西伯利亚鲟β-actin基因cDNA全长克隆、序列分析及其作为内参基因的应用研究

Cloning and sequence analysis of Siberian sturgeon full-length β-actin gene cDNA and application of genes as a internal reference

采用同源克隆和RACE技术获得西伯利亚鲟(Acipenser baerii)β-actin基因cDNA全长,序列分析表明西伯利亚鲟β-actin基因cDNA全长1322bp,其包括的137bp的5′端非翻译区,57bp的3′端非翻译区和编码375个氨基酸残基的1128bp开放阅读框。与其他物种比对,该基因具极高的保守性。同时也利用同源克隆技术获得了西伯利亚鲟Sox2基因的同源保守区,该序列长768bp,编码256个氨基酸,也具较高的保守性,与其他物种的相似性在70%以上。以β-actin作为内参基因,对Sox2基因在西伯利亚鲟成鱼各组织及胚胎发育各时期进行实时荧光定量PCR检测,发现Sox2基因在不同组织中均有表达。其中,在肝、胰、肾、脑4个组织中表达量差距不大,均处于相对较低的水平,在肌肉组织中表达量最高,达到了肝脏的10.4倍。同时Sox2基因虽在西伯利亚鲟胚胎发育各时期均有表达,但表达也具明显的时间差异性。在受精卵期、囊胚期Sox2基因的相对表达量较低,在原肠期、神经胚期、视泡形成期和心脏形成期Sox2基因相对表达量较高,且在心脏形成期达到最高点。本研究为量化西伯利亚鲟发育分化过程中相关基因提供了坚实的参照工具,增加了后续试验的可信度,同时可为今后深入研究西伯利亚鲟Sox2在侧线神经节分化发育、细胞迁移过程中的作用提供基础参考依据。

β-actin is one of the actin family,especially in maintaining cell structure,movement and physical activity in cell division and playing an important role in quantitative genetic experiments.In this study,homology cloning and RACE techniques were used to amplify Siberian sturgeon（Acipense baerii）＇s β-actin gene cDNA full-length.Sequence analysis showed that the full-length of β-actin gene cDNA had 1322 bp,which consisted of a 1128 bp open reading frame（ORF） encoding 375 amino acids,a 137 bp 5＇untranslated region（5＇UTR） and a 57 bp 3＇untranslated region（3＇UTR）.Multiple alignment analyses showed Siberian sturgeon was much closest to other fishes.Meanwhile,homology cloning method was used to obtain the Siberian sturgeon＇s Sox2 gene homology Sequence,which had 768 bp and shared over 70% similarity with other species.And then β-actin gene was used as a internal reference on detecting the expression of Sox2 gene in various tissues and various embryonic development periods of Siberian sturgeon by real-time fluorescence quantitative PCR.The results showed Sox2 gene had expressed in different tissues and the expression level of Sox2 were relatively low in liver,pancreas、kidney and brain.However,in muscle tissue,the expression level of Sox2 was highest in all of these tissues.Although Sox2 gene had expressed in various periods of the Siberian sturgeon embryo,the relative expression level of Sox2 gene was low in the fertilized egg period and blastocyst.In the gastrula,neurula period,formation of visual bubble and formation of heart,the expression of Sox2 gene reached to relatively high.At the same times,in the formation of heart,the expression of Sox2 gene reached to the highest.This research will provide a solid tool to quantify the expression of the related genes during the development of Siberian sturgeon embryo and lay a foundation for the further study on the role of Sox2 gene in the differentiation of Siberian sturgeon＇s ganglia and so on.