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。
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 KC-NJ12 through Lasergene software. According to the coding regions,we designed specific primers and conducted ex-perimental verification using Banna mini-pig inbred line （ BMI） . Meanwhile,the expression of KCNJ12 in 31 impor-tant 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 predic-ted 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 in-dicated 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 and＆amp;nbsp;sheep. 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 un-detected in submandibular gland, testis, thyroid, epididymis, lung, heart, spleen, adrenal gland, lymph nodes, kid-neys,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.
出处
《华北农学报》
CSCD
北大核心
2014年第1期41-49,共9页
Acta Agriculturae Boreali-Sinica
基金
国家自然科学基金项目(31160439)
云南农业大学学生科技创新创业行动基金项目
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
Potassium inwardly-rectifying channel, subfamily J, member 12 （ KCNJ12 ）
In silico cloning
Ban-na mini-pig inbred line （BMI）
Bioinformatics
Tissue expression
