Insecticidal Effect And Mechanism Of Vip3Aa On Cockroach And Application In Construction Of Transgenic Biocontrol Bacteria

Cockroach is one of the "four injurious insect",and can threaten human health through the pathogenic bacteria they carry.At present,the main control method against cockroaches is chemical control,but chemical insecticides show three major problems:drug resistance,recurrence of pests,and residual pesticides.Biological control has the advantages of being difficult to produce drug resistance,being able to spread naturally,having certain selectivity to pests,and having no pollution.Bacillus thuringiensis(Bt)can secrete a new class of insecticidal toxin protein during the vegetative growth stage-vegetative insecticidal protein(Vips).Vips is a potential insecticidal protein due to rare reports of drug resistance,there are no reports about Vip3Aa protein in cockroach control.At present,there is no report about Vip3Aa protein in cockroach control.In the mechanism of Vip3Aa killing Lepidoptera insects,the N-terminal sequence of the full-length Vip3Aa protein can be removed by insect midgut protease to form an active protein,then the active protein cross the peritrophic membrane and bind to some receptors on the apical membrane of midgut epithelial cells,resulting in the formation of pores in the midgut epithelial cells,which ultimately leads to insect death.The effect of N-terminal sequence on Vip3A protein activity is significantly different in different insects.At present,the research on Vip3Aa insecticide is still on the research of its insecticidal mode,and there is a lack of research on its molecular mechanisms.In the field of biological control,due to the instability of proteins,virulence proteins generally used in construction of transgenic crops and microorganismsIn this study,the vip3Aa gene was amplified from Bt strains,and a new N-terminal truncated vip3Aa gene(j-vip3Aa)was designed.Vip3Aa and J-Vip3Aa proteins were successfully prepared and purified by prokaryotic expression system.Bioassay activity showed that Vip3Aa protein has insecticidal activity against Periplaneta americana and Blattella germanica.After removing the N-terminal protein,the insecticidal activity of J-Vip3Aa was lower than that of Vip3Aa protein,and J-Vip3Aa was more sensitive to midgut digestive juice than Vip3Aa protein.He staining and Scanning Electron Microscopy showed that Vip3Aa could damage the midgut and columnar colon of Periplaneta americana,but had no effect on crop.The activities of detoxification enzymes and proteolytic enzymes in Periplaneta americana were increased by Vip3Aa protein.Vip3Aa protein can be hydrolyzed to about 62 kDa protein by trypsin,chymotrypsin,midgut digestive juice of Periplaneta americana.The BBMV pore formation experiment showed that Vip3Aa protein could form holes in the midgut and columnar colon.Fluorescence localization experiments showed that Vip3Aa protein and J-Vip3Aa protein had no binding receptors in the midgut and columnar colon.Analysis of transcriptome differences in midgut of Periplaneta americana showed that 1539 genes were up-regulated and 763 genes were down regulated after administration of Vip3Aa.Through the analysis of apoptosis and lysosome differential genes,transmission electron microscopy and Hoechst staining results,Vip3Aa protein may mediate apoptosis of midgut epithelial cells of Periplaneta americana through the lysosomal-p38MAPK signaling pathway,thereby playing a role in killing cockroaches.Using egfp as an indicator gene,a lithium acetate-mediated transformation method of Metarhizium anisopliae sporulation spores was constructed.After the vip3Aa gene was introduced into Metarhizium anisopliae,the killing activity of Periplaneta americana and Blattella germanica increased by 3.75 and 4.13 times respectively.A preliminary attempt was made to prepare Vip3Aa toxin bacteria by yeast display system,the wild-type yeast had no cockroach killing activity and could play a certain role in killing cockroaches after introducing vip3Aa gene.In conclusion,Vip3Aa protein can kill cockroaches.The mechanism of killing cockroaches by Vip3Aa protein is different from that of killing Lepidoptera.Vip3Aa can kill cockroaches directly without removing the N-terminal sequence,and does not depend on binding to midgut or columnar colon receptors.After N-terminal protein excision,the activity of ip3Aa protein decrease.N-terminal of Vip3Aa protein can be used to protect Vip3Aa protein from degradation of digestive juice and prolong the action time of Vip3Aa protein.Vip3Aa protein may mediate apoptosis of midgut epithelial cells of Periplaneta americana through the lysosomal-p38MAPK signaling pathway,thereby playing a role in killing cockroaches.After Vip3Aa protein was constructed into Metarhizium anisopliae and yeast respectively,the cockroach killing activity of Metarhizium anisopliae was enhanced,and yeas has the ability to kill cockroach.Through the above research,it is helpful to understand the effect and mechanism of Vip3Aa protein to kill cockroaches.By constructing transgenic biocontrol bacteria,it is expected to provide a new choice and a new research idea for the control of cockroaches.