鳜UCP1,2基因结构、序列分析及组织表达

从分子水平上探讨了鳜解偶联蛋白1、2 (UCP1, 2)基因结构、组织表达水平及与产热、脂肪代谢等生理机能的关系。通过与脊椎动物UCP1、UCP2基因序列进行比对,设计简并引物与特异引物进行PCR和RACE扩增、测序、拼接序列,获得UCP1、UCP2的基因组序列和内含子/外显子结构。基因组步行法克隆鳜肝脏UCP1、UCP2 基因5′侧翼序列。应用半定量RT-PCR的方法,以β-肌动蛋白作为外参照,在其指数期增长的范围内得到鳜不同组织UCP1、UCP2的相对表达水平。结果表明,UCP1基因组全序列为3 146 bp, 5′侧翼调控区为1 333 bp,含有5个内含子和6个外显子,开放阅读框(ORF)长942 bp,编码一个大小为313个氨基酸的蛋白质。UCP2基因组全序列为2 890 bp, 5′侧翼调控区为1 800 bp,含有7个内含子和8个外显子,ORF长939 bp,编码一个大小为312个氨基酸的蛋白质。UCP1、UCP2间隔外显子的内含子皆符合“GT-AG”规则,内含子的数目与哺乳动物一致。系统进化分析表明,鳜UCP1、UCP2氨基酸序列分别与鱼类UCP1、UCP2氨基酸序列聚为一支,且与UCP3、UCP4、UCP5分支区分明显。鳜UCP1、UCP2基因不同组织表达水平的高低可能与鳜本身的生态习性及各器官在产热、脂质代谢中的作用相关,但明确的分子机制尚待进一步研究。

Cloning, sequence analysis and tissue expression of UCP1, UCP2 in Chinese perch (Siniperca chuatsi)

To better understand the relation of UCP1 ,UCP2 to the physiology mechanism of Chinese perch (Siniperca chuatsi) , UCP1, UCP2 gene sequence and exon/intron structure were obtained from Chinese perch by PCR and RACE method. Using p-actin as external control, six tissues UCP1, UCP2 gene expression specificity of Chinese perch were also studied by RT-PCR. The UCP1, UCP2 gene sequences were 3 146 bp, 2 890 bp in length. ORF were 942 bp, 939 bp and the deduced protein sequences are 313, 312 amino acids respectively. UCP1 included 5 introns and 6 exons and UCP2 has 7 introns and 8 exons. In the deduced amino acid sequence of UCP1, UCP2, three mitochondrial carrier protein motifs, the UCP signature motifs, six transmembrane α-helix domains and the putative purine-nucleotide binding domain (PNBD) both were found. The UCP1 mRNA is primarily expressed in liver, and low level of expression can also be detected in intestine. The UCP1 mRNA was not detectable in spleen, muscle, brain and adipose tissue. In contrast, the UCP2 mRNA is ubiquitous in four selected tissues. The high abundance was observed in adipose tissue, muscle and intestine, and low abundance was observed in liver. Brain has no expression. The data indicated that tissue specificity of Chinese perch UCP1 ,UCP2 mRNA probably relates to their feed habits, physiology role, and mRNA expression level may be greatly affected by ROS production in different tissues.