产Zwittermicin A的苏云金芽孢杆菌菌株的筛选及抗性基因zmaR的克隆与表达

 通过PCR方法筛选160株苏云金芽孢杆菌，其中94株含有zmaR基因。测定了含zmaR基因的菌株培养物上清液对草生欧文氏杆菌（Erwinia herbicola）的抑菌活性，结果表明有67株菌有抑菌活性，其中21株抑菌活性较高。经鉴定，抑菌活性较高的G03菌株含有cry1Ac、cry1Aa、cry1Ca和cry2Ab等高毒力杀虫基因，并从G03中克隆了zmaR全长基因，完成了序列测定。该基因编码区为1 125 bp，由核苷酸序列推导的氨基酸残基组成为375个，分子量为43.5 kD，等电点pI4.945。通过载体pET-21b将zmaR基因导入大肠杆菌BL21，可正常表达43.5 kD蛋白，并使宿主菌产生对Zwittermicin A的抗性。

Screening of Zwittermicin A-Producing Strains of Bacillus thuringiensis and Cloning and Expression of zmaR Gene

One hundred and sixty isolates of Bacillus thuringiensis were screened by PCR,and 94 of these isolates were zmaR-positive.It was found that 67 zmaR-positive isolates showed inhibitory effects on the growth of Erwinia herbicola OS,and the inhibitory activity of 21 isolates were higher than those of others.Bacillus thuringiensis isolate G03,which has cry1Ac,cry1Aa,cry1Ca and cry2Ab genes,was identified with zmaR gene and highly active to Erwinia herbicola OS.The zmaR gene was cloned from G03,and sequence analysis showed that zmaR consisted of 1 125 bp DNA fragment and encoded a protein containing 375 amino acids with 43.5 kD.The isoelectric point was pI4.945.The zmaR gene was inserted into pET-21b to generate pET-21b::zmaR,and then this recombinant plasmid was introduced into E.coli BL21 (DE3) for high-level expression.The 43.5 kD peptide could be detected by SDS-PAGE in the transformants with Zwittermicin A resistance.