ÃÞ »¨ ѧ ±¨ Cotton Science 2009£¬21(5)£º346-350
Cloning and Expression of a Cotton Verticillium Wilt Antibacterial Peptide Gene 25a2 Abstract£ºIn order to clone the gene which encodes the cotton Verticillium wilt antibacterial peptide A2, 15 amino acid residues of N-terminal of the antibacterial peptide A2 was inputted in NCBI to blast and the result showed a protein contained a fragment which was the same with the 15 amino acid residues of A2. Following the protein sequence, the gene of the protein was found and a pair of primers was designed according to the gene. A DNA fragment about 300 bp was obtained by PCR amplification using the genome of strain Bacillus amyloliquefaciens RS-25 as template. The PCR production was ligated into pET -28a plasmid by two restriction enzyme sites of BamH ¢ñ and Hin d ¢ó . After colony PCR and restriction enzyme-digestion analysis, the PCR production was sequenced and sequencing analysis indicated that the PCR production was 354 bp and coded for a protein of 117 amino acid residues. This gene is named 25a 2. The constructed expression vector pET -28a -25a 2 was transformed into Eschrichia coli BL21 and colony PCR analysis was used to select the positive clones. The positive single clone was inoculated into LB medium and cultivated overnight with the kanamycin concentration about 100 mg¡¤L-1. The cultivated bacteria was transfered into fresh medium with 10% inoculum and cultured at 37 ¡æ without antibiotics until the OD600 reached 0.5-0.7. Then IPTG was added into the culture with the final concentration about 1 mmol¡¤L-1 to induce the expression. Five hours later, the cultured cells were collected and ultrasonic crushed. SDS-PAGE analysis on expression product shows the molecular weight is about 15 kDa and coincides with the theory analysis.
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