【作者】 刘小云；

【导师】 王敦；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2011， 硕士

【摘要】 本文通过对灰斑古毒蛾幼虫进行生物测定和组织病理切片观察,研究HearNPV ChiA和BtCry1Ab基因的原核表达产物,与OrerNPV以一定的浓度混合,饲喂3龄灰斑古毒蛾幼虫,观察记录不同处理灰斑古毒蛾幼虫的累计死亡率和喂毒后的体重变化,以及灰斑古毒蛾幼虫细胞的生理生化变化,研究HearNPV ChiA和BtCry1Ab对OrerNPV的增效作用,并对不同添加剂的增效机理进行初步推测,获得的主要结果为:1.原核表达菌BL21-32a-chiA及Bl21(DE3)-28a-BtCry1Ab在最优表达条件下,目的蛋白HearNPV ChiA和BtCry1Ab的量分别占表达菌总蛋白量的56%和12.6%.2.HearNPV ChiA和BtCry1Ab基因的原核表达产物,分别或者同时作为添加物质,在较高浓度都显著能提高OrerNPV的杀虫活性。当OrerNPV浓度为2.0×107 PIBs/ml、添加的HearNPV ChiA和BtCry1Ab浓度都为2.0μg/ml时,LT50比单用OrerNPV的处理缩短2.9d,LT90缩短3.7d;喂食BtCry1Ab的处理,5天后灰斑古毒蛾幼虫的体重是相同时间其他处理试虫体重的1/5。3. OrerNPV感染灰斑古毒蛾幼虫,12h气管细胞最先发现病毒发生基质,3d后在脂肪体组织发现复制产生的病毒粒子,5d真皮细胞中充满的多角体病毒粒子。说明OrerNPV易感染的组织为脂肪体细胞和表皮细胞。4.HearNPV ChiA和BtCry1Ab对OrerNPV的增效机理不同:BtCry1Ab能够一定程度地破坏中肠的组织结构,导致中肠对病原微生物的防御功能减弱;同时抑制灰斑古毒蛾幼虫的取食,导致幼虫发育迟缓;而HearNPV ChiA能加快病毒的侵染速度和病毒的增殖过程,从而提高杀虫活性。二者共同作用的结果可增强OrerNPV的杀虫能力。更多还原

【Abstract】 To investigate the enhancement on virulence of OrerNPV by HearNPV chitinase and BtCry1Ab, the HearNPV chiA gene and BtCry1Ab gene were cloned into Prokaryotic expression vector pET32a and pET28a and expressed in Escherichia coli BL21-32a and BL21-28a,respectively. The two proteins expressed in E. coli were mixed with OrerNPV and fed to the third instar larvae of Orgia ericae Germar for bioassay. The tissue and cell pathology were also observed on infected larvae by transmission electron microscope (TEM). The results are follows:1.The target proteins HearNPV ChiA and BtCry1Ab accumulated up to about 58.1 % and 12.6% of the total cellular proteins in prokaryotic expression BL21-32a-chiA and BL21-32a-BtCry1Ab,separately.2. The bioassays results showed that the lethal time of the third instar larvae of Orgia ericae are all significantly shorter than that of control treatment. When feeding with 2.0×107 PIBs/ml OrerNPV together with 2.0μg/ml HearNPV ChiA and BtCry1Ab, the median lethal time LT50 will be 2.9d shorter than inoculation of OrerNPV, while LT90 reduced 3.7d. When fed with BtCry1Ab protein, the weights of larvae were only about 1/5 of other treatments.3. When O. ericae third larva infected by OrerNPV, the virogenic stroma tracheal cell was found 12 h.p.i.. A number of nucleocapsids were synthesized in fat body cells in 3d.p.i. . While in 5d.p.i., dermal cells were filled with occluded virus. It means that fat body and leather system of insects are the most sensitive tissues to OrerNPV.4. Laboratory experiments show that BtCry1Ab have a strong effect on restraining feeding and growth of third instar larva of O. ericae, and make the midgut O. ericae be destroyed. Finally the defensive ability of midgut were lowered. HearNPV ChiA can enhance the invasion and reproductive of OrerNPV in O. ericae cells. These results illustrated that HearNPV ChiA and BtCry1Ab together can be used as the synergist of OrerNPV upon controlling population of O. ericae.更多还原