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为获得新的鳞翅目害虫杀虫基因,根据已知cry1I类基因编码区设计简并引物,采用直接克隆法,以苏云金芽胞杆菌(Bacillus thuringiensis,Bt)菌株BN23-5质粒DNA为模板进行扩增,并对得到的基因进行鉴定和分析。结果表明:克隆得到一个完整的cry1Ie基因,全长2 160 bp,由719个氨基酸组成,该氨基酸序列与已知的4种Cry1Ie蛋白不同,与Cry1Ie2和Cry1Ie3的氨基酸序列同源性最高,为95.4%,被国际Bt杀虫晶体蛋白基因命名委员会命名为Cry1Ie5(登录号为KJ710646)。将该基因插入表达载体p ET-28a,转化大肠杆菌BL21(DE3),IPTG低温诱导成功表达,SDS-PAGE电泳验证其大小为81 k D,与预测的分子量相符合。生物活性测定表明,Cry1Ie5表达的包涵体蛋白对小菜蛾和亚洲玉米螟具有杀虫活性,LC_(50)分别为0.43μg/m L和48.39μg/m L;对棉铃虫的致死率不高,但能明显抑制其生长;对甜菜夜蛾没有杀虫活性。
In order to obtain a new insecticidal gene of lepidopteran pests, a degenerate primer was designed according to the coding region of known cry1I gene and the direct cloning method was used to amplify the plasmid DNA of Bacillus thuringiensis (Bt) strain BN23-5 as a template Increase, and the resulting gene identification and analysis. The results showed that a full-length cry1Ie gene was cloned, which was 2 160 bp in length and consisted of 719 amino acids. The amino acid sequence was different from the known four Cry1Ie proteins and had the highest homology with Cry1Ie2 and Cry1Ie3 of 95.4 %, By the International Bt Insecticidal Crystal Protein Gene Nomenclature Committee named Cry1Ie5 (accession number KJ710646). The gene was inserted into the expression vector p ET-28a and transformed into E. coli BL21 (DE3). The recombinant plasmid was successfully induced by low temperature induction of IPTG. The size was 81 kD by SDS-PAGE and was consistent with the predicted molecular weight. Bioassay showed that Cry1Ie5 protein had insecticidal activity against Plutella xylostella and Asian corn borer, with LC 50 of 0.43μg / m L and 48.39μg / m L, respectively. The lethality to cotton bollworm was not high, But could obviously inhibit its growth; insecticidal activity to beet armyworm.