摘要
试验旨在对陆川猪黑皮质激素受体4(melanocortin-4receptor,MC4R)基因进行克隆及相关信息学分析。通过提取陆川猪背最长肌总RNA,采用RT-PCR、克隆等方法获得含目的基因MC4R的质粒pMD18-T-MC4R,经菌落PCR和测序鉴定正确后,应用相关生物信息学软件对陆川猪MC4R基因的理化性质、蛋白质的结构、修饰结构和亚细胞定位等进行预测分析。结果表明,MC4R基因CDS区长999bp,编码332个氨基酸,与NCBI上公布的野猪MC4R基因序列中的CDS区存在4个碱基差异,其中175和906bp处为同义突变,110和278bp处为错义突变,分别引起第37位谷氨酸变为甘氨酸和第93位缬氨酸变为丙氨酸。同源性比对结果发现,MC4R基因在不同物种及进化的过程中具有较高的保守性。陆川猪MC4R蛋白有明显的疏水区域,不存在信号肽,但有7个跨膜结构域,其编码蛋白的二级结构元件有α-螺旋、延伸链、β-转角和无规则卷曲。修饰结构预测表明,MC4R蛋白存在多处N糖基化位点,但无O糖基化位点,可能主要分布于内质网和囊泡。本研究成功克隆了陆川猪MC4R基因,为更好地开发利用地方品种陆川猪及其繁育奠定理论基础。
This study was aimed to clone melanocortin-4 receptor（MC4R）gene of Luchuan pig and analyze its genetic structure with bioinformatic software.The recombination plasmid pMD18-T-MC4R was constructed with RT-PCR and cloning methods,and detected by colony PCR and sequencing.After successfully constructed the recombination plasmid pMD18-T-MC4R,the further analysis of physical and chemical properties,structure of protein and modification and the subcellular localization were proceeded.The results showed that the cloned MC4R gene fragment included a 999 bp whole length CDS and encoded 332 amino acids.Compared with MC4R gene of Sus scrofasubmission in NCBI,there were four base mutations of MC4R gene in Luchuan pig,two mutations at 175 and 906 bp were the same sense mutations,the other two mutations at 110 and 278 bp were the mis-sense mutations,causing glutamic acid converted to glycine and valineconverted to alanine in 37 and 93 amino acid residues,respectively.Homology analysis indicated that MC4R was evolutionarily conserved in different species.Seven transmembrane helical regions,several N-glycosylation sites and distinct hydrophobic region were found,but no signal peptide and potential phosphorylation exited in MC4R protein,which might distribute mainly in the endoplasmic reticulum and vacuolar.The elements in the second structure of encoded protein wereα-helix,extended strand,β-turn and random coil.The Luchuan pig MC4R gene was successfully cloned in this study,and provided a foundation for further studying on developing and utilizing the local species Luchuan pig and its breeding.
出处
《中国畜牧兽医》
CAS
北大核心
2018年第2期320-329,共10页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金(31360544)
作者简介
谢婉（1994-），女，广西贺州人，硕士，研究方向：动物遗传育种，E—mail：xiew2281@163．com;通信作者：谢炳坤（1979-），男，广西平南人，博士，副研究员，研究方向：动物遗传育种，E-mail：bkxie@163．com
