Identification And Functional Verification Of Dz-daf-36 Gene Of Heterorhabditidoides Chongmingensis

Entomopathogenic nematodes(EPNs)are one kinds of insect parasitic nematodes carrying symbiotic bacteria in vivo.They have been widely concerned and studied due to their potential of wide insecticidal spectrum,high insecticidal-efficiency and environment-friend factor.Heterorhabditidoides chongmingensis is an EPN of the family Rhabditidae being symbiotic with Serratia nematodiphila.In our previous studies we found that DZ0503CMFT(DZ),the type strain of H.chongmingensis,showed a significant weakening in many aspects of growth and pathogenicity when co-cultured with non-cognate symbiotic bacterial strains.Further study of the digital gene expression profiling(DGE)sequencing libraries of the two monoxenic nematode-bacterium complexes DZ/S1 and DZ/186 explored that a majority numbers of genes closely related to the growth and development of DZ were significant up-regulation or down-regulation in DZ/186,the recombined DZ and non-cognate bacterial strain complex.In order to reveal the regulation mechanism of these differentially expressed genes on the growth and development of DZ,the present study selected dz-daf-36,a differentially expressed gene homologous to C.elegans daf-36 gene for study.The complete sequences of dz-daf-36 were amplified from cDNA template of DZ/S1.The obtained sequences were compared with transcriptome sequences and detected its open reading frames(ORFs)by Blast analysis in NCBI.Bioinformatics and phylogenetic analysis of DZ-DAF-36 protein were carried out.The results show that dz-daf-36 contain a conservative Rieske domain,and the related protein was a hydrophilic stable protein with an isoelectric point of 6.78 and made up of 5 a-helices and 22 β-sheets.In the evolutionary position,the DZ-DAF-36 protein is more homologous to parasitic nematodes.The specific fragment of dz-daf-36 gene was selected as the interference fragment by homology analysis.First,we constructed the cloning vector of the interfering fragment,and then the dsRNA expression vector of the interfering fragment by L4440 plasmid vector transformed into E.coli HT115(DE3).The RNAi fragement was thransformed into DZ by feeding method.The effect of RNAi interference was detected by real-time quantitative PCR,and the phenotypes of dz-daf-36 RNAi type DZ nematodes were observed and counted.Quantitative results showed that the expression of dz-daf-36 gene in DZ was significantly knocked down after RNAi with the highest reduction of 56.65%compared with the control group.Life span statistics analysis showed that the average lifetime of DZ nematodes were reduced to 9.03 days from 12.07 days in wild-type and 10.76 days in empty vector groups,and decreased by 25.19%and 16.03%,respectively.There was no significant difference in pregnancy,egg hatching rate,larval development rate and female-male ratio,but the larval diapause of the disturbance group increased by 5.93%.The growth rates of the larvae in the disturbance groups were significantly slower than that in the wild-type and empty vector groups,and need a longer development time(48 h)to adult.There was no significant difference in body length of each group.In this study we successfully obtained the sequences of dz-daf-36 gene and analyzed its physical and chemical properties and biological characteristics.The biological functions of dz-daf 36 gene to DZ nematode were confirmed by RNAi method which will be benefited to the study of other regulatory genes of the nematodeDZ or other EPNs.