Function Analysis Of Tetraspanin AglaTSP In Anoplophora Glabripennis Innate Immunity

Anoplophora glabripennis is a typical borer pest of forest trees,which has caused great harm to world forestry production and ecological environment.The role of A.glabripennis tetraspanin AglaTSP in innate immune system was examed and identified in this paper to set up a theoretical foundation for its biological control.After the gene sequence of AglaTSP was analyzed by bioinformatics method,the spores and spore lysates of Beauveria bassiana,Bacillus thuringiensis(Bt)and bacterial lysates were used as immune interfering reagents to challenge A.glabripennis adults,by real-time fluorescent quantitative PCR(qRT-PCR),RNA interference(RNAi)and other techniques,the relative gene expression of tetraspanin AglaTSP and five immune-related proteins of A.glabripennis and activity of phenol oxidase were detected,and the activity of the tested insects caused by immune interference were monitored,finally,according to KEGG pathway about insect immunity and experimental results,the immunity pathway of A.glabripennis with AglaTSP was drawn.The main results are as follows:(1)AglaTSP had the typical tetraspanins structure,its aa sequence included of CCG(Cys-Cys-Gly)and GC conserved sequences.AglaTSP was relatively conservative in evolution,but the similarity of gene sequences between AglaTSP and other tetraspanins was low.NCBI Blastx analysis revealed that AglaTSP belonged to CD63 subfamily.KEGG pathway analysis revealed that AglaTSP was CD63 antigen and was involved in the ko04142(lysosome)pathway.(2)By immune interference,in A.glabripennis adults,the AglaTSP gene relative expression quantity was significantly up-regulated within 24h(P<0.01),especially at 6h after B.bassiana interference(P<0.01),and the relative espression was highest at 18h(P<0.01).After B.bassiana spore lysates interference,the relative expression of gene was significantly up-regulated at 6h(P<0.05),up-regulated at 18h(P<0.01),and after 24 h it was down-regulated.The relative expression of AglaTSP gene was significantly up after 12 h of immunization with B.thuringiensis and bacterial lysates(P<0.01).(3)AglaTSP dsRNA was synthesized according to the specific sequence fragment of AglaTSP.After RNAi,the relative expression of AglaTSP gene in A.glabripennis significantly decreased at 48h(P<0.01),and after RNAi treatment 72 h,the gene expression was silenced at the highest level(66.5%).(4)Four immune interfering reagents were used to challenge before RNAi and after RNAi A.glabripennis,and the activity of samples were monitored for 24 h.The five treatments mortality about adults of before RNAi challenged by four immune interfering reagents and after RNAi stimulated by sterile water rates were 0,while all the AglaTSP RNAi samples were killed after Bt and bacterial lysates challenged at 12 h and 18 h,and the mortality were 50% and 40% in 24 h after infection with B.bassiana and spore lysates.(5)The phenoloxidase activity of A.glabripennis was detected.The phenoloxidase activity of the pests without RNAi increased first and then decreased.The phenoloxidase activity of RNAi adults increased significantly after 3h by Bt and its lysates challenged(P<0.01),then decreased,and at 12 h there was no significant difference between CK(P<0.05).The highest phenoloxidase activity of A.glabripennis with AglaTSP RNAi in each group was significantly lower than that non-RNAi samples(P<0.01),revealed that AglaTSP influence the phenoloxidase activity of A.glabripennis in the immune regulation process.Real-time fluorescence was used to detect the genes relative expression quantity of peptidoglycan recognition protein,?-1,3-glucan recognition protein(Signal recognition),serine protein(signal modulation and amplification),Toll receptor protein(signal transduction),phenoloxidase(immune effect)in non-and AglaTSP RNAi samples.The results showed that AglaTSP RNAi in A.glabripennis cannot affect recognition of peptidoglycan recognition protein and ?-1,3-glucan recognition protein.And the other three functional protein genes relative expression was significantly lower than that of the insects without RNAi.AglaTSP was responsible for receiving the intrusion signal of non-self substances recognized by signal recognition proteins in the immune response of A.glabripennis,and transferring the signal to signal regulation related proteins.(6)Combining the KEGG pathway of insect immunity with five proteins and the above experimental results,AglaTSP was located in immune response process of A.glabripennis between the signal recognition and signal modulation processes.And the related immune pathway of A.glabripennis with tetraspanin AglaTSP was mapped.In conclusion,the tetraspanin AglaTSP participates in the innate immune regulation process of A.glabripennis,and transmit intrusion signals from recognition molecules to signal modulators.This study improved the further understanding of the immune molecular mechanism and ecological adaptation mechanism of A.glabripennis,and proposed a new strategies and targets for pollution-free control of similar pests.