猪硫辛酸合成酶基因的克隆及组织表达

Cloning and Tissue Expression of Pig Lipoic Acid Synthase Gene

本试验通过电子延伸及RT-PCR技术克隆猪硫辛酸合成酶(lipoic acid synthase,Lias)基因,并进一步研究了该基因在猪体内组织的表达分布。基于电子延伸序列设计1对引物,成功克隆了猪Lias基因(GenBank登录号:JN797612.1),并进行了序列分析,同时利用RT-PCR方法分析了Lias基因的组织表达规律。克隆的猪Lias基因长度为1119 bp,包括1个完整的开放阅读框,编码372个氨基酸;克隆的猪Lias基因与小鼠、褐鼠、人、牛的核苷酸序列同源性分别为71.5%、69.0%、77.3%、70.4%;推导的cDNA编码区(CDS)的氨基酸序列与小鼠、褐鼠、人、牛的同源性分别为91.5%、79.4%、89.4%、88.2%;构建的系统进化树结果显示,猪与小鼠、褐鼠的亲缘关系最近;组织表达分布结果表明,Lias基因在心脏、肝脏、脾脏、肺脏、肾脏、十二指肠、空肠、直肠、回肠、脂肪、皮肤和肌肉中都有表达;Gel-Pro软件分析结果表明,Lias基因在肾脏和脂肪组织中表达丰度最高,其次是直肠和回肠。

The objective of this expriment was to clone pig lipoic acid synthase （Lias） gene by using electronic extension and RT-PCR and to study its tissue distribution in pig. A pair of cloning primers was designed based on the in silico sequence information and was used to clone and analyse the gene of pig Lias （No：JN797612.1）. The positive clone was identified and sequenced, at the same time the Lias gene organization distribution rule was analyzed by RT-PCR. The length of pig Lias was 1119 bp, including an ORF of 1119 bp, encoding 372 amino acids. Identity analysis showed that the Lias nucleotide sequence in pig shared 71.5%, 69.0%, 77.3%, 70.4% homology with that of Mus musculus, Rattus norvegicus, Homo sapiens, Bos taurus, respectively, the predicted peptide shared 91.5%, 79.4%, 89.4%, 88.2% homology with that of Mus musculus, Rattus norvegicus, Homo sapiens, Bos taurus, respectively. The phylogenetic tree demonstrated that pig was the closest to Mus musculus and Rattus norvegicus. Semi-quantitative RT-PCR analysis showed that Lias expressed in heart, liver, kidney, adipose tissue, spleen, skin, rectum, lung, duodena jejunum, ileum and muscle, but it was higher in the kidney and fat, and then expressed in rectun and ileum.