水牛SIRT6基因克隆及其在组织中的表达分析

doi:10.16431/j.cnki.1671-7236.2019.09.004

Abstract

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

CLC Number:

CUI Jiayu, CHENG Juanru, HUANG Jianqiang, ZHANG Ruimen, SHI Deshun, YANG Sufang, DENG Yanfei. Cloning and Tissue Expression Analysis of SIRT6 Gene in Buffalo[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2526-2534.

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Cloning and Tissue Expression Analysis of SIRT6 Gene in Buffalo

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