| 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. |
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