Research On Cloning And Expression Of The Antimicrobial Peptides Gene Of Cotton Verticillium Wilt

Cotton Verticillium wilt which influences the growth of cotton is one of the most dangerous fascicular diseases caused by Verticillium dahliae. Bacillus amyloliquefaciens RS-25 which showed high antibiosis activity to Verticillium dahliae was screened by Wu Cuihong. It was separated and purified a 9 kD protein named A2. Edman degradation showed that 15 amino acids of N-terminal sequences of A2 were ASGGTVGIYGANMRS. The further research was performed based on these works. It was mainly studied the cloning of the mature peptides and whole peptides of the antimicrobial peptides gene of cotton Verticillium wilt and their expression in E.coli and P.pastoris.The protein sequences with 100% homology were found by the program of BLAST on NCBI GenBank database as well as the result of the 16S rDNA sequences of RS-25. The primers of the whole gene of the new antimicrobial peptides were designed. The recombinant plasmid pET-30a-a2q1 was constructed. Then gene a2q1 was sequenced. The result indicated that this gene contained 354 bp nucleotides and encoded 117 amino acids, and the amino acids from 42 to 56 of a2q1 were the same as 15 amino acids of N-terminal of antimicrobial peptides A2 which was separated and purified by Wu. The protein was bigger than the antimicrobial peptides A2. The more 41 amino acids might be the regulatory regions. And the antimicrobial peptides A2 which was separated and purified by Wu may be the mature peptides. The recombinant plasmids pET-30a-a2q1 were transformed into host bacteria of E.coli BL21. SDS-PAGE analysis was performed after being induced by IPTG. The relative molecular weights of the expressed products were 19 kD. The expressed products were purified by Nickel-affinity chromatography column. SDS-PAGE showed that there was a single band at 19 kD. A2Q1 which was cloned and expressed did not show antibiosis activity to Verticillium dahliae by testing. The protein A2Q1 was bigger than the antimicrobial peptides A2. The research will be performed on mature peptides in order to test if the antibiosis activity of protein is destroied by the 41 amino acids and obtain recombinant protein which shows antibiosis activity to Verticillium dahliae.The primers of mature peptides gene were designed. The recombinant plasmids pET-30a-a2c were constructed and transformed into host bacteria E.coli BL21. And the expressed products were purified by Nickel-affinity chromatography column. SDS-PAGE showed that there was a single band at 14 kD. But it did not show antibiosis activity to Verticillium dahliae too. The antimicrobial peptides A2 is glycoprotein. The E.coli expression system did not show the function of ptotein glycosylation. So the recombinant proteins A2Q1 and A2C did not show the antibiosis activity to Verticillium dahliae.Pichia pastoris which shows the function of ptotein glycosylation. So the further research was performed with Pichia pastoris as host. The primers of whole gene of antimicrobial peptides were designed with His tag. The recombinant plasmids pPIC9K-a2q2 were constructed and digested by Sac I, then transformed into Pichia pastoris GS115. And the expressed products were purified by Nickel-affinity chromatography column after being induced by methanol and low temperature. SDS-PAGE showed that there was a single band at 19 kD. And it showed antibiosis activity to Verticillium dahliae. So, the protein A2Q2 could show the antibiosis activity to Verticillium dahliae after being glycosylated, and it was proved as a glycoprotein.In this study, the antimicrobial peptides A2 was cloned and expressed, and it showed antibiosis activity to Verticillium dahliae. It will offer stuff for further research of antimicrobial peptides of cotton Verticillium wilt, and provide a necessary theory and practical foundation for transgenic cotton and the genetic engineering of cotton disease resistance.