Cloning Of The Immune-related Gene Of Laodelphax Striatellus And Its Functional Analysis Against RSV Immune Response

The Laodelphax striatellus can damage rice plants by acts as vector for the transmission of rice stripe virus(RSV), which can cause even more serious rice yield loss. The traditional method of chemical control of this pest was ineffective, not only polluted the environment, but also led to the increased resistance to pesticides. It is need to explore the new aspects to control this pest. It is the key to know the interaction mechanisms between RSV and host L. striatellus.The innate immune system of insects is important to defense against pathogenic microorganisms and is promptly activated by the recognition of pathogen-associated molecular patterns(PAMPs), which can be recognized by specialized pattern recognition receptors(PRRs), Toll-like receptors(TLRs), Peptidoglycan recognition protein (PGRP) and gram-negative binding protein(GRP) are three major protein families member of the PRR families. Therefore, to further elucidate the mechanism of anti-RSV immunity response of L. striatellus, our study based on the EST database of L.striatellus and use it as a research object,using RACE-PCR,we cloned and characterized eight genes from L.striatellus; Quantitative RT-PCR analysis was used to detect the expression profiles of genes transcripts in different individuals;we injected E.coli to L. striatellus with RSV infection to analyzed the expression of LsToll-8, LsToll-13 and RSV-RNP; RNAi was used to explore the function of the related genes in L.striatellus by feeding dsRNAs; In addition, the bioinformatic analysis of LsToll-13 was made. The result is as follows:(1) Eight full-length cDNAs were cloned by RT-PCR and RACE-PCR from L.striatellus, including two PGRPs, three GRPs, two TLRs, Defensin B,designated as LsPGRP-LB,LsPGRP-LC,LsGRP2,LsGRP4,LsGRP5,LsToll-8,LsToll-13, LsDefensin B. Which laid a foundation for the exploration of genes functions.(2) Quantitative RT-PCR was employed to quantify the expression profiles of genes transcripts in female adults and male adults of high-viruliferous(RSV-infected)and naive strains, respectively. The results showed that the genes were ubiquitously expressed in all vector types of L. striatellus, and the expression levels of LsToll-8,LsToll-13, LsGRP2 in L.striatellus with RSV infection were significantly suppressed than Naive strains control, but, the expression levels of LsGRP5, LsPGRP-LB,LsPGRP-LC were significantly increased.(3) Quantitative RT-PCR was used to detect the expression of LsToll-8, LsToll-13 and RSV-RNP of viruliferous(RSV) nymphs of L.striatellus after E.coli stimulation,respectively. The results showed that the expression level of LsToll-13 was significantly up-regulated, and the expression level of RSV-RNP mRNA was significantly decreased at 6h after E.coli infection, but, there was no change of LsToll-8.(4) RNA interference was used to explore the function of five genes in L.striatellus by feeding dsRNAs. The results showed that only upon silencing LsToll-13 using RNAi in L.striatellus , the expression level of RNP was significantly increased, the results demonstrate that LsToll-13 play an important role in anti-RSV immune response of L. striatellus. By the bioinformatic analysis,modling the 3D structure of LsToll-13 protein and compared with the overall structure of a dimeric TLR13 complex of mouse,elucidating the recognition mechanism of LsToll-13 protein involved in the recognition of ssRNA.In summary,this study not only cloned and functionally characterized eight genes in L.striatellus for the first time, but also provided a new insight into further elucidating the molecular mechanism of antiviral immune response in L.striatellus.the results demonstrate that LsToll-13 play an important role in anti-RSV immune response of L. striatellus.