对鳞翅目害虫高毒力的Bt cry1Aa基因的分离克隆及表达

Bt菌Ly30株是我国自行分离的对多种害虫具有高毒力的苏云金芽孢杆菌，经CAPS(cleaved amplified polymorphic sequences)系统鉴定，它含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法, 设计一对特异引物，分别引入NcoⅠ和BamHⅠ/NcoⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Aa全长基因,与表达载体Pkk233-2相应酶切产物连接，转化大肠杆菌，获得含有cry1Aa基因重组质粒pKKLy1Aa。完成了该基因的亚克隆和序列测定，结果表明，该基因的编码区为3 531 bp，编码蛋白分子量为133.2kD，含1.176个氨基酸，等电点Pi为4.99。该基因序列已在GenBank中登记注册，登录号为AF384211，并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Aa12。对重组菌KKLy1Aa进行诱导表达研究。在0.6 mmol/L IPTG、37℃、8 h培养条件下，该基因获得高效表达，SDS-PAGE电泳检测到明显的133.2 kD蛋白带。室内生测结果表明，Cry1Aa蛋白对不同的小菜蛾品系均有较高的杀虫活性，其LC₅₀值分别为0.203 μg/mL和0.554 μg/mL。

Cloning and expression of a Bt cry1Aa gene with high toxicity against lepidopterous pests

Bacillus thuringiensis strain Ly30, isolated from Lianyungang, Jiangsu Province, was highly toxic to several kinds of insect pests. Results of CAPS (cleaved amplified polymorphic sequences) analysis indicated that Ly30 contained the cry1Aa gene. According to the 5' and 3' end nucletiode sequence of the known cry1Aa gene, one pair of primers was designed and the full-length cry1Aa gene (3.5 kb) was thereby obtained by PCR amplification from strain Ly30. Sequencing analysis showed that this cry1Aa gene contained one open reading frame of 3 531 bp, and encoded 1 176 amino acid residues deduced from its nucletiode sequence with molecular mass of 133.2 kD. This gene was registered in GenBank (Accession number is AF384211) and designated as cry1Aa12, a novel cry gene, by the international Nomenclature Committee of Bt δ-endotoxin genes. Expression of cry1Aa12 had been performed by inserted it into E. coli expression vector Pkk233.2, and a 133.2 kD protein band could be easily detected by SDS-PAGE. Results of bioassays proved that the expression products of cry1Aa12 gene had high toxicity against two Plutella xylostella colonies,with LC₅₀ of 0.203 μg/mL，0.554 μg/mL respectively.