CN1477110A - Adverse-resistance transcription factor from tomato, its coding gene and application - Google Patents

Abstract

The present invention discloses an adverse-resistance transcription factor from tomato, its coding gene and application. The name of said adverse-resistant transcription factor is LeDREB2, and it is a protein which has amino acid residue sequence of sequence 2 in sequence table or the protein derived from sequence 2. The coding gene of the adverse-resistant transcription factor LeDREB2 is one of the following nucleotide sequences: 1) DNA sequence of sequence 1 in sequence table; and 2). DNA sequence which and DNA sequence limited by sequence in sequence table have above 90% of homology, and can be used for coding protein with identical function.

Description

Derive from resistance transcription factor and encoding gene and the application of tomato

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 land tomato.

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.The arid dehydration causes ICW content and behavior pattern to change, and high salt causes intracellular osmotic stress that ionic concn is changed, and low temperature freezing-disaster produces ice crystal comes to harm cell.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.

Tomato is understood fully its disease-resistant degeneration-resistant mechanism, and then improves its disease resistance and resistance of reverse to have important theory and realistic meaning as a kind of important edible vegetable plant.

Summary of the invention

The purpose of this invention is to provide transcription factor that derives from tomato and encoding gene thereof with strong adverse-resistant characteristic.

Anti-reverse transcription factor names provided by the present invention is LeDREB2, 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 299 amino-acid residues in the sequence table.The AP2/EREBP function that is transcription factor LeDREB2 from the nitrogen end to 86-143 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 LeDREB2 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 1238 based compositions in the sequence table, and the reading frame of this gene is that it is expressed and mainly is subjected to inducing of arid, low temperature, high salt and external dormin, ethene from 3 ' end the 218th to the 1114th bit base.

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 LeDREB2 provided by the present invention, can obtain transgenic cell line and transfer-gen plant that arid, low temperature and high-salt stress tolerance are enhanced.Expression vector pBI121-LeDREB2 is the expression vector that has LeDREB2 cDNA of the present invention that utilizes conventional molecular biology method to make up, and its gene mapping as shown in Figure 1.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 LeDREB2 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 LeDREB2 expression vector.

Fig. 2 is the vivoexpression of LeDREB2 gene in intestinal bacteria.

Fig. 3 is Southern hybridization analysis result.

Fig. 4 is the LeDREB2 allelic expression that Northern hybridization detects under the different stress conditions.

Embodiment

The clone and the sequential analysis of embodiment 1, tomato LeDREB2 transcription factor cDNA

The tomato seedling of growing 10 days is at room temperature handled after washing only.It is that seedling was placed on the filter paper of adequate thickness dehydration 5 hours that arid is handled, collecting plant is ground to fine degree, places the 4mol/L guanidinium isothiocyanate in the mortar that liquid nitrogen is housed, use acid phenol/chloroform extracting mixture again, get supernatant liquor, with the total RNA of isopropanol precipitating, repeat above-mentioned steps more once, after after 75% the washing with alcohol drying RNA is dissolved in the DFPC 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).By Promega Technical Manual-PloyATtract ^MRNA Isolation Systems (on the internetat www.promega.com) separation obtains ploy (A)+mRNA, after gel reclaims check analysis, by EcoRI and two adaptors of XhoI purpose mRNA is connected to the corresponding multiple clone site of expression vector pB42AD, and be transformed in the intestinal bacteria and increase, the requirement in the equal by specification of all programs is carried out.Library titre after the amplification detects with pcr analysis and shows that the library reaches requirement.Utilize yeast-one-hybrid system screening tomato seedling cDNA expression library to isolate a cDNA clone, called after LeDREB2 (sequence 1 in the sequence table).It is 1238bp that this gene inserts fragment, the open reading frame (218 initial-1114 stop) that contains 897bp, the polypeptide (sequence 2 in the sequence table) that 299 amino acid of encoding are formed, contain a nuclear signal location conservative amino acid residues sequence and an activation domain conservative amino acid residues sequence that is rich in acidic amino acid that is rich in basic aminoacids respectively at aminoterminal and carboxyl terminal, its 5 ' end is 217bp, and 3 ' end is 118bp.

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

The structure of LeDREB2 cDNA expression vector molecular biology method routinely carries out.The LeDREB2 coding region is inserted in a 35S promoter back, be packaged on the pBI121 binary expression vector that contains the TMV translational enhancer, obtain a fusion expression vector pBI121-LeDREB2 (its collection of illustrative plates as shown in Figure 1), through enzyme cut identify to insert fragment errorless after, transform in Agrobacterium, extract plasmid again and cut affirmation through enzyme and successfully transform in Agrobacterium, this complex body can directly be used for plant by transgenic technology, particularly the conversion of tomato.

Embodiment 3, the vivoexpression of tomato LeDREB2 gene in a big bacillus are identified

Utilize a pair of Auele Specific Primer LeDREB2F-317 5 '-AAAA GAATTCCTTCTTCAGCCACTT-3 ' 331, LeDREB2R-945 5 '-AAAA CTCGAGCGTAACTCGATCCCA-3 ' 931 obtains a cDNA fragment (Size-624bp) that comprises the AP2/EREBP structural domain by pcr amplification, and this cDNA fragment is inserted among the protein expression vector pGEX-4T-1 on the corresponding EcoRI and XhoI site, transform then in intestinal bacteria, induce 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 LeDREB2 gene in the tomato dna group.

Utilize LeDREB2 cDNA as probe, with through different restriction enzyme (E-EcoRI, V-EcorV, P-PstI and X-XhoI) postdigestive tomato dna group DNA hybridizes under 65 ℃ of conditions, and under the rigorous degree of height, wash film, (condition of high rigorous degree is: 0.5 * SSC, 0.1 * SDS, 65 ℃), the result as shown in Figure 3, except that the EcoRI digested genomic dna presented faint two hybrid belts, other all had many hybrid belts to occur, show that the LeDREB2 gene is a multi-copy gene in the tomato dna group, perhaps in its genome, exist the LeDREB2 autoploid of low copy.

Embodiment 5, the expression characteristic of tomato LeDREB2 gene under the environment stress condition.

The tomato seedling in 2 week of growth is placed 4 ℃ of water respectively, carry out illumination cultivation in the 250mM NaCl aqueous solution and 100 μ M dormin (ABA) solution, the arid processing is that the tomato seedling in 2 week of growth is at room temperature dewatered through washing to be placed on only on the filter paper that is enough to suck dry moisture, and respectively at 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 12 hours and sampling in 24 hours, extract total RNA and utilize the primer shown in the embodiment 3 to carry out quantitatively (1 μ gRNA) RT-PCR amplification purpose fragment, transfer to nylon membrane then and LeDREB2 cDNA probe carries out the Northern hybridization analysis, and under the rigorous degree of height, wash film.Identical step is used for LeUbi3 (LeUbi3: the ubiquitin gene that a kind of tomato exists) analyze.The primer is: LeUbi.3Fd-1463 5 '-GTGAAAGCCAAGATCCAGGAC-3 ' 1484; LeUbi.3Re-1857 5 '-CAATCGCCAGCCTCCTTGTTG-3 ' 1878 (394bp) (Hoffman et al, 1991, Ubi3:accession number-X58253), LeUbi3 is used as external control.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, D is the acid-treated result that comes off, and as can be seen from the figure, transcribing of LeDREB2 mainly is subjected to low temperature, salt and drought-induced expression, and external dormin and second is rare also can induce this expression of gene.

＜160〉2＜210〉1＜211〉1238＜212〉DNA＜213〉 ( Lycopersicon esculentum L. )＜400〉1tctctctcat caaccctcct ctcctctccc ctctctctct atattctatt ttcacacact 60aaaacagagc agctcgtcat ttcttcaaca tctctgtgta tacactgcgt tcagtttatt 120gagagtttcg caaatttaca gacatagttt tttcgcgaaa aaagagagag aagaaaacag 180agcaaacgta acaaaaacag agcaacacaa ttcatacatg ataataatgt ctacagagca 240accaaattgt tcagaaagta ctgaatctag ctgcaactct tcttcttctt cgtcgccctc 300atcgccatct tccgttcttc ttcagccact tccacaaatc aattcaaaaa atcgactcaa 360aagatgcaga ggtgaagagg aagtagaaga agaagaagac gtggtcgtta ataatccaaa 420tccgaagaag atgaataaaa acaacaacaa tggcagcagt actagtgttg tttcttatgt 480aggtgtacga atgagagcat ggggaaaatg ggtatccgaa atccgtgaac ctaaaaaaaa 540atcacggatt tggttaggta cctttgctac cccggaaatg gcggcgcgtg ctcacgacgt 600cgccgccatg tccattaaag gtacctcagc tatactcaat tttccccaat tttcacattt 660actgccccga cccgtcacgt gctccccacg tgacatccaa aacgccgccg taaaagccgc 720tcacatggat cacctaaatc caaaattctc aatattaccc gaaacctctg cggcgacgat 780gacctcatcg tcatcgtcgc tgtcgttagt ttcgggcgtt acatcctcct cgtcgtcgtt 840tcaagacgac gaggagtcaa gaccgtcgcc tccggagctg attccggagg cgacggggca 900gttgagtgag atagtggagc tgccaaaatt gggatcgagt tacgaattgg ttgaatcaac 960tcagagttta tttgaatcag atgaatggtg ggataataat tatggaaatt gtgaatattt 1020ttttggacaa gataattata ttagtagtaa tatggaattt acaggattag aaaatgtggt 1080ttcaaccagt tttgagagtt ttttatggca acattagaat aaaaaaaata atgttttttc 1140ttttgataat taattttaca ctttattttt ttttatgtaa tttaggtgga aattattgat 1200gaactttata cgtttcttgc aaaaaaaaaa aaaaaaaa 1238＜210〉2＜211〉299＜212〉PRT＜213〉 ( Lycopersicon esculentum L. )＜400〉2Met Ile Ile Met Ser Thr Glu Gln Pro Asn Cvs Ser Glu Ser Thr

5 10 15Glu?Ser?Ser?Cys?Asn?Ser?Ser?Ser?Ser?Ser?Ser?Pro?Ser?Ser?Pro

20 25 30Ser?Ser?Val?Leu?Leu?Gln?Pro?Leu?Pro?Gln?Ile?Asn?Ser?Lvs?Asn

35 40 45Arg?Leu?Lys?Arg?Cys?Arg?Gly?Glu?Glu?Glu?Val?Glu?Glu?Glu?Glu

50 55 60Asp?Val?Val?Val?Asn?Asn?Pro?Asn?Pro?Lys?Lys?Met?Asn?Lys?Asn

65 70 75Asn?Asn?Asn?Gly?Ser?Ser?Thr?Ser?Val?Val?Ser?Tyr?Val?Gly?Val

80 85 90Arg?Met?Arg?Ala?Trp?Gly?Lys?Trp?Val?Ser?Glu?Ile?Arg?Glu?Pro

95 100 105Lys?Lys?Lys?Ser?Arg?Ile?Trp?Leu?Gly?Thr?Phe?Ala?Thr?Pro?Glu

l10 115 120Met?Ala?Ala?Arg?Ala?His?Asp?Val?Ala?Ala?Met?Ser?Ile?Lys?Gly

125 130 135Thr?Ser?Ala?Ile?Leu?Asn?Phe?Pro?Gln?Phe?Ser?His?Leu?Leu?Pro

140 145 150Arg?Pro?Val?Thr?Cys?Ser?Pro?Arg?Asp?Ile?Gln?Asn?Ala?Ala?Val

155 160 165Lys?Ala?Ala?His?Met?Asp?His?Leu?Asn?Pro?Lys?Phe?Ser?Ile?Leu

170 175 180Pro?Glu?Thr?Ser?Ala?Ala?Thr?Met?Thr?Ser?Ser?Ser?Ser?Ser?Leu

185 190 195Ser?Leu?Val?Ser?Gly?Val?Thr?Ser?Ser?Ser?Ser?Ser?Phe?Gln?Asp

200 205 210Asp?Glu?Glu?Ser?Arg?Pro?Ser?Pro?Pro?Glu?Leu?Ile?Pro?Glu?Ala

215 220 225Thr?Gly?Gln?Leu?Ser?Glu?Ile?Val?Glu?Leu?Pro?Lys?Leu?Gly?Ser

230 235 240Ser?Tyr?Glu?Leu?Val?Glu?Ser?Thr?Gln?Ser?Leu?Phe?Glu?Ser?Asp

245 250 255Glu?Trp?Trp?Asp?Asn?Asn?Tyr?Gly?Asn?Cys?Glu?Tyr?Phe?Phe?Gly

260 265 270Gln?Asp?Asn?Tyr?Ile?Ser?Ser?Asn?Met?Glu?Phe?Thr?Gly?Leu?Glu

275 280 285Asn?Val?Val?Ser?Thr?Ser?Phe?Glu?Ser?Phe?Leu?Trp?Gln?His

290 295 299

Claims (10)

1, anti-reverse transcription factor LeDREB2, 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 LeDREB2 from the nitrogen end to 86-143 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 LeDREB2 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 LeDREB2 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 218th to the 1114th 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-LeDREB2.

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

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

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