鳜Shh基因分子特征和时空表达规律及其对肌肉生长的调控

MOLECULAR CHARACTERIZATION AND SPATIO-TEMPORAL EXPRESSIONPATTERN OF THE SHH GENE AND ITS REGULATION IN MUSCLEGROWTH IN SINIPERCA CHUATSI

研究从鳜(Siniperca chuatsi)基因组中获得Shh(Sonic hedgehog)基因序列,对该基因编码的蛋白质和同源进化特征进行了分析。鳜Shh基因的开放阅读框(ORF)为1242 bp,编码413个氨基酸,分子量为46.01 kD,等电点(pI)为6.57,脂溶系数为82.83,亲水性平均系数为−0.292,拥有一个跨膜区,被推定为亲水性膜结合蛋白。Shh蛋白具有两个结构域,即Hh-N和Hh-C结构域。鳜Shh蛋白与自身所属的鲈形目鱼类Shh蛋白同源性较高。通过实时荧光定量PCR技术检测了鳜Shh基因的时空表达水平。结果显示:Shh的表达量在神经胚期表达显著上调,并在胚胎发育中后阶段保持较高水平。Shh在鳜不同组织中存在一定的表达差异,在脑、肠道中表达量较高,而在红肌、白肌等组织中表达量相对较低。通过环巴胺(Cyclopamine)处理鳜胚胎来抑制Shh信号通路的实验表明,Shh信号通路被抑制后,Pax3、Pax7、Myomaker、生肌调节因子及肌球蛋白重、轻链等基因的表达均显著降低,推测Shh参与调控鳜肌细胞的早期分化和发育过程。研究将有助于从分子水平了解Shh信号调控鱼类发育的分子机理,为鱼类发育生物学以及健康养殖提供参考依据。

Sonic hedgehog(Shh)is one of the important signaling molecules in the Hedgehog gene family that regulates cell proliferation and differentiation during the early embryo development.In this study,the DNA sequence of the Shh gene was obtained from Siniperca chuatsi genome,and the protein encoded by the gene and the homologous evolutionary features were analysed.The Siniperca chuatsi Shh gene has an open reading frame(ORF)of 1242 bp,encoding 413 amino acids,with molecular weight of 46.01 kD,isoelectric point(pI)of 6.57,lipolysis coefficient of 82.83,hydrophilic mean coefficient of−0.292,and one transmembrane structure,making it a hydrophilic membrane-bound protein.The Shh protein has two structural domains,Hh-N and Hh-C.The Siniperca chuatsi Shh protein shows a high degree of homology with that of the perciforms.The spatio-temporal expression pattern of the Shh gene in Siniperca chuatsi was analysed by using Real-time fluorescence quantitative PCR.The results showed that the expression of Shh was significantly up-regulated at the neurula stage and remained at a high level during the middle and later stages of embryonic development.Shh was differentially expressed in different tissues of Siniperca chuatsi,with higher expression in brain and intestine,and lower expression in fast muscle,slow muscle and other tissues.The expression of muscle development related genes was analysed in Siniperca chuatsi embryos which treated with Cyclopamine.The results showed that the expression of Pax3,Pax7,Myomaker,MRFs,MyLC,MyHC and MLCK,were significantly reduced after inhibiting the Shh signalling pathway,suggesting that Shh is involved in regulating the early differentiation and development of Siniperca chuatsi muscle cells.The study will help us to understand the molecular mechanism of Shh signalling in regulating fish muscle development at the molecular level and provide a reference for fish developmental biology and healthy breeding.