The Effects Of Gonadotropin-releasing Hormone Analogues On Gonadal Development Of Penaeus Monodon

Penaeus monodon is one of the most important aquaculture species in the coastal areas of our country.In the past ten years,our group has overcome the difficult problem of artificial breeding on Penaeus monodon and successfully realized the whole artificial breeding.However,the mechanism of gonadal development and endocrine regulation on maturation progress of broodstock shrimp was not fully understood,which restricted the upgrading of artificial breeding techniques.Therefore,the study on the mechanism of gonadal development based on small peptide hormones will be helpful to solve the above problems.Especially,gonadotropin-releasing hormone(GnRH)is a gonadotropin-releasing hormone secreted from the hypothalamus of vertebrates,it is a decapeptide hormone that regulates the synthesis and secretion of mature hormones by activating the signal transduction pathway associated with GnRH receptor.To regulate the development and maturation of gonad in vertebrates.Current studies have demonstrated the presence of GnRH in the nervous system of Penaeus monodon,but it is few on how to regulate gonadal maturation and development jointly with GnRHR.In this study,the transcriptome library was constructed with ovary and nerve tissue before and after maturation of Penaeus monodon to analyse the differential expression gene changed during the different ovarian development stages,the candidate genes of GnRH and its receptor were screened from them,at the same time,the effects of GnRH analogues on gonadal maturation and development of Penaeus monodon were determined by tissue primary culture in vitro and injection of prokaryotic expression GnRH protein of The regulation of GnRH analogue on gonadal maturation and development.The specific findings are as follows: 1.Constructing the gonad and nervous system transcriptomes on Penaeus monodonThe ovary and nerve system samples of female shrimp at different developmental stages were collected to build the transcriptome library by using Illumina Hi SeqTM High-throughput sequencing technique.After analyzing the library,the total data amount is 44.63 Gb.Thereinto,after the original sequences were clustered and assembled,246206 transcripts and 125806 Genes was found,after doing the homology searching,alignment and functional annotation between the genes sequence and the seven databases,7728 Genes can be blasted,there were 56 genes differentially expressed found in ovary while 164 differentially expressed genes were found in nerve tissues,and a total of 6912 differentially expressed genes were found among ovarian and nerve tissues at different development stages.2.The function of G protein-coupled receptor gene of Penaeus monodonThe candidate gene of hormone receptor: G protein-coupled receptor was obtained by transcriptome library screening,and its gene characteristics and expression characteristics were studied.According to bioinformatics analysis,the Pm GPCR protein has seven transmembrane domains(7TM)and a DRY motif,which confirm us wihch belong to the rhodopsin receptor family.Phylogenetic tree also showed that Pm GPCR and Pc GPCR converged into one branch.The results of real-time fluorescence quantitative PCR(Q RT-PCR)showed that Pm GPCR was involved in the process of growth and development and immune stress resistance of Penaeus monodon.3.Optimizing the condition of primary cells culture of Penaeus monodonThree kinds of improved culture medium were used to investigate the cell growth state and activity and the expression of related genes after the tissue was cultured by mechanical isolation and enzyme digestion respectively.The results showed that the method of mechanical separation was suitable for observing the changes of tissues and easy to operate.The enzyme digestion method was suitable for observing the morphological changes of single cells and the localization of protein.By observing the growth of cells,the sections of gonadal tissue after HE staining and the cell viability values in three kinds of media,the enzymatic hydrolysis was confirmed more suitable for in vitro culture of gonadal cells of Penaeus monodon.The effect of culture medium No.1 or 2 was better than No.3 under specific culture conditions.There was a positive correlation between the expression of c-Mos gene and the growth of cultured tissues and cell.4.The effect of GnRH analogues on gonadal tissue cultured in vitroThe primary culture with GnRH analogues,including Oct GnRH,Pc GnRH and Mro GnRH were studied in vitro to know their effects on gonadal development of Penaeus monodon.The results showed that the gonadal development of male and female shrimp was significantly affected by 50 ng·m L-1 Oct GnRH,the diameter of oocytes was increased significantly(P < 0.01),the accumulation of vitellogenin in excised ovaries of female shrimps treated with 50 ng·m L-1 Oct GnRH,500 ng·m L-1 Pc GnRH and brain nerve was significantly increased with the prolongation of culture time.Therefore,the sequence of Oct GnRH was selected for expression of recombinant protein in vitro.5.The effect of injection of GnRH analogues on gonadal maturation of Penaeus monodonThe core amino acid sequence of Oct GnRH analogue was inserted into the p GEX-2T expression plasmid in the form of multiple copies.The fusion proteins of soluble 8 copies and 16 copies of Oct GnRH-p GEX-2T were obtained by prokaryotic expression system.The fusion protein was purified by affinity purification with GST tag.The 8 copies and 16 copies of Oct GnRH fusion protein and the chemically synthesized polypeptide of Oct GnRH and Pc GnRH were injected into male and female prawns in the early stage of maturation.The results showed that the gonadal index of 16 copies of Oct GnRH increased significantly at the last stage of the experiment,and the expression pattern of Vg gene was consistent with the rule of development and maturation of Penaeus monodon.The expression of GnRHR suggests that there may be a second type of GnRHR in Penaeus monodon,which lays a foundation for the further study of the existence of GnRHR.The expression of c-JUN and CREB gene indicate that there is also a GnRH signal transduction pathway in Penaeus monodon,moreover,the process of regulation and control is diverse and complex,so it needs to be studied more deeply.