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为了扩充新的鳞翅目杀虫蛋白基因,本研究采用限制性片段长度多态性PCR(PCR-restricted fragment length polymorphisms,PCR-RFLP)方法对苏云金芽胞杆菌(Bacillus thuringiensis)菌株BN23-5中所含杀虫晶体蛋白基因进行鉴定和分析。结果表明,该菌株中含有晶体蛋白(crystal,cry)cry2Ab类基因。根据已知cry2Ab类基因编码区设计简并引物,以菌株BN23-5全质粒DNA为模板进行扩增并测序。结果显示,扩增产物为一个完整的cry2Ab类基因,全长1 902 bp,由634个氨基酸组成,氨基酸序列与Cry2Ab1同源性最高(96.5%),命名为cry2Ab32(Gen Bank登录号:KJ710647)。将该基因插入表达载体p ET-28a,构建重组质粒p ET-2Ab并转化大肠杆菌(Escherichia coli)BL21(DE3),异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)诱导成功表达蛋白,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)验证其相对分子质量为65 k D左右。表达的包涵体蛋白采用饲喂法进行生物活性测定,结果表明,Cry2Ab32对小菜蛾(Plutella xylostella)、玉米螟(Ostrinia fumacalis)和棉铃虫(Helicoverpa armigera)均有杀虫活性,半致死浓度(median lethal concentration,LC50)分别为8.99、19.34和21.24μg/m L,对甜菜夜蛾(Laphygma exigua)的致死率不高,但能明显抑制甜菜夜蛾的生长。cry2Ab32新基因的发现将为重要农业害虫的防治提供更多选择,为进一步研究Cry2Ab类蛋白结构和杀虫机理提供基础资料。
In order to expand the new Lepidopteran insecticidal protein gene, this study used PCR-restricted fragment length polymorphism (PCR-RFLP) method to detect Bacillus thuringiensis strain BN23-5 Insecticidal crystal protein containing genes were identified and analyzed. The results showed that the strain contains the crystal (cry) cry2Ab gene. Degenerate primers were designed according to the coding region of the known cry2Ab gene. The full-length plasmid BN23-5 was used as a template for amplification and sequencing. The results showed that the amplified product was a complete cry2Ab gene, which was 1 902 bp in length and consisted of 634 amino acids with the highest homology (96.5%) with Cry2Ab1, which was named cry2Ab32 (Gen Bank accession number: KJ710647) . The gene was inserted into the expression vector p ET-28a to construct the recombinant plasmid p ET-2Ab and transformed into Escherichia coli BL21 (DE3), isopropyl-β-d-thiogalactopyranoside -thiogalactoside (IPTG), and the relative molecular mass was about 65 kD by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that Cry2Ab32 had insecticidal activity against Plutella xylostella, Ostrinia fumacalis and Helicoverpa armigera, and the median lethal concentration (median lethal concentration (LC50) were 8.99, 19.34 and 21.24μg / m L, respectively. The lethal concentration (LC50) was not high to lethal to Laphygma exigua, but significantly inhibited the growth of beet armyworm. The discovery of the novel cry2Ab32 gene will provide more options for the prevention and treatment of important agricultural pests and provide the basic information for further study on the structure and insecticidal mechanism of Cry2Ab proteins.