苏云金杆菌营养期蛋白杀虫活性及Vip3A-LS1基因克隆

Bioactivity of vip3A Protein from Bacillus thuringiensis Isolates and Cloning of Vip3A-LS1 Gene from Bt LS1

对营养期高活性杀虫菌株进行筛选,得到高效菌株BtLS1,对其营养期杀虫蛋白活性及发酵特性进行了系统研究,克隆了该菌株的vip3A新基因。测定了31株vip3A基因阳性菌株营养期杀虫蛋白的活性,发现BtLS1和BtLS8菌株对甜菜夜蛾Spodopteraexigua生长的抑制作用明显高于其他菌株。进一步研究表明,BtLS1菌株营养期杀虫蛋白对初孵和2龄甜菜夜蛾幼虫的体重增长抑制率分别为95.3%±2.1%和90.7%±6.6%;对棉铃虫Helicoverpaarmigera初孵幼虫的校正死亡率为22.1%,对2龄幼虫的体重增长抑制率为78.7%±6.6%。发酵液中以胞内可溶性物质为主。设计vip3A全长基因特异引物PCR扩增,插入质粒pBluescriptSK(+),克隆测序证实该菌株中存在vip3A新基因,命名为Vip3A-LS1,GenBank登录号为DQ016968。按该序列推断的蛋白与同类蛋白的8个氨基酸之间存在差异。

Vegetative insecticidal protein （VIP）, which was highly toxic to Lepidoptera and Coleoptera,was found from some Bt strains in recent years. Bioassay of the VIPs from 31 isolates, with vip3A genes detected by PCR, was carded out. The results showed that the strain of Bt LS1 had higher toxicity to Spodoptera exigua and Helicoverpa armigera. The morality rates against S. exigua and H. arrnigera （ 1 st instar） were 9. 7% and 22. 1% respectively, and the inhibiting rates of larval weight against S. exigua and H. armigera （2nd instar） were 90.7% ±6.6% and 78.7% ±6.6% respectively. VIP was mostly soluble in cell of Bt LS1. The vip3A gene amplified from Bt LS1 was cloned into pBluescript SK（ ＋ ） and Vip3A-LS1 gene was obtained. The GenBank accession number was DQ016968. 8 AAs in Vip3A-LS1 were different from other deduced vip3A proteins.