Functional Analysis Of Chitin Synthase Gene 1 From Plutella Xylostella

Diamondback moth,Plutella xylostella is a very notorious insect pest which attacks the cruciferous crops worldwide.Functional analysis of chitin synthase 1 gene(Px CHS1)of Plutella xylostella was done in this study.The gene silencing effects of chitin synthase 1 were investigated by feeding bacterially expressed target ds RNA(ds Px CHS1)to the DBM larvae.Gene expression analysis indicated that chitin synthase relative expression level in different tissues and developmental stages were not unique,suggesting that regulation of chitin synthesis in different tissues and different developmental stages are different.The highest chitin synthase 1 expression was observed in fourth insatar larvae whereas pupa and adults showed the lowest relative gene expression.RNAi targeting four different regions of the gene showed different efficacy against DBM larvae.ds RNA targeting 5? end 3? end showed a significant efficacy and the ds RNA targeting 5? end showed the highest efficacy.Mortality data revealed that the mortality rate of ds Px CHS1 fed larvae was increased by ~50% than control after continuous feeding for four days.Feeding bacterially expressed ds RNA resulted in abnormal phenotypes in pupa and relatively longer duration of pupariation.The caudal segment of pupa remained attached to the body disturbing pupal adult ecdysis.Interruptions occurred in pupariation caused a significant effect on adult emergence rate,lowering by 43.33% than that of control.There were no morphological differences in larval and adult stages due to ds RNA feeding.q PCR results indicated that feeding of chitin synthase gene-specific ds RNA caused lower relative gene expression level than that of control.The gene expression level was reduced by 53.5% than control at the 24 th hour after feeding ds RNA.However,at 48 th hour relative expression level has risen up showing a transient effect of RNAi by ingestion.Illumina sequencing results found 1348 new genes in DBM larvae and were annotated in COG(140),GO(406),KEGG(308),KOG(488),Pfam(573),Swiss-Prot(402),egg NOG(1003)and nr(1335)databases.The Transcriptome data revealed that RNA degradation and Spliceosome pathway genes are down-regulated in ds Px CHS1 fed larvae.Lsm proteins are found to be down-regulated in ds Px CHS1 feeding larvae suggesting that feeding chitin synthase specific ds RNA has caused reduction of m RNA decaying.However,more studies are needed to understand the effects of ds Px CHS1 on regulation of biochemical pathways in DBM.