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

Abstract

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

CLC Number:

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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Cloning, Expression and Subcellular Localization of SIRT2 Gene in Wenshan Cattle

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