Genetically Modification On HearNPV With Cry2Ab And Virulence Enhancement On CypoGV By Bt Toxin

Baculovirus is natural pathogen in insect population. Some of them have been used asinsecticide for its host-specificity and safety to environment, etc. Like other microbialinsecticide, their utility has been limited by their disadvantages, especially their relativelyslow speed of kill and narrow specificity compared with chemical peptides. To cover thedisadvantages of baculoviruses, a lot of genetic modified approaches have been taken. In thispaper, we use Cry1Ab and Cry2Ab as enhancer to improve the virulence of NPV and GV.First, we introduced Bt Cry2Ab toxin into Helicoverpa armigera nucleopolyhedrovirus（HearNPV） polyhedra to generate a novel recombinant HearNPV, Cry2Ab-HearNPV, toimprove insecticidal activity of HearNPV. Besides, the prokaryotic expressed BtCry1Abprotein was used as a virulence enhancer for C. pomonella granulovirus Kashgar strain1（CypoGV-KS1） at a trace level dose that was lower than sublethal to enhance the power ofCypoGV. The results are as following:1. Construction and biological characterization of recombinant HearNPV producingpolyhedra that contain bacillus thuringiensis Cry2ab crystal protein（1） Generation of recombinant virusThe recombinant virus was produced by the Bac-to-Bac system. Firstly, a transfer vector,Ha.p-ph I-cry2Ab-ph II-HTb, was constructed by digestion and ligase. To constructrecombinant bacmid, the transfer vector was transformed into E. coli BW25113（containHaBacHZ8）. With the enzyme encoded by the helper plasmid pMON7124, the recombinantbacmid cry2Ab-HaBacHZ8was generated by inserting Ha.p-ph I-cry2Ab-ph II fragment（from transfer vector） into HaBacHZ8.To generate Cry2Ab-HearNPV, cry2Ab-HaBacHZ8-ph was transfected into Hz-AM1cells. To further confirm the polyhera was recombinant virus, viral DNA was extracted fromthe infected cells and analyzed by PCR.（2） TEM analysis To further analyze whether expression of Cry2Ab could infect the occlusion of polyhedra,we used transmission electron microscopy to compare Cry2Ab-HearNPV polyhedra withthose from wild-type HearNPV. The results show that both the shape and size ofCry2Ab-HearNPV polyhedra are similar to wild-type HearNPV. Besides, the ODV ofCry2Ab-HearNPV are also formed normally.（3） Analysis of viral growth curve and DNA replicationTo assess the effect of Cry2Ab insertion on viral replication and viral DNA replication,we performed one-step growth curves and DNA replication curve, respectively. The resultsindicated that there were no significant differences in viral replication and DNA replicationbetween wild-type HearNPV and Cry2Ab-HearNPV.（4） In vivo activity of Cry2Ab-HearNPVTo determine the insecticidal properties of Cry2Ab-HearNPV, the does-and time-mortality responses of Cry2Ab-HearNPV were detected in3instar larva of H. armigera.Compared to wild-type HearNPV, the LD50and LD₉₀were reduced by40.3%、46.1%,respectively. Besides, the LT₅₀and LT₉₀of Cry2Ab-HearNPV were shorter than that ofwild-type HearNPV （17.2%、21.6%decreased, respectively）. These results demonstrated thatCry2Ab-HearNPV is powerful than wild-type HearNPV.2. The enhancement on Cydia pomonella granulovirus virulence by BtCry1AbThe LT₅₀and LT₉₀values of CypoGV-KS1were reduced by14.42%and10.06%,respectively with a dose of0.5μg/mL BtCry1Ab; and they were reduced by21.51%and14.49%respectively with a dose of1.5μg/mL BtCry1Ab, The results showed that the LT₅₀and LT₉₀values of CypoGV-KS1were improved significantly when the trace level doseBtCry1Ab protein was supplemented slightly.