山羊RPL29基因克隆和序列分析及其对肌内脂肪细胞脂质生成的影响

Gene cloning and sequence analysis of the RPL29 gene and its effect on lipogenesis in goat intramuscular adipocytes

摘要:

本研究旨在克隆简州大耳羊RPL29基因并分析其分子特性及对肌内脂肪细胞脂质生成的影响。利用反转录-聚合酶链式反应（reverse transcription-polymerase chain reaction，RT-PCR）法克隆山羊RPL29基因序列，用生物信息学方法分析该基因序列特征，实时荧光定量PCR （quantitative real-time PCR，qRT-PCR）法检测其在山羊各种组织和不同分化阶段肌内脂肪细胞中的mRNA表达水平。利用基因重组法构建的pEGFP-N1-RPL29过表达载体转染进山羊肌内前体脂肪细胞并诱导分化，用油红O和Bodipy染色方法从形态学上揭示过表达RPL29对脂滴积聚的影响，用qRT-PCR检测脂代谢相关基因表达水平的变化。结果显示，山羊RPL29长为507 bp，其中氨基酸编码区（coding sequence，CDS）为471 bp，编码156个氨基酸，是一种主要分布在细胞核、带正电荷且较稳定的亲水性蛋白质。组织表达谱表明该基因在山羊皮下脂肪组织及腹部脂肪组织中的表达水平显著高于其他组织（P<0.05）。时序表达谱表明该基因在山羊肌内脂肪细胞分化第84小时表达水平最高，极显著高于未分化时期（P<0.01）。过表达RPL29促进山羊肌内脂肪细胞中的脂质积聚，且油红O染色后OD值显著增加（P<0.05）。此外，过表达RPL29基因后ATGL和ACC基因表达量极显著增加（P<0.01），FASN基因的表达量显著增加（P<0.05）。总之，山羊RPL29基因可能通过上调FASN、ACC和ATGL来促进山羊肌内脂肪细胞脂质沉积。

Abstract:

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P<0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P<0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P<0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P<0.01) and a significant increase in FASN gene expression (P<0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.