过表达MAT2A基因促进猪肌内脂肪细胞分化的研究

doi:10.16431/j.cnki.1671-7236.2018.09.010

摘要/Abstract

摘要：

本研究通过构建腺病毒介导的体外超表达载体，探究腺苷甲硫氨酸转移酶2A（methionine adenosyltransferase 2A，MAT2A）基因在猪肌内脂肪细胞分化中的作用。根据GenBank中猪MAT2A基因mRNA序列（登录号：NM_001167650.1）设计引物，提取猪脂肪组织细胞总RNA并反转录获得cDNA，以此为模板进行PCR扩增并连接到pAdTrack-CMV腺病毒穿梭载体中，对重组质粒pAd-MAT2A进行测序鉴定；pAd-MAT2A载体经PacⅠ限制酶酶切线性化，经质粒大片段回收纯化后转染293A细胞进行病毒包装；采用实时荧光定量PCR检测MAT2A基因表达情况，并提取蛋白进行Western blotting分析，取分化第8天的细胞进行油红O染色。结果表明，穿梭载体pAdTrack-CMV-MAT2A构建成功，并能与骨架载体pAdEasy-1实现同源重组；腺病毒载体pAd-MAT2A转染293A细胞后，病毒滴度达到1E+6 PFU/mL，可满足侵染猪肌内脂肪细胞的需要。实时荧光定量PCR和Western blotting结果显示，MAT2A基因mRNA和蛋白水平均显著上调。油红O染色结果显示，过表达MAT2A基因可促进猪肌内脂肪细胞内脂滴聚积。结果表明，腺病毒介导的MAT2A基因过表达在猪肌内脂肪细胞中呈上调趋势，MAT2A基因可促进脂质积累。

关键词: 腺苷甲硫氨酸转移酶2A(MAT2A)基因; 重组腺病毒; 猪; 肌内脂肪细胞

Abstract:

This study was aimed to explore the role of methionine adenosyltransferase 2A (MAT2A) in porcine intramuscular adipocyte differentiation by constructing the adenovirus mediated overexpression vector in vitro.The primers was designed according to the MAT2A gene mRNA sequence in GenBank (accession No.:NM_001167650.1),the total RNA sequence of porcine adipose tissue cell was extracted and reversed to get cDNA,which was as the template to proceed PCR amplification and connected to the pAdTrack-CMV adenovirus shuttle vector for sequencing identification,the recombinant plasmid was named pAd-MAT2A.pAd-MAT2A was digested by Pac Ⅰ and transfected into 293A cell.The expression level of MAT2A gene was detected by Real-time PCR,and the MAT2A protein was analyzed by Western blotting.The cells differentiated on the 8th day were used for Oil red O staining.The results showed that the shuttle vector (pAdTrack-CMV-MAT2A) was successfully constructed and homologous recombinated with backbone vector (pAdEasy-1).The virus titer was 1E+6 PFU/mL after transfecting the 293A cell with adenovirus vector pAd-MAT2A.Real-time PCR and Western blotting showed that the mRNA and protein levels of MAT2A gene were significantly upregulated by transfecting pAd-MAT2A in porcine intramuscular preadipocytes.Oil red O staining results showed that the overexpression of MAT2A gene promoted lipid accumulation in porcine intramuscular adipocytes.In conclusion,MAT2A gene was overexpressed in expression vector mediated by adenovirus and promoted the lipid increasement in porcine intramuscular adipocytes.

Key words: MAT2A gene; recombinant adenovirus; porcine; intramuscular preadipocyte

中图分类号:

赵存真, 吴海港, 唐雪峰, 焦凤超, 马云, 刘纪成. 过表达MAT2A基因促进猪肌内脂肪细胞分化的研究[J]. 《中国畜牧兽医》, 2018, 45(9): 2425-2432.

ZHAO Cunzhen, WU Haigang, TANG Xuefeng, JIAO Fengchao, MA Yun, LIU Jicheng. Study on Overexpression of MAT2A Gene Promotes Porcine Intramuscular Preadipocyte Differentiation[J]. , 2018, 45(9): 2425-2432.

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