苏云金杆菌辅助蛋白P20对营养期杀虫蛋白Vip3A表达的影响

Effects of helper protein P20 from Bacillus thuringiensis on Vip3A expression

为检测苏云金杆菌辅助蛋白P20对Vip3A表达和杀虫活性的影响,将p20基因与vip3A基因相连构建了重组质粒pHVP20,然后电激转化至Bt中进行了共表达,以仅携带vip3A基因的质粒pHPT3作为对照质粒。Westernblot结果显示,当vip3A基因和p20基因在Bt无晶体缺陷株CryB中共表达时,Vip3A蛋白的最大表达量约是其在CryB(pHPT3)菌株中单独表达的1.5倍。生物测定结果表明,CryB(pHVP20)和CryB(pHPT3)菌株对初孵斜纹夜蛾幼虫的LC50值分别为48.79μg/mL和78.00μg/mL,这说明P20蛋白可以促进vip3A基因在Bt中的表达,但对提高Vip3A蛋白的杀虫毒力没有显著性帮助。

Insecticidal crystal proteins （ICPs） produced in Bacillus thuringiensis accumulate as crystalline inclusions that represent up to 30 % of total dry weight the cell produces. The mechanisms of in vivo crystalhzation of these insecticidal proteins remain interests, yet unclear. A 20-kDa protein （P20）, the product of the third open reading frame of cry 11 A operon in B. thuringiensis subsp, israelensis has been defined to be an important molecular chaperone （helper protein） for forming Cyt1A crystal and enhancing Cry11A expression. The novel vegetative insecticidal proteins （VIPs） are secreted outside the cell of B. thuringiensis during mid-logarithmic growth. VIP3A shows activity against many lepidopteran insect larvae in a different mechanism from that of ICPs.To investigate the influence of helper protein P20 on Vip3A production and its insecticidal activity,P20 was coexpressed with Vip3A protein in B. thuringiensis and the yields and insecticidal toxicity of Vip3A were also analyzed.The recombinant plasmid pHVP20 was constructed by inserting a 5.4kb foreign fragment containing both vip3A gene and p20 gene into the shuttle vector pHT3101. The plasmid pHPT3 only containing vip3A gene was used as control, pHVP20 and pHPT3 were transformed into the B.thuringiensis acrystalliferous strain CryB not containing vip3A gene by electroporation. The obtained B. thuringiensis transformants were CryB（pHVP20） and CryB（pHPT3） respectively.Western blot showed that Vip3A protein reached its maximum yield after 48h of CryB （pHVP20） growth and remained high expression level during the soorulation. The maximum yield of Vip3A protein in CryB（pHVP20） was about 1.5 fold as compared with that in CryB （pHPT3） by the mean of ImageMaster VDS software.it is considered that P20 might combine with the native Vip3A protein during the sporulation, stabilize Vip3A and protect Vip3A from unspecific furl proteolysis. Bioassay showed that the cell pellets of CryB （pHVP20） and CryB （pHPT3） performed high insecticidal toxicity against the first instar larvae of Spodoptera litura.Their LCsos of were 48.79/zg/mL and 78.00/~g/mL respectively and were not significantly different. Cell supernatants of two strains containing small amounts of secreted Vip3A were not toxic to the tested insect. It suggestes that p20 can enhance the expression of Vip3A,but not improve its insecticidal toxicity remarkably.