Cloning And Functon Analysis Of Myestatin Gene In Grass Carp (Cteaopharyagedon Idellus)

In the field of fish breeding, gene function and transgene studies are the mostimportant research methods in aquaculture. The approach, through the study of majorgene on the growth performance so as to breeding improved variety, has obviousadvantages for breeding of large fish.Grass carp (Ctenopharyngodon idellus), which is specific economic fish in China,has long been one of the most important farmed fish. Adult grass carp has relatively bigbody and long sexual maturation cycle, ripe fish need 5-6 years in the eastern Chinaregion, so breeding 4 generations requires 20-24 years or even longer, it's difficult tobreed superior varieties in the short term using the traditional methods.The myostatin gene (MSTN or GDF8) belongs to the transforming growth factorTGF-βsuperfamily. Myostatin is a potent negative regulator of muscle growth in manyanimals, including fish. It contains all of the structural components which arerepresentative to the superfamily, including a signal peptide for secretion, an inactiveN-terminal propeptide, and an active C-terminal region including 9 conserved cysteineresidues and a tetrapeptide (RXRR) cleavage site. Mammals and birds have single copyof the myostatin gene, but in some fish, two forms of myostatin (I and II) have beendetected; even in carp there is four distinct myostatin genes (1a, 1b, 2a and 2b). Itsexpression is limited primarily to skeletal muscle in mammals, but occurs in differenttissues of adult fish. This suggests that myostatin gene also have other functions inaddition to participating in the regulation of muscle growth and development as inmammals.Grass carp MSTN-1 cDNA was obtained by RACE. Grass carp MSTN-1 cDNA is2200 bp in length, encoding 376 a.a.. The same with zebrafish myostatin-1, grass carpmyostatin-1 mature peptide has two TGF-βprotein domains, 9 conserved cysteineresidues and RIRR of protease sites of C-terminus. Sequence alignment showed that grass carp myostatin-1 is highly conserved throughout evolution, with 96% identity withzebrafish myostatin-1. RT-PCR of different embryonic stages showed that the level ofgrass carp myostatin-1 gene is low at early embryonic stages, but increased significantlyafter 36hpf, which indicates the gene may be related to the somite differentiation. By insitu hybridization, expression of myostatin-1 mRNA was mainly observed in thenotochord and brain of grass carp, but with the development of embryos the position ofexpression emerge immigration. At the adult stage, grass carp myostatin-1 mRNApredominated in brain, muscle, eye, and was weakly expressed in the liver, spleen,heart , while other tissues have not been found.To investigate more about the functional aspects, we overexpressed the MSTN-1and observed embryos with bent morphology and lower activity. In the myostatin-1mRNA-injected embryos can be founded the body section of the performance of theformer - the rear axle longer, dorsal - ventral axis became shorter and the formation ofspindle-shaped, coated layer atrophy. And cause high mortality and deformity rates,its performance is similar to muscular dystrophy. Whole embryo in situ hybridizationresults showed that overexpression of myostatin-1 gene causes the downstream genes,such as: MyoD and ntl abnormal expression of genes, resulting in abnormal embryonicdevelopment.Collectively, these studies suggest that myostatin signaling plays a critical role inmyoblast differentiation and in midline formation and notochord development. Theseresults not only provide new insights into the special features in fish, but also reveal anovel role of myostatin signaling in midline formation and notochord development in avertebrate model.