Identification Of Differentially Expressed Genes In Early Developmental Ovary Of Yellow River Carp (Cyprinus Carpio Var) Using Suppression Subtractive Hybridization

In the lifecycles of higher eukaryotes,the expression of genes is carried out in an orderly and spatial order,and thus the diversity and complexity of life activities.Fish gonadal development at different stages have a strict and accurate mechanism of gene expression regulation,different developmental period of the specific genes are also different.By comparing the differences in gene expression at different developmental stages of ovary,it is possible to find genes that are closely related to ovarian developmental biological traits and functions.Therefore,we selected the ovaries of different developmental stages of Yellow River carp for different genes.In this study,Suppression Subtractive Hybridization(SSH)DNA libraries in two species of Yellow River carp were analyzed.Primordial gonads and stage II ovaries were used as testers,and adult ovaries as drivers.The sequences in the library were screened by Southern blot hybridization and verified by real-time quantitative qRT-PCR and GO signal pathway analysis.The expression of 14 genes of Bmp family was analyzed by fluorescence quantitative qRT-PCR,Bmp2,Bmp4 and Bmp15 were selected for futher study of their location of chromosome by means of genome information of carp.And analyzed their location by hybridization and immunohistochemically staining.The effect of Bmp signaling pathway on the differentiation and early ovarian development of the Yellow carp was studied by using Bmp signal pathway inhibitor DSM.The main results of the study are as follows:1.The construction of the Yellow River carp primordial gonads and stage II ovaries suppression subtractive hybridization library after the transformation,two library received a total of about 1200 monoclonal,screened by nested PCR,about 600 positive clones;after using dot blot hybridization,150 clones were selected to further verify the differences in the larger the company sent sequencing.2.Sequencing by bioinformatics analysis,the blast ratio in genebank,to remove duplicate and some microsatellite sequences,two libraries were obtained 78 Unigene after GO cloud platform analysis and classification of the biological pathway.Library of gene sequences belonging to 17 categories of functions,many of which belong to ESTs gene sequence of protein hydrolysis,signal transduction,cell differentiation,transformation and growth.The 22 genes(Bmp2,PV,Mid1ip1 l,Pax2,Bmp4,zgc,desmin,et al)related to ovarian development were selected to verify the function of the genes and their functions were analyzed3.The 14 Bmp gene family may be involved in ovarian development in-depth research,in the the Yellow River carp primordial gonads,larval and adult male and female gonads.The expression pattern of quantitative analysis found that Bmp2,Bmp4 and Bmp15 significantly in the stage II ovaries,primordial gonad and high expression in mature ovaries,respectively.Then we select Bmp2,Bmp4 and Bmp15 for further study,the Bmp2,Bmp4 and Bmp15 sequence were verified 2068,2037,2165,each encoding 410,399 and 384 amino acids,and contains TGF-beta carboxy terminal domain.Analysis of Bmp4 gene in the genome of the skeleton has a repeat,and its linear analysis of chromosomal location and other species.Bmp2 and Bmp15 were located on fortieth chromosome and fifth chromosome of Cyprinus carpio,and the linear analysis showed a high similarity to the zebrafish.4.Localization by in situ hybridization and immunohistochemistry,we found that Bmp2,Bmp4 and Bmp15 three gene expression in a similar position,expressed in the nucleus of stage II ovaries,ovarian mature period expressed in follicle around cortical vesicles,with its expression suggest that Bmp2 is an important regulation of early development of the Yellow River carp;Bmp15 gene as early follicular growth regulatory genes may be involved in follicular development in the ovary size of the Yellow River carp.5.The design of Dorsomorphin inhibitors to suppress the signal pathway,suppression of Bmp signal pathway of carp undifferentiated gonads by DSM successfully inhibited female gene Cyp19,Foxl2 expression was significantly decreased,partial male differentiation trend,indicating that Bmp signaling pathway for undifferentiated gonadal differentiation to play an important role in the regulation of ovarian development;two after treatment also showed inhibition of ovarian development,we hypothesized that the Bmp signaling pathway to maintain the normal development of the ovary early plays an important role.In summary,the construction of differential gene expression library inYellow River carp ovarian development has an important significance to find genes related with sex differentiation and development,to understand the process of gonad development of the Yellow River carp,to thoroughly aware of the molecular mechanism of the carp ovarian development.At the same time,it provided a rich resource for gene identification for the study of the molecular mechanism of female development in carp.