The Basic Research Of The Role Of MYOZ1 During The Porcine Muscle Fiber Development

As we know, the basic unit of muscle is muscle fibers which affect the meat quality by theirs different types and the proportion in muscle. The quality of the meat which contains more slow muscle fibers will be better. Different types of muscle fibers can be transformed between each other. Calcineurin is a calmodulin dependent,calcium-activated protein phosphatase that plays an important role in myofiber transformation. Many studies have showed that the biological signal pathway CaN/NFAT played a key role in the myofiber development which can lead to the increase of slow muscle fibers. A set of studies showed that MYOZl gene has an inhibition with CaN which can effect myofiber growth by influencing the CaN/NFAT signaling pathway. In order to study the function of MYOZl in the myofiber development, we carried out the research by the technology of QRT-PCR, RNAi and gene overexpression. The major findings are as follows:1. The 1144bp cDNA sequence including CDS of MYOZ1 was cloned, we found that the sequence was conserved by sequence alignment between Yorkshire, Landrace, Tongcheng and Meishan.2. Using real-time qPCR, the expression trend of MYOZ1 gene in Tongcheng at the stage of 57d,70d,90d before birth, newborn, and 3d,21 d,35d,60d,90d,120d after birth were studied. The results showed that the expression trend of MYOZ1 gene in Tongcheng increased before birth but decreased within the 21 days after birth, while the expression increased again during the 35d to 90d after birth, but there was almost no change in 90days. The expression of MYOZ1 decreased sharply within the 21 days after birth which was the key period of myofiber-switch. This results suggested that MYOZ1 gene may be involved in muscle fiber transformation process.3. Using real-time qPCR, the expression trend of MYOZ1 gene in Yorkshire at the stage of 3d,21d,35d,60d,90d and 120d after birth were studied. The results showed that the expression of MYOZ1 gene decreased within the 21 days after birth, and there was almost no change after 35days. The results further indicated that MYOZ1 gene may be involved in muscle fiber transformation process. 4. LD(Longissimus dorsi muscle) and MA(Masseter muscle) of 90 days of Tongcheng pig were collected to study the relative expression of gene MYOZ1 and PPP3CA,PPP3CB, PPP3R1 genes which were regulated by MYOZ1. The results showed that the expression of MYOZ1 gene and PPP3R1 gene in LD were lower than in MA, but the expression of PPP3CA gene and the PPP3CB gene in MA were lower than in LD. The results suggested that there maybe some inhibition interaction between MYOZ1 gene and CnA.5. We designed and synthesized three small RNAi fragments of mouse MYOZ1 gene and transfected them into mouse C2C12 cells, the results showed that the expression of PPP3CA was increased significantly, and the expression of PPP3CB, PPP3R1 were also increased, but the increase were not significant. The results indicated that MYOZ1 gene maybe inhibit the expression of calcineurin in mouse.6. We construct the over-expression plasmid of pig MYOZ1 gene and transfected it into mouse C2C12 cells, the results showed that the expression of mouse PPP3CB gene and PPP3R1 gene decreased after the transfection of over-expression plasmid of pig MYOZ1 gene, but the expression of mouse PPP3CA gene increased non-significantly. The results illustrated that MYOZ1 gene maybe also inhibit the activity of calcineurin in pigs.