CN1477109A - Transcription factor capable of regulating and controlling soybean adverse resistance, its coding gene and application - Google Patents

Abstract

The present invention discloses a transcription factor for controlling and regulating adverse resistance of soya ben, its coding gene and application. The name of the adverse-resisting transcription factor is GmDREB, and it is a protein having amino acid residue sequence of sequence 2 in sequence table or the protein derived from sequence 2. The coding gene of the adverse-resisting transcription factor GmDREB is one of the following nucleotide sequences: 1). DNA sequence of sequence 1 in sequence table; and 2). DNA sequence which has homology with above 90% with DNA sequence limited by sequence 1 in sequence table and can be used for coding protein with identical function.

Description

The transcription factor of regulating and controlling soybean resistance and encoding gene thereof and application

Technical field

The present invention relates to plant transcription factor and encoding gene thereof and application, particularly derive from transcription factor and encoding gene and the application of soybean.

Background technology

Biologies such as cause of disease, arid, saline and alkaline, low temperature, freeze injury, waterlogging and the abiotic environment factor of coercing have material impact to growth and development of plant.Improve the resistance of reverse of crop, except utilizing traditional breeding method, one of field that the molecular genetic breeding has become present scientific worker to be paid close attention to.Under the environment stress environment, a series of Physiology and biochemistry can take place in the plant materials usually change.Plant experienced the variation of external environment before this by number of ways, and extracellular signal transferred to cell interior, chain reaction passes to transcription factor with signal through a series of phosphorylation level, transcription factor interacts by its specific function amino acid and goal gene again, startup produces the destination gene expression of replying to environment stress, thereby improves the resistance of plant.Verified, some transcripton of DREB class can be accepted the environment-stress signal and start the adverse circumstance response gene, improves the resistance of reverse of plant.In plant, the conservative territory of AP2/EREBP function is the specificity structure of DREB class transcription factor.

Soybean is understood fully its disease-resistant degeneration-resistant mechanism, and then improves its disease resistance and resistance to have important theory and realistic meaning as a kind of important oil plant and food crop.

Summary of the invention

The purpose of this invention is to provide a kind of more sensitive anti-reverse transcription factor and encoding gene thereof that derives from soybean of signal of coercing to external world.

Anti-reverse transcription factor names provided by the present invention is GmDREB, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

The protein that sequence 2 amino acid residue sequences are made up of 174 amino-acid residues in the sequence table.The AP2/EREBP function that is transcription factor GmDREB from the nitrogen end to 44-101 amino-acid residue conserved domain of carbon teminal in the described sequence 2 is guarded the territory.

The encoding gene of anti-reverse transcription factor GmDREB is one of following nucleotide sequences:

1) dna sequence dna of sequence 1 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.

The dna sequence dna of sequence 1 is by 910 based compositions in the sequence table, and the reading frame of this gene is that its expression mainly is subjected to low temperature from 3 ' end the 155th to the 676th bit base, and high salt and dormin are induced.

Utilize any carrier that can guide foreign gene in plant, to express, derive and next pBI101 as pBIN19 and by it, pBI121 and pBI221 serial carrier (Bevan, 1984 nucleic acids research, 12:8711-8721), with the gene transfered plant cell of encoding transcription factor GmDREB provided by the present invention, can obtain low temperature and high-salt stress tolerance enhanced transgenic cell line and transfer-gen plant.Expression vector pBI121-GmDREB is the expression vector that has GmDREB cDNA of the present invention that utilizes conventional molecular biology method to make up, its gene mapping as shown in Figure 1, this expression vector can be used for dicotyledonss such as soybean transformation.Gene of the present invention can add any enhancing promotor or inducible promoter, as cauliflower mosaic virus (CaMV 35S) and Ubiquitin promotor etc. in being building up to plant expression vector the time before its transcription initiation Nucleotide.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing (gentamicin, kantlex etc.) that adds the alternative mark (gus gene, luciferase genes etc.) of plant or have resistance.Carry that GmDREB expression carrier of the present invention can conventional biotechnological means imports vegetable cell by using that Ti (the Tumor-induced-cancer is induced) plasmid, Ri (Root-induced-root induction) plasmid, plant viral vector, directly DNA conversion, microinjection, electricity are led etc., by the plant transformed host both can be monocotyledons, also can be dicotyledons.Gene pairs of the present invention is cultivated the adversity resistant plant kind, particularly cultivates the plant variety of drought resisting, cold-resistant and anti-salt, and it is significant to improve crop yield.

Description of drawings

Fig. 1 is the structure of GmDREB expression vector.

Fig. 2 is the vivoexpression of GmDREB gene in intestinal bacteria.

Fig. 3 is Southern hybridization analysis result.

Fig. 4 is the GmDREB allelic expression that Northern hybridization detects under the different stress conditions.

Embodiment

The clone of embodiment 1, soybean GmDREB transcription factor cDNA and sequential structure analysis

The soybean seedling of growing 10 days at room temperature placed on the filter paper of adequate thickness dehydration 5 hours after cleaning, collect plant and in liquid nitrogen, be ground to fine degree, place the 4mol/L guanidinium isothiocyanate, use acid phenol/chloroform extracting mixture again, get supernatant liquor, with the total RNA of isopropanol precipitating, repeat above-mentioned steps more once, RNA is dissolved in the DEPC water, be stored in-80 ℃ of (Davis etc., molecular biological basic skills: [Basic Methods in MolecularBiology], pp.777, APPLETON ﹠amp; LANCE, Norwalk, Connecticut, USA, 1994).

Get the total RNA of 1 μ g and by 12 (three forward primer and four reverse primer combinations) special degenerated primers is carried out separately an independently step reverse transcription chain type amplified reaction (PCR), 3 forward primers (Forwardprimers) corresponding to the conservative territory N-end of AP2/EREBP 5 '-GIRMRK-3 ' are:

GDF1：5’-GGIAT(A/C/T)CGIATGCGIAA(A/G)-3’；

GDF2：5’-GGIAT(A/C/T)CGIATGAG(A/G)AA(A/G)-3’；

GDF3：5’-GGIAT(A/C/T)AG(A/G)ATGCGIAA(A/G)-3’。

Article 4, the reverse primer (Reverse primers) corresponding to the conservative territory C-end of AP2/EREBP 5 '-ARLNFP-3 ' is:

GDR1：5’-IGG(A/G)AA(A/G)TTIAGICGIGC-3’；

GDR2：5’-IGG(A/G)AA(A/G)TTIAG(C/T)CTIGC-3’；

GDR3：5’-IGG(A/G)AA(A/G)TT(T/C)AA(T/C)CTIGC-3’；

GDR4：5’-IGG(A/G)AA(A/G)TT(T/C)AAICGIGC-3’。

I is Trophicardyl (inosine) in the above-mentioned primer, and the PCR condition is: 4 ℃, and 3 minutes; 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1.5 minutes-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations.

The PCR product is connected to and is used for order-checking on the pMD18-T carrier, be that template is utilized a pair of Auele Specific Primer GmDF1R:5 '-CTTCCGCATCCTTATTCC-3 ' and GmDR1F:5 '-GCGCGCCTTAACTTCCCC-3 ' again with the positive colony, (the PCR program is seen kit D6122 to 5 ' end by reverse transcription PCR reaction amplifying target genes, Takara Biotech. (Dalian) Co.Ltd) and 3 ' end (the PCR program is seen kit D6121, TakaraBiotech. (Dalian) Co.Ltd).External then packing In vitro-GmDREB gene order.Design a pair of Auele Specific Primer GmDREBFd:165 again, 5 '-AAAA GAATTCATGGAAGACAGGGATCACTG-3 ', 172; GmDREBRe:676,5 '-AAAA CTCGAG657,94 ℃ of ATCTTGAAGCTCTTCGAGTT-3 ', 3 minutes; 94 ℃ 1 minute, 58 ℃ 1 minute, 72 ℃ 1.5 minutes-30 the circulation; 72 ℃ 10 minutes; 4 ℃ of preservations, amplification mDREB-cDNA, the PCR product is connected to the pMD18-T carrier and checks order, and has obtained one and the on all four cDNA clone of external packaging sequence, i.e. the cDNA fragment of sequence 1GmDREB.It is 910bp that this gene inserts fragment, contains the open reading frame of 522bp, the polypeptide that 174 amino acid of encoding are formed, and its 5 ' end is 156bp, 3 ' end is 192bp.

The structure of embodiment 2, the fusion expression vector that is used to transform.

The structure of GmDREB cDNA expression vector molecular biology method routinely carries out.The SacI site that utilizes GmDREB cDNA to carry out pcr amplification and BamHI by the forward primer end and reverse primer end as template the GmDREB coding region is inserted into a CaMV35S promotor back in the pBI121 binary expression vector, obtain a fusion expression vector pBI121-GmDREB, its collection of illustrative plates as shown in Figure 1, through enzyme cut identify to insert fragment errorless after, transform in Agrobacterium, extracting plasmid again cuts affirmation through enzyme and successfully transforms in Agrobacterium, this expression vector can be directly used in the conversion of plant, particularly soybean.

Embodiment 3, the vivoexpression of soybean GmDREB gene in a big bacillus are identified

GmDREB cDNA coding region is connected on the expression vector pGEX-4T-1, induces in intestinal bacteria through IPTG, SDS-PAGE detects and shows that this gene can carry out external accurate translation in intestinal bacteria.And there do not have protein expression to take out of without the inductive bacterial strain to be existing.The polyacrylate hydrogel electrophoretic band as shown in Figure 2, among the figure, M is Marker; 1, the 3rd, through the inductive bacterial strain; The 2nd, without the inductive bacterial strain.As can be seen from the figure, there is protein band to occur, do not have protein band to occur without the inductive bacterial strain through the inductive bacterial strain.

Embodiment 4, the analysis of GmDREB gene in the soybean gene group.

GmDREB cDNA with digoxin (DIG) mark is a probe, with through different restriction enzyme (EcoRI, EcoRV, P-PstI and XhoI) postdigestive cotton genomic dna carries out the Southern hybridization analysis under 65 ℃ of conditions, and (condition of high rigorous degree is: 0.5 * SSC to wash film under the rigorous degree of height, 0.1 * SDS, 65 ℃), the result as shown in Figure 3, there are many hybrid belts to occur, show the GmDREB gene that exists multiple copied in the soybean gene group, perhaps exist the GmDREB autoploid of low copy.

Embodiment 5, the expression characteristic of soybean GmDREB gene under the environment stress condition.

The soybean seedling in 2 week of growth places the aqueous solution of 4 ℃ of water, 250mM NaCl respectively, and 100 carry out illumination cultivation in μ M dormin (ABA) solution, it is that soybean seedling with 2 week of growth is placed on dehydration at room temperature on the filter paper that is enough to suck dry moisture only through washing that arid is handled, and, extract total RNA and the analysis of GmDREB cDNA probe hybridization respectively 1 hour, 3 hours, 5 hours, 7 hours, 12 hours and sampling in 24 hours.The result as shown in Figure 4, wherein A is the result that arid is handled, B is the result of 4 ℃ of subzero treatment, C is the result that 250mM NaCl handles, and D is the acid-treated result that comes off, as can be seen from the figure, transcribing of GmDREB induced by low temperature and salt mainly, and different degree is induced by arid and ABA.

＜160〉2＜210〉1＜211〉910＜212〉DNA＜213〉 ( Glycine max ( L. ) Merr. )＜400〉1gcacctctat atatacatag acacatgtaa atggttgcaa agaaagataa gataggtacc 60agctttcaca tagtttattt aatccgttat atgccacctg atatataggc atattagcta 120gttaggtagg tatagtagtt aagttaattc aaccatggaa gacagggatc actgttgttc 180caacaattca acgatgatca caacaacaaa gaaaagaacg ggtagaagaa gtccaacatc 240ggataagctc aagaatcaac accgcgagaa gcagtcgatg aaaccttacc gtggaataag 300gatgcggaag tgggggaagt gggtggcgga gatcagagaa cccaacaaaa ggtcgaggat 360atggttgggt tcttacacga cacccgtggc cgccgcacgt gcctacgaca ccgctgtctt 420ttacctccgg ggtcccaccg cgcgccttaa cttccccgaa ctcttgttcc aggacgacga 480ccaggagggc agtgattcgg tgcagcacgg cgcagcaggg aacatgtccg ctgattccat 540tcgccgaaaa gccacgcaag tcggcgccag agtcgacgct ctccaaaccg cgcttcacca 600ccacgcgcca agtaccaact ctctcaatct caagcccgac ttgaacgagt ttccaaaact 660cgaagagctt caagattgat ataaatcaaa tatcaatatc aatcaatata tttaatttcc 720taagttcttt attaatatat agttttatgt gtgtatatat agatgatgat gcctcggagt 780tggggcttga aactaattaa ccccttccct tccccttaat ttagatatat cccttttctt 840gtttttccgt atcttcaatc ataatatcaa atcaaagaag tattattatt ttctaaaaaa 900aaaaaaaaaa 910＜210〉2＜211〉174＜212〉PRT＜213〉 ( Glycine max ( L. ) Merr. )＜400〉2Met Glu Asp Arg Asp His Cys Cys Ser Asn Asn Ser Thr Met Ile

5 10 15Thr?Thr?Thr?Lys?Lys?Arg?Thr?Gly?Arg?Arg?Ser?Pro?Thr?Ser?Asp

20 25 30ys?Leu?Lys?Asn?Gln?His?Arg?Glu?Lys?Gln?Ser?Met?Lys?Pro?Tyr

35 40 45Arg?Gly?Ile?Arg?Met?Arg?Lys?Trp?Gly?Lys?Trp?Val?Ala?Glu?Ile

50 55 60Arg?Glu?Pro?Asn?Lys?Arg?Ser?Arg?Ile?Trp?Leu?Gly?Ser?Tyr?Thr

65 70 75Thr?Pro?Val?Ala?Ala?Ala?Arg?Ala?Tyr?Asp?Thr?Ala?Val?Phe?Tyr

80 85 90Leu?Arg?Gly?Pro?Thr?Ala?Arg?Leu?Asn?Phe?Pro?Glu?Leu?Leu?Phe

95 100 105Gln?Asp?Asp?Asp?Gln?Glu?Gly?Ser?Asp?Ser?Val?Gln?His?Gly?Ala

110 115 120Ala?Gly?Asn?Met?Ser?Ala?Asp?Ser?Ile?Arg?Arg?Lys?Ala?Thr?Gln

125 130 135Val?Gly?Ala?Arg?Val?Asp?Ala?Leu?Gln?Thr?Ala?Leu?His?His?His

140 145 150Ala?Pro?Ser?Thr?Asn?Ser?Leu?Asn?Leu?Lys?Pro?Asp?Leu?Asn?Glu

155 160 165Phe?Pro?Lys?Leu?Glu?Glu?Leu?Gln?Asp

170 174

Claims (10)

1, anti-reverse transcription factor GmDREB, be protein, or the amino acid residue sequence of sequence 2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence 2 deutero-protein with the amino acid residue sequence of sequence 2 with sequence 2 amino acid residue sequences in the sequence table.

2, transcription factor according to claim 1 is characterized in that: it is the protein with sequence 2 amino acid residue sequences in the sequence table.

3, transcription factor according to claim 2 is characterized in that: the AP2/EREBP function that is transcription factor GmDREB from the nitrogen end to 44-101 amino-acid residue conserved domain of carbon teminal in the described sequence 2 is guarded the territory.

4, the encoding gene of anti-reverse transcription factor GmDREB is one of following nucleotide sequences:

1) dna sequence dna of sequence 1 in the sequence table;

2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and the identical function protein DNA sequence of encoding.

5, gene according to claim 4 is characterized in that: the encoding gene of described anti-reverse transcription factor GmDREB is the dna sequence dna of sequence 1 in the sequence table.

6, gene according to claim 5 is characterized in that: the reading frame of this gene is for holding the 155th to the 676th bit base from 3 '.

7, contain the described expression carrier of claim 4.

8, carrier according to claim 7 is characterized in that: described expression vector is pBI121-GmDREB.

9, the clone that contains the described gene of claim 4.

10, the application of the described gene of claim 4 in cultivating cold-resistant, salt-resistant plant kind.

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