Preliminary Study On The Regulatory Effect Of MicroRNA-184 On The Expression Of Insulin-like Androgenic Gland Hormone In Macrobrachium Rosenbergii

The giant freshwater prawn,Macrobrachium rosenbergii(M.rosenbergii),which is one of the important freshwater economic aquaculture prawn species in China.Similar to other crustaceans,M.rosenbergii exhibits sexual dimorphism,with males having more advantages than females in terms of growth rate.At present,gonadal differentiation and gonadal development are the hot issues in M.rosenbergii.Artificial regulation of sex to achieve monosexual culture can create greater economic benefits to aquaculture.Therefore,it is particularly necessary to develop the mechanism of sex-related gene of M.rosenbergii.Insulin-like androgenic gland hormone(IAG)is secreted by androgenic gland that plays a key role in regulating sexual differentiation in crustaceans.As a single-stranded,non-coding small RNA molecule that regulates gene expression at the post-transcriptional level,the regulation of miRNA on sex-related genes has gradually become a hot topic for scholars in relevant fields,and a large number of relevant research results have emerged.However,miRNA has not been reported in the regulation of Mr IAG gene in M.rosenbergii,so it is necessary to carry out this studyThis experiment was based on the published full-length sequence of Mr IAG gene and the miRNA library constructed by our laboratory.RNAhybird software was used to preliminarily predict the miRNA with regulatory relationship with Mr IAG.And,the regulatory relationship between miRNA and target m RNA was verified by in vitro and in vivo experiments.RNA-seq was used to screen out differentially expression genes(DEGs)after miRNA interference.GO and KEGG analysis of DEGs was performed to verify their expression.The results can help to clarify the regulatory of miRNA on the growth and development of M.rosenbergii.The main results are as follows:1、Prediction of miRNA regulating IAG gene in M.rosenbergiiRNAhybird predicted that there were three miRNAs binding to the 3’UTR of Mr IAG gene,and a total of seven binding sites.Since the MEF value of miR-184 is the smallest,it indicates that the combination of the two genes is more stable.Therefore,we selected miR-184 as the candidate miRNA with the most potential,and speculated that Mr IAG is the target gene of miR-184.The regulatory relationship and binding target between the two need to be verified.2、In vitro validation of the regulatory relationship between miR-184 and Mr IAG gene expressionCo-transfection of miR-184 agomir with double fluorescent plasmids containing Mr IAG gene sequences into 293 T cells,after 24 h,double luciferase activity was detected to determine whether miR-184 had knockdown effect on Mr IAG gene.Dual-luciferase reporter assays showed that co-transfection of miR-184 agomir and wild-type recombinant plasmids into 293 T cells could significantly depress the activity of firefly luciferase compared with co-transfection miRNA agomir NC and wild-type plasmids.To further confirm direct targeting effect of miR-184 on Mr IAG,we constructed the mutant plasmid.The mutant plasmid was co-transfected with miR-184 agomir to 293 T cells,and the double fluorescent enzyme activity was detected after 24 h.The result displayed that the activity of luciferase was not affected compared to the control after cotransfection of mutant plasmids and miR-184 agomir.These results indicated that miR-184 could directly regulate Mr IAG and reduced the expression of Mr IAG.3、miR-184 was injected in vitro and transcriptome sequencing was performed in juvenileFour healthy and active males were selected from the experimental group(miR-184)and the control group(PBS)for Mr IAG gene detection.The results showed that the expression of Mr IAG gene could not be detected in 3/4 of the experimental group after overexpression of miR-184,while the control group all expressed Mr IAG gene.Subsequently,we mixed three individual RNA of the experimental group that did not detect Mr IAG gene expression,and three individual RNA of the control group were mixed,and transcriptome sequencing was performed on two samples.15.05 Gb of clean data were obtained,with the percentage of Q30 base up to 93.4 %,and 57729 unigene and 72676 transcripts were assembled.Through RNA-seq of carapace of experiment group and control group,1510 DEGs were screened(P < 0.05),including 1117 downregulated genes and 393 up-regulated genes.DEGs analysis revealed many changes in gene expression related to sex,growth and metabolism,such as Fem-1,FTZ-F1,cuticle protein,and PEPCK.Eight randomly selected DEGs were used for q RT-PCR validation.The results showed that the overall trend of q PCR expression profiles was consistent with RNA-Seq sequencing results.Functional and pathway enrichment analysis was performed for DEGs showed that they were mainly participated in carbohydrate metabolism,insulin resistance and phagosome.In addition,enrichment analysis also found that some pathways responded to the miR-184 interference,such as hippo signaling pathway,ovarian steroidogenesis and pyruvate metabolism.