苏云金芽胞杆菌杀虫晶体蛋白基因cry1Ab16的克隆及表达

通过对已知cry1类基因以及已发表的cry1Ab的序列进行分析，分别设计了引物P1、P2、P3和P4，首次从无晶体的芽胞杆菌AC11中扩增到一个苏云金芽胞杆菌杀虫晶体蛋白（Insecticidal crystal protein, ICP）cry1Ab类基因。测序结果显示该基因与已知的cry1Ab1基因有8个核苷酸不同，编码的蛋白有7个氨基酸差异。此基因已登录GenBank，并命名为新亚型基因cry1Ab16 (Ac. NO. AF375608)。Southern杂交结果进一步证实该基因存在于菌体的质粒上。将cry1Ab16基因克隆到Escherichia coli表达载体pQE30上并转化E. coli M15。Western印迹分析表明，E. coli M15表达了130 kD的Cry1Ab16蛋白，但此蛋白不稳定，大部分降解成65 kD的蛋白。将表达Cry1Ab16 蛋白的大肠杆菌用涂布法对三龄小菜蛾（Plutella xylostella）毒力测定，其LC50为258.3mg/L；对其他夜蛾科害虫的生长发育也有明显的抑制作用。

Cloning and Expression of ICP cry1Ab16 Gene of Bacillus thuringiensis

With two pairs of primers designed on the basis of the sequence of cry 1 and cry1 Ab gene from Bacillus thuringiensis (Bt), a novel insecticidal crystal protein cry 1Ab gene of Bt was amplified from bacillus strain AC-11 by using Long Template PCR System. Southernblot further confirmed that the novel gene existed in plasmid of the strain. The amplified fragment was sequenced and compared with cry 1Ab1 gene in EMBL/GenBank. The results showed that eight nucleotides and seven amino acid residues were different from that of cry1 Ab1, suggesting that it was a new cry1 Ab gene, which was designated cry1 Ab16 in GenBank (Accession No. AF375608). The cry1 Ab16 gene was cloned into the E.coli expression vector pQE30, creating the recombinant plasmid pQCT, which was then transformed into E.coli M15. Westernblot analysis showed that Cry1Ab16 protein, induced by IPTG was expressed in the strain M15 (pQCT) with the molecular mass of approximate 130 kD, but Cry1Ab16 was unstable and was mostly degraded into about 65 kD protein. Bioassay showed that the LC _ 50 of Cry1Ab16 against the third instar lavae of Plutella xylostella with a spreaded method was 258.3 mg/L, and it could also inhibit the growth of Spodoptera larvae.