Molecular Cloning And Function Analysis Of 6 BHLH Family Genes In Brown Planthopper,Nilaparvata Lugens

The rice brown planthopper(BPH),Nilaparvata lugens St?l,is one of the most destructive insect pests of rice crops in China.The insect damages rice plant by directly sucking the phloem sap and transmitting plant viruses,which causes serious economic losses.Basic helix-loop-helix(bHLH)transcription factors play important roles and control a wide array of developmental processes in eucaryotic organisms.The completion of the genome(field population)sequencing at 2014 and the transcriptome sequencing in 2013 by our group in BPH provided us with a basis to investigate the roles of bHLH proteins in the growth development processes and virulence.Based on 6 expressed sequence tags(EST)of BPH bHLH genes,we cloned and identified their coding sequence(CDS),termed as NlTai?NlTgo?NlTrh?NlSage?NlCyc and NlClk,respectively.Analysis of conserved domain and phylogenetic tree revealed that NlTai?NlTgo?NlTrh?NlCyc and NlClk comprises one bHLH domain and two PAS domains,which belong to group C,while NlSage only includes one bHLH domain.Using real-time PCR,the development and tissue-specific expression patterns of 6 bHLH genes were analyzed for revealing the role of these genes in BPH.For instance,NlTai were highly expressed in egg and female adult ovary,indicating that NlTai may be related to juvenile hormone(JH)pathway and regule vitellogenesis maturation.In addition,the expression level in 3,4 and 5 instar decreased gradually,which was similar to JH.The expression patterns revealed that bHLH transcription factors regulate several important development events in BPH.To determine the development effect of the 6 bHLH genes,dsRNA for each of these genes were injected into the appropriate instar nymphs by RNAi.The RNAi results showed that 90% BPH nymphs injected with dsNlTai,ds NlTgo and ds NlTrh died in this stage with different phenotypes,such as failing to shed their old cuticle during molting and wing deformities.By contrast,the nymphs injected with ds NlClk,dsNlCyc and dsNlSage can molt to normal adults.Those results showed that members of bHLH family were involved in different development processes.To cofirm the role of NlTai in JH pathway,we also examined the expression level of JH response gene Krüppel-homolog 1(Kr-h1),which is shown to be direct target of Tai.The level of Kr-h1 decreased in dsNlTai injected nymphs and so do the amount of lipids was reduced.The results revealed that NlTai may regulate nutrition-dependent growth rates in BPH by participating in insulin pathway and Target of Rapamycin(TOR)pathway to affect metamorphosis development processes.The data on structures,expression patterns and biological functions of bHLH family genes in BPH will facilitate further research about bHLH family genes and new pest management strategies.