Molecular Characteristics And Functional Analysis Of Halloween In Locusta Migratoria

Locusta migratoria belongs to Orthoptera.it can be found in Africa,the East Indies,and most parts of China.L.migratoria mainly harms wheat,sorghum and crops,which seriously affects the agricultural and animal diversity in the world.L.migratoria is incomplete metamorphosis insect which metamorphosis accompanied by molting.Normal molting is vital to insects throughout their life cycle.Insect molting and metamorphosis are regulated by two important hormones which are juvenile hormone(JH)and 20-hydroxyecdysone(20E),therefore,the Halloween genes involved in 20 E synthesis have attracted widespread attention from scientists,and they are potential new targets for pest control.In this study,we depict here the cloning and characterization of Lm Nvd,Lm Spookier(CYP307A2 spok),Lm Phantom(CYP306A1;phm),Lm Disembodied(CYP302A1;dib),Lm Shadow(CYP315A1;sad)and Lmshade(CYP314A1;shd)from L.migratoria.Expression characteristics of six Halloween genes were evaluated by RT-q PCR.Ultimately,the influence of six Halloween genes to nymphs development were well studied by RNAi.1)Molecular characteristics and functional analysis of Lm NvdIn this study,we identified and characterized a c DNA of Lm Nvd which encoding ecdysteroid biosynthesis enzyme according to L.migratoria transcriptomics data.The open reading frame(ORF)of Lm Nvd is 1,311 bp encoding a putative protein of 436 amino acids RT-q PCR revealed that Lm Nvd was highly expressed in prothoracic gland and the seventh day of prothoracic gland of the fifth-instar nymphs.After RNAi of Lm Nvd with ds RNA,the nymphs showed obvious molting delay,molting failure and curled wing pads which can be rescued by 20-hydroxyecdysone(20E)or 7-deoxycholesterol(7-DC).In addition,H&E staining of integument also indicate that apolysis inthe ds Lm Nvd and 20E-treated nymphs were earlier than in the ds Lm Nvd-treated nymphs.Finally,RT-q PCR analysis showed that the transcription level of Lm E75(20E signalling pathway gene)and Lm CHT5(chitin degradation genes)were dramatically declined and that of Lm CHSI(chitin synthesis genes)substantially up-regulated based on RNAi of Lm Nvd compared to those of ds GFP-injected nymphs.All results implying that Lm Nvd can affect molting and wing pads development of L.migratoria by regulating 20 E synthesis.2)Identification of five Halloween genes and functional analysis of Lm Spookier in L.migratoria.Here we analyzed the characteristics and functional of Lm Spookier,Lm Phantom,Lm Disembodied,Lm Shadow and Lmshade from L.migratoria.we first identified and characterized five Halloween genes according to L.migratoria transcriptome data.The ORFs of Lm Spookier,Lm Phantom,Lm Disembodied,Lm Shadow and Lmshade are 1,578 bp,1,470 bp,1,581 bp,1,461 bp and 1,605 bp,which encoding putative proteins of 525,489,526,486 and 534 amino acids,respectively.RT-q PCR results revealed that transcription level of 4 Halloween genes are highest in the prothoracic gland except to Lm Shade is highly expressed in the ovary and that 5 Halloween genes all have high level expression from fifth to seventh days of fifth instar nymphs.The RNAi was used to explore function of five Halloween genes from L.migratoria.After RNAi of five Halloween genes,we analyzed silencing efficiency and effects on development of nymphs revealed that ds Lm Spookier-injested nymphs performed obvious molting delay,molting failure and curled wing pads which also can be rescued by 20 E.H&E staining of integument also confirmed this conclusion.Finally,RT-q PCR revealed that Lm CHS1(chitin synthesis gene)was significantly up-regulated and that Lm CHT5(chitin degradation gene)and Lm CHT10(chitin degradation gene)were significantly down-regulated.Results reveals that Lm Spookier can affect molting andwing pads development of L.migratoria by regulating 20 E synthesis.