AcMNPV chiA基因表达产物对棉铃虫围食膜的破坏及对Bt和NPV的增效作用研究

采用PCR方法扩增出苜蓿银纹夜蛾核型多角体病毒（AcMNPV）几丁质酶基因（chiA）编码区1.6kb全长片段，并将该片段分别克隆至原核表达载体pET30a和杆状病毒Bac to Bac表达系统转移载体pFastBac中，分别在大肠杆菌（E. coli）BL21(DE3)和草地贪夜蛾细胞系Sf-9中进行了表达。SDS-PAGE分析表明，在大肠杆菌和昆虫细胞中均有效表达了60kDa的蛋白。将表达产物饲喂5龄棉铃虫幼虫后取其围食膜，扫描电镜结果显示，围食膜结构遭到破坏形成大量孔洞。生物测定结果表明，以上两种表达产物对Bt和NPV均具有增效作用。以AcMNPV ChiA在大肠杆菌和细胞系Sf-9中的表达产物分别与Bt Cry2Ac蛋白混合饲喂棉铃虫幼虫，增效率分别为33.4％和54.5％，其LT50较对照处理分别缩短了17.8h和20.6h；当AcMNPV ChiA在大肠杆菌和细胞系Sf-9中的表达产物分别与甘蓝夜蛾核型多角体病毒（MbNPV）混合处理棉铃虫幼虫时，其LT50与对照比较分别缩短了16.6h和22.4h。

AcMNPV ChiA disrupts the peritrophic membrane and enhances insecticidal activity of Bt and NPV to Helicoverpa armigera

To investigate the synergism of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase with insect pathogens for pest control, the chitinase A (chiA) gene was amplified from the AcMNPV genome by PCR and expressed in E. coli BL21 and Spodoptera frugiperda cell line Sf-9 respectively. The results of SDS-PAGE showed that chiA gene could be expressed as a 60 kDa protein in both E. coli and Sf-9 successfully. When the fifth instar larvae of Helicoverpa armigera were fed with the expressed product of chiA gene, the perforations on the peritrophic membrane (PM) were observed by the scanning electron microscope (SEM). When the expressed product of chiA gene was mixed with Bt and with Mamestra brassica nuclear polyhedrosis virus (MbNPV) fed to larvae of Helicoverpa armigera respectively. Then bioassay indicated that when compared with their corresponding controls, the LT50 of Helicoverpa armigera larvae fed by a mixture of Bt Cry2Ac protein and expressed product of chiA gene in E. coli and in Sf-9 cells were shortened 17.8 and 20.6 hours, with rates of enhancement were 33.4% and 54.5%, respectively. Another LT50 of Helicoverpa armigera larvae fed with MbNPV and expressed product of chiA gene in E. coli and in Sf-9 cells were shortened 16.6 and 22.4 hours, respectively. These results suggested that AcMNPV Chitinase may enhance insecticidal activity of Bt and NPV for pest control.