文山牛SIRT2基因的克隆、表达及亚细胞定位

doi:10.16431/j.cnki.1671-7236.2018.06.001

摘要/Abstract

摘要：

试验旨在克隆文山牛SIRT2基因，检测SIRT2基因在文山牛不同组织中的表达谱并进行序列分析，鉴定其在293A细胞系中的亚细胞定位。参照GenBank中牛SIRT2基因序列（登录号：NM_001113531.1），在其保守区设计1对特异性引物，以cDNA为模板克隆文山牛SIRT2基因，进行同源性和系统进化分析；以GAPDH基因为内参分析SIRT2基因在文山牛心脏、肝脏、肾脏、肺脏、胃、结肠、肌肉、脂肪8个组织中的表达水平；构建pDsRed-SIRT2真核表达载体并转染到293A细胞系中表达，观察融合蛋白的亚细胞定位。结果表明，文山牛SIRT2基因开放阅读框全长1 173 bp，编码390个氨基酸，分子质量为43.3 ku，理论等电点为4.985；SIRT2编码蛋白整体表现为亲水性，带有负电荷。文山牛与水牛、白豚、蹄蝠等哺乳动物具有较高的同源性，在系统发育树中距离最近，但与树袋熊距离稍远，推测有袋目哺乳动物在进化过程中过早地与其他哺乳动物产生了遗传差异，在哺乳动物遗传相关的研究中需特殊考虑有袋目哺乳动物。实时荧光定量PCR结果显示，SIRT2基因在文山牛8个组织中均有表达，在肌肉和肝脏中的表达水平较高，与其他组织存在显著差异（P<0.05）。pDsRed-SIRT2融合蛋白主要定位于293A细胞的细胞质中，可能参与部分细胞器的运作及细胞内物质的运输，调节细胞稳态并在细胞繁殖过程中提供必要的能量物质。本试验结果为进一步探究牛SIRT2基因功能提供了一定的理论依据。

关键词: 文山牛; SIRT2基因; 表达谱; 序列分析; 亚细胞定位

Abstract:

This study was aimed to clone silent information regulator 2 (SIRT2) gene in Wenshan cattle,detect the expression patterns of SIRT2 gene in different tissues of Wenshan cattle, and investigate the subcellular localization of SIRT2 protein in 293A cell lines.One pair of specific primers was designed in the conserved region which based on the bovine SIRT2 gene sequence in GenBank (accession No.:NM_001113531.1),using cDNA as a template,the SIRT2 gene of Wenshan cattle was cloned for homology and phylogenetic analysis.The expression level of SIRT2 gene in heart,liver,kidney,lung,stomach,colon,muscle and fat of Wenshan cattle was analyzed using GAPDH gene as internal reference gene.The pDsRed-SIRT2 eukaryotic expression vector was constructed and transfected into 293A cell line to observe the subcellular localization of the fusion protein.The results showed that the open reading frame of SIRT2 gene in Wenshan cattle was 1 173 bp,encoded 390 amino acids,the molecular weight was 43.3 ku,the theoretical isoelectric point was 4.985.SIRT2 protein was hydrophilic and negatively charge.Homology analysis indicated that SIRT2 gene sequence of Wenshan cattle was highly homologous to mammals such as buffalo,dolphin and hoof,but the distance from the phascolarctos cinereus tree was long.It was surmised that marsupial mammals were prematurely separated from other mammals during evolution,so the marsupial mammals need to be special considered in the genetic research.Real-time quantitative PCR result showed that SIRT2 gene was expressed in all detected eight tissue samples of Wenshan cattle,the SIRT2 gene expression levels in muscle and liver were significantly higher than those of other tissues (P<0.05).The fusion expression vector pDsRed-SIRT2 was mainly located in the cytoplasm of 293A cell line,which might involve in the operation of some organelles and the transport of intracellular substances,regulate cell homeostasis and provide the necessary energy material during cell propagation.This study provided a theoretical basis for further exploring the fuction of SIRT2 gene in cattle.

Key words: Wenshan cattle; SIRT2 gene; expression profile; sequence analysis; subcellular localization

中图分类号:

卢徐斌, 绪欣, 王文强, 李明勋, 陈志, 黄必志, 亐开兴, 张慧敏, 毛永江, 杨章平. 文山牛SIRT2基因的克隆、表达及亚细胞定位[J]. 《中国畜牧兽医》, 2018, 45(6): 1427-1436.

LU Xubin, XU Xin, WANG Wenqiang, LI Mingxun, CHEN Zhi, HUANG Bizhi, QU Kaixing, ZHANG Huimin, MAO Yongjiang, YANG Zhangping. Cloning, Expression and Subcellular Localization of SIRT2 Gene in Wenshan Cattle[J]. , 2018, 45(6): 1427-1436.

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