【摘要】 Bt菌Ly30株是中国自行分离的对多种害虫具有高毒力的苏云金芽孢杆菌(Bacillusthuringiensis,Bt),经CAPS(cleavedamplifiedpolymorphicsequences)系统鉴定,它含有cry1Ac基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,分别引入SalⅠ和BamHⅠ酶切位点。以Ly30质粒DNA为模板扩增cry1Ac全长基因,与表达载体pET-21b相应的酶切产物连接,转化大肠杆菌(Eschrichiacoli),获得含有cry1Ac基因重组质粒pEKLy1Ac。该基因的亚克隆和序列测定结果表明,其编码区为3534bp,编码蛋白分子量为133.5kD,含1177个氨基酸,等电点为4.8,与Cry1Ac3同源性最高,存在4个氨基酸的差异,与Cry1Ac10之间则有6个氨基酸的不同。该基因序列已在GenBank中登记注册为AF482767,并被国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Ac14。该基因经诱导获得高效表达,SDS-PAGE电泳检测到明显的133.5kD蛋白带。室内生物测定结果表明,诱导表达的Cry1Ac蛋白对棉铃虫、甜菜夜蛾等鳞翅目害虫幼虫均有较高的杀虫活性,其LC50值分别为19.236和3.276μg/g饲料。更多还原

【Abstract】 Ly30 strain of Bacillus thuringiensis screened from China was highly toxic to several kinds of pests. And results of the CAPS (cleaved amplified polymorphic sequences) identification indicated that it contained a cry1Ac gene. According to cry1A gene sequences of 5’-terminal and 3’-terminal, a pair of primers for full-length cry1Ac gene was designed. PCR was performed to produce a 3.6 kb full-length cry1Ac fragment, using plasmid DNA from Bt Ly30 as the template. Based on the results of sequence analysis, the open reading frame of the cry1Ac gene containing 3 534 bp encoded a 133.5 kD protein composed of 1177 amino acids, and its IE point was 4.82. The cry1Ac14 gene was registered in GenBank (Accession number is AF482767) as novel gene, named by International Nomenclature Committee of Bt δ-endotoxin genes and expressed by inserted into an expressing vector pET-21b. The 133.5 kD protein could be identified obviously by SDS-PAGE. Results of bioassay proved that the expression products of cry1Ac14 gene had high toxicity against Helicoverpa armigera and Spodotera exigua with LC 50 of 19.236 μg / g and 3.276 μg / g artificial diet respectively.更多还原