Characterization And Reproductive Function Analysis Of Neuroparsin 1 (MrNP1) And Neuroparsin 2 (MrNP2) Of The Female Shrimp Macrobrachium Rosenbergii

Neuroparsins(NP)are cysteine-rich small size neuropeptides discovered in the crustacean.They are known to have reproductive functions in insects.There was little research on freshwater shrimp.Macrobrachium rosenbergii is one of the important freshwater shrimps in China.It is important in shrimp hatchery that we understand the endocrine control of reproduction as control by this group of newly discovered neuropeptides.The result of this study may provide some theoretical reference for the function research of NP gene.The specific research contents and results for this study are as follows:1.Cloning,bioinformatics analysis and expression analysis of MrNP1 and MrNP2 in M.rosenbergiiThe gene sequences of NP1 and NP2 were obtained from transcriptome data in M.rosenbergii.The two complete open reading frame c DNA sequences of NP1,NP2 were cloned by PCR amplification technology,The MrNP1 and MrNP2 have the sizes of 336 bp and 378 bp,respectively.By Blast and sequence analysis,two genes were named MrNP1 and MrNP2.The expression characteristics of two genes in different tissues and different developmental stages were monitored by Quantitative Real-time PCR.The results showed that MrNP1 expression level was high in ovary and MrNP2 expression level was highly in the heart in the previtellogenic females.In different developmental stages,there was no significant difference in the expression of MrNP1 gene,and the expression of MrNP2 gene was highly expressed in the early stage of vitellogenesis.Based on the results from expression study,it was preliminarily concluded that two genes were involved in vitellogenesis.2.Using RNA interference technology to explore the influence of MrNP1 and MrNP2 genes on Vg,VgR,Cyclin B.The ds RNA of MrNP1,MrNP2 genes were obtained by in vitro transcription.For RNAi study,in vivo injection experiments were performed.The results showed that the knockdown of MrNP1 and MrNP2 genes could significantly down-regulate the expression of Mr Vg and Mr VgR genes,but had no significant effect on the expression of Mr Cyclin B genes.Also,knockdown of MrNP1 could significantly down-regulate the expression of MrNP2 genes in various tissues,and the knockdown of MrNP2,MrNP1 genes were only down-regulated in hepatopancreas,but had no significant influence in other tissues.To extend further,in vitro tissue incubation experiment was carried out,and ds MrNP1 was added to the experimental group.The results showed that in hepatopancreas,the konckdown of MrNP1 could significantly down-regulate the expression of MrNP2 gene,but had no significant influence on Mr Vg expression.In ovary fragments,MrNP1 gene knock down could significantly down-regulate MrNP2,Mr Vg,Mr VgR and Mr Cyclin B gene expression.For RNAi of MrNP2 with ds MrNP2,d the results showed that the MrNP2 gene was not knocked down successfully in hepatopancreas,but it was knockdown successfully in ovary.At the same time,the expression of MrNP1,Mr Vg,Mr Cyclin B genes decreased significantly,but the expression of Mr VgR gene had no significant influence.3.Production of recombinant protein forMrNP1,MrNP2 and bioassay for their functionsThe recombinant proteins rMrNP1,rMrNP2 were obtained by constructing in prokaryotic expression vectors,and the recombinant proteins were injected into females at previtellogenic stage.The experimental group was injected with rMrNP1 for 24 h,and the results showed that the expression of Mr Vg and Mr VgR genes were up-regulated,and the degrees of expression(i.e.up-regulation)of the two genes was different with different injection doses.In vivo injection of rMrNP1 for 24 h,the results showed that the expression of Mr Vg gene was significantly down-regulated in hepatopancreas,and in ovarian tissue,a low-dose injection caused a significant down-regulation of Mr Vg gene in ovary,but had no effect on Mr VgR and Mr Cyclin B gene.With the increase of injection dose,the expression of Mr Vg,Mr VgR and Mr Cyclin B gene was significantly up-regulated.In vitro tissue incubation experiment was carried out on M.rosenbergii,which continued its ovarian development stage I,II and III.The results showed that the expression of Vg increased when two recombinant proteins were added to ovarian tissues in vitro.