Protein coding sequence of protein kinase 3 activated by cell division protoplasm for brassia rape
A cabbage rape, cell division technology, applied in the field of protein coding sequences, can solve the problems of unclear gene salt tolerance, drought resistance and insect resistance environmental factors and other problems
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Embodiment 1
[0123] Cloning of BnMPK3 gene of Brassica napus
[0124] 1. Tissue isolation (isolation)
[0125] Brassica napus (variety "Huyou 15") was purchased from the market. The Brassica napus was placed at 28°C to germinate for 24 hours, and then sown in the greenhouse. When the number of Brassica napus leaves was 3-5, it was ready to be extracted. DNA or RNA.
[0126] 2. RNA isolation (RNA isolation)
[0127] Take part of the tissue, grind it with a mortar, add it to a 1.5mL EP tube containing the lysate, shake it sufficiently, and then transfer it into a glass homogenizer. After homogenization, it was transferred to a 1.5 mL EP tube, and total RNA was extracted (TRIzol Reagents, GIBCO BRL, USA). The total RNA quality was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
[0128] 3. Cloning of Full-length cDNA
[0129] According to the amino acid conserved sequence of the Arabidopsis AtMPK3 gene, using the ...
Embodiment 2
[0141] Sequence information and homology analysis of BnMPK3 gene in Brassica napus
[0142] The novel Brassica napus BnMPK31 full-length cDNA of the present invention has a length of 1464 bp, and the detailed sequence is shown in SEQ ID NO. 3, wherein the open reading frame is located at 154-1263 nucleotides (1114 nucleotides). The amino acid sequence of Brassica napus BnMPK3 was deduced from the full-length cDNA, with a total of 370 amino acid residues, a molecular weight of 42588.74 Daltons, and an isoelectric point (pI) of 5.79. The detailed sequence is shown in SEQ ID NO.3.
[0143] The full-length cDNA sequence of Brassica napus BnMPK3 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 database. Homology search, it was found that it has 90% identity with the Arabidopsis gene AtMPK3 at the nucleotide level (Supplementary Table 2); at the amino acid level, it al...
Embodiment 3
[0145] Eukaryotic expression of Brassica napus gene BnMPK3 protein or polypeptide in yeast and identification of stress resistance in transformed yeast
[0146] Construction of Expression Vector Containing Target Gene (Brassica napus gene BnMPK3 Gene)
[0147] According to the full-length sequence of Brassica napus gene BnMPK3 (SEQ ID NO. 3), primers for amplifying the complete coding reading frame were designed, and restriction endonuclease sites were introduced into the upstream and downstream primers respectively (this can be optional vector) in order to construct an expression vector. Taking the amplification product obtained in Example 1 as a template, after PCR amplification, the Brassica napus gene BnMPK3 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. The expression vector was identified under the correct premise, and then transformed into yeast INVSIC by Li...
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