《中国畜牧兽医》 ›› 2019, Vol. 46 ›› Issue (5): 1273-1282.doi: 10.16431/j.cnki.1671-7236.2019.05.003

• 生物技术 • 上一篇    下一篇

牦牛SMPX基因CDS区克隆及组织表达分析

郭梁1, 王吉坤1, 王会1, 柴志欣1, 信金伟2, 姬秋梅2, 钟金城1   

  1. 1. 西南民族大学青藏高原研究院, 青藏高原动物遗传资源保护与利用教育部重点实验室, 四川省重点实验室, 成都 610041;
    2. 西藏自治区农牧科学院, 省部共建青稞和牦牛种质资源与遗传改良国家重点实验室, 拉萨 850009
  • 收稿日期:2018-09-27 出版日期:2019-05-20 发布日期:2019-05-20
  • 通讯作者: 钟金城 E-mail:zhongjincheng518@126.com
  • 作者简介:郭梁(1993-),男,河北邢台人,硕士生,研究方向:动物遗传学,E-mail:1964055572@qq.com
  • 基金资助:

    西南民族大学硕士研究生创新课题重点项目(CX2018SZ116);国家肉牛牦牛产业技术体系项目(CARS-37)

CDS Cloning and Tissue Expression Analysis of SMPX Gene in Bos grunniens

GUO Liang1, WANG Jikun1, WANG Hui1, CHAI Zhixin1, XIN Jinwei2, JI Qiumei2, ZHONG Jincheng1   

  1. 1. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Sichuan Provincial Key Laboratory, Institute of Qinghai-Tibetan Platean, Southwest Minzu University, Chengdu 610041, China;
    2. State Key Laboratory of Barley and Yak Germplasm Resources and Genetic Improvement, Tibet Academy of Agricultural and Animal Husbandry Sciences, Lhasa 850009, China
  • Received:2018-09-27 Online:2019-05-20 Published:2019-05-20

摘要:

本研究旨在克隆牦牛X染色体相关小肌肉蛋白(small muscle protein X-link,SMPX)基因的CDS区序列,分析该序列所编码蛋白的结构与功能,并检测SMPX基因在牦牛不同组织中的表达情况。运用RT-PCR技术扩增并克隆SMPX基因CDS区序列,分析其氨基酸序列相似性并构建系统进化树;通过在线软件对其理化性质、二级结构和三级结构进行生物信息学分析;采用实时荧光定量PCR方法检测SMPX基因在牦牛右心室、臀大肌、肺脏和大脑4个组织中的表达情况。结果表明,牦牛SMPX基因CDS区全长515 bp,开放阅读框(ORF)长261 bp,编码86个氨基酸。牦牛SMPX氨基酸序列与野牦牛、水牛、家犬、人、白尾鹿德克萨斯亚种、绵羊、黑猩猩、藏羚羊、野猪的相似性分别为100%、97.7%、96.5%、96.5%、95.3%、95.3%、96.5%、94.2%和91.9%,说明其在不同物种间具有较高的保守性。生物信息学分析发现,SMPX蛋白是一种不稳定的亲水性蛋白,二级结构以无规则卷曲和α-螺旋为主,为膜内蛋白,无信号肽和跨膜蛋白;SMPX氨基酸序列共有4个磷酸化位点。亚细胞定位结果表明,SMPX蛋白的分布于细胞核(52.2%)、线粒体(43.5%)和细胞质(4.3%)。实时荧光定量PCR检测结果显示,SMPX基因在牦牛右心室中表达量最高,显著高于其他组织(P<0.05)。本试验结果为深入研究SMPX基因在牦牛中的生理功能和调控机制提供了参考数据。

关键词: 牦牛; SMPX基因; 克隆; 生物信息学; 组织表达

Abstract:

This study was aimed to clone the CDS sequence of small muscle protein X-link (SMPX) gene,analyze its potential structure and function,and explore the expression levels of SMPX gene in different tissues of Bos grunniens.The CDS sequence of SMPX gene was amplified and cloned by RT-PCR,the amino acid sequence homology of SMPX gene was analyzed,and the phylogenetic tree was constructed.The physical and chemical properties,secondary structure and tertiary structure of SMPX protein were analyzed by bioinformatics softwares.The expression patterns of SMPX gene in right ventricle,gluteus maximus,lung and brain of Bos grunniens were detected by Real-time PCR.The results showed that the length of SMPX gene CDS and ORF sequences were 515 and 261 bp,respectively,which encoded 86 amino acids.The amino acid sequence homology of SMPX gene in Bos grunniens with Bos mutus,Bubalus bubalis,Canis lupus familiaris,Homo sapiens,Odocoileus virginianus texanus,Ovis aries,Pan troglodytes,Pantholops hodgsonii and Sus scrofa were 100%,97.7%,96.5%,96.5%,95.3%,95.3%,96.5%,94.2% and 91.9%,respectively,indicating that it was highly conserved in different species.Bioinformatics analysis represented that SMPX protein was unstable and hydrophilic.The secondary structure of SMPX mainly contained random coils and alpha helix.The SMPX protein had no signal peptide and transmembrane protein, and belonged to intramembrane protein.There were four phosphorylation sites in SMPX protein sequence.The results of subcellular localization showed that SMPX protein was located in the nucleus (52.2%),mitochondria (43.5%) and cytoplasm (4.3%).Real-time PCR results exhibited that the expression level of SMPX gene in right ventricle was significantly higher than gluteus maximus,lung and brain (P<0.05).This study might provide reference data for further study of physiological functions and regulatory mechanisms of SMPX gene in Bos grunniens.

Key words: Bos grunniens, SMPX gene, cloning, bioinformatics, organizational expression

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