Research Of Myostatin And Its Pathway Related Genes In Muscle Growth And Development Of Chinese Tongue Sole(Cynoglossus Semilaevis)

Chinese tongue sole(Cynoglossus semilaevis)is an important target of mariculture in China.The growth of Chinese tongue sole has obvious sex dimorphism.The body length and body weight of sexually mature females are significantly higher than that of males.myostatin(mstn)is a negative regulator of muscle growth and development and has an inhibitory effect on muscle growth and development.The mstn/smad signaling pathway plays an important regulatory role in muscle growth and development.In this study,RT-qPCR,tissue section staining and in situ hybridization were used to explore the temporal and spatial expression patterns of mstn,a negative regulator of muscle growth and development,and its pathway related genes in female and male Chinese tongue sole during muscle growth and development,so as to provide a basis for further study on the growth differences between male and female Chinese tongue sole.The main results are as follows:The RT-qPCR results of mstn gene in 9 tissues of 1year Chinese tongue sole show that the expression of mstn gene in muscle tissues is the highest,while that in other tissues is lower.The expression level of mstn in testis and brain of male fish is second only to muscle.The expression levels in muscle and brain of female fish are significantly higher than those in other tissues.The results of gender differential expression analysis show that there are significant differences in mstn expression levels in spleen,intestine,kidney,gonad,brain and muscle tissues(P < 0.05),but there is no significant difference in the expression levels in liver,gill and heart tissues.At different stages of muscle development,the expression level of male fish was significantly higher than that of female fish at one year old(P < 0.05),but there was no significant difference between male and female fish at 40 d,60d,120 d,180d and 2y.The results of muscle tissue section preparation showed that the cross section of muscle fibers presented irregular polygonal shape.The results of in situ hybridization show that the expression of mstn is at the edge of myoblasts,located between muscle fibers,that is,at the site of proliferation and differentiation of myoblasts.The coding regions of smad2,smad3 a,smad3b and smad7 genes of smad family are cloned by PCR.The amino acid sequences encoded by smad2,smad3 a,smad3b and smad7 all contain the typical domains of the SMAD protein family MH1 and MH2.RTqPCR results showed that smad2 expression was the highest in male gonads,followed by heart and gill tissues.The expression was most abundant in female ovary,followed by gill and brain tissue.The expression level of smad2 in gonads of female fish was significantly higher than that of male fish,and there was a significant difference(P <0.05).smad3 a gene expression was the highest in muscle tissue of male fish,followed by brain and heart.In female fish,the highest expression is in muscle tissue,followed by brain.The expression of smad3 a gene in liver,kidney,gonad,heart,brain and muscle tissues of male fish was higher than that of female fish,and there was a significant difference(P < 0.05).There was no significant difference in the expression of smad3 a in other tissues.RT-qPCR results showed that the expression of smad3 b gene was the highest in muscle tissue of Chinese tongue sole,and the expression of smad3 b gene in male fish was higher than that in female fish,and the difference was significant(P <0.05).The expression level of smad7 gene was the highest in gill tissue of Chinese tongue sole,and the expression level of smad7 gene in gill,liver,spleen,intestine,kidney,heart,brain and muscle tissue of male fish was higher than that of female fish,with significant differences(P < 0.05).