山羊RPL26基因生物信息学分析及对肌内脂肪细胞分化的影响

doi:10.11843/j.issn.0366-6964.2022.04.007

摘要/Abstract

摘要： 旨在克隆山羊RPL26基因序列并对其在山羊各组织中的表达情况和对山羊脂肪细胞分化的调控作用进行探究。本研究以1周岁简州大耳羊公羊作为试验对象(健康生长状态良好，体重约50 kg，n=3)，利用RT-PCR等方法克隆RPL26序列，对基因及蛋白质序列进行生物信息学分析；以山羊各组织cDNA为模板，利用qPCR方法构建组织表达谱；利用双酶切方法构建RPL26过表达载体，通过脂质体转染在山羊肌内脂肪细胞中过表达RPL26，诱导分化后利用油红O染色和Bodipy染色观察细胞形态学变化，qPCR检测过表达效率及成脂相关基因的表达情况，以阐明其对肌内脂肪细胞分化的影响。结果表明，克隆获得山羊RPL26序列544 bp，CDS区为438 bp，编码蛋白质为带负电的不稳定亲水性蛋白质，主要定位于细胞核。RPL26在山羊各组织广泛表达，其中以腹部皮下脂肪和背部皮下脂肪表达量较高，极显著高于其他组织(P<0.01)。在山羊肌内脂肪细胞中过表达RPL26，肌内脂肪细胞的脂滴明显增多，油红O染色OD值极显著上升；qPCR结果显示，Pref1表达水平极显著下降(P<0.01)，PPARγ等成脂标志基因及LPL等脂代谢标志基因的表达都极显著上升(P<0.01)。本研究克隆获得了山羊RPL26序列544 bp，CDS区为438 bp，在腹部皮下脂肪中表达量最高，RPL26对山羊肌内前体脂肪细胞的分化有着正向调控作用。

关键词: 山羊, 核糖体蛋白, 克隆, 肌内脂肪细胞, 细胞分化

Abstract: The purpose of this study was to clone the sequence of goat RPL26 gene and explore its expression in tissues and its regulation on adipocyte differentiation of goat. The one-year old male Jianzhou da’er goat (with good healthy growth status, weight of 50 kg, n=3) was selected as the experimental object, the RPL26 sequence was cloned by RT-PCR, and the sequence of gene and protein were analyzed by bioinformatics; Goat tissue cDNA was used as template to construct tissue expression profiles by qPCR; The overexpression vector of RPL26 was constructed by double enzyme digestion method. RPL26 was overexpressed in goat intramuscular adipocytes by liposome transfection. The morphological changes were observed by Oil red O staining and Bodipy staining after induction. The overexpression efficiency and expression of adipogenesis related genes were detected by qPCR to clarify its effect on intramuscular adipocyte differentiation. The results showed that the cloned goat RPL26 sequence was 544 bp, the CDS region was 438 bp, and the coding protein was negatively charged unstable hydrophilic protein, which was mainly located in the nucleus. RPL26 was widely expressed in goat tissues, and highly expressed in abdominal subcutaneous fat and back subcutaneous fat, which were extremely significantly higher than that in other tissues (P<0.01). RPL26 was overexpressed in goat intramuscular adipocytes, and the lipid droplets in intramuscular adipocytes increased significantly, the OD value detected by Oil red O staining showed the same result; qPCR results showed that the expression level of Pref1 decreased significantly(P<0.01), the expression levels of adipocytes differentiation marker genes such as PPARγ, and lipid metabolism marker gene such as LPL showed an extremely significantly upregulated expression(P<0.01). The cloned goat RPL26 sequence is 544 bp, and the CDS region is 438 bp, its highest expression was in abdominal subcutaneous fat. RPL26 has a positive regulatory effect on the differentiation of goat intramuscular preadipocytes.

Key words: goat, ribosomal protein, clone, intramuscular adipocytes, cell differentiation

中图分类号:

S827.2

张浩, 王永, 李艳艳, 罗成, 李鑫, 李志雄, 朱江江, 林亚秋. 山羊RPL26基因生物信息学分析及对肌内脂肪细胞分化的影响[J]. 畜牧兽医学报, 2022, 53(4): 1064-1076.

ZHANG Hao, WANG Yong, LI Yanyan, LUO Cheng, LI Xin, LI Zhixiong, ZHU Jiangjiang, LIN Yaqiu. Bioinformatics Analysis of Goat RPL26 Gene and Its Effect on Intramuscular Adipocyte Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1064-1076.

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