剑尾鱼P-糖蛋白基因全长cDNA克隆、分析及组织分布

P-GLYCOPROTEIN OF SWORDTAIL FISH XIPHOPHORUS HELLERI: cDNA CLONING, BIOINFORMATIC AND TISSUE-SPECIFIC EXPRESSION ANALYSIS

P-糖蛋白(P-glycoprotein,P-gp)为ABC转运体超家族的主要成员,分布于细胞膜,依赖ATP供能,可将细胞内的亲脂性毒物或药物逆浓度泵出胞外,在机体解毒上具重要功能。研究采用RT-PCR和RACE法对剑尾鱼P-gp基因进行克隆并获得了全长cDNA序列4301 bp,该基因编码1286个氨基酸残基,估算编码蛋白的分子质量为141.64 kD。生物信息学分析表明,剑尾鱼P-gp不含信号肽序列,具ATP转运家族蛋白的典型结构域,包括2个疏水性的跨膜区(Transmembrane domains,TMD)和2个位于细胞浆内的核苷酸结合区(Nucleotide-binding domains,NBD),为全转运子,每个跨膜区都有6个跨膜α螺旋,而每个NBD包含1个ABC转运体家族标记序列;跨膜性质预测结果显示该蛋白属膜蛋白,具P-gp的典型特征。序列同源性分析发现,不同进化地位动物P-gp存在高度同源性,显示P-gp在系统进化上高度保守。P-gp在剑尾鱼肝脏、肾脏、脑等不同组织均有表达,其中以肝脏的相对表达量最高,是P-gp检测的代表组织。QRT-PCR实验表明,苯并(a)芘胁迫可明显诱导P-gp mRNA的表达。剑尾鱼P-gp的表达水平或可作为一个生物标记物指标,应用于环境持久性有机污染物的监测研究中。

P-glycoprotein is one of the major members of the ATP-binding cassette （ABC） superfamily which prevented in plasma membrane, utilizes the energy of ATP hydrolysis to transport various substrates across celluar membranes, functions as an export pump that decreases intracellular concentrations of xenobiotic agents. In our experiment, RT-PCR and RACE-PCR were used for P-gp gene cloning in swordtail fish Xiphophorus helleri. The full-length of P-gp cDNA for swordtail fish was 4301 bp with open reading frame （ORF） of 3861 bp encoding a polypeptide of 1286 amino acids. The calculated MW was of 141.64 kD and a theoretical pI of 7.06. Bioinformatics analysis indicated that the deduced amino acid sequence contained striking features of the P-gp including four core regions, two transmembrane domains （TMD） and two nucleotide-binding domains （NBD） but no signal peptide. Each TMD had six transmembrane helices while NBD contained the highly conserved motif Walker A, Walker B, ABC transporters family signature, D-loop, H-loop, Q-loop and Signature C （L-S-G-G-Q）. The deduced amino acid sequence aligned with those of P-gp genes from different species also displayed high degree of sequential homology as was shown by the phylogenic tree. The gene of P-gp was expressed in all examined organs in swordtail fish and had a significantly high expression in liver, so it will be major organ for P-gp examination. Our results also showed that the hepatic P-gp expression of swordtail fish can be significantly upregulated with the exposure of benzo （a） pyrene. Therefore, the reaction of P-gp in the fish to the expo- sure of various xenobiotics can thus be used as a potential biomarker for monitoring environmental pollution.