Identifying Target Genes Of Differentially Expressed Novel Micrornas In Two Monoxenic Nematode Complexes Of Heterorhabditidoides Chongmingensis

Entomopathogenic nematodes(EPNs)are a kind of parasitic nematodes which can use insects as the hosts.The pathogenicity of EPNs is generated by the symbiotic bacteria that have the mutualistic symbiosis relationship with them.To date,three kinds of EPNs have been found,Heterorhabditidoides,Heterorhabditidae and Steinernematidae.DZ0503CMFT(DZ),the type strain of Heterorhabditidoides chongmingensis was isolated from the Chongming Island in Shanghai and has a stablely mutual relationship with its symbiotic bacteria strain DZ0503SBS1T(S1)of Serratia nematodiphila.Previous studies found that the expression of miRNAs in DZ-S1 and DZ-186,the two monoxenic nematode-bacterial complexes,were significantly different revealed by Solexa sequencing technology.In order to understanding the relationship between miRNAs and symbiotic bacteria in the two monoxenic nematode combinations,we obtained the precursor sequences of the two differentially expressed novel miRNA n-miR-9 and n-miR-10 by PCR amplification.Their sequences were 293bp and 330bp,respectively.Overexpression vector of n-miR-9 and n-miR-10 was successfully constructed by molecular biology.The overexpression vector was transfected into DZ-S1 nematode and cultured for 3 days.Total RNA was extracted and inversely transcripted into cDNA.The expression of candidate target genes was detected by qRT-PCR.The results showed that the expression of n-miR-9 candidate target genes was significantly different,showing up-regulation.n-miR-10 candidate target genes were significantly down-regulated.Growth and development experiments showed that n-miR-9 inhibited the development of DZ nematodesFor further identification the target genes of n-miR-9 and n-miR-10,were cloned and inserted into the plasmid pPD95.67 containings green fluorescent protein to construct recombinant plasmid which was then cotransformed into DZ nematodes with the overexpression vector of n-miR-9 and n-miR-10 by feeding method.Results showed that three days later,there was no significant difference of green fluorescence in the control group,whereas the green fluorescence of the experimental group of target genes DZMN9-3 was weakened in the DZ nematodes.We speculated that n-miR-9 also had other target sites,that could regulate the transcription factor such as promoters for improving the expression of genes.N-miR-10 had a significant decrease in the fluorescence of the potential target gene DZMN10-1 in experimental group,which correspond to the results of quantitative.There were no significant changes in the potential target genes DZMN10-3 and DZMN10-5 in the experimental group.Our results indicated that DZMN9-3 inhibited the development of DZ nematodes which was consistent with the effect of n-miR-9 on DZ nematodes'development rate,and DZMN10-1 inhibited the growth of DZ nematodes' body length by transfecting the over-expression vectors pPD95.67::DZMN9-3 and pPD95.67::DZMN10-1.