九孔鲍α-淀粉酶基因克隆、表达分析及与生长性状相关性

摘要

We cloned the alpha-amylase gene cDNA of Haliotis diversicolor supertexta by rapid amplification of cDNA ends method (RACE) and reverse transcription polymerase chain reaction (RT-PCR) to analyze its tissue expression and correlation with growth traits.The results show that the full length of alpha-amylase gene cDNA was 2 260 bp with an open reading frame (ORF) of 2 088 bp encoding 695 amino acids.The alpha-amylase contained a signal peptide with 18 amino acid residues (MWAQYGIVSALLVLSASA).Two functional structure domains (domain A,domain C) were found in the protein of alpha-amylase.Domain A contained one catalytic center between 28th and 396th amino acids.The alpha-amylase mRNA expression in seven tissues (adductor muscle,intestine,digestive gland,stomach,mantle,rostral,foot) was observed,highest in stomach (326.803) and lowest in mantle (0.35).Significant difference was found in the expression of alpha-amylase between digestive and non-digestible tissues (P ＜ 0.05).Significant positive correlation existed between alpha-amylase gene expression and growth traits (P ＜0.05).%实验采用cDNA末端快速扩增(RACE)和反转录聚合酶链式反应(RT-PCR)技术对九孔鲍(Haliotis diversicol-or supertexta)α-淀粉酶基因cDNA进行克隆,并分析该基因的组织表达及其与生长性状的相关性.结果表明,九孔鲍α-淀粉酶基因cDNA全长为2 260 bp,其中开放阅读框长度为2 088 bp,共编码695个氨基酸.经SingalP分析,α-淀粉酶多肽链中含有信号肽结构,且长度为18个氨基酸(MWAQYGIVSALLVLSASA),由该多肽链折叠成的三级结构中含有domain A、domain C 2个结构域,并且在domain A第28～第396氨基酸处存在催化中心.该基因在九孔鲍右侧壳肌、肠、肝脏、胃、外套膜、吻、腹足7种组织中均有不同程度表达.其中在胃中表达量最高,表达量高达326.803;而在外套膜中表达量最低,表达量仅为0.35.经ANOVA分析,消化组织与非消化组织间的α-淀粉酶基因表达量存在显著差异(P＜0.05).α-淀粉酶基因mRNA表达量与生长性状呈显著正相关(P＜0.05).