大珠母贝(Pinctada maxima)α-淀粉酶基因cDNA及内含子克隆分析

doi:10.11978/j.issn.1009-5470.2013.03.008

热带海洋学报 ›› 2013, Vol. 32 ›› Issue (3): 52-58.doi: 10.11978/j.issn.1009-5470.2013.03.008cstr: 32234.14.j.issn.1009-5470.2013.03.008

大珠母贝(Pinctada maxima)α-淀粉酶基因cDNA及内含子克隆分析

潘俐玲^{1, 2}, 黄桂菊¹, 成书营^{1, 2}, 王晓宁^{1, 2}, 喻达辉¹

1. 农业部南海渔业资源开发利用重点实验室, 中国水产科学研究院南海水产研究所, 广东广州 510300; 2. 上海海洋大学水产与生命学院, 上海 201306

收稿日期:2012-01-13 修回日期:2012-12-10 出版日期:2013-08-27 发布日期:2013-08-28
通讯作者: 现代农业产业技术体系建设专项资金(CARS-48)
作者简介:潘俐玲(1986―)，女，硕士研究生，从事海洋生物技术研究。E-mail：lingli8611@yahoo.com.cn

Cloning and characterization of alpha amylase cDNA and its introns in the pearl oyster Pinctada maxima

PAN Li-ling^{1, 2}, HUANG Gui-ju¹, CHENG Shu-ying^{1, 2}, WANG Xiao-ning^{1, 2}, YU Da-hui¹

1. Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture, P. R. China, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China; 2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China

Received:2012-01-13 Revised:2012-12-10 Online:2013-08-27 Published:2013-08-28
Contact: 喻达辉，博士，博导，研究员。E-mail：pearlydh@163.com

摘要/Abstract

摘要：

α-淀粉酶广泛分布于动物、植物和微生物中，是贝类软体动物的主要消化酶，对贝类生长有重要影响。文章首次获得大珠母贝α-淀粉酶基因(命名为pmAMY, Pictada maxima alpha amylase)，其cDNA全长1732bp，其中5'UTR 25bp，ORF 1554bp，编码518个氨基酸，3'UTR 153bp，分子量为57.7KDa，等电点7.63。氨基酸序列分析表明，pmAMY的氨基酸序列包括16个氨基酸组成的信号肽序列(MLLIVCSIAFFHSVYG)、8个半胱氨酸位点(Cys₄₆、Cys₁₀₄、Cys₁₅₇、Cys₁₇₆、Cys₃₉₂、Cys₃₉₈、Cys₄₆₄、Cys₄₇₆)、3个活性催化位点(Asp₂₁₃、Glu₂₄₉、Asp₃₁₄)、4个钙结合位点(Asn₁₁₈、Arg₁₇₄、Asp₁₈₃、His₂₁₇)、3个氯离子结合位点(Arg₂₁₁、Asn₃₁₂、Arg₃₅₀)和4段保守序列(Ile₁₁₁—Val₁₁₆、Val₂₀₇—Ala₂₁₅、Phe₂₄₇—Val₂₅₁、Val₃₀₈—Asn₃₁₅)。pmAMY的氨基酸序列与企鹅珍珠贝(Pteria penguin)同一性最高，为82%；与超嗜热古菌(Thermococcus hydrothermalis)同一性最低仅为27%；与其他物种的同一性在57%—79%之间。克隆获得大珠母贝pmAMY基因的2个内含子，长度分别为846bp、162bp。2个内含子都起始于GT，终止于AG，符合内含子共同剪接位点序列。组织表达分析表明pmAMY只在肝胰脏中表达。本研究为α-淀粉酶基因的功能分析、单核苷酸多态性(SNP, single nucleotide polymorphism)位点分离及其与生长性状的关联分析奠定了基础。

关键词: 大珠母贝, α-淀粉酶基因, 外显子, 内含子, 序列特征

Abstract:

Alpha amylase is widely distributed in animals, plants and bacteria, and is one of the important digestive enzymes and has a significant effect on the growth of mollusk. It is the first time the alpha amylase gene in silver lipped pearl oyster Pinctada maximais cloned. The full length of the cDNA (named as pmAMY) is consisted of 1732 bp, including a 5 UTR of 25 bp, an open reading frame (ORF) of 1554 bp encoding 518 amino acids and a 3 UTR of 153bp. The estimated isoelectric point and molecular mass of deduced amino acid residues are 7.63 and 57.7 kD, respectively. Amino acid sequence analysis indicates that pmAMY contains a signal peptide with 16 aimino acid residues (MLLIVCSIAFFHSVYG), 8 cysteine residues

Key words: Pinctada maxima, alpha amylase, exon, intron, sequence characteristics

中图分类号:

P735.54

潘俐玲, 黄桂菊, 成书营, 王晓宁, 喻达辉. 大珠母贝(Pinctada maxima)α-淀粉酶基因cDNA及内含子克隆分析[J]. 热带海洋学报, 2013, 32(3): 52-58.

PAN Li-ling, HUANG Gui-ju, CHENG Shu-ying, WANG Xiao-ning, YU Da-hui. Cloning and characterization of alpha amylase cDNA and its introns in the pearl oyster Pinctada maxima[J]. Journal of Tropical Oceanography, 2013, 32(3): 52-58.

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大珠母贝(Pinctada maxima)α-淀粉酶基因cDNA及内含子克隆分析

Cloning and characterization of alpha amylase cDNA and its introns in the pearl oyster Pinctada maxima

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