Heterologous Expression Of Helicoverpa Armigera HaVipR And 8 Midgut Membrane Proteins And Their Functional Identification In Vip3Aa Insecticidal Mechanism

The cotton bollworm（Helicoverpa armigera Hübner）is one of the most important agricultural pests in the world,chemical control is the main way to control it in the field over the years.Since the planting of Bt cotton in the world from 1996,the first generation Bt cotton with the Cry1Ac gene and the second generation Bt cotton with the CrylAc+Cry2Ab gene have achieved good results in the control of H.armigera.However,under the long-term selection pressure of Bt protein,the field population of H.armigera developed high resistance to Cry1Ac,which seriously affected the control of H.armigera.The third generation of Bt cotton which simultaneously expresses CrylAc+Cry2Ab+Vip3Aa protein has been used to control H.armigera in the field and has been widely cultivated in Australia.Vip3Aa protein is a non-crystalline protein secreted extracellularly during the vegetative growth stage of Bacillus thuringiensis,and has a good effect on the control of a variety of lepidopteran insects.Vip3Aa has a low homology similarity with Cry-like proteins.It is speculated to have different modes of action,and hard to produce cross-resistance.It has a broad application prospect in gene-stacked transgenic Bt crops.In the previously study,by the reverse genetics analysis it was proved that the knockout of the H.armigera VipR（HaVipR）gene is genetically linked to the resistance of Vip3Aa,and the knockout strain has over 250-fold resistance to Vip3Aa.However,due to the lack of understanding of the basic characteristics of HaVipR,the specific molecular mechanism of knockout resistance to Vp3Aa is still unclear.Here,firstly the sequence characteristics of HaVipR genes in the 3 populations of H.armigera SCD,SW and AY were analyzed,and phylogenetic analysis of the orthologous genes from 5 lepidopteran insects was preformed;then the cellular localization and characteristics of H.armigera VipR protein was identified.Finally,through a series of experiments such as ligand binding,saturation binding,Pull-down,Co-IP and cytotoxicity experiment,the interaction with Vip3Aa and the role in the Vip3Aa toxicity of HaVipR and other 8 midgut membrane proteins were verified.All results provide an important foundation for the study of insecticidal mechanism of Vip3Aa.The results are as follows:1.Polymorphism analysis of HaVipR of H.armigera and phylogenetic relationship of VipR in 5 lepidopteran insectsUp to now,only the HaVipR gene of H.armigera has been proved to be directly related to Vip3Aa resistance.In this study,the sequence characteristics of HaVipR in 3 sensitive populations of H.armigera,SCD,SW and AY,were analyzed.The a mino acid sequence polymorphism of SW in the 3 populations was highest,while the polymorphism of SCD and AY was low.At the same time,the VipR genes of 5 species of lepidopteran insects:H.armigera,Spodoptera frugiperda,Spodoptera exigua,Spodoptera litura,and Plutella xylostella,were cloned and phylogenetic analysis were performed.The identification of a mino acid sequences between these 5 lepidopteran VipR was～73.64%,and there are multiple conserved domains among these orthologs.Finally,ExPASy,TMHMM-2.0,SignalP,TargetP and PredGPI programs were used to predict the physical and chemical properties and protein characteristics of VipR protein from 5 lepidopteran insects.The results indicate that the molecular weight of VipR protein is about 47 KDa,which are all acidic hydrophilic proteins with weak stability.Siganl peptides are found in the protein but no transmembrane regions exist,they are not blong to GPI anchored proteins families.Therefore,it is supposed that VipR is a secreted protein.This study identifide the a mino acid sequence,sequence polymorphism,phylogenetic relationship and protein properties of VipR in multiple lepidoptera insects,and laid the foundation for the functional study of HaVipR.2.Heterologous expression and subcellular localization of HaVipR proteinHaVipR is presumed to be a secreted protein.Therefore,after fusion with GFP marker,the wildtype H.armigera HaVipR and the mutant HaVipR-KO were expressed in vitro by insect cell baculovirus expression system（Bac-to-Bac）and insect select BSD insect cell transient expression system simultaneously.The results of cell fluorescence localization and Western Blot indicated that:HaVipR and HaVipR-KO proteins could be successfully expressed in both systems;recombinant HaVipR protein could be detected in cell supernatant medium,cell membrane and cytoplasm.Recombinant HaVipR-KO protein cannot be detected outside the cell,it only exists inside the cell and on the cell membrane.This study successfully expressed HaVipR in vitro and identified the cellular localization of HaVipR,providing clues for the further study of HaVipR.3.Interaction between HaVipR protein and Vip3Aa proteinPrevious studies supposed that HaVipR might be a specific binding receptor for Vip3Aa protein,or may play a key role as a bridge in the cellular toxicity of Vip3Aa.In this sduty,it was found that HaVipR can not degrade Vip3Aa toxin after incubating the two proteins in vitro.The results of ligand binding,saturation binding,Pull-down and Co-IP showed that neither Vip3Aa protoxin nor activated toxin can bind to recombinant HaVipR protein directly.The activated Vip3Aa toxin and recombinant HaVipR can not cause the death of Sf9 cells.Therefore,it can be basically ruled out that HaVipR is a specific binding receptor for Vip3Aa protein.It is suspected that there are other proteins involved in the insecticidal effect of Vip3Aa except HaVipR.4.The role of HaVipR and 8 midgut membrane proteins in the cellular toxicity of Vip3AaPrevious studies have shown that the HaVipR of H.armigera cannot directly interact with Vip3Aa protoxin and activated toxin.Based the secretory characteristics of HaVipR,there might be other key factors involved in the toxicity of Vip3Aa.The membrane protein might be those factors.In this study,8 membrane proteins within 23 Vip3Aa binding proteins obtained from BBMV of H.armigera midgut are investigated.They are Neutral ceramidase,APN1,APN2,APN3,APN4,Leucine-rich repeat neuronal protein 3,ALP2 and Integral membrane protein.The 8 genes were cloned and the GFP tag was fused with them before heterologous expression.At the same time,HaVipR of H.armigera fused with mCherry red fluorescence gene was also successfully expressed in vitro.Cytotoxicity experiments were conducted with protoxin and activated toxin of Vip3Aa,and it was found that Vip3 Aa was not toxic to Sf9 cells under the co-expression of 8 membrane proteins and HaVipR.Therefore,these 8 midgut membrane proteins are not the key factor as supposed previously.This study provides an important basis for the further study of insecticidal mechanism of Vip3Aa protein.