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首页> 外文期刊>Genomics >Bioinformatics analysis and transcriptional regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes
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Bioinformatics analysis and transcriptional regulation of TORC1 gene through transcription factors NRF1 and Smad3 in bovine preadipocytes

机译：通过转录因子NRF1和Smad3在牛前脂肪细胞的生物信息分析和Torc1基因的转录调节

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Intramuscular fat is the an important factor that defines meat quality; however, enhancing its deposition without increasing the other three adipose depots (subcutaneous, visceral, and intermuscular fat) is a challenge for animal science and the meat industry. The TORC1 is a key regulator of adipogenesis and its regulation in bovine intramuscular preadipocytes has not been studied. The TORC1 is a member of the gene family that codes for a binding proteins which regulate transcription of cAMP which, is a key regulator of adipogenesis. In the present study, expression and sub-cellular localization of the TORC1 gene was analyzed in bovine preadipocytes. Bioinformatics tools were applied to characterize TORC1. To investigate the molecular mechanism of bovine TORC1 gene regulation, we cloned a 1008?bp of the 5’UTR regulatory region into a luciferase reporter vector. Different fragments were amplified using 5’UTR unidirectional deletion of the TORC1 promoter. Site directed mutation, dual luciferase reporter assay, RNAi interference and DNA-protein interaction (EMSA) were used to validate the regulatory roles of Smad3 and NRF1 in the regulation of TORC1 gene in bovine preadipocytes. The core promoter region of the TORC1 gene was identified at a location ?410 to ?155?bp upstream of transcription start site. Different vectors were constructed through serial deletion of the 5’UTR flanking region and in combination with site directed mutations and transcription interference through siRNA or shRNA, two transcription factors of NRF1 and Smad3 were found to be repressors in the promoter of the TORC1 gene. These findings were further confirmed through Electrophoretic Mobility Shift Assay (EMSA) within nuclear extracts of bovine adipocytes. The core promoter region of the TORC1 gene, spanning from ?410 to ?155?bp upstream of the transcription start site was specified in this study and this information will provide opportunity for the improvement of intramuscular fat in cattle.
机译：肌内脂肪是定义肉质的重要因素;然而，增强其沉积而不增加其他三个脂肪仓库（皮下，内脏和胚胎脂肪）是动物科学和肉类工业的挑战。 Torc1是脂肪发生的关键调节因子，并且其在牛肌内前脂肪细胞中的调节尚未研究。 Torc1是基因系列的成员，其代码调节营地转录的结合蛋白是脂肪发生的关键调节因子。在本研究中，在牛前脂肪细胞中分析了TORC1基因的表达和亚细胞定位。生物信息学工具用于表征TORC1。为了探讨牛Torc1基因调节的分子机制，我们将5'UTR调节区的1008bp克隆到荧光素酶报告量载体中。使用TORC1启动子的5'UTR单向缺失扩增不同的片段。网站定向突变，双荧光素酶报告器测定，RNAI干扰和DNA-蛋白质相互作用（EMSA）用于验证SMAD3和NRF1在牛前脂肪细胞中Torc1基因调节中的调节作用。在转录起始位点上游的位置α410至α110〜155β1110〜155℃的核心启动子区域。通过连续缺失来构建不同的载体，并且通过SiRNA或ShRNA与位点定向突变和转录干扰结合，发现NRF1和Smad3的两个转录因子是Torc1基因启动子中的阻遏物。通过牛脂肪细胞的核提取物中的电泳迁移率移位测定（EMSA）进一步证实了这些发现。在这项研究中指定了从α410到410送到410〜410的核心启动子区域，跨越转录开始部位的BP，这些信息将为牛内肌肉脂肪的改善提供机会。

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