CN1919866B - Soybean Trihelix transcription factor, encode gene and application thereof - Google Patents
Soybean Trihelix transcription factor, encode gene and application thereof Download PDFInfo
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- CN1919866B CN1919866B CN2006100892112A CN200610089211A CN1919866B CN 1919866 B CN1919866 B CN 1919866B CN 2006100892112 A CN2006100892112 A CN 2006100892112A CN 200610089211 A CN200610089211 A CN 200610089211A CN 1919866 B CN1919866 B CN 1919866B
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Abstract
The invention discloses a soybean Trihelix transfer factor and encoded gene and application, which is comprises the following parts: possessing one of amino acid residue sequence proteins: (1) SEQ IDNo.:2 in the sequence table; (2) replacing and/or deleting and/or adding SEQ ID No.:2 in the sequence table amino acid residue sequence through one or several sequence table amino acid residue and proteins in related to plant difficulty resistant.
Description
Technical field
The present invention relates to a kind of soybean Trihelix transcription factor and encoding gene and application.
Background technology
The variation of physics, chemical factor in the environment, the factors of coercing such as for example arid, saline and alkaline, low temperature have material impact to growth and development of plant, can cause the extensive underproduction of farm crop when serious, therefore cultivating the high crop of resistance of reverse is one of major objective of plant husbandry.At present, using gene engineering technique carries out breeding and has become one of important method that improves the crop resistance of reverse.Higher plant cell has many approach and replys various environment stresses in the environment, and wherein transcription factor plays a part the regulation and control effector of anti-the retrocorrelation and expresses.Now in plant, found the multiclass transcription factor relevant, for example: the DREB class among the EREBP/AP2, bZIP, MYB and PHD or the like with plant stress tolerance.The Trihelix transcription factor is the little transcription factor family of plant specific one class.The Trihelix transcription factor has 3 alpha-helixs (helix-loop-helix-loop-helix) to gain the name because of the DNA in its protein structure in conjunction with the territory.The member of this protein family is divided into 3 albumen subfamilies according to the specificity of its DNA binding member, be GT-1, GT-2 and GT-3 (Ayadi et al., 2004 Analysis of GT-3a identifies a distinctsubgroup of trihelix DNA-binding transcription factors in Arabidopsis, FEBSLetters 562,147-154.).In conjunction with regard to the number of territory, GT-1 class and GT-3 class only have a trihelix territory at its N end with regard to DNA in the protein structure, and the GT-2 proteinoid then has two trihelix territories, lay respectively at C end and N and hold.
The expression of Trihelix transcription factor regulation and control light response gene.Report is arranged recently, PETAL LOSS in the Arabidopis thaliana is the albumen of a GT-2 class, it participates in normal development (the Brewer et al. of leaf and floral organ, 2004, PETALLOSS, a trihelix ranscription factor gene, regulates perianth architecturein the Arabidopsis flower.Development 131,4035-4046.).And whether in plant tolerance abiotic stress mechanism, play a role about the Trihelix transcription factor, do not appear in the newspapers as yet.
Soybean is one of China five big crops, understands fully its anti-abiotic stress molecule mechanism, and then provides the basis for improving its resistance of reverse, has important theory and realistic meaning.
Summary of the invention
The purpose of this invention is to provide a kind of soybean Trihelix transcription factor and encoding gene and application.
Soybean Trihelix transcription factor provided by the present invention, name is called GmGT-2B, derives from Glycine soybean (Glycine max (L.)), is the protein with one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 2;
2) with SEQ ID № in the sequence table: 2 amino acid residue sequence through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with the protein of the anti-retrocorrelation of plant.
Wherein, the sequence in the sequence table 2 is made up of 581 amino-acid residues.GmGT-2B is a kind of Trihelix transcription factor, and it has two trihelix territories, lays respectively at C end and N end.The trihelix territory of N end is aminoterminal the 90th (Leu)-150 (Tyr) the amino acids residue sequence from sequence 2, and the trihelix territory of C end is aminoterminal the 428th (Arg)-520 (Tyr) the amino acids residue sequence from sequence 2.
The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or the disappearance and/or the interpolation of no more than ten amino-acid residues.
The encoding gene (GmGT-2B) of above-mentioned soybean Trihelix transcription factor also belongs to protection scope of the present invention.
The cDNA gene of above-mentioned soybean Tribelix class transcription factor can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;
3) with sequence table in SEQ ID №: 1 dna sequence dna has 90% above homology, and the identical function protein DNA sequence of encoding;
4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, the SEQ ID № in the sequence table: 1 is made up of 1746 deoxynucleotides, classifies encoding sequence as from 5 of sequence 1 ' end 1-1746 position nucleotides sequence, and coding has the protein G mGT-2B of the amino acid residue sequence of sequence 2 in the sequence table; Hold 268-450 position nucleotide sequence and 1282-1560 position nucleotides sequence to classify the encoding sequence of 2 trihelix structural domains among the GmGT-2B as from 5 '.
The recombinant expression vector, transgenic cell line and the engineering bacteria that contain gene of the present invention all belong to protection scope of the present invention.
Can utilize plant expression vector that the encoding gene of GmGT-2B of the present invention is imported vegetable cell, obtain environment stress tolerance enhanced transgenic cell line and transfer-gen plant.
When being building up to GmGT-2B in the plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as adding selected marker's (gus gene, luciferase genes etc.) that can in plant, express or antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry GmGT-2B gene of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as cucumber, tomato, willow, turfgrass, lucerne place.
Experimental result shows that the expression of GmGT-2B of the present invention is subjected to inducing of high salt, arid, low temperature and dormin ABA.ABA replys relevant plant hormone with the plant abiotic stress, so the regulation and control that GmGT-2B and plant are replied abiotic stress are relevant.The transgenic arabidopsis of overexpression GmGT-2B is compared with not genetically modified wild-type Arabidopis thaliana, and its salt tolerance, drought tolerance and low temperature resistant ability are significantly increased, and illustrates that transcription factor GmGT-2B has participated in the regulation and control that plant is replied abiotic stress.
GmGT-2B gene pairs of the present invention is cultivated the particularly anti-contrary soybean varieties of plant with adverse resistance kind, as salt tolerant, drought-enduring and low temperature resistant soybean, particularly the output of soybean is significant to improve farm crop, and proved the regulation and control that the certain involved in plant of some members the transcription factor Trihelix family is replied abiotic stress from the molecular biology angle, it is expressed and the anti-abiotic stress of plant is proportionate.
The present invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the part-structure synoptic diagram that contains the plant expression vector of GmGT-2B
Fig. 2 is the expression characterization figure of GmGT-2B under abiotic stress is coerced
Fig. 3 A for the Arabidopis thaliana T1 that changes the GmGT-2B gene for strain be L1 and L2 with the wild-type plant under the salt stress with the normal growth condition under the growth comparison diagram
Fig. 3 B is L1 and L2 and the growing state of wild-type plant under natural arid for the Arabidopis thaliana T1 that changes the GmGT-2B gene for strain
Fig. 3 C is L1 and L2 and the growing state of wild-type plant under cold condition for the Arabidopis thaliana T1 that changes the GmGT-2B gene for strain
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The screening of embodiment 1, soybean GmGT-2B gene and the clone of cDNA thereof
At first each conservative DNA that intends the research transcription factor family is compared in conjunction with the sequence in territory sequence and the soybean est database, obtain this fragment of EST of 13 Trihelix family members respectively with blast program.Agricultural 1138-2 is a material with the cultivated soybean kind south, and the big bean seedlings of getting 15 days seedling ages carry out cold (4 ℃) respectively, salt (150mMNaCl solution), and ABA (100 μ M solution), atmospheric drought is handled and is promptly blotted root moisture content with filter paper, places air.Above-mentioned processing is after 0,1,3,6,12 hour, and the leaf of gathering respectively extracts RNA.
According to above-mentioned 13 Trihelix family members' EST, design special separately RT-PCR primer.With the RT-PCR method responsing reaction of said gene under salt, arid, cold, ABA coerce analyzed.In above-mentioned 13 genes, 2 genes are arranged,, coerce equal abduction delivering down, satisfy, further carried out gene clone and functional analysis thereof research as candidate gene at high salt, arid, low temperature and ABA comprising GmGT-2B.
Utilize the method for RACE, design RACE primer, the wherein special nested primer 1 (GSP of 5 ' RACE on the est sequence basis of artificial further splicing
1) be 5 '-CTTGCAGGGGACGAAGTAGTGGCTGAGA-3 ', special nested primer 2 (GSP
2) 5 '-CATCATGTCCTCAAGGAGCTTGTTGTGC-3 '; The special nested primer 1 (GSP of 3 ' RACE
3) 5 '-GAGGAAGCTTGGAAGAAGCAAGAGATGG-3 ', special nested primer 2 (GSP
4) 5 '-GTCCCCTGCAAGCCACACCCTCAAGAAG-3 ', nido universal primer sequence (NUP) is 5 '-AAGCAGTGGTATCAACGCAGAGT-3 '.
5 ' RACE, the first chain cDNA's is synthetic: get the RNA of the agricultural 1138-2 in 1 μ g soybean varieties south, add 5 '-CDS primcr[5 ' of 1 μ l-(T)
25N-1N-3 ' (N=A, C, G or T; N
-1=A, G or C)], the SMART II A oligo that adds 1 μ l,, add water to final volume 5 μ l, 70 ℃ of water-baths 2 minutes, put then 2 minutes on ice, and added 2 μ l, 5 * first strand buffer then respectively, 1 μ l DTT (20mM), 1 μ l dNTP mix (10mM), 1 μ lPowerScript reverse transcriptase, mixing then, 42 ℃ of reverse transcriptions 1.5 hours, add ultrapure water to 250 μ l then,, obtain 5 ' RACE, the first chain cDNA 72 ℃ of following sex change 7 minutes.It is stand-by to put-20 ℃ of refrigerators.
5 ' RACE response procedures: divide the amplification of 2 steps.The first step amplification reaction system 25 μ l: 17.25 μ l ultrapure waters, 2.5 μ l, 10 * GC buffer I, 1 μ l dNTP Mix (10mM), 1 μ l LA-Taq enzyme, 1.25 μ l, 5 '-RACE, the first chain cDNA, 2.5 μ l UPM (10 *), 0.5 μ l GSP
1(10 μ M), mixing covers Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃ of 5sec; 68 ℃ of 10sec, 72 ℃ of 3min, 25 circulations; 4 ℃ of preservations.
Nido reaction system 50 μ l: 36 μ l ultrapure waters, 5 μ l, 10 * GC buffer I, 1 μ l dNTP Mix (10mM), 1 μ l LA-Taq enzyme, 5 μ l the first step amplified productions (50 *), 1 μ l NUP (10 μ M), 1 μ l GSP
2(10 μ M), mixing covers Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃, 5sec; 68 ℃, 10sec; 72 ℃, 3min, 30 circulations.After the reaction product recovery, connect PMD18-T vector, order-checking.
3 ' RACE, the first chain cDNA's is synthetic: get the RNA of the agricultural 1138-2 in 1 μ g soybean varieties south, add 3 '-CDS primer[5 '-AAGCAGTGGTATCAACGCAGAGTAC (T) of 1 μ l
30N-1N-3 ' (N=A, C, G or T; N-1=A, G or C)], the SMART II A oligo that adds 1 μ l, add water to final volume 5 μ l, 70 ℃ of water-baths 2 minutes were put 2 minutes on ice then, add 2 μ l, 5 * first strand buffer then respectively, 1 μ l DTT (20mM), 1 μ l dNTP mix (10mM), 1 μ l PowerScript reverse transcriptase, mixing then, 42 ℃ of reverse transcriptions 1.5 hours add ultrapure water to 250 μ l then, 72 ℃ of following sex change 7 minutes, obtain 3 ' RACE, the first chain cDNA.It is stand-by to put-20 ℃ of refrigerators.
3 ' RACE response procedures: divide the amplification of 2 steps.The first step amplification reaction system 25 μ l: 17.25 μ l ultrapure waters, 2.5 μ l 10 * GC buffer I, 1 μ l dNTP Mix (10mM), 1 μ l LA-Taq enzyme, 1.25 μ l 3 '-RACE first chain cDNA, 2.5 μ l UPM (10 *), 0.5 μ l GSP1 (10 μ M), mixing covers Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃ of 5sec; 68 ℃ of 10sec, 72 ℃ of 3min, 25 circulations; 4 ℃ of preservations.
Nido reaction system 50 μ l: 36 μ l ultrapure waters, 5 μ l, 10 * GC buffer I, 1 μ l dNTP Mix (10mM), 1 μ l LA-Taq enzyme, 5 μ l the first step amplified productions (50 *), 1 μ l NUP (10 μ M)), 1 μ l GSP2 (10 μ M), mixing covers Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃, 5sec; 68 ℃, 10sec; 72 ℃, 3min, 30 circulations.After the reaction product recovery, connect PMD18-T vector, order-checking.
5 ' the end and the 3 ' terminal sequence that are clearly increased by checking order belong to the goal gene component, then by artificial splicing, find the maximum frame of reading, again by NCBI BLAST, determine it is the total length of goal gene, extend to 5 ' non-translational region from the initiator codon of this gene then, design forward primer 5 '-GTCGGATCCATGTTCGATGGAGTACCAGACC-3 ', from terminator codon to 3 ' the end non-translational region extend design reverse primer 5 '-CGAGAGCTCTTAAAACTGATCAAAATCCAAAG-3 ', RNA with the agricultural 1138-2 in soybean varieties south is a template, get the total RNA of 5 μ g and carry out reverse transcription by the method for test kit with reverse transcription test kit (Promega company), obtain the cDNA fragment as template, the reaction system of PCR is 50 μ l, comprising 4 μ l, one chain cDNA (0.05 μ g), 6 μ l primers (20 μ M), 5 μ l, 10 * pfu PCR Buffer, 1.0 μ l dNTP (10mM) and 1U pfu Taq archaeal dna polymerase, the surplus ultrapure water polishing of using.Cover Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃ of sex change 4min; 94 ℃ of sex change 30sec, 57 ℃ of renaturation 30sec, 72 ℃ of extensions 3min30sec, totally 30 circulations; 72 ℃ are extended 10min then.The goal gene that is increased is connected pMD18-T vector, order-checking, the result shows, obtain the total length ORF sequence of candidate gene, called after GmGT-2B, the total length of GmGT-2B is 1746bp, has the nucleotide sequence of sequence 1 in the sequence table, classify encoding sequence as from 5 of sequence 1 ' end 1-1746 position nucleotides sequence, coding has the protein G mGT-2B of the amino acid residue sequence of sequence 2 in the sequence table; Hold 268-450 position nucleotide sequence and 1282-1560 position nucleotides sequence to classify the encoding sequence of 2 trihelix structural domains among the GmGT-2B as from 5 '.Sequence 2 is made up of 581 amino-acid residues in the sequence table.GmGT-2B has two trihelix territories, lays respectively at C end and N end.Trihelix structural domain amino-acid residue is very conservative, and the sequence variations of middle portion is bigger.The tribelix territory of N end is aminoterminal the 90th (Leu)-150 (Tyr) the amino acids residue sequence from sequence 2, the trihelix territory of C end is aminoterminal the 428th (Arg)-520 (Tyr) the amino acids residue sequence from sequence 2, so GmGT-2B belongs to the GT-2 subfamily.
Embodiment 2, the expression characterization of GmGT-2B gene under abiotic stress
The agricultural 1138-2 in soybean south is carried out arid, NaCl, low temperature stress and ABA processing be used to analyze the expression of soybean GmGT-2B under abiotic stress.The seed kind of the agricultural 1138-2 in south in basin, after 2 weeks of growing, is carried out cold (4 ℃) respectively to seedling, salt (150mM NaCl solution), atmospheric drought, ABA handles.In the different treatment period, the leaf of gathering respectively is used to extract RNA.Concrete grammar is as described below:
Followingly coerce processing:
Arid is handled: the agricultural 1138-2 seedling in soybean south is taken out the moisture that blots on the root from soil, place on the exsiccant filter paper, arid is cultivated sampling after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours under illumination condition respectively.
Salt is handled: the root system of the agricultural 1138-2 seedling in soybean south is placed 150mM NaCl solution, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
Subzero treatment: the agricultural 1138-2 seedling in soybean south is placed 4 ℃ of incubators, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
ABA handles: the root system of the agricultural 1138-2 seedling in soybean south is placed 100 μ M ABA solution, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.
Extract the total RNA of the above-mentioned processing soybean agricultural 1138-2 blade in south respectively.Be template with different soybean samples of handling with aforesaid method and the RNA that does not have process to coerce the soybean sample (contrast) of processing respectively, get the total RNA of 5 μ g and carry out reverse transcription by the method for test kit, obtain the cDNA fragment as template with reverse transcription test kit (Promega company); According to known est sequence, it is right to design special RT-PCR primer, wherein forward primer be 5 '-GTTTTTGCGAGAGCATTGTG-3 ', reverse primer is 5 '-AACTAGGGTTCTGGGGAGGA-3 '.Adopt the RT-PCR method.The reaction system of RT-PCR is 20 μ l, comprising 1 μ l, one chain cDNA (0.05 μ g), 1 μ l primer (20 μ M), 2 μ l, 10 * PCR Buffer, 0.5 μ l dNTP (10mM) and 1U Taq archaeal dna polymerase, the surplus ultrapure water polishing of using.Cover Witco 70 above.Entire reaction is carried out on PE9600 type PCR instrument, and its program is 94 ℃ of sex change 4min; 94 ℃ of sex change 30sec, 56 ℃ of renaturation 30sec, 72 ℃ of extensions 1min, totally 30 circulations; 72 ℃ are extended 10min then; 4 ℃ of preservations.With the product of the constructive expression's of amplification soybean Tubulin gene in contrast, its primer be 5 '-AACCTCCTCCTCATCGTACT-3 ' and 5 '-GACAGCATCAGCCATGTTCA-3 '.Amplified production is at 1.0% agarose gel (containing ethidium bromide), and 1 * TAE electrophoretic separation is observed under the ultraviolet lamp and taken a picture.To said gene salt, arid, cold coerce and the responsing reaction of ABA under handling analyzed.As shown in Figure 2, the GmGT-2B expression of gene is induced by high salt, arid and low temperature induction and ABA, so GmGT-2B possibility involved in plant is to the responsing reaction of abiotic stress.
The Function Identification of embodiment 3, GmGT-2B albumen and encoding gene thereof
One, the acquisition and the salt tolerance thereof of changeing GmGT-2B gene Arabidopis thaliana strain system identified
Design primer according to the GmGT-2B gene order: GmGT-2B ORF forward primer: 5 '-GTC
GGATCCATGTTCGATGGAGTACCAGACC-3 ' (
BamH I), GmGT-2B ORF reverse primer: 5 '-CGA
GAGCTCTTAAAACTGATCAAAATCCAAAG-3 ' (
Sac I), use the RT-PCR method, amplification GmGT-2B gene from the total RNA of soybean.Concrete grammar is as described below: get Nan Nong 1138-2 blade, place liquid nitrogen to grind, be suspended from the different sulphur hydracid of the 4mol/L guanidine, and with acid phenol, chloroform extracting, add dehydrated alcohol and precipitate in supernatant, will precipitate total RNA that obtains soluble in water at last.Getting the total RNA of 5 μ g and carry out reverse transcription with reverse transcription test kit (Promega company) by the method for test kit, is that template is carried out pcr amplification reaction with the cDNA fragment that obtains.20 μ l PCR reaction systems are: 1 μ l, one chain cDNA (0.05 μ g), 1 μ l primer (20 μ M), 2 μ l, 10 * PCR damping fluid, 0.4 μ l dNTP (10mM) and 1U Taq archaeal dna polymerase, supply 20 μ l with ultrapure water, and liquid level covers Witco 70.Be reflected on the PE9600 type PCR instrument and carry out, its program is 94 ℃ of sex change 5min; 94 ℃ of 30sec again, 56 ℃ of 30sec, 72 ℃ of 2min, 30-32 circulation altogether; 72 ℃ are extended 10min then; 4 ℃ of preservations.The sequential analysis after reclaiming of PCR product, the result shows that this PCR product is 1764bp, contains the nucleotide sequence of sequence 1 in the ordered list, clones then in the multiple clone site of pMD18-T plasmid, obtains recombinant vectors pMDGmGT-2B.
With BamHI and SacI double digestion pMDGmGT-2B, obtain comprising the fragment of the complete encoder block of GmGT-2B, its forward is inserted between the BamHI and SacI enzyme recognition site of pBI121 plant expression vector, sequence verification then, the plant expression vector of GmGT-2B will be confirmed to contain, called after pBI121-GmGT-2B, its part-structure synoptic diagram as shown in Figure 1.PBI121-GmGT-2B is by the mediated transformation Arabidopis thaliana of agrobacterium tumefaciens GV3101, the PCR detected result shows 31 transformed plants of acquisition, the RT-PCR analysis revealed wherein at least 3 strains expression amount of GmGT-2B very high, 2 strains that expression amount is high are called after L-1, L-2 respectively.The seed kind of L-1 and L-2 is gone into the MS substratum, is seedling called after L1, L2 respectively with the T1 of the L-1, the L-2 that obtain for strain, after 5 days and simultaneously not transgenic arabidopsis (WT) seedling of sowing is transplanted to respectively in the MS substratum that contains 0mM, 75mM, 125mM, 150mM and 180mM NaCl and cultivated 16 days, and each handles each 30 strain.The result shows, under the normal growth condition, wild-type and commentaries on classics pBI121-GmGT-2B plant do not have difference, also difference is little under 75mM NaCl condition, but the blade warp appears in the wild-type plant under the 125mMNaCl condition, and it is normal substantially to change the pBI121-GmGT-2B plant, and the wild-type plant leaf is rolled up fully and cuddled up in a heap under the 150mMNaCl condition, plant presents red-purple, changes the pBI121-GmGT-2B plant and slight warp phenomenon occurs.The wild-type plant strain growth stops under 180mM NaCl condition, and beginning is dead, has in the pBI121-GmGT-2B plant that 80%-90%'s can keep slow growth yet change.
The seedling (each handle each 30 strains) that to handle under 75mM, 125mM, 150mM and 180mM NaCl condition 16 days is transferred in the vermiculite of normal condition and recovers growth, recovers to observe after 8 days and 21 days it respectively and recovers growing state.The result as shown in Figure 3A, the result shows, under higher NaCl condition, the Arabidopis thaliana surviving rate of wild-type is lower (in 75mM and 125mM NaCl processing, 30 young plants of wild-type all can recover growth, but under 150mM NaCl processing and 180mM NaCl processing, have 10 strains and 5 strains can recover growth respectively), and growth has been subjected to inhibition; And commentaries on classics pBI121-GmGT-2B plant recovery well-grown, the surviving rate height is (in 75mM and 125mM NaCl processing, 30 young plants of transfer-gen plant all can recover growth, and under 150mM NaCl processing and 180mM NaCl processing, have 24 strains and 26 strains can recover growth respectively).The result shows that the GmGT-2B gene is relevant with salt tolerance, and its overexpression can improve the salt tolerance of plant.
Two, the drought tolerance of changeing GmGT-2B gene Arabidopis thaliana strain system is identified
The wild-type that to grow 7 days in the MS substratum and commentaries on classics pBI121-GmGT-2B Arabidopis thaliana T1 are for seedling (each 30 strain) atmospheric drought in 26-28 ℃ of L1, L2 strain system, observations after promptly stopping to feed water 16 days is contrast with wild-type and the commentaries on classics pBI121-GmGT-2B Arabidopis thaliana T1 that cultivates under normal operation for the seedling that L1, L2 strain are.The result is shown in Fig. 3 B, and the result shows, changes pBI121-GmGT-2B plant no matter seedling stage or flowering period, all shows tangible drought tolerance.Wild-type plant survival rate reaches 43% (13 strains of surviving), and transfer-gen plant still has energy normal growth more than 80%, and is solid, and wherein, T1 has 24 strains energy normal growth for L1 strain system, and is solid; There is 26 strains energy normal growth in L2 strain system, and is solid.
Three, the frost resistance of changeing GmGT-2B gene Arabidopis thaliana strain system is identified
The wild-type (contrast) that to grow in the MS substratum 7 days and commentaries on classics pBI121-GmGT-2B Arabidopis thaliana T1 are for L1, the seedling of L2 strain system (each 20 strain), be transplanted in the normal vermiculite growth after 5 days, to contrast and change pBI121-GmGT-2B Arabidopis thaliana T1 for L1, the L2 plant places-20 ℃ of refrigerator-freezers to handle 105min, observations after recovering 8 days under the normal growth condition afterwards, the result is shown in Fig. 3 C, the result shows, wild-type contrast survival rate is 20% (4 strains of surviving), and change the pBI121-GmGT-2B plant still have (T1 is 17 strains for the L1 strain, and the L2 strain is 18 strains) 80% or more can recover grow.
Sequence table
<160>2
<210>1
<211>1746
<212>DNA
＜213〉Glycine soybean (Glycine max (L.))
<400>1
atgttcgatg?gagtaccaga?ccagttccac?caattcataa?ctccaaggac?ctcacaacct 60
cttcacttac?ctttccctct?ccatgcttca?ggaaccccca?ataccacctt?cccttccaat 120
tttgatcctt?ataacaaccc?ttctcatcaa?ttgccactcc?aacccaacaa?cctcttgcac 180
ccattacacc?acaaagatga?agagaaggaa?gaaaacacaa?ccaccgtccc?aatgaacttc 240
gaaatccaaa?gagatcaaag?gcaacaactt?cctgaactaa?ttgatccctg?gactaccgat 300
gaagtgctta?ccctcttgag?gatcagatct?agcatggaaa?gttggttccc?agaactcact 360
tgggaacatg?tctcaaggaa?actagcaggg?cttggatata?agaggagtgc?agagaagtgc 420
aaggagaagt?ttgaagaaga?gagtagatat?ttcaacaaca?acatcaatta?cgccaagaat 480
aacaataata?gtaccagcaa?ttatcggttt?cttagtgagc?ttgaacaact?ttatcaccaa 540
caagggagta?gtggtgatca?tcttgaaaag?atgactcagc?caccactgca?gaaacaaggc 600
aggatggacc?accatgcatt?agaattggaa?gaggaagaag?gggattcgag?aaacgttatt 660
gttgacgcat?cggtgacaaa?aatacaaagc?gacgaggcac?ttgcggtgga?aaagatcaca 720
aaggaccgca?agaggaagag?gtctgatagg?tttgagatgt?tcaagggttt?ttgcgagagc 780
attgtgcaca?agatgatgac?tcagcaagaa?gagatgcaca?acaagctcct?tgaggacatg 840
atgaagagag?atgaagagaa?gttcaccaga?gaggaagctt?ggaagaagca?agagatggag 900
aagatgaaca?aggagcttga?gatgatggca?cgtgagcagg?ctgttgcagg?tgatagacaa 960
gccaaaatta?tccaaatcct?gaacaaattc?tcagccacta?cttcgtcccc?tgcaagcctc 1020
accctcaaga?aggtaaacac?ccacatttca?caaaacccta?atccttcaca?aacagaaaac 1080
cctactttaa?gtgttgcaca?agacacactg?attccttcta?cttcttctac?ttcaactcca 1140
gctccagctc?ctccccagaa?ccctagttct?tgttctctaa?atagccagaa?taataatcat 1200
attaataata?atattccagt?agagaaaaat?tcgattttga?ataaggggtc?ctcatcaaat 1260
gagaaagacg?atgttgggag?aaggtggcca?aaggatgaag?tgttggcact?cataaacctg 1320
aggtgcacca?gtgtgaacaa?caacaacaac?aatgaggaga?aggaagggaa?caacaaggtc 1380
cctctgtggg?agagaatctc?acaagggatg?tcggagttga?gatacaagag?gagtgcaaag 1440
aggtgcaaag?agaagtggga?gaacataaac?aagtacttca?ggaaaaccaa?ggatattact 1500
aagaagaggt?cccttgactc?aagaacttgt?ccctactttc?atcagttgag?cagtctctac 1560
aatcaaggaa?aactcgttct?acaatctgaa?agccacttga?ataatactcc?acctgatcag 1620
aatcccgagc?aagtaaaacc?agatcaaaca?acacaagctc?atgagtcttc?ttcgcaggtg 1680
gggtctggta?gtggtttcag?tgttcagcaa?cagcaggtac?cttctttgga?ttttgatcag 1740
ttttaa 1746
<210>2
<211>581
<212>PRT
＜213〉Glycine soybean (Glycine max (L.))
<400>2
Met?Phe?Asp?Gly?Val?Pro?Asp?Gln?Phe?His?Gln?Phe?Ile?Thr?Pro?Arg
1 5 10 15
Thr?Ser?Gln?Pro?Leu?His?Leu?Pro?Phe?Pro?Leu?His?Ala?Ser?Gly?Thr
20 25 30
Pro?Asn?Thr?Thr?Phe?Pro?Ser?Asn?Phe?Asp?Pro?Tyr?Asn?Asn?Pro?Ser
35 40 45
His?Gln?Leu?Pro?Leu?Gln?Pro?Asn?Asn?Leu?Leu?His?Pro?Leu?His?His
50 55 60
Lys?Asp?Glu?Glu?Lys?Glu?Glu?Asn?Thr?Thr?Thr?Val?Pro?Met?Asn?Phe
65 70 75 80
Glu?Ile?Gln?Arg?Asp?Gln?Arg?Gln?Gln?Leu?Pro?Glu?Leu?Ile?Asp?Pro
85 90 95
Trp?Thr?Thr?Asp?Glu?Val?Leu?Thr?Leu?Leu?Arg?Ile?Arg?Ser?Ser?Met
100 105 110
Glu?Ser?Trp?Phe?Pro?Glu?Leu?Thr?Trp?Glu?His?Val?Ser?Arg?Lys?Leu
115 120 125
Ala?Gly?Leu?Gly?Tyr?Lys?Arg?Ser?Ala?Glu?Lys?Cys?Lys?Glu?Lys?Phe
130 135 140
Glu?Glu?Glu?Ser?Arg?Tyr?Phe?Asn?Asn?Asn?Ile?Asn?Tyr?Ala?Lys?Asn
145 150 155 160
Asn?Asn?Asn?Ser?Thr?Ser?Asn?Tyr?Arg?Phe?Leu?Ser?Glu?Leu?Glu?Gln
165 170 175
Leu?Tyr?His?Gln?Gln?Gly?Ser?Ser?Gly?Asp?His?Leu?Glu?Lys?Met?Thr
180 185 190
Gln?Pro?Pro?Leu?Gln?Lys?Gln?Gly?Arg?Met?Asp?His?His?Ala?Leu?Glu
195 200 205
Leu?Glu?Glu?Glu?Glu?Gly?Asp?Ser?Arg?Asn?Val?Ile?Val?Asp?Ala?Ser
210 215 220
Val?Thr?Lys?Ile?Gln?Ser?Asp?Glu?Ala?Leu?Ala?Val?Glu?Lys?Ile?Thr
225 230 235 240
Lys?Asp?Arg?Lys?Arg?Lys?Arg?Ser?Asp?Arg?Phe?Glu?Met?Phe?Lys?Gly
245 250 255
Phe?Cys?Glu?Ser?Ile?Val?His?Lys?Met?Met?Thr?Gln?Gln?Glu?Glu?Met
260 265 270
His?Asn?Lys?Leu?Leu?Glu?Asp?Met?Met?Lys?Arg?Asp?Glu?Glu?Lys?Phe
275 280 285
Thr?Arg?Glu?Glu?Ala?Trp?Lys?Lys?Gln?Glu?Met?Glu?Lys?Met?Asn?Lys
290 295 300
Glu?Leu?Glu?Met?Met?Ala?Arg?Glu?Gln?Ala?Val?Ala?Gly?Asp?Arg?Gln
305 310 315 320
Ala?Lys?Ile?Ile?Gln?Ile?Leu?Asn?Lys?Phe?Ser?Ala?Thr?Thr?Ser?Ser
325 330 335
Pro?Ala?Ser?Leu?Thr?Leu?Lys?Lys?Val?Asn?Thr?His?Ile?Ser?Gln?Asn
340 345 350
Pro?Asn?Pro?Ser?Gln?Thr?Glu?Asn?Pro?Thr?Leu?Ser?Val?Ala?Gln?Asp
355 360 365
Thr?Leu?Ile?Pro?Ser?Thr?Ser?Ser?Thr?Ser?Thr?Pro?Ala?Pro?Ala?Pro
370 375 380
Pro?Gln?Asn?Pro?Ser?Ser?Cys?Ser?Leu?Asn?Ser?Gln?Asn?Asn?Asn?His
385 390 395 400
Ile?Asn?Asn?Asn?Ile?Pro?Val?Glu?Lys?Asn?Ser?Ile?Leu?Asn?Lys?Gly
405 410 415
Ser?Ser?Ser?Asn?Glu?Lys?Asp?Asp?Val?Gly?Arg?Arg?Trp?Pro?Lys?Asp
420 425 430
Glu?Val?Leu?Ala?Leu?Ile?Asn?Leu?Arg?Cys?Thr?Ser?Val?Asn?Asn?Asn
435 440 445
Asn?Asn?Asn?Glu?Glu?Lys?Glu?Gly?Asn?Asn?Lys?Val?Pro?Leu?Trp?Glu
450 455 460
Arg?Ile?Ser?Gln?Gly?Met?Ser?Glu?Leu?Arg?Tyr?Lys?Arg?Ser?Ala?Lys
465 470 475 480
Arg?Cys?Lys?Glu?Lys?Trp?Glu?Asn?Ile?Asn?Lys?Tyr?Phe?Arg?Lys?Thr
485 490 495
Lys?Asp?Ile?Thr?Lys?Lys?Arg?Ser?Leu?Asp?Ser?Arg?Thr?Cys?Pro?Tyr
500 505 510
Phe?His?Gln?Leu?Ser?Ser?Leu?Tyr?Asn?Gln?Gly?Lys?Leu?Val?Leu?Gln
515 520 525
Ser?Glu?Ser?His?Leu?Asn?Asn?Thr?Pro?Pro?Asp?Gln?Asn?Pro?Glu?Gln
530 535 540
Val?Lys?Pro?Asp?Gln?Thr?Thr?Gln?Ala?His?Glu?Ser?Ser?Ser?Gln?Val
545 550 555 560
Gly?Ser?Gly?Ser?Gly?Phe?Ser?Val?Gln?Gln?Gln?Gln?Val?Pro?Ser?Leu
565 570 575
Asp?Phe?Asp?Gln?Phe
580
Claims (5)
1. soybean GmGT-2B gene, its base sequence is shown in SEQ ID NO:1.
2. the recombinant expression vector that contains the described gene of claim 1.
3. the transgenic cell line that contains the described gene of claim 1.
4. the engineering bacteria that contains the described gene of claim 1.
5. the application of the described gene of claim 1 in cultivating the resistance of reverse plant, described resistance of reverse is salt tolerance and/or drought tolerance and/or winter hardiness.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006100892112A CN1919866B (en) | 2006-08-09 | 2006-08-09 | Soybean Trihelix transcription factor, encode gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006100892112A CN1919866B (en) | 2006-08-09 | 2006-08-09 | Soybean Trihelix transcription factor, encode gene and application thereof |
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| Publication Number | Publication Date |
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| CN1919866A CN1919866A (en) | 2007-02-28 |
| CN1919866B true CN1919866B (en) | 2010-06-09 |
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| CN2006100892112A Expired - Fee Related CN1919866B (en) | 2006-08-09 | 2006-08-09 | Soybean Trihelix transcription factor, encode gene and application thereof |
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| CN (1) | CN1919866B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101456909B (en) * | 2009-01-13 | 2012-07-04 | 南京农业大学 | Soja bean HKT protein and coding gene thereof and application |
| CN103588867B (en) * | 2012-08-16 | 2015-01-28 | 中国科学院遗传与发育生物学研究所 | Soybean transcription factor GmMYB174a, and coding gene and applications thereof |
| CN103626856B (en) * | 2012-08-24 | 2016-04-13 | 中国科学院遗传与发育生物学研究所 | Transcription factor AtGT4 and encoding gene thereof and application |
| CN105462990A (en) * | 2015-12-25 | 2016-04-06 | 中国农业科学院油料作物研究所 | Protein BnSIP1 gene related to rape drought resistance and ABA tolerance and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477109A (en) * | 2002-08-19 | 2004-02-25 | 清华大学 | Transcription factor capable of regulating and controlling soybean adverse resistance, its coding gene and application |
| CN1583789A (en) * | 2003-08-21 | 2005-02-23 | 中国科学院遗传与发育生物学研究所 | Soybean transcripting factor, its coding gene and use thereof |
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2006
- 2006-08-09 CN CN2006100892112A patent/CN1919866B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1477109A (en) * | 2002-08-19 | 2004-02-25 | 清华大学 | Transcription factor capable of regulating and controlling soybean adverse resistance, its coding gene and application |
| CN1583789A (en) * | 2003-08-21 | 2005-02-23 | 中国科学院遗传与发育生物学研究所 | Soybean transcripting factor, its coding gene and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| O＇Grady, K., Goekjian, V., Nairn, C., Nagao, R. and Key, J..The transcript abundance of GmGT-2, a new member of theGT-2 family of transcription factors from soybean, isdown-regulated by light in a phytochrome-dependent manne.Plant Molecular Biology47.2001,47367-378.. * |
| Smalle J,Kurepa J,Haegman M,et al..The trihelix DNA-binding motif in higher plants is not restrictedto the transcription factors GT-1 and GT-2..Proc Natl Acad Sci USA95.1998,953318-3322.. * |
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| CN1919866A (en) | 2007-02-28 |
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