Cloning Of Grass Carp CTGF?ROCK1 And Expression Of TGF?1-CTGF Signaling Pathway And The Collagen Genes Of Grass Carp After Taking Broad Beans

Exp.1 Cloning,molecular characterization and tissues differential expression of connective tissue growth factor(ctgf)from grass carpConnective tissue growth factor(ctgf)is involved in the proliferation,migration,adhesion of cell and the constituent of extracellular matrix,which plays an important role in embryogenesis,angiogenesis,wound repair and fibrosis diseases.In this study,the c DNA sequence of grass carp ctgf gene was cloned by rapid amplification of c DNA ends(RACE)method,the characteristics of this gene and its predicted protein sequence were analyzed by bioinformatics methods,and the tissues differential expression pattern was detected by the quantitative real-time PCR.The results showed that the full-length of grass carpctgf gene was 2223 bp,encoding 343 amino acids.The deduced CTGF protein was a hydrophilic and secretary protein with a molecular mass of 37978.2 Da and an isoelectric point of 8.22.The signal peptide located between residue position 1 to 22 of the polypeptide chain.The protein included ?-helixs,?-strand and loops.The CTGF protein of grass carp showed a homology of 98%?96%?91% and 91% with Wuchang bream(Megalobrama amblycephala),zebrafish(Danio rerio),common carp(Cyprinus carpio)and mexican tetra(Astyanax mexicanus).The grass carp ctgf gene expressed significantly higher in blood and spleen than that in other tissues(P<0.05).The low expression tissues included heart,gill,skin,muscle,kidney,brain,intestinal,and the lowest expression tissue was liver.The results are consistent with the function of this gene.Exp.2 Cloning,Molecular Characterization and Tissues Differential Expression of Rho-associated Coiled-Coil-Containing Protein Kinase1(ROCK1)of Grass CarpROCK1 is one member of the ROCK(Rho-associated coiled-coil-containing protein kinase)family which is downstream targets of the small GTP-binding protein Rho.ROCK is involved in cell contraction,adhesion,migration,and proliferation via activated by Rho and play an improtent role in tissue fibrosis,cancer and cardiovascular disease.In this study,the c DNA sequence of grass carp ROCK1 gene was cloned by rapid amplification of c DNA ends(RACE)method,and the characteristics of this gene and its predicted protein sequence wereanalyzed by bioinformatics methods.Used the quantitative real-time PCR,the tissues differential expression pattern was detected.The results showed that the full-length of grass carp ROCK1 gene was 4604 bp,encoding 1361 amino acids.The grass carp ROCK1 protein showed a homology of 95% ? 94% and 94% with golden-line barbell(Sinocyclocheilus graham),zebrafish(Danio rerio)and common carp(Cyprinus carpio).The deduced ROCK1 protein was a hydrophilic and non-secretary protein with a molecular mass of 158035.73 Da and an isoeletronic point of 5.69.And it included ?-helixs,?-strand and loops.The grass carp ROCK1 gene in blood and brain expressed significantly higher than that in other tissues(P<0.05)followed by the gills,heart,kidneys,spleen and intestinal.The lower expression tissues included liver and muscle,and the lowest expression tissue was skin.Those results are consistent with the function of this gene.Exp.3 Expression of TGF?1-CTGF signaling pathway andthe collagen related genes of grass carp muscular and skin after taking broad beans.The control fishwere fed with formulafeed and the experiment fishwere fed with broad beans which were soaked for 24 h before thefeeding,At the 0,4th,8th and 12 th week of the feeding trial,the muscle and skinwere sampled to measure the spatio-temporal expression of TGF?1,CTGF,ROCK1,COL1A1 and COL1A2 by RT-PCR.The results showed that the expression of TGF?1,CTGF,COL1A1 and COL1A2 in muscle and skin of experiment group were significantly higher than those of control group at the 4th week,and then decresed at the 8th and 12 th week.The expression of ROCK1 in muscle of experiment group reached the peak at the 4th week,but the expression of ROCK1 was lower than that of control group at the 4th,8th,12 th week.The expression of ROCK1 in skin of experiment group deceased gradually at the 4th,8th and 12 th week,but it was higher than those of the control group at the same time point.The results indicated that broad bean affected the expression of TGF?1?CTGF?ROCK1?COL1A1 and COL1A2,and TGF?1,CTGF and ROCK1 were involved in the expression modulation of COL1A1 and COL1A2.According to the results,we speculated that broad bean may stimulate the high expression of CTGF by the TGF?1/CTGF pathway in the earlier stage,and then promoted the synthesis of collagen.But in the late stage,broad bean reduced the expression of ROCK1,then furtherly suppressed the TGF?1/CTGF signaling pathways and reduced the synthesis of collagen.The molecular mechanism of broad bean modulating the flesh quality of grass carp needs a further study in the future.