CN1477110A - Stress resistance transcription factor derived from tomato and its coding gene and application - Google Patents

Abstract

The invention discloses a stress-resistant transcription factor derived from tomato and its coding gene and application, and aims to provide a transcription factor derived from tomato and its coding gene with strong stress resistance characteristics. The name of the antiretroviral factor provided by the present invention is LeDREB2, which is a protein having the amino acid residue sequence of Sequence 2 in the sequence table, or the amino acid residue sequence of Sequence 2 is replaced, deleted or replaced by one or several amino acid residues. A protein derived from Sequence 2 added and having the same activity as the amino acid residue sequence of Sequence 2. The coding gene of the anti-reverse transcription factor LeDREB2 is one of the following nucleotide sequences: 1) the DNA sequence of sequence 1 in the sequence listing; 2) having more than 90% homology with the DNA sequence defined in sequence 1 in the sequence listing, and A DNA sequence that encodes a protein of the same function. The gene of the invention is of great significance for cultivating stress-resistant plant varieties, especially for cultivating drought-resistant, cold-resistant and salt-resistant plant varieties, and improving crop yield.

Description

Stress resistance transcription factor derived from tomato and its coding gene and application

technical field

The invention relates to a plant transcription factor and its coding gene and application, especially a transcription factor derived from land tomato and its coding gene and application.

Background technique

Biological and abiotic environmental stress factors such as pathogens, drought, salinity, low temperature, freezing damage, and waterlogging have important effects on plant growth and development. Drought and dehydration lead to changes in water content and behavior patterns in cells, high salt causes osmotic stress in cells to change ion concentrations, and low temperature freezing damage produces ice crystals that damage cells. To improve the stress tolerance of crops, in addition to using traditional breeding methods, molecular genetic breeding has become one of the areas of concern to scientific and technological workers. Under adversity stress, a series of physiological and biochemical changes usually occur in plants. Plants first sense changes in the external environment through a variety of ways, and transfer the extracellular signal to the inside of the cell, and then transmit the signal to the transcription factor through a series of phosphorylation chain reactions, and the transcription factor then communicates with the target through its specific functional amino acid. Gene interaction can initiate the expression of target genes that respond to adversity stress, thereby improving the resistance of plants. It has been confirmed that certain transcripts of the DREB class can receive environmental stress signals and activate stress response genes to improve the stress tolerance of plants. In plants, the AP2/EREBP functionally conserved domain is the specific structure of DREB transcription factors.

As an important edible vegetable plant, tomato has important theoretical and practical significance to clarify its disease resistance and stress resistance mechanism, and then improve its disease resistance and stress tolerance.

Contents of the invention

The purpose of the present invention is to provide a tomato-derived transcription factor and its coding gene with strong stress resistance.

The name of the antiretroviral factor provided by the present invention is LeDREB2, which is a protein having the amino acid residue sequence of Sequence 2 in the sequence table, or the amino acid residue sequence of Sequence 2 is replaced, deleted or replaced by one or several amino acid residues. A protein derived from Sequence 2 added and having the same activity as the amino acid residue sequence of Sequence 2.

The sequence 2 amino acid residue sequence in the sequence listing is a protein consisting of 299 amino acid residues. The 86th-143rd amino acid residue conserved domain from the nitrogen end to the carbon end in the sequence 2 is the AP2/EREBP function conserved domain of the transcription factor LeDREB2.

The coding gene of the antiretroviral factor LeDREB2 is one of the following nucleotide sequences:

1) DNA sequence of sequence 1 in the sequence listing;

2) A DNA sequence having more than 90% homology with the DNA sequence defined in Sequence 1 in the sequence listing and encoding the same functional protein.

The DNA sequence of Sequence 1 in the sequence listing consists of 1238 bases. The reading frame of the gene is from the 218th to the 1114th base at the 3' end, and its expression is mainly affected by drought, low temperature, high salt and external abscisic acid, Ethylene induction.

Utilize any carrier that can guide exogenous gene expression in plants, such as pBIN19 and derived pBI101, pBI121 and pBI221 series vectors (Bevan, 1984 Nucleic Acid Research, 12:8711-8721), the present invention The provided gene encoding transcription factor LeDREB2 is introduced into plant cells, and transgenic cell lines and transgenic plants with enhanced tolerance to drought, low temperature and high salt stress can be obtained. The expression vector pBI121-LeDREB2 is an expression vector with the LeDREB2 cDNA of the present invention constructed by means of conventional molecular biology, and its gene map is shown in Figure 1. When the gene of the present invention is constructed into a plant expression vector, any enhanced promoter or inducible promoter can be added before its transcription start nucleotide, such as cauliflower mosaic virus (CaMV 35S) and Ubiquitin promoter wait. In order to facilitate the identification and screening of transgenic plant cells or plants, the vectors used can be processed, such as adding plant selectable markers (GUS gene, luciferase gene, etc.) or antibiotic markers with resistance (qing Damycin, Kanamycin, etc.). The expression vector carrying the LeDREB2 gene of the present invention can be obtained by conventional biotechnologies such as Ti (Tumor-induced-cancer induction) plasmid, Ri (Root-induced-root induction) plasmid, plant virus vector, direct DNA transformation, microinjection, and conductance. The method is introduced into plant cells, and the transformed plant host can be a monocotyledonous plant or a dicotyledonous plant. The gene of the invention is of great significance for cultivating stress-resistant plant varieties, especially for cultivating drought-resistant, cold-resistant and salt-resistant plant varieties, and improving crop yield.

Description of drawings

Figure 1 shows the construction of LeDREB2 expression vector.

Fig. 2 is the in vitro expression of LeDREB2 gene in Escherichia coli.

Figure 3 shows the results of Southern hybridization analysis.

Figure 4 shows the expression characteristics of LeDREB2 gene detected by Northern hybridization under different stress conditions.

Detailed ways

Example 1, Cloning and Sequence Analysis of Tomato LeDREB2 Transcription Factor cDNA

The 10-day-old tomato seedlings were washed with water and treated at room temperature. The drought treatment is to dehydrate the seedlings on filter paper of sufficient thickness for 5 hours, collect the plants, grind them to a fine degree in a mortar filled with liquid nitrogen, place them in 4mol/L guanidine isothiocyanate, and then use acid phenol/chloroform The mixture was extracted, the supernatant was taken, the total RNA was precipitated with isopropanol, and the above steps were repeated once more. Finally, after washing and drying with 75% ethanol, the RNA was dissolved in DFPC water and stored at -80°C (Davis et al., Molecular Biology Basic Methods of Molecular Biology: [Basic Methods in Molecular Biology], pp.777, APPLETON & LANCE, Norwalk, Connecticut, USA, 1994). The ploy(A)+mRNA was isolated by Promega Technical Manual- ^{PloyATtract®} mRNA Isolation Systems (on the internetat www.promega.com). After gel recovery verification and analysis, the target mRNA was connected to the expression The corresponding multiple cloning site of the vector pB42AD was transformed into Escherichia coli for amplification, and all procedures were carried out according to the requirements in the instructions. The titer detection and PCR analysis of the amplified library showed that the library met the requirements. The tomato seedling cDNA expression library was screened by yeast one-hybrid system to isolate a cDNA clone named LeDREB2 (sequence 1 in the sequence listing). The gene insertion fragment is 1238bp, contains an open reading frame of 897bp (218 start-1114 stop), encodes a polypeptide consisting of 299 amino acids (sequence 2 in the sequence listing), and contains a base-rich The nuclear signal localization conservative amino acid residue sequence of the sex amino acid and a conserved amino acid residue sequence of the activation domain rich in acidic amino acid, its 5' end is 217bp, and the 3' end is 118bp.

Example 2. Construction of fusion expression vectors for transformation.

The construction of the LeDREB2 cDNA expression vector was carried out by conventional molecular biology methods. The LeDREB2 coding region was inserted behind a 35S promoter and packaged in the pBI121 binary expression vector containing the TMV translation enhancer to obtain a fusion expression vector pBI121-LeDREB2 (its map is shown in Figure 1). The insertion was identified by enzyme digestion After the fragment is correct, it is transformed into Agrobacterium, and then the plasmid is extracted and confirmed to be successfully transformed into Agrobacterium through enzyme digestion. The complex can be directly used for plant transformation, especially tomato transformation, through transgenic technology.

Example 3, In vitro expression identification of tomato LeDREB2 gene in Field Bacillus

A cDNA fragment ( Size -624bp), and insert this cDNA fragment into the corresponding EcoRI and XhoI sites in the protein expression vector pGEX-4T-1, then transform in Escherichia coli, induce in Escherichia coli through IPTG, SDS-PAGE detection shows The gene can be translated and expressed in Escherichia coli in vitro. However, no protein expression band appeared in the uninduced strain. Polypropylene gel electrophoresis bands are shown in Figure 2. In the figure, M is Marker; 1 and 3 are induced strains; 2 is uninduced strain. It can be seen from the figure that the induced strains have protein bands, but the uninduced strains have no protein bands.

Example 4. Analysis of LeDREB2 gene in tomato genome.

Using LeDREB2 cDNA as a probe, it was hybridized with tomato genomic DNA digested with different restriction endonucleases (E-EcoRI, V-EcorV, P-PstI and X-XhoI) at 65°C, and at high stringency Wash the membrane under high stringency (conditions for high stringency: 0.5×SSC, 0.1×SDS, 65°C), the results are shown in Figure 3, except for two weak hybridization bands in EcoRI digested genomic DNA, there are many others The appearance of hybridization bands indicated that the LeDREB2 gene was a multi-copy gene in the tomato genome, or there was a low-copy LeDREB2 homologue in the tomato genome.

Example 5. Expression characteristics of tomato LeDREB2 gene under adversity stress conditions.

The two-week-old tomato seedlings were placed in 4°C water, 250 mM NaCl aqueous solution and 100 μM abscisic acid (ABA) solution for light culture, and the drought treatment was to wash the two-week-old tomato seedlings with water and place them in sufficient Dehydrate at room temperature on the filter paper that absorbs moisture, and take samples at 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 12 hours and 24 hours respectively, extract total RNA and use the primers shown in Example 3 for Quantitative (1 μg RNA) RT-PCR amplified target fragment, then transferred to nylon membrane and LeDREB2 cDNA probe for Northern hybridization analysis, and washed the membrane under high stringency. The exact same procedure was used for LeUbi3 (LeUbi3: an ubiquitin gene present in tomato) analysis. The primers used are: LeUbi.3Fd-1463 5'-GTGAAAGCCAAGATCCAGGAC-3'1484; LeUbi.3Re-1857 5'-CAATCGCCAGCCTCCTTGTTG-3'1878 (394bp) (Hoffman et al, 1991, Ubi3: accession number-X58253), LeUbi3 Used as an external control. The results are shown in Figure 4, where A is the result of drought treatment, B is the result of 4°C low temperature treatment, C is the result of 250mM NaCl treatment, and D is the result of abscisic acid treatment. It can be seen from the figure that the transcription of LeDREB2 The expression is mainly induced by low temperature, salt and drought, and the expression of this gene can also be induced by external abscisic acid and ethylene.

序列表<160>2<210>1<211>1238<212>DNA<213>番茄属陆地番茄(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>番茄属陆地番茄(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 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 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 Ser Leu

185 190 195Ser Leu Val Ser Gly Val Thr Ser 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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