Protein MtCBF4 associated with stress tolerance of plants, encoding gene and application thereof
A stress tolerance, gene technology, applied in plant genetic improvement, botanical equipment and methods, applications, etc., can solve problems such as significant differences in expression patterns
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Embodiment 1
[0043] Embodiment 1, the discovery of MtCBF4 gene
[0044] Establish a perfect alfalfa seed germination and seedling salt stress gene expression profile of truncated alfalfa through two microarray experiments, and perform cluster analysis and GO annotation analysis on genes significantly induced by salt stress, and perform GeneBins on the probes of interest Horizontal pathway classification analysis basically constructed alfalfa salt tolerance regulatory network framework. On this basis, a salt stress expression database of alfalfa truncation was established. A transcription factor whose expression was significantly up-regulated after salt induction was found, and the expression of this transcription factor was up-regulated by 64 times after induction with 200mM NaCL for 5 hours.
[0045] The protein composed of amino acid residues shown in Sequence 1 is named MtCBF4 protein, and the coding gene of MtCBF4 protein is named MtCBF4 gene, and its coding region is shown in the 11t...
Embodiment 2
[0046] Embodiment 2, the acquisition of transgenic plants and the identification of stress tolerance
[0047] 1. Cloning of MtCBF4 gene
[0048] Specific primer pairs were synthesized by Invitrogen.
[0049] MtCBF4_5': 5'-TAC CAT GGA CAT GTT TAC TAT GAA TCA ATT-3';
[0050] MtCBF4_3': 5'-ATA CTA GTT TAA AAT GAG TAA CTC CAC A-3'.
[0051] Using the truncated alfalfa cDNA as a template, PCR amplification was carried out with specific primers. The PCR amplification product was inserted into the pMD18-T simple vector (purchased from TaKaRa Bioengineering Company) to obtain the recombinant vector pMD18-Tsimple-MtCBF4. After sequencing verification, the PCR amplification product has the nucleotide (MtCBF4 gene) shown in sequence 2 in the sequence listing.
[0052] 2. Construction of recombinant expression vector (p1302-MtCBF4)
[0053] See the build process figure 1 .
[0054] 1. Digest the vector pMD18-T simple-MtCBF4 with restriction enzymes Nco I and Spe I, and reclaim the...
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