苏云金杆菌辅助蛋白P20对杀虫晶体蛋白Cry1Ab表达的影响

Bacillus thuringiensis Helper Protein P20 Affects the Formation of Cry1Ab

苏云金杆菌 (Bacillusthuringiensis,简称Bt)杀虫晶体蛋白Cry1Ab因其C 半端缺少了一段含 4个半胱氨酸的氨基酸序列而导致蛋白的不稳定 ,报道苏云金杆菌辅助蛋白P2 0帮助Cry1Ab蛋白的表达及晶体的形成。利用穿梭载体pHT310 1构建 3个表达质粒 ,即pT1B、pP1B和pDP1B ,3个质粒都含有cry1Ab基因 ,不同在于pT1B没有p2 0基因 ,pP1B含有p2 0全基因 ,而pDP1B不仅含有p2 0全基因 ,且在p2 0基因前插入cry1A(c)启动子。分别将这 3个表达质粒经电转化到苏云金杆菌晶体缺陷型菌株CryB中 ,获得转化菌株T1B、P1B和DP1B。Westernblot表明cry1Ab基因在这 3株菌中均表达了 130kD的蛋白 ,部分降解为大约 6 0kD的蛋白。蛋白定量分析显示 ,3株菌 130kD蛋白量的比为 1∶1.4∶1 5 ,降解后的 6 0kD蛋白量的比为 1∶1.1∶1.6 ,Cry1Ab蛋白总量的比为 1∶1∶2∶1 6。镜检发现 ,Cry1Ab在 3株菌中都形成典型的菱形晶体 ,其晶体大小为T1B <P1B <DP1B。生物测定结果显示 ,3个菌株对棉铃虫 (Helicoverpaarmigera)均具有明显的杀虫活性 ,三者的LC50 差异不显著。研究表明 ,P2 0对cry1Ab基因的表达和晶体形成均有帮助 ,P2

The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p 20 gene, pP1B carries p20, and pDP1B contains p 20 with cry 1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that cry1Ab was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1∶1.4∶1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1∶1.1∶1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B<P1B<DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC 50 . This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.