联系我们 加入收藏
 
年期检索 高级检索
33
2025年8月13日 星期三
  2016, Vol. 24 Issue (10): 1560-1568    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
鸡Perilipin1基因的克隆及亚细胞定位
秦菲悦1,周纬男2,王彦博1,程博涵1,李辉1,王宇祥3
1. 东北农业大学动物科学学院
2.
3. 东北农业大学动物科学技术学院
Cloning and Subcellular Distribution of Chicken (Gallus gallus) Perilipin1 Gene
全文: PDF (831 KB)   HTML (1 KB) 
输出: BibTeX | EndNote (RIS)      
摘要 本研究旨在克隆鸡(Gallus gallus)脂滴包被蛋白基因(Perilipin1)的cDNA序列,并检测其在鸡前脂肪细胞分化过程中的亚细胞定位。本研究以7周龄肉鸡腹部脂肪组织为材料,采用RT-PCR和RACE的方法扩增并克隆了鸡Perilipin1基因的5'UTR、CDS和3'UTR片段,分析了该基因的结构;以鸡原代前脂肪细胞为材料,利用免疫荧光及激光共聚焦技术,在诱导分化的不同时间点(12~120 h),检测了鸡Perilipin1在前脂肪细胞中的表达位置。结果表明,本研究获得的鸡Perilipin1基因5'UTR、CDS和3'UTR序列总长度为2 379 bp,该基因由9个外显子、8个内含子组成(ATG位于第二外显子上);在鸡原代前脂肪细胞诱导分化过程中,Perilipin1始终包被在脂滴周围。综上,本研究成功克隆了鸡Perilipin1基因的完整cDNA序列并确定了Perilipin1在鸡前脂肪细胞分化过程中与脂滴的空间位置关系,该结果为深入开展鸡Perilipin1基因的功能研究,揭示鸡脂肪代谢的分子遗传机理提供了重要的理论依据。
服务
把本文推荐给朋友
加入我的书架
加入引用管理器
E-mail Alert
RSS
作者相关文章
秦菲悦
周纬男
王彦博
程博涵
李辉
王宇祥
关键词 脂滴包被蛋白克隆亚细胞定位    
Abstract:Surrounding the membrane surface of lipid droplets, Perilipin1 plays important roles in the formation and maturation of lipid droplets, and regulation of fat mobilization (lipolysis). Considered as one of the important marker genes of adipocyte differentiation, many Perilipin1 studies in mammals have been carried out, while in chickens (Gallus gallus) relatively few. The objectives of the present study were to clone full length mRNA of chicken Perilipin1 gene, and analyze its subcellular location during the progression of chicken preadipocyte differentiation. We extracted total RNA of abdominal adipose tissue from chickens at 7 weeks of age. RT-PCR and rapid amplification of cDNA ends (RACE) methods were used to clone the 5'-untranslated region (5'-UTR), the coding domain sequence (CDS) and the 3'-untranslated region (3'-UTR) sequences of chicken Perilipin1 gene, and then the gene structure was analyzed. The subcellular localizations of Perilipin1 in chicken preadipocyte was examined by immunofluorescence confocal microscopy at different time points (12~120 h). Results showed that the whole mRNA length of chicken Perilipin1 gene was 2 379 bases (bp), consisted of nine exons and eight introns (with the start codon ATG located in the second exon). The CDS length of chicken Perilipin1 gene was 1 551 bp, encoding 517 amino acids, and lengths of 5'-UTR and 3' UTR were 94 and 733 bp, respectively. Sequences have been submitted to NCBI database, and assigned the serial number (Accession No. GU327532). In addition, we performed the multiple alignment for the chicken Perilipin1 CDS sequence with mouse (NM_175640) and human (NM_001145311) sequences deposited in NCBI database by the Clustalw software (http://www.genome.jp/tools/Clustalw/), and obtained the homology percentages, 38% and 41%, respectively. Therefore, sequence divergence at nucleotide level was quite large, suggesting that the role of chicken Perilipin1 in adipocye differentiation may not be fully consistent with mammals. We used immunofluorescence method combined with laser confocal microscopy to check the subcellular location of chicken Perilipin1 in the process of preadipocyte differentiation, which was induced by oleate at different time points (12, 24, 48, 72, 96 and 120 h), started on primary chicken preadipocytes cultured by the digestion method. At the first 12h of induction, few small lipid droplets were visualized in the preadipocytes, and Perilipin1 located on the surface of neonatal lipid droplets. As time passed on (24 to 48 h), more lipid droplets emerged, and Perilipin1 surrounded the lipid droplets. At late stage (72 to 120 h), several large lipid droplets formed through the fusion of small lipid droplets, and Perilipin1 convened and packaged on the surface of lipid droplets. Therefore, our results indicated that chicken Perilipin1 surrounded the membrane surface of lipid droplets during the whole process of adipocyte differentiation, and we speculated that basically, the function of chicken Perilipin1 might help in forming a protective barrier separating lipase from lipids stored in the lipid droplet. In conclusion, we have successfully cloned the full length mRNA of chicken Perilipin1 gene, and confirmed the subcellular position of Perilipin1 during chicken preadipocyte differentiation. The current study provided some molecular information for further investigating the function of chicken Perilipin1 gene.
Key wordsChicken    Perilipin1    Clone    Subcellular localization
收稿日期: 2016-03-10      出版日期: 2016-08-06
基金资助:国家自然科学基金;高等学校博士学科点专项科研基金;现代农业产业技术体系建设专项资金
通讯作者: 王宇祥     E-mail: wyx2000@neau.edu.cn
引用本文:   
秦菲悦 周纬男 王彦博 程博涵 李辉 王宇祥. 鸡Perilipin1基因的克隆及亚细胞定位[J]. , 2016, 24(10): 1560-1568.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I10/1560
 
版权所有 © 2014 《农业生物技术学报》编辑部   京ICP备11035905号-3
地址:北京市海淀区圆明园西路2号中国农业大学生命科学楼1053室 邮编:100193
电话:010-62733684 传真:010-62731615 E-mail: nsjxb@cau.edu.cn