Arabidopis thaliana DnaJ-like of claim 2 albumen, it is characterized in that the described proteic aminoacid sequence of claim 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residues and has identical active derived protein with the described amino acid residue sequence of claim 2.
8. the application of the described Arabidopis thaliana DnaJ-like of claim 1 protein gene in cultivating drought-enduring, anti-dormin and low temperature resistant plant variety.
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| CN105017393A (en) * | 2014-04-29 | 2015-11-04 | 中国科学院植物研究所 | Protein BhDNAJC2 relevant to plant adverse resistance and encoding gene and application thereof |
| CN106754961A (en) * | 2016-12-27 | 2017-05-31 | 内蒙古蒙草生态环境（集团）股份有限公司 | A kind of regulation plant is to arid induced signal calcium ion and the gene of hormone abscisic acid reaction |
| WO2018234191A1 (en) * | 2017-06-19 | 2018-12-27 | University Of Copenhagen | Increased drought resistance in plants |
| CN109678940A (en) * | 2017-10-18 | 2019-04-26 | 中国科学院植物研究所 | Protein B hDnaJ6 and its encoding gene and application |
| CN110272911A (en) * | 2019-07-05 | 2019-09-24 | 四川大学 | Application of the AOX1a gene in terms of improving drought resistance in plants |
| CN110484542A (en) * | 2019-07-30 | 2019-11-22 | 中国科学院华南植物园 | Arabidopsis disease-resistant related gene EIJ1 and its application |
| CN113121663A (en) * | 2019-12-31 | 2021-07-16 | 中国农业大学 | Application of corn CRR1 protein and coding gene thereof in regulating and controlling low-temperature stress tolerance of corn |
| CN114561391A (en) * | 2022-03-21 | 2022-05-31 | 河南师范大学 | Clone and application of rice meiosis specific expression promoter |
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| 《NCBI GenBank》 20030513 Town,C.D.et al. chaperone protein dnaJ-like protein [Arabidopsis thaliana],NCBI Reference Sequence: NP_568623.1 , 2 * |
| 《Plant Cell》 20061231 Shan Lu et al. The Cauliflower Or Gene Encodes a DnaJ Cysteine-Rich Domain-Containing Protein That Mediates High Levels of ß-Carotene Accumulation 第18卷, 第12期 2 * |
| 《Tair：www.arabidopsis.org》 20030527 Ecker et al. Sequence: AT5G43260.1 and Tair Accession Sequence:3709926 , 2 * |
| 《中国优秀硕士学位论文全文数据库（农业科技辑）》 20090215 杜志如 水稻热激蛋白Hsp90基因的克隆及互作蛋白的筛选 , 第2期 2 * |
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