摘要： 通过NCBI下载猪近缘物种的KCNJ12基因序列,在线BLAST比对获得猪EST序列、参考基因组序列和高通量基因组序列。利用Lasergene软件电子克隆猪KCNJ12基因编码区序列及部分侧翼序列。针对编码区设计特异引物并以版纳微型猪近交系BMI为研究材料进行试验验证,同时应用半定量RT-PCR技术对BMI 31种重要组织进行了表达分析。研究获得了猪KCNJ12 1 290 bp的编码区序列(GenBank登录号:KC505155,对应的氨基酸登录号:AGK36093)。生物信息学分析表明,该基因编码429个氨基酸,预测的蛋白质分子量(Mw)为48.63 kDa,等电点(pI)为5.66。蛋白质结构分析显示,KCNJ12存在2个保守域,3个跨膜结构域,无信号肽;疏水性分析表明其N端和C端均具有亲水性;亚细胞定位显示,该蛋白位于细胞质的概率是94.1%。系统进化分析表明,猪与牛、羊的亲缘关系最近。组织表达分析表明,KCNJ12基因在31个被检组织中存在明显的表达差异,在小脑中高表达;在肌肉、结肠、盲肠、食管、皮肤、大脑、下丘脑及脊髓中中度表达;在胸腺、肝、直肠及脑干中低表达;在颌下腺、睾丸、甲状腺、附睾、肺、心、脾、肾上腺、淋巴结、肾、胰脏、十二指肠、空肠、回肠、胃、垂体、松果体及舌下腺中不表达。研究结果为该基因在猪中的功能研究提供依据。

关键词: 内向整流型钾离子通道亚家族J成员12, 电子克隆, 版纳微型猪近交系, 生物信息学, 组织表达

Abstract: The KCNJ12 sequences of allied species of pig were downloaded from NCBI and compared by online BLAST to obtain the EST sequences,the referenced genomic sequences and high-throughput genomic sequences of pig. Subsequently we used silicon cloning strategy to gain the coding sequence and a few flanking sequence of KCNJ12 through Lasergene software. According to the coding regions,we designed specific primers and conducted experimental verification using Banna mini-pig inbred line(BMI). Meanwhile,the expression of KCNJ12 in 31 important tissues of BMI was analyzed by semi-quantitative RT-PCR. The research obtained 1 290 bp coding sequence(GenBank accession number: KC505155,the corresponding amino acid accession number: AGK36093) of swine KCNJ12. Bioinformatics analysis showed that the KCNJ12 gene encoded a protein of 429 amino acids with a predicted molecular weight(Mw) of 48. 63 kDa and isoelectric point(pI) 5. 66. Protein structural analysis suggested that it contained two conserved domains,three transmembrane regions and no signal peptide; hydrophobicity analysis indicated that both N-terminal and C-terminal of the protein were hydrophilic; and it was a cytosolic protein with a probability of 94. 1%. Phylogenetic analysis demonstrated that pig had the closest relationship with cattle andsheep. The analysis of tissue expression showed that the expression of KCNJ12 examined in 31 tissues had obvious differences. It was highly abundant in the cerebellum; moderately expressed in muscle,colon,cecum,esophagus, skin,brain,hypothalamus and spinal cord; and weakly expressed in thymus,liver,rectum and brain stem; while undetected in submandibular gland,testis,thyroid,epididymis,lung,heart,spleen,adrenal gland,lymph nodes,kidneys,pancreas,duodenum,jejunum,ileum,stomach,pituitary,pineal gland and sublingual gland. These results will provide a basis for the study of KCNJ12 function in swine.

Key words: Potassium inwardly-rectifying channel, subfamily J, member 12(KCNJ12), In silico cloning, B an-na mini-pig inbred line(BMI), Bioinformatics, Tissue expression

中图分类号: 

• Q78