苏云金杆菌cry1Ib基因的克隆、表达与活性研究

Cloning expression and activity of cry1Ibgene from Bacillus thuringiensis isolate

根据cry1Ib类基因的全长序列设计引物,以苏云金芽胞杆菌(Bacillus thuringiensis)GS8菌株的质粒DNA为模板扩增出片段长为2.16kb的cry1Ib全长基因,插入大肠杆菌(Escherichia coli)表达载体pET-21b,转化大肠杆菌BL21(DE3)菌株,诱导表达出81.20kD的蛋白。推导的分子量为81.20kD,生物信息学分析表明其由719个氨基酸组成,氨基酸序列与Cry1Ib1蛋白的相似性为99.58%,不同于已知的10种Cry1Ib蛋白,是一种新的Cry1Ib蛋白,其基因已被国际基因命名委员会正式命名为cry1Ib3。杀虫活性测定结果表明:Cry1Ib3对小菜蛾(Plutella xylostella)有很强的杀虫活性,LC50为0.076 3μg/mL,而对甜菜夜蛾(Spodoptera exigua)、粉纹夜蛾(Trichoplusia ni)、家蚕(Bombyx mori)、棉铃虫(Helicoverpa armigera)和榆兰叶甲(Pyrrhalta aenescens)基本上没有活性。该基因的获得为我国抗虫转基因作物和工程菌的研制提供新的基因来源,亦为筛选延缓昆虫抗性产生的基因组合提供了重要依据。

A full-length cryllb gene fragment, which obtained by PCR amplification with a pair of primers designed according to cryllb-type gene sequences and DNA from Bacillus thuring- iensis GS8 as template, was introduced into expression vector pET-21b and transformed into Escherichia coli BL21 （DE3）. Molecular weight of the induced express product was 81 kD. The encoded protein was composed of 719 amino acid residues and the predicted MW was 81.20 kD. The amino acid sequence of the CrylIa was very different from those of 10 known CrylIb- type proteins. This gene with accession number EU677422 was designated as cryllb3 by Inter- national Bt Insecticidal Gene Nomenclature Committee. The bioassay results indicated that the CrylIb3 toxin protein showed distinctly insecticidal activity against Plutella xylostella with LCso of 0.076 3 btg/mL, but no activity against Spodoptera exigua, Trichoplusia ni and Bom- byx mori , Helicoverpa armigera , Pyrrhalta aenescens. The novel cryllb3 gene will be new resource for the construction of genetically engineered bacterium and transgenic plant for blo- control of insect pests. It is also available for screening gene stacks to delay the resistance pro-duce of the pests.