西伯利亚鲟Sox9基因cDNA全长克隆、序列分析及其表达检测

Cloning,analysis and expression detection of the full-length cDNA of Sox9 from the Siberian sturgeon (Acipenser baerii Brandt)

为了研究Sox9基因在西伯利亚鲟侧线神经发育过程当中所起的调控作用,本研究利用RT-PCR和RACE方法获得了西伯利亚鲟Sox9基因cDNA全长序列,经序列分析表明,所克隆Sox9cDNA全长为1409bp,包括开放阅读框(open reading frame,ORF)1083bp,5′端非翻译区(untranslate dregion,UTR)19bp和3′端非翻译区(untranslate dregion,UTR)307bp。1083bp的ORF共编码360个氨基酸,相对分子质量为41221.7U。序列分析表明,其与施氏鲟的亲缘关系较近,其次是斑马鱼。经实时荧光定量PCR技术对西伯利亚鲟Sox9基因在各组织以及在胚胎发育各时期中的相对表达量检测发现,Sox9在西伯利亚鲟中的表达具明显的组织差异性和时间差异性。其中,在肝,肾,肌肉,胰中的相对表达量较低。然而,大脑组织中的表达量与以上4个组织相比,处于极高水平,达到胰脏中相对表达量8.9倍,达到表达量最低点肾脏中的近21倍。在胚胎发育的不同阶段,Sox9均有表达。在原肠期、神经胚期及视泡形成期,Sox9基因的表达量均较高,特别是在原肠期其表达量达到西伯利亚鲟整个胚胎发育期的最高点,相当于表达量最低点心脏形成期的1.9倍。在神经胚期和视泡形成期,Sox9的相对表达量也相对处于较高水平,仅比原肠期略低,其相对表达量是心脏形成期1.2倍和1.4倍。本研究将为今后深入研究Sox9基因在西伯利亚鲟侧线神经节分化发育、细胞迁移过程中所起的调控作用积累资料。

In order to further research the role of Sox9 in the development of the Siberian sturgeon （Acipenser baerii Brandt）’s lateral line neuromast,the full-length Sox9 cDNA of Siberian sturgeon had been successfully cloned by RT-PCR and rapid amplification of cDNA ends （RACE） methods. The full-length Sox9 cDNA of Siberian sturgeon had 1 409 bp,which consisted of a 1 083 bp open reading frame （ORF） encoding 360 amino acids,a 19 bp 5′untranslated region （5′UTR） and a 307 bp 3′untranslated region （3′UTR）. The molecular weight of this mature peptide was estimated to be 41 221. 7U. Neighbor-Joining （NJ） method had been used for multiple alignment analyses,and Siberian sturgeon was closest to Amur sturgeon （ Acipenser schrenckii）. The Siberian sturgeon’s Sox9 gene was highly conservative to other species. These results were much similar to other studies that had already been done in blue tilapia （Oreochromis aurea）,yellow catfish （Pelteobagrus fulvidraco） and swamp eel （Moncpterus albus）. Meanwhile,in order to study the connection between the feature of the expression of the Sox9 to the development of the Siberian sturgeon’s lateral line neuromast,the fluorescent quantitative RT-PCR analysis was used for detecting Sox9 relative expression levels in the different tissues and different embryonic development stages. Sox9 had relatively low expression levels in liver,pancreas,kidney and muscle. However,in the brain,Sox9 had very high expression levels,and the relative expression levels of the brain reached 8. 9 times the quantity of the relative expression in the pancreas and 21 times the quantity of the relative expression in the kidney. In the different stages of the Siberian sturgeon embryonic development,Sox9 expression had different relative expression levels. Its relative expression had no major differences and reached relatively high in gastrula stage,neurula stage and formation of visual bubble. Especially in the gastrula stage and neurula stage,Sox9 relative expression reached 1. 2 times and 1. 4 times the quantity of formation of heart. All of these results were nearly same as the researches of zebrafish （ Danio rerio）,chicken （ Gallus gallus） and mouse （ Mus musculus）. At the same time,the expression features showed Sox9 might play a very important role in the development of the neuromast of the Siberian sturgeon which had already been found in the development processes of the zebrafish neuromast. This research will lay a foundation for the future study on the role of Siberian sturgeon’s Sox9 gene in the differentiation of lateral line ganglion development,cells migration processes and so on. At the same time,the study on the role of the Sox9 in the cell signaling pathway of the development of the Siberian sturgeon’s lateral line neuromast is required too.