Function Exploration Of Two Porcine Skeletal Muscle Expressed Genes And Establishment Of A RNAi Strategy

Pork is the main source of animal protein for Chinese and pork industry is the critical part of our domestic husbandry. Moreover, it is the constant purpose of the animal breeding researchers to improve the quality and quantity of pork. Most of porcine carcass lean meat comes from the skeletal muscle, a detailed understanding of the mechanisms of skeletal muscle development and growth will provide an essential foundation to improve the pork industry. The skeletal musculature is a complex organ system, composed of functionally discrete units that are generated by a series of distinct morphogenetic events during embryogenesis. The process of generating muscle is highly regulated by series of factors such as growth factors, transcription factors as well as signaling molecules. So far, although many skeletal muscle expressed genes have been cloned and function characterized, the myogenesis process is not clear enough, and more genes need to be discovered and given annotation.IRS-1 (Insulin receptor substrate-1), the first member of the IRS family, has important role in skeletal muscle and brown fat tissue.PRDM16 (PR domain-containing protein 16), a transcription factor, controls the switch between skeletal muscle and brown fat. RNAi is a convenient technology for gene function research, and the core of this tech is the vector construction. The siRNA expression vector with converged promoters is an efficient tool for RNAi tech. The results of this study are shown as follows:1. By using the semi-quantitative RT-PCR tech, the porcine IRS-1 gene expression pattern in tissues was obtained. This gene expressed highly in the kidney, dorsal muscles and the brain of pigs. A SNP at site 3257(C>A), was detected by PCR-BseGⅠ-PFLP tech in Tongcheng experimental pig population and Wen’s Large White pig population and the association analysis implied that IRS-1 gene is associated to the carcass straight length in Tongcheng experimental population(P<0.05).2. Based on the semi-quantitative RT-PCR technology the expression profile of porcine PRDM16 gene in various tissues was assayed. And PRDM16 gene expression was up-regulated in the brain, lung, kidney and interior fat. A SNP was identified in this gene (c.-3284+171C>T) and was deposited in the SNP database (dbSNP acc. no. ss136268362). Then porcine PRDM16 gene was genotyped in Tongcheng pig population and ISU Berkshire×Yorkshire F2 population by using the PCR-AciⅠ-PFLP tech. This gene is related to the loin muscle area (p<0.01), backfat and Carcass weight (p<0.05) in the ISU Berkshire x Yorkshire F2 population which the association analysis showed.3. A siRNA expression vector with a converged H1 promoter and a U6 promoter, named as PH1U6-GFP, was constructed. The results suggest that this siRNA expression vector is valid after it was transfected into the BHK-21 cells.