农业生物技术学报2011，Vol.19Issue(5)：932-937,6.DOI:10.3969/j.issn.1674-7968.2011.05.021
苏云金芽胞杆菌新型vip3Aa基因的克隆、表达与活性分析
Cloning, Expression and Analysis of Insecticidal Activity of a Novel vip 3A a-type Gene from Bacillus thuringiensis
摘要
Abstract
To find novel vip3 genes, the vip3-type genes in 72 Bt stains were identified by PCR method and high-resolution melting analysis (HRMA) system. The results indicated that three vip3 gene types, vip3Aa, vip3Af and vip3Ba were found in 18 positive strains. A full-length vip3 gene fragment, which obtained by PCR amplification with a pair of primers designed according to lupJ-type gene sequences and DNA from T03B001 (B. Thuringiensis subsp. Sumiyoshiensis) as template, was introduced into expression vector pEB and transformed into Escherichia coli Rosetta (DE3). At low temperatures, a peptide of 88 kD was expressed by IPTG induction. Theencoded protein was composed of 789 amino acid rsidues. It shared a 96% sequence homology with Vip3Aa protein. This gene designated as vip3A a39 by International Nomenclature Committee of Bt endotoxin, GenBank accession No. Was HMI17631. Vip3Aal 1 protein was obtained in our previous study, 39 amino acid residues were found to be different between the Vip3Aa39 and Vip3Aal 1 proteins. Insecticidal activities of soluble expressed products of vip3Aa39 and vip3Aall genes were tested against Agrotis ipsilon, Plutella xylostella, Helicoverpa armigera and Spodoptem exigua by feeding method. The bioassay results indicated that Vip3Aa39 had insecticidal activity against A.ipsilon with 50% lethal concentration (LCX) of 5.43 |xg/mL compared to 73.62 p-g/mL for Vip3Aal 1; Vip3Aa39 showed insecticidal activity against P.xylostella with LCX of 140.64 (xg/mL while Vip3Aal 1 protein had no activity against P.xylostella; Vip3Aal 1 demonstrated insecticidal activity against H.armigera with LCX of 35.18 |xg/mL compared to 286.99 |xg/mL for Vip3Aa39; Vip3Aa39 had insecticidal activity against S. Exigua with LCX of 2.02 |xg/mL similar to 2.04 |xg/mL for Vip3Aal 1. The results indicated that insecticidal activity of Vip3Aa39 and Vip3Aal 1 was different from each other against A.ipsilon, P.xylostella and H.armigera. Compared with the Vip3Aall, Vip3Aa39 had high insecticidal activity against A.ipsilon and P.xylostella while showed low activity agaist H.armigera. Vip3Aa39 will provide more chocies to control agricultural pest, and it provide materials to study structure and insecticidal mechanism of the Vip3 protein.关键词
苏云金芽胞杆菌/克隆/活性/Vip3Key words
Bacillus thuringiensis/ Cloning/ Insecticidal activity/ Vip3引用本文复制引用
何晓明,束长龙,刘晓垒,李海涛,高继国,张杰,宋福平..苏云金芽胞杆菌新型vip3Aa基因的克隆、表达与活性分析[J].农业生物技术学报,2011,19(5):932-937,6.基金项目
本研究由国家基础研究发展规划(973)项目(No.2009CBll 8902)、国家转基因生物新品种培育科技重大专项(No.2009ZX08009-030B)和国家高科技研究发展计划(863)项目(No.2011AA10A203)共同资助 （973）
