马氏珠母贝细胞内视黄酸结合蛋白表达及其与类胡萝卜素的相关性分析

摘要

[目的]明确细胞内视黄酸结合蛋白(CRABP)对马氏珠母贝吸收代谢类胡萝卜素的影响,为培育富含类胡萝卜素的马氏珠母贝养殖新品系打下基础.[方法]采用RACE克隆马氏珠母贝CRABP基因(PmCRABP)cDNA全长序列,并以实时荧光定量PCR检测分析PmCRABP基因在各组织中的表达特征及其在黄白闭壳肌中的表达量.[结果]PmCRABP基因cDNA全长1778 bp,开放阅读框(ORF)长366 bp,编码121个氨基酸,其5'非翻译区(5'UTR)长125 bp,3'UTR长1287 bp.PmCRABP的分子量13.51 kD,理论等电点(pI)5.68,脂溶性系数62.81,不稳定指数35.38,总平均亲水性-0.517,其第5~120个氨基酸为Lipocalin家族的结构域.PmCRABP氨基酸序列与新对虾(Metapenaeus ensis)CRABP氨基酸序列的相似度最高,同属于CRABP蛋白,且二者的空间结构极其相似.PmCRABP基因在马氏珠母贝肝胰腺中的表达量最高,其后依次是外套膜、鳃和闭壳肌,各组织的表达量差异显著(P<0.05,下同).黄白闭壳肌个体的类胡萝卜素含量与PmCRABP基因相对表达量存在显著差异,且二者间呈明显的正相关.[结论]PmCRABP参与了马氏珠母贝的类胡萝卜素代谢.%[Objective]Effects of intracellular retinoic acid-binding protein(CRABP) on absorption and metabolism of carotenoid in Pinctada fucata martensii were investigated to lay a foundation for breeding carotenoid-rich strains.[Method]In this study, cDNA full length sequence of PmCRABP was obtained using RACE technology. Expression cha-racters of PmCRABP gene in different tissues and its expression in white and yellow colored adductor muscle were detected by real-time fluorescence quantitative PCR. [Result]The full length of PmCRABP gene was 1778 bp, including 366 bp of open reading frame(ORF), encoded 121 amino acids, a 5'untranslated region(5'UTR) of 125 bp and a 3'UTR of 1287 bp. The molecular weight was 13.51 kD, theoretical isoelectric point(pI) was 5.68, aliphatic index was 62.81 ,instability index was 35.38 and the grand average of hydropathicity (GRAVY) was -0.517. The 5th to the 120th amino acids of the Pm-CRABP protein were the domains of Lipocalin family. Comparative analysis between amino acid sequence of PmCRABP and that of CRABP of other species indicated that, the similarity between PmCRABP and Metapenaeus ensis CRABP was the highest. Expression of PmCRABP gene in hepatopancreas of P. fucata martensii was the highest, and followed by mantle, gill and adductor muscle. There was significant difference between expressions in different tissues (P<0.05, the same beow). Carotenoid contents and PmCRABP gene expression in yellow and white adductor muscles were signifi-cantly different, and there was a significant positive correlation between carotenoid content and PmCRABP expression.[Conclusion]The results showed that PmCRABP gene is involved in carotenoid metabolism of P. fucata martensii.