Gene Cloning And Functional Analysis Of Endophilins From Nilaparvata Lugens (Stal)

The brown planthopper(BPH),Nilaparvata lugens(Stal)(Hemiptera:Delphacidae),is the most destructive insect pest of rice in Asia,with the characteristics of migratory,easily-outbreaking and gregarious.N.lugens causes serious harm to Chinese rice production through feeding rice,spawning and transmitting rice virus.The long-term use of chemical pesticides makes N.lugens develop strong resistance.Now,the promotion and cultivation of resistant rice varieties is one of the most important ways to control N.lugens.With the virulence change of brown planthopper,the resistance of resistant rice varieties is gradually lost.The virulence variation is associated with yeast-like symbionts(YLSs).YLSs play vital roles in the growth and development of N.lugens.YLSs are gathered in the abdomen fat body of N.lugens,and they are ovarially transmitted between the generations of N.lugens.In this study,two endophilin genes of N.lugens were cloned,and their roles in the ovarial development of N.lugens and the invasion of YLSs into N.lugens ovary were studied through bioinformatics analysis,expression pattern analysis and RNAi.The results are concluded as follows:1.Sequence analysis of endophilin A.endophilin B from N,lugensTwo endophilin genes,endophilin A(Endo A)and endophilin B(Endo B),were cloned from N.lugens,and their GenBank accession numbers are KY126096 and KY126095,respectively.The cDNA sequence of Endo A contains an entire 1164 bp open reading frame,encoding 387 amino acids with a predicted molecular weight of 43.075 kDa.The cDNA sequence of Endo B contains an entire 1059 bp open reading frame,encoding 352 amino acids with a predicted molecular weight of 39.003 kDa.The results of the amino acid alignment and the phylogenetic tree analysis indicate the conservation of Endo A and Endo B in insects.The predictions of the secondary and tertiary structures of Endo A and Endo B showed that they both contain the typical BAR domain and SH3 domain,but with big differences in BAR domain,indicating that Endo A and Endo B may be different in functions.2.Expression feature of Endo A and Endo BBy qRT-PCR,the expression of Endo A and Endo B at the different development stages and in the various tissues of N.lugens were analyzed.The results showed that Endo A and Endo B were expressed at the different development stages of N.lugens.The expression level of Endo A in the 1?5 instar nymphs was relatively stable,but in adult the expression level was generally increased within days after emergence.The expression level of Endo B in the 1?5 instar nymphs and adults of N.lugens was relatively stable.Both Endo A and Endo B had the highest expression in the ovary among detected tissues of N.lugens3.Prokaryotic expression,polyclonal antibody preparation and expression localization of Endo A and Endo BThe prokaryotic expression systems of Endo A and Endo B were built,and fusion proteins were purified to prepare polyclonal antibody.The titer of the obtained antibodies were estimated as high as 1:1000000 dilution ratio through ELISA,and the antibodies had good specificity as shown by western blot.The results of immunofluorescence showed that Endo A and Endo B were widely expressed in N.lugens ovary,and the distributions of Endo A and Endo B were similar to that of lipids.Endo A and Endo B were also colocalized with YLSs invading into N.lugens ovary.These results indicated that Endo A and Endo B were related to the absorption of lipids and maturation of N.lugens ovary,as well as the invasion of YLSs into N.lugens ovary.4.RNAi of Endo A and Endo BDsendo A and dsEndo B were synthesized in vitro,and were injected into the late 5th instar nymphs,respectively.After RNA interference,Endo A and Endo B were significantly downregulated at the transcriptional level,and the expression of Endo A and Endo B in the ovarial follicle cells was correspondingly decreased.After RNA interference,the ovary development of the females at the 3rd days after emergence was delayed compared with the control,and meanwhile,YLSs did not enter the ovary.The results confirmed the important roles of Endo A and Endo B in the ovary development of N.lugens and the invasion of YLSs into N.lugens ovary.