摘要
通过NCBI下载猪近缘物种的PFN3基因序列,在线BLAST比对获得猪EST序列,利用Lasergene软件电子克隆猪PFN3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滇南小耳猪为研究材料进行实验验证,同时应用半定量RT-PCR技术对30个组织进行了表达分析。研究获得了猪PFN3 414 bp的编码区序列(GenBank登录号:JX457348,对应的氨基酸登录号:AFU74013)。生物信息学分析表明,该基因编码137个氨基酸,预测的蛋白质分子量(Mw)为14.73 ku,等电点(pI)为9.30。蛋白质结构分析显示,PFN3存在1个保守域,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,猪与牛亲缘关系最近。组织表达分析表明,PFN3基因在30个被检组织中均有表达,在睾丸、肝、肾、结肠、盲肠、直肠及食管中高表达,在胸腺、甲状腺、附睾、淋巴结、十二指肠、空肠、回肠、胃、皮肤、大脑、小脑、垂体及下丘脑中中度表达,在颌下腺、肺、心、脾、肾上腺、肌肉、胰脏、脑干、舌下腺及脊髓中低表达。研究结果将为该基因在猪中的进一步功能研究奠定基础。
The PFN3 sequences of allied species of pig were downloaded from NCBI and compared by online BLAST to obtain the EST sequences.Subsequently we used silicon cloning strategy to gain the coding sequence and a few flanking sequences of PFN3 through Lasergene software.According to the coding regions,we designed specific primers and conducted expcrimental verification using Diannan small-ear pig.Meanwhile,the expression level of the PFN3 in 30 important tissues of Diannan small-ear pig was analyzed by semi-quantitative RT-PCR.We obtained 414 bp coding sequence （accession number：JX457348,the corresponding amino acid sequence accession number：AFU74013） of swine PFN3.Bioinformatics analysis showed that the PFN3 gene encoded a protein of 137 amino acids with a predicted molecular weight （Mw） of 14.73 ku and isoelectric point （pI） 9.30.Protein structural analysis suggested that it contained one conserved domain and possessed 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 than with other species.The analysis of tissue expression showed that PFN3 was expressed in all 30 tissues of Diannan small-ear pig.It was highly abundant in the testis,liver,kidneys,colon,cecum,rectum and esophagus,moderately expressed in thymus,thyroid,epididymis,lymph nodes,duodenum,jejunum,ileum,stomach,skin,brain,cerebellum,pituitary and hypothalamus,and weakly expressed in the submandibular gland,lung,heart,spleen,adrenal gland,muscle,pancreas,brain stem,sublingual gland and spinal cord.The results will provide a basis for the study of PFN3 function in swine.
出处
《云南农业大学学报（自然科学版）》
CSCD
北大核心
2014年第2期179-186,共8页
Journal of Yunnan Agricultural University:Natural Science
基金
国家自然科学基金项目(31160439)
