中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (9): 2526-2534.doi: 10.16431/j.cnki.1671-7236.2019.09.004

• 生物技术 • 上一篇    下一篇

水牛SIRT6基因克隆及其在组织中的表达分析

崔佳瑜, 程隽如, 黄坚强, 张瑞门, 石德顺, 杨素芳, 邓彦飞   

  1. 广西大学, 亚热带农业生物资源保护与利用国家重点实验室, 南宁 530004
  • 收稿日期:2018-12-10 出版日期:2019-09-20 发布日期:2019-09-21
  • 通讯作者: 杨素芳, 邓彦飞 E-mail:85997362@qq.com;yanfei-dun@163.com
  • 作者简介:崔佳瑜(1994-),女,广西桂林人,硕士,研究方向:动物遗传育种与繁殖,E-mail:569560329@qq.com
  • 基金资助:
    国家自然科学基金项目(31760334、31860644);广西自然科学基金(AA17204051、2015GXNSFAA139080、2018GXNSFAA281007)

Cloning and Tissue Expression Analysis of SIRT6 Gene in Buffalo

CUI Jiayu, CHENG Juanru, HUANG Jianqiang, ZHANG Ruimen, SHI Deshun, YANG Sufang, DENG Yanfei   

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Univesity, Nanning 530004, China
  • Received:2018-12-10 Online:2019-09-20 Published:2019-09-21

摘要: 本研究旨在克隆水牛SIRT6基因,构建真核表达载体,并对该基因进行生物信息学及组织表达谱分析。以牛SIRT6基因编码区为种子序列(GenBank登录号:NM_001098084.1),应用Oligo 7.0软件设计引物序列,用RT-PCR方法扩增水牛SIRT6基因完整编码区序列,测序鉴定后进行生物信息学分析,构建逆转录病毒载体pMXs-SIRT6-IRES-GFP,并在HEK-293T细胞和水牛胎儿成纤维细胞上进行重组载体表达鉴定;采集水牛心脏、肝脏、脾脏、肺脏、肾脏、肠、脑、皮肤、生殖嵴分别提取RNA,反转录后以cDNA为模板进行实时荧光定量PCR,检测SIRT6基因在各组织中的表达差异。结果表明,水牛SIRT6基因编码区全长1 080 bp,编码359个氨基酸,其中亮氨酸及脯氨酸含量最高(10.3%),酪氨酸含量最低(1.1%)。水牛SIRT6基因核苷酸序列与牛、人和小鼠的同源性分别为98.4%、86.7%和78.5%,物种间同源性较低。系统进化树结果表明,水牛与牛聚为一支,再与人、小鼠聚为一大支,亲缘关系相对较近,与果蝇亲缘关系较远。SIRT6蛋白理论分子质量为39.47 ku,分子式为C1737H2783N509O516S13,等电点为8.48,不存在跨膜区,为膜内蛋白;含有Sirtuin家族特有的Sir2去乙酰化酶的催化结构。二级结构预测结果显示,水牛SIRT6蛋白包含13个α-螺旋、27个β-折叠、24个T-转角和20个无规则卷曲。逆转录病毒载体介导的SIRT6基因能在HEK-293T细胞和水牛胎儿成纤维细胞中表达。实时荧光定量PCR结果显示,SIRT6基因在水牛心脏、肝脏、脾脏、肺脏、肾脏、肠、脑、皮肤、生殖嵴中均有表达,其中在生殖嵴中表达量最高,在皮肤中的表达量次之,而在肝脏中表达量最低。本试验结果将为水牛SIRT6基因的功能研究提供参考。

关键词: 水牛; SIRT6基因; 克隆; 载体构建; 表达谱

Abstract: This study was aimed to clone buffalo SIRT6 gene,construct retroviral expression vector,and conduct bioinformatics and tissue expression profile analysis.Primers were designed by Oligo 7.0 according the coding sequence(CDS) of cattle SIRT6 gene(GenBank accession No.:NM_001098084.1),the SIRT6 gene CDS was amplified by RT-PCR,and analyzed by bioinformatic after sequencing.The recombinant retroviral vector pMXs-SIRT6-IRES-GFP was constructed,and expressed in HEK-293T cells and buffalo fetal fibroblasts (BFF).RNA was extracted from heart,liver,spleen,lung,kidney,intestine,brain,skin and genital ridge in buffalo,respectively,the relative expression of SIRT6 gene in different tissues was detected by Real-time quantitative PCR using cDNA as template.The results showed that the coding region of SIRT6 gene in buffalo was 1 080 bp in length and encoded 359 amino acids,the contents of leucine and proline were the highest (10.3%) and tyrosine was the lowest (1.1%).The homology of nucleotide sequence of SIRT6 gene in buffalo were 98.4%,86.7% and 78.5% with Bos taurus,Homo sapiens and Mus musculus,respectively,the homology among species was low.The phylogenetic tree results showed that buffalo and Bos taurus were clustered into one branch,and then Homo sapiens and Mus musculus were clustered into one large branch,the relationship was relatively close,but far from that of Drosophila melanogaster.The theoretical molecular weight of SIRT6 protein was 39.47 ku,the molecular formula was C1737H2783N509O516S13,and the isoelectric point was 8.48,there was no transmembrane region,and it was an intramembrane protein.SIRT6 protein contained the catalytic structure of Sirtuin family-specific SIRT2 deacetylase.Secondary structure prediction showed that buffalo SIRT6 protein contained 13 α-helix,27 β-sheet,24 T-turn and 20 random coil.The retroviral vector mediated SIRT6 gene could be expressed in HEK-293T cells and BFF.Real-time quantitative results showed that SIRT6 gene was expressed in heart,liver,spleen,lung,kidney,intestine,brain,skin and genital ridge of buffalo,and the expression of SIRT6 gene was the highest in genital ridge,followed by in skin,and was the lowest in liver.This results would lay a foundation for the functional study of SIRT6 gene in buffalo.

Key words: buffalo; SIRT6 gene; cloning; vector construction; expression profile

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