The Effects Of Estrogen-related Receptor (ERR) On Reproducation Of Female Giant Freshwater Prawn,Macrobrachium Rosenbergii

Estrogen-related receptor(ERR)is an eukaryotic transcription factor that is involved in many physiological processes in organisms such as growth,reproduction and metabolism.The previous study in our laboratory showed that ERR had the highest expression level in ovary in Macrobrachium rosenbergii and its expression in ovary was significantly higher than that in testis,suggesting that ERR might be involved in the reproductive process in female M.rosenbergii.In order to investigate the role and responsible mechanism of ERR in the reproductive process in female M.rosenbergii,we detected the expressions of ERR in the ovaries at different development stages firstly.Then the expressions of ERR in M.rosenbergii were interfered by injecting ERR double-stranded RNA(ERR-dsRNA)Synthesized in vitro and the comparative transcriptome sequencing was curried out on the ovaries from the ERR-dsRNA interference and control groups.The differential expression genes related to reproduction were screened and they expressions were detected in the ovaries from the ERR-dsRNA interference and control groups.The expressions of some of these genes during ovarian development were detected.In addition,in order to evaluate the interference effect of ERR-dsRNA in the protein level,the ERR protein was expressed using the prokaryotic expression system and the ERR antibody was prepared.The main results are as follows: 1.The prokaryotic expression of ERR protein of M.rosenbergii and the detection of specificity for ERR antibody The prokaryotic expression plasmid pET-32a-on-ERR of ERR protein was successfully constructed and the ERR protein was expressed by inducing.The optimal conditions for inducing to express the ERR protein were respectively 0.1 mM/L IPTG,35 °C and 8 h.The ERR protein was obtained through purifying and the ERR antibody that had the stronger specificity was obtained.2.The expressions of ERR in the ovaries at different development stages in M.rosenbergii ERR was highly expressed in the ovaries at the early development stages(0 and I)and the highest expression level occurred in the ovaries at development stage I.The expressions of ERR in the ovary at development stage II and III decreased.These results indicated that ERR was involved in the regulation of ovarian development in M.rosenbergii.3.Transcriptome sequencing analysis screen reproduction-associated genes regulated by ERR The levels of ERR mRNA and ERR protein were significantly decreased after injecting ERR-dsRNA.5407 differential expression Unigenes were obtained from ERR-dsRNA interference and control groups by transcriptome sequencing,of which 2490 Unigenes were up-regulated and 2557 Unigenes were down-regulated.Among these differential expression Unigenes,9 Unigenes were enriched to reproductive GO term,3 Unigenes were enriched to reproductive process GO term and 21 were enriched to oocyte meiosis Pathway term.There are 4 Unigenes with large differential fold and well-defined annotation in these differential expression Unigenes related to reproduction and they were verified to increase after the expressions of ERR were interfered by qPCR,which were consistent with the results of transcriptome sequencing.Among the 4 Unigenes,2 Unigenes were differentially expressed during ovarian development processes,of which 1 Unigenes(Unigene4600_All)were associated with the oocyte meiosis and 1 unigene(CL4888.Contig2_All)was related to the synthesis and release of reproduction-associated hormones.These results indicated that ERR is likely to regulate ovarian development by regulating the oocyte meiosis and the synthesis and release of reproduction-associated hormones in M.rosenbergii.4.The effects of RNA interfering the expressions of ERR on the expressions of reproduction-associated genes in ovary in M.rosenbergii After RNA interfering the expressions of ERR in M.rosenbergii,the expression levels of reproduction-associated genes vitellogenin(VTG),Cathepsin L,Cyclin B and(EcR)were significantly increased,indicating that could influence the ovarian development of M.rosenbergii by regulating the expressions of VTG and EcR(regulating generation of yolk protein)and Cyclin B(involved in oocyte meiosis).These results indicate ERR may be involved in ovarian development in M.rosenbergii by regulating oocyte meiosis,the synthesis and release of reproduction related hormones and vitellogenesis.