Cai Qi-Liang, Liu Zi-Duo, Sun Ming, Wei Fang, Yu Zi-Niu
College of Life Science and Technology, Key Laboratory of Agricultural Microbiology, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China.
Sheng Wu Gong Cheng Xue Bao. 2002 Sep;18(5):578-82.
Three kinds of Bacillus thuringiensis serotype-subsp. Leesis(H33) strain YBT-833, subsp. Aizawai(H7) strain YBT-1416 and subsp. Kurstaki(H3ab) strain YBT-1535, which were isolated by our lab, are chosen as original strain to clone vegetative insecticidal protein gene. Southern hybridization showed that vip genes are all localized at roughly 4-5 kb size-fractionated XbaI fragments of total DNA from YBT-833, YBT-1416 and YBT-1535. Three subgenomic libraries containing the vip gene fragment, were constructed with pUC19 as vector. Then, three vegetative insecticidal protein gene vip83, vip14 and vip15 are obtained from the libraries through the methods of colony-blot-in-situ screening and enzyme-cut detection. Comparision of DNA sequence made out that only vip83 gene exist five different base pairs with known vip genes. Because the sequences of vip14 and vip15 are the same, two of the three genes, vip83 and vip14, were subcloned to shuttle vehicle pHT315 to get recombinant plasmids pBMB8901 and pBMB8902 in turn. The plasmids were separately transformed into vip Bt. receptors BMB171 and 4Q7 to obtain four engineered strains BMB8901-171, BMB8902-171, BMB8901-4Q7 and BMB8902-4Q7. SDS-PAGE results indicated that all recombinant strains express 88 kD vegetative insecticidal protein. Bioassay also showed that the proteins of genes vip83 and vip14 both have certain toxicity to Lepidopteran insect larvae such as Heliochis armigera, Spodotera exigua and Plutella xylostella. While the toxicity of vip protein from four engineered strains to Plutella xylostellas are highest, whose LC50 value is 28.6, 31.6, 45.4 and 37.6 microL/mL respectively. This study will contributed to construct high efficacy and wide spectrum engineered strains on theory and reality.
选用本实验室分离的苏云金芽孢杆菌库斯塔克亚种（H3ab）YBT - 1535菌株、库尔斯塔克亚种（H3ab）YBT - 1416菌株和鲇泽亚种（H7）YBT - 833菌株作为原始菌株，克隆营养期杀虫蛋白基因。Southern杂交表明，vip基因均定位在YBT - 833、YBT - 1416和YBT - 1535总DNA经XbaI酶切后大小约为4 - 5 kb的片段上。以pUC19为载体，构建了3个含有vip基因片段的亚基因组文库。然后，通过菌落原位杂交筛选和酶切检测等方法，从文库中获得了3个营养期杀虫蛋白基因vip83、vip14和vip15。DNA序列比较发现，只有vip83基因与已知vip基因存在5个不同的碱基对。由于vip14和vip15序列相同，将其中2个基因vip83和vip14分别亚克隆到穿梭载体pHT315上，依次获得重组质粒pBMB8901和pBMB8902。将质粒分别转入vip Bt受体菌BMB171和4Q7中，获得4株工程菌BMB8901 - 171、BMB8902 - 171、BMB8901 - 4Q7和BMB8902 - 4Q7。SDS - PAGE结果表明，所有重组菌株均表达88 kD的营养期杀虫蛋白。生物测定结果还表明，vip83和vip14基因的蛋白对棉铃虫、甜菜夜蛾和小菜蛾等鳞翅目昆虫幼虫均有一定毒性。其中4株工程菌的vip蛋白对小菜蛾的毒性最高，其LC50值分别为28.6、31.6、45.4和37.6 μL/mL。本研究将为高效广谱工程菌的构建提供理论和实际依据。
