青石斑鱼抗细胞凋亡因子DAD1的克隆及表达特征分析

Molecular cloning and characterization of a homologue of defender against cell death 1 in Epinephelus awoara

抗细胞凋亡因子(Defender against cell death 1,DAD1)是一种重要的内源性细胞凋亡抑制因子.采用RACE-PCR方法,结合SMART cDNA合成方法、抑制性差减杂交技术,从青石斑鱼脾脏中克隆DAD1并研究其在正常的青石斑鱼和注射了脂多糖的青石斑鱼各组织中的表达模式.结果表明:DAD1基因cDNA全长721 bp,包含一个342 bp的完整阅读框,编码一个由113个氨基酸组成的蛋白.RT-PCR半定量分析显示DAD1在正常的青石斑鱼和注射了脂多糖的青石斑鱼的脾、心、头肾、肾、肝中都有转录,在脾、肝中表达水平较高,而在肌肉中未见明显表达.DAD1在注射了脂多糖的青石斑鱼各组织中的表达量均高于正常的青石斑鱼.

Defender against cell death 1 （DAD1） is a negative regulator of apoptosis and a subunit of the oligosaccharyltransferase complex. We cloned DAD1 from the spleen of Epinephelus awoara using the comhinative methods of suppressive subtraction hybridization, SMART cDNA synthesis and RACE--PCR. The full--length cDNA of DAD1 is 721 bp, containing a 342 bp open reading frame that encodes a 113 amino acid protein. Semi-quantitative RT--PCR analyses showed that mRNA of DAD1 can be detected in the spleen, heart, liver, anterior kidney and kidney of normal E. awoara and lipopolysaccharide--stimulated E. awoara . The expressions of DAD1 in all these tissues showed some increase in the LPS- treated E. awoara, compared with normal E. awoara, which indicates that lipopolysaccharide might be a stimulator of programmed cell death.