Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene.
Molecular Characterization and Transcriptional Regulation Analysis of the Bovine PDHB Gene.
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牛PDHB基因的分子特征及转录调控分析
DOI：
10.1371/journal.pone.0157445
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Zan L
中科院分区：
文献类型：
--
作者：
Li A;Zhang Y;Zhao Z;Wang M;Zan L
丙酮酸脱氢酶β亚基（PDHB）是丙酮酸脱氢酶（E1）的一个亚基，催化丙酮酸转化为乙酰辅酶A，并在三羧酸循环（TCA）和糖酵解途径之间提供连接。以往的研究表明PDHB与肌内脂肪（IMF）含量呈正相关。然而，PDHB的转录调控仍不清楚。本研究克隆了牛PDHB的cDNA，并对其基因组结构进行了分析。系统进化树显示，牛PDHB与山羊和绵羊的亲缘关系较近，与鸡的亲缘关系最小。空间表达谱分析表明，牛PDHB蛋白在睾丸脂肪组织中表达量最高。为了了解牛PDHB的转录调控，克隆了1899 bp的5 '调控区。序列分析在牛PDHB基因5 '端侧翼区未发现共有TATA盒和CCAAT盒。然而，从核苷酸-284至+117预测CpG岛。在双荧光素酶报告基因分析中评估的5 '侧翼区的系列缺失构建体揭示核心启动子位于转录起始位点上游490 bp（+1）。电泳迁移率变动分析（EMSA）和染色质免疫沉淀分析（ChIP）结合定点突变实验表明，肌细胞生成素（MYOG）和CCAAT/增强子结合蛋白β（C/EBP 3）是牛PDHB在骨骼肌细胞和脂肪细胞中的重要转录因子。本研究结果为进一步研究牛PDHB的功能和调控提供了重要依据。
The pyruvate dehydrogenase beta subunit (PDHB) is a subunit of pyruvate dehydrogenase (E1), which catalyzes pyruvate into acetyl-CoA and provides a linkage between the tricarboxylic acid cycle (TCA) and the glycolysis pathway. Previous studies demonstrated PDHB to be positively related to the intramuscular fat (IMF) content. However, the transcriptional regulation of PDHB remains unclear. In our present study, the cDNA of bovine PDHB was cloned and the genomic structure was analyzed. The phylogenetic tree showed bovine PDHB to be closely related to goat and sheep, and least related to chicken. Spatial expression pattern analysis revealed the products of bovine PDHB to be widely expressed with the highest level in the fat of testis. To understand the transcriptional regulation of bovine PDHB, 1899 base pairs (bp) of the 5’-regulatory region was cloned. Sequence analysis neither found consensus TATA-box nor CCAAT-box in the 5’-flanking region of bovine PDHB. However, a CpG island was predicted from nucleotides -284 to +117. Serial deletion constructs of the 5’-flanking region, evaluated in dual-luciferase reporter assay, revealed the core promoter to be located 490bp upstream from the transcription initiation site (+1). Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) in combination with asite-directed mutation experiment indicated both myogenin (MYOG) and the CCAAT/enhancer-binding protein beta (C/EBPß) to be important transcription factors for bovine PDHB in skeletal muscle cells and adipocytes. Our results provide an important basis for further investigation of the bovine PDHB function and regulation in cattle.