| Differentiated embryo chondrocyte1(DEC1), a member of basic-helix-loop-helix transcription factor, play an important role in the regulation of neurogenesis, tumorigenesis, chondrogenesis, response to hypoxia and development of metabolic syndrome. Previous studies suggested that DEC1was involved both in skeletal muscle development and adipogenesis. Considering skeletal muscle and adipose are most important tissues for meat animal production, and DEC1was suggested to play role in both myogenesis and adipogenesis, more functional studies of DEC1in livestock will significantly contribute to animal breeding and genetics. In the present study, qRT-PCR, gene clone, packaging of adenovirus, Western blot, immunostaining, cell culture, and luciferase reporter assay were performed to study the role and molecular mechanism of DEC1played in the myogenesis and adipogenesis. The research contents isolation and identification of bovine satellite cells, developmental expression patterns of DEC1gene in bovine tissues and process of bovine satellite cells, CoCl2induce DEC1and down regulated expression of myogenic factors, cloning of bovine DEC1gene and its role in differentiation of bovine satellite cells, over-expression of DEC1inhibits the MyoG promoter activity, role of DEC1played in differentiation of preadipocyte and its regulation mechanism, polymorphisms of bovine DEC1gene and their association with growth traits. The main results of the present study including:1. Isolation and identification of bovine satellite cellsBovine satellite cells were isolated by using type I collagenase digestion method, and immunostaining results showed that the expression of satellite cell maker gene Pax7was detected. The isolated bovine satellite cell could be induced to myotube by2%horse serum differentiation medium, which suggested it could be used as cell model of study on the regulatory mechanism of skeletal muscle developmemt.2. Expression patterns of DEC1gene in bovine tissues and process of bovine satellite cellsNine different tissues of cattle, including heart, liver, spleen, lung, kidney, skeletal muscle, fat (newborn calves and adult cattle), stomach and small intestine were removed from fetuses, young calves and adults of Qinchuan cattle. Q-RT-PCR was performed to detect the mRNA expression of DEC1gene in these tissues, results showed that DEC1 was ubiquitously expressed in most tissues of cattle. In addition, the expression of bovine DEC1gene showed a decreasing trend in skeletal muscle and fat during the developmental stages. Then, we detected the expression of DEC1during the process of bovine satellite cells differentiation. We found the expression of DEC1was down-regulated during differentiation of bovine satellite cells.3. CoCl2induce DEC1and down regulated expression of myogenic factorsEither on growth or differentiation condition, CoCl2induced the expression of DEC1, which suggested CoCl2could be use an activator of DEC1gene. Although CoCl2did not affect the expression of myogenic factors on growth condition, CoCl2down-regulated the expression of myogenic factors on differentiation condition.4. Cloning of bovine DEC1gene and its role in differentiation of bovine satellite cellsThe coding region of bovine DEC1was cloned from skeletal muscle of Qinchuan cattle, and then sequence alignment with the10species results showed that DEC1was conserved gene, genetic distance of DEC1between cattle and sheep was closest. Then, adenovirus vector of DEC1overexpressed was successfully constructed, and DEC1overexpressed adenovirus was collected after the process of packaging and propagation. When bovine satellite cells were infected by DEC1overexpressed adenovirus, differentiation was inhibited and the expressions of myogenic factors were down regulated.5. Promoter activity analysis of cattle MyoG gene and DEC1regulating the MyoG promoter activityThe fragment of MyoG promoter was cloned from Qinchuan cattle and the activity of5truncated fragments were analyzed. The fragment of pGL3-MyoGpro1117was a functional and more active promoter. When DEC1was over-expressed, the activity of MyoG promoter was significantly down regulated, which suggested DEC1inhibit the expression of MyoG through down-regulated its promoter activity.6. Over-expression of DEC1inhibits differentiation of preadipocyte and down regulated expression ofPPARgThe differentiation of bovine preadipocyte was inhibited after infected by DEC1over-expressed adenovirus, and the expression of PPARg, FABP4and HSL were significantly down regulated. The promoter activity study showed DEC1inhibit the expression of PPARg through down-regulated its promoter activity.7. Polymorphisms of bovine DEC1gene and their association with growth traitsTwo novel mutations (EX5+114T>C and EX5+733A>G) of bovine DEC1gene were identified by BamHI and Apal ACRS PCR-RFLP in1226individuals from five indigenous cattle breeds in China. Association analysis revealed BamHI locus was significantly associated with the average daily gain at18months in Nanyang cattle (P<0.05). |
