摘要
应用RACE法从刺参Apostichopus japonicus体腔细胞中克隆出26S蛋白酶体组成19S亚复合体亚基之一的S7亚基基因(GenBank登录号:JQ922514)。该基因的cDNA序列全长为2 445 bp,其中5’UTR为70bp,3’UTR为1 070 bp,开放阅读框为1 305 bp,编码435个氨基酸;保守区具有典型的ATP结合和水解基序,氨基酸序列分别为GPPGTGKT和DEID,具有MAT和VRPGRLDR的RNA/DNA螺旋酶的特征性基序;刺参26S蛋白酶体S7亚基基因与紫海胆的氨基酸序列同源性最高(95%),与脊椎动物、节肢动物的同源性较高(88%~89%),与线虫、拟南芥和酵母同源性较低(85%、77%和73%);S7亚基编码的蛋白没有信号肽,也没有跨膜结构域,为非跨膜蛋白,定位于细胞中的液态基质中。采用实时定量PCR方法检测了26S蛋白酶体S7亚基基因在刺参5种组织中的表达情况,结果显示,在肠、呼吸树、表皮、体腔细胞、纵肌中26S蛋白酶体S7基因均有明显表达,且体腔细胞中表达量最高,其次是纵肌和肠,在体壁和呼吸树中表达量较低。本研究中首次在刺参体内发现并克隆了26S蛋白酶体S7亚基基因,同时采用Realtime PCR技术对该基因的表达部位进行了研究,所得结果将为研究棘皮动物蛋白质代谢途径提供新的着眼点,为刺参生理功能的调控研究奠定了基础。
In this study,S7 subunit gene in 26S proteasome was cloned from coelomocytes in sea cucumber Apostichopus japonicus using RACE method for the first time. It was found that the cDNA of the gene had a full length of 2 445 bases in all,including 70 bp of the 5' UTR,1 070 bp of 3' UTR,and open reading frame of 1 305 bp encoding 435 amino acids. The S7 subunit was shown to have typical sequences of ATP-binding motif GPPGTGKT, ATP hydrolytic region DEID and RNA / DNA helicase region MAT and VRPGRLDR,sharing 95% amino acid identity with those in sea urchin,88%-89% with vertebrate,arthropodand and nematode,73%-85% with yeast and Arabidopsis thaliana. There was not any signal peptides and trans-membrane domain in the protein coded by S7 gene. The real-time PCR quantification revealed that the expression of S7 subunit gene was observed in 5 tissues of sea cucumber,the maximal level in the coelomocytes,followed by longitudinal muscle and intestine,and the minimum in body wall and respiratory tree. The findings provide the molecular bases for understanding of the function of 26S proteasome in physiological regulation,regeneration and immune mechanisms in sea cucumber.
出处
《大连海洋大学学报》
CAS
CSCD
北大核心
2013年第6期528-534,共7页
Journal of Dalian Ocean University
基金
国家自然科学基金资助项目(30371099)
辽宁省教育厅实验室专项(2008S064)
关键词
刺参
26S蛋白酶体S7亚基
基因克隆
组织表达
Apostichopus japonicus
S7 subunit of 26S proteasome
molecular clone
tissue expression
