Cloning,expression And Binding Characterization Of Empoasca Flavescens Potential Receptor APN 7 Against Cry Toxin

Tea plant is one of the most important economic crops in China.However,it is endangered by a variety of pests and diseases,which are difficult to be controlled and cause great economic losts.Among these pests,Empoasca flavescens is a dominant species.Because of chemical control,this insect is resistant to many chemical agents.Therefore,it is of great significance to develop biological pesticide to control Empoasca flavescens.Bacillus thuringiensis is one of the most popular and widely studied biopesticides in the world.However,it is not high toxicity on Empoasca flavescens.As we know,there are two main steps on Bt insecticidal mechanism:enzymolysis of protoxin in the insect midgut and the specific binding of intestinal receptor.In our previous research,enzymolysis of Cry protoxin has been detected in Empoasca flavescens.In order to explore whether potential receptors exist in Empoasca flavescens or whether binding happends between Cry and potential receptors,research of Bt mechanism on Empoasca flavescens has been focused on receptors in this study.Firstly,many potential receptor genes such as ALP,APN and Cadherin are detected according to the Empoasca flavescens transcriptome data obtained in our previous reseach.The relatively expression level from some of these candidate genes were verified by RT-qPCR.According to the values of FPKM,eight ALP genes,seven APN genes and eight Cadehrin genes were identified with high expression level and significant different expression between in the intestine and other tissues.Then,APN 7 with the highest expression level in the midgut was selected according to the message of NJ evolutionary tree for intraspecific homology analysis and GPI targeting site.In order to know whether it could bind with Cry,the target gene APN 7 was further cloned,expressed and purified successfully.Result of far-western blot showed that APN 7 could bind to Cryl A and Cry11A,which could both be hydrolyzed into activated mature peptides by gut lumen and membrane fraction of Empoasca flavescens,but the binding ability against Cry11A was stronger than that of Cry1 A.Meanwhile,binding analysis was also carried out between APN 7 and three molecular modified Cryl Ab toxins after short peptide R replacement,whose toxicities were some higher than that of CrylA protoxin.Results of far-western blot showed that bindings could all be detected between APN 7 and Cryl Ab-1R,Cryl Ab-2R as well as Cryl Ab-3R.However,the binding of the modified toxins was all a little stronger than Cryl A toxins,expecially Cryl Ab-3R.It could be deduced that APN 7 might be a candidate receptor for Cry toxins,and the toxicity improvement of the modified Cryl A might relate to the binding ability beween APN 7 and Cry toxins in a certain degree,but their limited binding could not make tested Cry with higher toxicity.To sum up,we found some Bt potential receptors existed in the transcriptome of Empoasca flavescens,such as APN,ALP and Cadherin.Because of the highest expression level and GPI targeting site,APN 7 was cloned,expressed and purified.Binding assay between APN 7 and different tested Cry toxins showed that it could be a candidate receptor for some Cry toxins,which will great enrich the knowledge of Bt mechanism against Empoasca flavescens.