苏云金芽胞杆菌cry1Ie新基因的克隆、表达与生物活性测定

Cloning,expression,and biological activity of a new cry1Ie gene from Bacillus thuringiensis strain

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 novel lepidopteran insecticidal genes,PCR method was used for identification of cry1I-type gene from Bacillus thuringiensis（Bt） strains BN23-5.According to the NCBI open cry1I-type gene encoding area,a pair of degenerate primers was designed.A full-length cry1 Ie gene was cloned by PCR amplification with B.thuringiensis strain BN23-5 as template.It contained an open reading frame of 2 160 bp nucleotides encoding a protein of 719 amino acids.Compared with other Cry1 Ie protein,it shared 95.4% amino acid homology with Cry1Ie2 and Cry1Ie3.It was designated as Cry1Ie5 by Bacillus thuringiensis Insecticidal Crystal Proteins Nomenclature Committee and the NCBI accession number was KJ710646.It was constructed into the recombinant plasmid p ET28 a,and transformed into Escherichia coli BL21（DE3）.The result of SDS-PAGE indicated that cry1Ie5 could be expressed as 81 k D protein in E.coli BL21（DE3） strain induced by IPTG.The bioassay results indicated that the Cry1Ie5 protein was highly toxic to the larvae of Plutella xylostella and Ostrinia furnacalis with a LC50 value of 0.43 μg /m L and 48.39 μg /m L,respectively;and had low activity against cotton bollworm,butcould obviously inhibit the growth of it;it had no activity against beet armyworm.