Functional Identification Of Gsa-1 And Goa-1 Genes Of Heterorhabditidoides Chongmingensis

Entomopathogenic nematodes(EPNs)are a new type of highly effectivenon-polluting biological control agents received worldwide attention as an environment-friendly alternative to chemical pesticides.EPNs have a highly specific lifestyle and share mutual symbiosis with the symbiotic bacterium carried in their intestines.EPNs mainly use the allelopathic systems such as chemosensory and humidity sensing to actively search for the host.Therefore,the study of biological traits such as olfactory chemotaxis and adaptation is of great significance for the commercial application of EPNs.Heterorhabditidoides chongmingensis DZ0503CMFT(DZ)is an entomopathogenic nematode identified and preserved in the early stage of this lab.According to previous studies,DZ nematode associated with its own symbiotic bacteria Serratia nematodephila DZ0503SBS1T(S1)or its non-native bacterial strain S.nematodiphila DR186(186)formed two monoxenic nematode combinations DZ-S1 and DZ-186,show significant difference in the body length,lifespan,reproduction rate and female ratio.This study further found that there are also a significant difference in olfactory chemotaxis and adaptation.In order to explore the molecular regulation mechanism of olfactory of nematodes,this study screened two differentially expressed gene,G protein-coupled receptor Ga subunits gsa-1 and goa-1,from digital gene expression profiles(DGE)libraries of the two monoxenic nematode combine DZ-S1 and DZ-186.The functions of genes gsa-1 and goa-1 were identified.The main research methods taken and the main findings obtained are as follows:Infected juveniles of DZ-S1 and DZ-186 had significant differences in olfactory chemotaxis,and accordingly,the relative expression levels of gsa-1 and goa-1 in DZ-S1 were significantly down-regulated comparing with DZ-186.The olfactory chemotaxis index of DZ nematodes to benzaldehyde and live Galleria mellonella larvae were higher in DZ-S1(2.99 and 8.62 times of that of DZ-186,respectively).The olfactory adaptation of nematodes to the benzaldehyde in the DZ-S1 group was also stronger than that in the DZ-186 group.The complete sequences of gsa-1 and goa-1 were amplified and verified by sequencing.The complete ORF(Open Reading Frame)sequences of the GSA-1 and GOA-1 proteins were predicted by the ORF Finder function in NCBI.BLAST was performed using NCBI based on its predicted amino acid sequence,which showed that GSA-1 and GOA-1 were highly conserved with the corresponding amino acid sequences of other parasitic nematodes.The phylogenetic trees of related nematodes were constructed based respectively on amino acids sequences of GSA-1 and GOA-1,which showed that DZ nematode are Caenorhabditis elegans and Haemonchus contortus’ sister group,respectively.Real-time PCR was used to detect the relative expression of gsa-1 and goa-1 in nematodes of DZ-S1 and DZ-186,which showed that the gsa-1 and goa-1 relative expression levels in DZ-S1 were significantly down-regulated(gsa-1 down-regulated 34.30%and goa-1 down-regulated 88.83%).According to the NCBI database,domain prediction and conserved region analysis of gsa-1 and goa-1 were performed,and part of the homologous fragments was selected for RNAi.The RNAi fragment was amplified and the PCR product was ligated into T-Vector pMD19(Simple)and transformed into DH5α competent cells.The positive clone-bearing transformants were amplified and subjected to plasmid extraction,ligated into the vector L4440 plasmid by double digestion and transformed into E.coli HT115(DE3)for amplification.The RNAi of genes of DZ nematodes was carried out by feeding method,and then the interference effect was detected by real-time PCR.After RNAi of gsa-1,its expression level was inhibited by 54.19%compared with the L4440 control group.After goa-1 RNAi,the goa-1 expression level was inhibited by 55.21%compared to the L4440 control group.Changes in olfactory chemotaxis,olfactory adaptation,body length,lifespan,spans of four physiological stages,reproduction rate,hatching rate and female rate of DZ nematodes after RNAi were recorded and statistically analyzed.After gsa-1 RNAi,the chemotaxis of DZ nematode to benzaldehyde and live Galleria mellonella larvae was significantly increased,and the chemotaxis index was 4.22 and 2.14 times of the control,respectively.The average number of eggs laid per female decreased significantly by 47.4%;the hatching rate was significantly reduced by 10.31%;there were no significant difference in olfactory adaptation,body length,lifespan,spans of four physiological stages.After goa-1 RNAi,the chemotaxis of DZ nematode to benzaldehyde and live Galleria mellonella larvae was significantly increased after RNAi with the chemotaxis index of 1.74 and 2.77 times of the control,respectively.The body length of goa-1 RNAi DZ nematode was significantly decreased.The lifespan of goa-1 RNAi DZ nematodes was significantly prolonged with a special style of prolonged rapid tail-swinging stage and rapid pumping stage of pharynx.The amount of eggs laid per female of the goa-1 RNAi nematodes was significantly increased by 60.73%.In addition,there were no significant difference in olfactory adaptation,hatching rate and female rate in goa-1 RNAi nematode.In this study,the biological functions of gsa-1 and goa-1 in DZ nematodes were identified by RNAi.The gene gsa-1 is involved in the regulation of the olfactory chemotaxis and egg formation of DZ nematodes.The gene goa-1 is involved in the regulation of the olfactory chemotaxis,growth and oviposition behavior of DZ nematodes.