腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化

doi:10.11843/j.issn.0366-6964.2017.05.003

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (5): 802-809.doi: 10.11843/j.issn.0366-6964.2017.05.003

腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化

赵存真¹, 李黄琨¹, 马云², 杨公社^3*

1. 信阳农林学院牧医工程学院, 信阳 464000;
2. 信阳师范学院生命科学学院, 信阳 464000;
3. 西北农林科技大学动物脂肪沉积与肌肉发育实验室, 杨凌 712100

收稿日期:2016-11-10 出版日期:2017-05-23 发布日期:2017-05-20
通讯作者: 杨公社,教授,E-mail:gongsheyang999@hotmail.com
作者简介:赵存真(1987-),女,河南安阳人,讲师,博士,主要从事畜禽脂肪细胞增殖及分化研究,E-mail:baizhenlmncool@126.com
基金资助:
信阳农林学院青年基金项目（201601005）；国家重点基础研究发展计划（2012CB124705）

MAT2B Overexprssion Mediated by Adenovirus Promoting Porcine Intramuscular Preadipocyte Differentiation

ZHAO Cun-zhen¹, LI Huang-kun¹, MA Yun², YANG Gong-she^3*

1. College of Animal Science and Veterinary Medicine, Xinyang Agriculture and Forestry University, Xinyang 464000, China;
2. College of Life Science, Xinyang Normal University, Xinyang 464000, China;
3. Laboratory of Animal Fat Deposition and Muscle Development, Northwest A&F University, Yangling 712100, China

Received:2016-11-10 Online:2017-05-23 Published:2017-05-20

摘要/Abstract

摘要：

本研究旨在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（MAT2B）对猪肌内脂肪细胞分化的影响。本研究以猪脂肪组织为试验材料，提取总RNA，并反转录得到cDNA，参考GenBank收录的猪MAT2B基因mRNA序列设计引物，PCR扩增并连接到腺病毒穿梭载体pAdTrack-CMV，进行测序鉴定。结果表明，构建的穿梭载体与骨架载体pAdEasy-1可以实现同源重组，即腺病毒重组载体pAd-MAT2B构建成功；用PacⅠ限制性内切酶酶切线性化pAd-MAT2B载体并回收质粒大片段转染293A细胞可以实现病毒的成功包装，病毒滴度测定可满足原代细胞侵染需要。转染猪原代肌内脂肪细胞后，MAT2B的mRNA和蛋白水平实现了显著的上调。油红O染色结果表明，过表达MAT2B促进了肌内脂肪细胞脂质积累；实时定量结果证明，MAT2B促进成脂标志基因PPARγ和aP2表达的显著上调。综上所述，腺病毒介导的体外表达载体可以成功的实现MAT2B基因超表达；MAT2B正向调控猪肌内脂肪细胞分化。

关键词: 腺苷甲硫氨酸转移酶2B, 重组腺病毒, 肌内脂肪细胞, 猪

Abstract:

The aim of this study was to explore the effect of MAT2B on porcine intramuscular preadipocyte differentiation by constructing overexpression vector in vitro mediated by adenovirus. A pair of exclusive primers was designed according to the GenBank sequence information of MAT2B gene, and MAT2B was amplified by PCR. The obtained PCR products were inserted into a shuttle vector pAdTrack-CMV, then the gene was identified by sequencing. The results showed that shuttle vector (pAdTrack-CMV) and backbone vector (pAdEasy-1) were in homologous recombination, and the adenovirus recombinant vector (pAd-MAT2B) was constructed successfully. Then the recombinant adenovirus DNA was digested by the Pac I, the pAd-MAT2B adenovirus was produced, and we used it to infect the porcine preadipocytes. The expression level of mRNA and protein of MAT2B significantly increased. Oil Red O staining assay showed that overexpression of MAT2B promoted lipid accumulation in intramuscular preadipocyte. Moreover, the expression of adipogenic gene PPARγ and aP2 mRNA were significantly promoted when infected by Ad-MAT2B. The results suggest that MAT2B is overexpressed in expression vector in vitro mediated by adenovirus, and MAT2B play a positive role during porcine intramuscular preadipocyte differentiation.

Key words: Methionine adenosyltransferase 2B, recombinant adenovirus, intramuscular preadipocyte, pig

中图分类号:

S828
S813.1

赵存真, 李黄琨, 马云, 杨公社. 腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化[J]. 畜牧兽医学报, 2017, 48(5): 802-809.

ZHAO Cun-zhen, LI Huang-kun, MA Yun, YANG Gong-she. MAT2B Overexprssion Mediated by Adenovirus Promoting Porcine Intramuscular Preadipocyte Differentiation[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(5): 802-809.

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腺病毒介导的MAT2B过表达促进猪肌内脂肪细胞分化

MAT2B Overexprssion Mediated by Adenovirus Promoting Porcine Intramuscular Preadipocyte Differentiation

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