Cloning And Expression Analysis Of Tgfbr2Gene In Erhualian Pig

Erhualian pig is one of the best varieties in China’s Taihu pigs, genetic mechanisms of its high-yield breeds is not yet clear. TGFBR2gene is an important member of transforming growth factor β superfamily, it plays an important role in mammalian ovarian granulosa cell proliferation, differentiation, follicular development and reproductive performance. At present, it has not been reported about the research of TGFBR2gene of Erhualian pig reproductive performance, this study made Erhualian pig which is high fecundity pig in China as the object, applying cloning and sequencing technology to obtain TGFBR2coding sequence of Erhualian pig, and by means of using bioinformatics methods to analyze its sequence features; applying RT-PCR and immunohistochemistry technology to analyze Erhualian pigs TGFBR2gene organization expression characteristics and ovarian tissue expression patterns,and adopting real-time PCR and western blot to detect Erhualian pig and commercial pig ovarian tissue mRNA and protein expression level of TGFBR2gene; Using real-time PCR to detect TGFBR2gene mRNA expression levels in the process of porcine follicular atresia. applying bioinformatics methods and luciferase reporter gene to select miRNA which regulates TGFBR2gene. Provide a theoretical basis to explore the relationship between the TGFBR2gene and the high fecundity traits of Erhualian pigs,in order to reveal the molecular mechanism of the high fecundity traits in Erhualian pigs. The conclusions of the paper are as following:1. TGFBR2gene cloning and sequence analysis of Erhualian pigs.This study has obtained Erhualian pigs TGFBR2coding sequence successfully.The gene sequence of a total length of1461bp, consistency with the other species of mammals as74-84%. Erhualian pig TGFBR2genes encode proteins containing486amino acid residues, and consistency with the other species of mammals as88-97%. Domain prediction TGFBR2prote in Erhualian pigs that it contains transmembrane domains, PKc domain typical functional domains, the same as human, mouse and other mammals, The tertiary structure prediction Erhualian pigs TGFBR2protein is composed of six a-helices, seven β-sheet structure and random coil. The phylogenetic tree made by TGFBR2amino acid sequence using neighbor-joining was consistented with the classical taxonomy of species. The results show that the TGFBR2gene of Erhualian pigs with other mammals and species were highly homologous. It suggested that TGFBR2plays an important role on follicular development in Erhualian pigs as it does in other mammal species.2. TGFBR2expression features and expression level of ovarian tissue of Erhualian pigsThe tissue expression of TGFBR2was detected using RT-PCR.The results showed that TGFBR2mRNA existed in hypothalamus, pituitary and other12kinds of organizations of Erhualian pig. In particular, it was highly expressed in the ovary. The immunohistochemical results showed that TGFBR2was expressed many developmental stages in porcine follicular.Using real-time PCR method to detected the TGFBR2gene mRNA expression levels of ovarian tissue in Erhualian pigs and Commercial pigs, the results show that TGFBR2mRNA expression levels was higher in Erhualian pigs ovarian tissue, the difference was significant (P<0.05). Then detected the TGFBR2protein levels By Western blot in Erhualian pigs and Commercial pigs ovarian tissue, we found the TGFBR2protein level of Erhualian pigs and Commercial pigs was significantly different (P<0.05). The results show that the TGFBR2gene and protein expression levels in ovarian tissue may be related to the high fecundity of Erhualian pigs. And it would be the candidate genes that affect the high fecundity Characters of Erhualian pigs.Using real-time PCR method to analyze TGFBR2gene mRNA expression levels of healthy follicles (H), early atretic follicles (EA) and progressively atretic follicles (PA), it suggestted that TGFBR2gene may play a role in the process of early porcine follicular atresia. It may affect the follicular fluid E2synthesis and thus affect follicular atresia3. Initial screening of pigs TGFBR2gene regulation of miRNAUsing internet site bioinformatics prediction, obtained29candidate miRNA that regulation TGFBR2gene, combined with the preliminary miRNA expression profiling using miRNA microarray detection of porcine follicular atresia process by our research group.It showed that miR-21is both TGFBR2gene candidate for regulation of miRNAs, it has also the differentially expressed miRNAs in the process of pig follicular atresia (P <0.000001). Using miR-21minics and build the TGFBR2gene3’UTR luciferase reporter gene vector were co-transfected into Hela cells and found that the reporter gene activity of miR-21minics group was significantly lower than the control group (minics NC group)(P <0.01) after transfected24h and48h later, indicating over-expression of miR-21can be a very significant inhibition of reporter gene activity, verify that the regulatory role of miR-21to TGFBR2gene,and it is the miRNA which regulates TGFBR2gene.