Protein coded sequence of protein kinase 9 activated by Brassica napus L. cell mitogen
A Brassica napus, coding sequence technology, which is applied in the field of protein kinase 9 protein coding sequences activated by cytomitogens in Brassica napus, can solve problems such as unclear, unclear salt tolerance, drought resistance and insect resistance.
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Embodiment 1
[0155] Cloning of BnMPK9 Gene in Brassica napus
[0156] 1. Tissue separation (isolation)
[0157] Brassica napus (the variety is "Huyou 15") is purchased from the market, and the Brassica napus is placed at 28°C to germinate for 24 hours, and then sowed in the greenhouse. When the Brassica napus leaves are 3-5 pieces, it is ready to extract DNA or RNA.
[0158] 2. RNA isolation (RNA isolation)
[0159] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCOBRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0160]3. Cloning of Full-length cDNA
[0161] According to the conserved amino acid sequence of the Arabidopsis AtMPK9 gene, using the principle of homologous gen...
Embodiment 2
[0173] Sequence Information and Homology Analysis of BnMPK9 Gene in Brassica napus
[0174] The novel Brassica napus BnMPK9 full-length cDNA of the present invention has a length of 1897bp, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 111-1613 nucleotides (1503 nucleotides). The amino acid sequence of Brassica napus BnMPK9 was deduced according to the full-length cDNA, with a total of 501 amino acid residues, a molecular weight of 57423.37 Daltons, and an isoelectric point (pI) of 8.57. See SEQ ID NO.3 for the detailed sequence.
[0175] The full-length cDNA sequence of Brassica napus BnMPK9 and its encoded protein were analyzed by BLAST program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases for nucleotide and protein Homology search, it was found that it has 90% identity with the Arabidopsis gene AtMPK9 at the nucleotide level (attached table 2); at the ...
Embodiment 3
[0177] Expression of Brassica napus gene BnMPK9 protein or polypeptide in eukaryotic cells and identification of stress resistance of transformed yeast
[0178] Construction of expression vector containing target gene (Brassica napus gene BnMPK9 gene)
[0179] According to the full-length sequence (SEQ ID NO.3) of the Brassica napus gene BnMPK9, design primers that amplify the complete coding reading frame, and introduce restriction endonuclease sites on the upstream and downstream primers respectively (this can be selected depending on the vector) in order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Brassica napus gene BnMPK9 gene cDNA was cloned into an expression vector (such as pYES2.1), and further transformed into Escherichia coli TOP10 or DH5α, in the guaranteed reading frame Under the correct premise, the expression vector was identified, and then it was transformed into yeast INVSIC...
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