Gene Cloning And Functional Analysis Of Clathrin Heavy Chain And Dynamin Genes From Nilaparvata Lugens(St(?)l)

The brown planthopper,Nilaparvata lugens Stal,is a kind of long-range migration pest of rice in Asia.Presently using agricultural and chemical prevetion methods cannot efficiently control the outbreak of N.lugens because of its strong adaptability.The yeast-like symbionts(YLSs),living in the abdomen of N.lugens,provide nutrition for N.lugens and play an important role in the development and virulence change of N.lugens.YLSs can be ovarially transmitted from mother N.lugens to its offsprings.The invasion of YLSs into N.lugens ovary is related to the endocytosis of ovarial follicle cells,and in this process clathrin heavy chain and dynamin are involved.In this study,four related genes,NICHC,NlDNM1,NlDNM1L-1 and NlDNM1L-2,were cloned and their bioinformatics were analyzed.Using quantitative real-time PCR(qPCR)technology,the relative expression levels of the four genes in different tissues and at different development stages of N.lugens were detected.Then immunofluorescence was conducted to detect the localization of target proteins in ovary.Finally,RNAi were carried out to explore their functions in N.lugens.The major results are concluded as follows:1.Cloning and functional analysis of clathrin heavy chain NICHC in N.lugensThe full-length of NICHC ORF was 5034 bp,encoding 1677 amino acids.PRO SITE predicted that NICHC had typical CHCR domains and the phylogenetic tree showed that NICHC was conserved in insects.The qPCR results showed that NICHC was highly expressed in the ovary of N.lugens,followed by the gut and the expression in fat body and other tissues was low.And NICHC was expressed higher in the female adults than in the nymphs,and the expression level increased in the adults along with age.Immunofluorescence results showed that NICHC was widely distributed in the cytoplasm and extracellular space of ovarial follicle cells of N.lugens,and colocalized with lipids and YLSs.After RNAi,the expression of NICHC was significantly decreased compared with the control,and the ovary development was delayed while YLSs were not detectable in oocyte after 3-day postinjection of dsNICHC.These results indicated that NICHC was important to the ovary development of N.lugens and the invasion of YLSs into N.lugens ovary.2.Cloning and functional analysis of dynamin 1 NlDNM1 in N.lugensThe full-length of NlDNM1 ORF was 2649 bp,encoding 882 amino acids.PROSITE predicted that NlDNM1 had classical GTPase,MD,GED,PH and PRD domains and the phylogenetic tree showed that NlDNM1 was conserved in insects.The qPCR results showed that NlDNM1 was highly expressed in the ovary of N.lugens,followed by the gut,fat body and other tissues.And the expression level was gently higher in the adults than in the nymphs.Immunofluorescence results showed that NlDNM1 was also widely distributed in the cytoplasm and extracellular space of ovarial follicle cells,and colocalized with lipids and YLSs.After RNAi,the expression of NlDNM1 was suppressed.On the 3rd day after RNAi,the ovary development of N.lugens was delayed and YLSs were not found in N.lugens oocyte.These results indicated that NlDNM1 played a vital role in the ovary development of N.lugens and the invasion of YLSs into N.lugens ovary.3.Cloning and functional analysis of two dynamin-l-like genes NlDNM1L-1 and NlDNM1L-2 in N.lugensThe ORF length of NlDNM1L-1 and NlDNM1L-2 was 2073 bp and 2148 bp,encoding 690 amino acids and 715 amino acids,respectively.PROSITE predicted that they both had classical GTPase,MD,GED and PH domains.The phylogenetic tree showed that NlDNM1L-1 and NlDNM1L-2 were conserved in insects.The qPCR results showed that NlDNM1L-1 and NlDNM1L-2 were both highly expressed in the ovary of N.lugens,but lower expressed in the gut,fat body and other issues.NlDNM1L-2 was expressed significantly higher in the adults than in the nymphs,while the expression of NlDNM1L-1 was relatively stable between adults and nymphs.NlDNM1L-1 and NlDNM1L-2 were prokaryotic expressed and their polyclonal antibodies with high titer and specificity were obtained.Using these antibodies,immunofluorescence was carried out and the results showed that NlDNM1L-1 was mainly expressed in the cytoplasm and extracellular space of ovarial follicle cells of N.lugens ovary,while NlDNM1L-2 was mainly expressed in the cytoplasm.Both of NlDNM1L-1 and NlDNM1L-2 were colocalized with lipids and YLSs.The expression of NlDNM1L-1 and NlDNM1L-2 were suppressed after injecting dsRNA of NlDNM1L-1 and NlDNM1L-2,and the ovary development of N.lugens was delayed on the 3rd after dsRNA injection compared with the control,and the oocyte was without YLSs.These results indicated that NlDNM1L-1 and NlDNM1L-2 was related to the ovary development ofN.lugens and the invasion of YLSs into N.lugens ovary.