Cloning And Identification Of Serine Protease Inhibitor Gene Of Nilaparvata Lugens And Functional Analysis Of Nlserpin2

Serine protease inhibitor(serpin)is a superfamily of protease inhibitors with conservative structure and diverse functions that widely exist in insects.It negatively regulates the immune defense response mainly by inhibiting the serine protease cascade.Brown Planthopper(BPH)is an important pest of rice in China.Microbial control based on pathogens and symbionts is an effective way to achieve control of BPH.The control efficacy of microbial insecticides is closely related to the immune defense of host insects.In-depth study on the immune defense mechanism of BPH is of great significance for the development of BPH based on microbial pathogens and symbionts.In this study,the serpin superfamily genes of BPH were cloned and identified,their temporal and spatial expression rules and microbial induced expression patterns were detected by fluorescence quantitative PCR,and then the biological functions of Nlserpin2 gene in the process of growth and immune defense of BPH were analyzed by RNAi technology.Our results will lay a theoretical foundation for the study of microbial immune defense mechanism of BPH,and provide scientific basis and genetic resources for the development of new microbial control technology of BPH.The main results are as follows:1.Cloning and sequence analysis of BPH serpin superfamily genes: Based on the transcriptome and whole genome data of BPH,eight serpin genes of BPH(Nlserpin1,Nlserpin2,Nlserpin3,Nlserpin4,Nlserpin5,Nlserpin6,Nlserpin7 and Nlserpin8)were successfully cloned.All of them have the conserved domain “serpin” that unique observed in serpin superfamily.The C-terminal contains a reactive central loop(RCL)composed of about 20 amino acids,and the predicted molecular weight is between44.9 k D(Nlserpin3)and 62.0 k D(Nlserpin5).Except Nlserpin8,the other seven BPH serpin proteins contain signal peptides at the N-terminal.Subcellular localization prediction shows that they are located in the cytoplasm or secreted outside the cell,indicating that they are likely to function in the form of secretory protein.Phylogenetic analysis showed that the insect serpin was relatively conservative in evolution,and the serpin of BPH(except Nlserpin8)was closely related to the serpins of other insects belonging to Hemiptera,suggesting that they may have similar biological functions.2.The expression pattern analysis of BPH serpin superfamily gene: The q RT-PCR analysis showed that BPH serpin superfamily genes had obvious temporal and spatial expression specificity.Eight serpin genes were expressed in all developmental stages of BPH,among which the expression of Nlserpin1,Nlserpin5 and Nlserpin8 in the nymphal stage was significantly higher than that in the adult stage,while the expression of Nlserpin2,Nlserpin3,Nlserpin4 and Nlserpin6 was relatively high in the adult stage,and the expression of Nlserpin7 was stable in the whole growth period of BPH,with no significant difference in each stage.The expression of Nlserpin1-5 in the throax was significantly higher than that in other tissues,while the expression level of Nlserpin6-8 was the highest in the head.The expression level of Nlserpin4 was the highest in the intestine,fat body and ovary,and the expression levels of Nlserpin7 and Nlserpin8 were lower in all tissues.The expression patterns of each gene induced by different types of microorganisms are different.The entomopathogenic fungus Metarhizium anisopliae can significantly induce the expression of Nlserpin2,Nlserpin3 and Nlserpin6.After inoculation with the symbiotic fungus Pichia guilliermon,the expression levels of Nlserpin2 and Nlserpin3 genes decreased significantly,while the expression of other genes showed an upward trend.The expression levels of Nlserpin4,Nlserpin5,Nlserpin6 and Nlserpin8 were up-regulated in varying degrees under the stress of pathogenic bacteria Escherichia coli(Gram-negative)and Staphylococcus aureus(Gram-positive),while the expression level of Nlserpin1 gene had no significant change.The above results showed that the contribution of each gene to the immune interaction between BPH and different types of microorganisms was different.3.Biological function analysis of Nlserpin2: Nlserpin2 that significantly induced by entomopathogenic fungus after stress but significantly down regulated after treatment by symbiotic fungus were selected for biological function analysis.The results showed that microinjection of ds Nlserpin2 could significantly inhibit the expression of Nlserpin2.RNA interference with Nlserpin2 could significantly reduce the survival rate of BPH.For example,after 5 and 8 days,the BPH survival rates in the Nlserpin2 interference group were 66.1% and 47.2% respectively,which were 31.7% and 49.0%lower than those in the control group(ds GFP).After Nlserpin2 knockdown,the number of eggs decreased by 80.7% significantly compared with the control group,but there was no significant difference in egg hatching rate.The bioassay results showed that Nlserpin2 knockdown significantly reduced the resistance of BPH to entomopathogenic fungus.For example,after microinjection of M.anisopliae,the cumulative corrected mortality of BPH on the 7th day was 70.9%,which was 15.5%higher than that of ds GFP control group.The corrected survival rates of Nlserpin2 silenced BPH were 42.0% and 23.9% after infected by M.anisopliae for 5 and 8 days,respectively,which were decreased by 28.4% and 31.0% respectively when compared with the control group.However,Nlserpin2 knockdown caused no significant change in the tolerance of BPH to the symbiotic fungus Pichia guilliermon.The above results show that Nlserpin2 plays important roles in the development and immune defense of BPH.