Study On The Function Of MiRNAs In The Regulating The Development Of Ovarian Of Portunus Trituberculatus

The swimming crab Portunus trituberculatus is widely distributed in coastal areas of Korea,Japan,China and Southeast Asia,and is an important marine aquaculture species in China.Micro RNAs are a class of functionally important non-coding RNAs that bind complementarily to the 3’ untranslated region of their target m RNA,resulting in m RNA instability or blocked protein translation.Previous studies have shown that mi RNAs are crucial in regulating the ovarian development and maturation of aquatic animals.However,the regulatory roles of mi RNAs in ovarian development of P.trituberculatus is largely unknown.In this study,we investigated the regulatory effects of mi RNAs on key reproductive hormone-methyl farnesyl(MF)and an important gene related to ovarian development-forkhead box protein L2(foxl2)in P.trituberculatus.The results of this study are helpful for understanding the molecular mechanism of reproductive regulation,and developing new techniques for reproduction manipulation of P.trituberculatus.The research contents of this thesis are as follows:1.The role of mi RNAs targeting methyl farnesyl metabolism pathway in ovarian development of P.trituberculatusIn this experiment,we investigated the regulatory effect of mi RNAs targeting MF metabolism pathway on ovarian development of P.trituberculatus.Four mi RNAs,including let-7-5p,mi RNA-141,mi R-276-5p and novel-72,were predicted to target MF degradation pathway by Target Scan software.The regulation of the selected mi RNAs on their predicted target genes was validated in vivo and in vitro.Firstly,results of dual-luciferase reporter assay showed that let-7-5p and mi R-141-5p can regulate the key enzymes of MF degradation pathway,namely,juvenile hormone esterase-like carboxylesterase 1(JHE-like CXE 1)and juvenile hormone esterase-like carboxylesterase 2(JHE-like CXE 2),respectively.In addition,the relationship between let-7-5p and mi R-141-5p and their potential target genes was verified in vivo.After injection of let-7-5p agomir and mi R-141-5p agomir,the expression of target genes was significantly decreased.After injection of Antagomir let-7-5p antagomir and mi R-141-5p antagomir,the expressions of target genes were significantly increased.These results indicated that let-7-5p and mi R-141-5p can regulate the genes in MF degradation pathway.After the regulation of let-7-5p and mi R-141-5p on JHE-like CXEs was confirmed,the roles of let-7-5p and mi R-141-5p in ovarian development was studied.Expression of the mi RNAs in different tissues using q RT-PCR showed that both let-7-5p and mi R-141-5pwere highly expressed in hepatopancreas,the key tissue of MF catabolism,which was significantly higher than other tissues.Expression analysis of let 7-5p,mi R-141-5p,JHE-like CXE 1 and JHE-like CXE 2 at different developmental stages were analyzed by real-time q RT-PCR,which showed that let-7-5p was significantly negatively correlated with JHE-like CXE 1 expression in hepatopancreas,and mi R-141-5p was significantly negatively correlated with JHE-like CXE 2expression in ovary and hepatopancreas.Over-expression and knockdown experiment showed that injection of let-7-5p agomir and mi R-141-5p agomir can significantly increase MF titer and Vtg,while injection of let-7-5p antagomir and mi R-141-5p antagomir can lead to an increased levels of MF titer and Vtg expression.These results suggest that let-7-5p and mi R-141-5p can regulate vitellogenesis by targeting the catabolic pathway of MF in P.trituberculatus.2.Study on the role of mi RNAs targeting foxl2 gene in ovarian development of P.trituberculatusTo investigate the regulation of mi RNA on foxl2,a key reproductive gene of P.trituberculatus.First,we cloned the full length c DNA of foxl2(Ptfoxl2)gene.The 5 ’and 3’non-coding regions(UTR)of the gene were 701 bp and 211 bp,respectively,and the open reading frame was 1590 bp.The results of gene expression analysis showed that Ptfoxl2 was expressed in different tissues of P.trituberculatus,but the expression level was the highest in ovary.There were significant differences in its expression at different stages of ovarian development,and the highest expression level was in V stage.The expression of the gene decreased significantly after the removal of the eyestalk.After knock-down of fox L2 gene,the expression of Vtg gene in ovary was significantly upregulated.These results suggest that Ptfoxl2 may play an important role in the regulation of ovarian development and inhibit the synthesis of Vtg in ovarian tissues.Bioinformatics methods were used to predict the mi RNAs that target to regulate Ptfoxl2,and the regulation of these mi RNAs on Ptfoxl2 was verified at the cellular level by dual luciferase reporter gene assay,and the expression patterns of these mi RNAs in different stages of ovarian development and after eyestalk resection were analyzed.The results showed that the ratio of firefly luciferase to renilla luciferase activity decreased significantly in the group of co-transfected mi R-9 mimics and pmir GLO plasmid containing Ptfoxl2 3 ’UTR,and the expression pattern of Ptfoxl2 was opposite during ovarian development and after eyestalk removal.These results preliminarily confirmed that mi R-9 regulates Ptfoxl2 gene expression in P.trituberculatus at the post-transcriptional level.This paper has 27 figures,2 tables and 107 references.