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。
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 maxima is 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 （Cys46、 Cys104、 Cys157、 Cys176、 Cys392、 Cys398、 Cys464、 Cys476）, 3 active sites （Asp213、 Glu249、 Asp314）, 4 calcium binding residues （Ash118, Arg174、 Asp183、 His217）, 3 chloride binding sites （Arg211、 Asn312、 Arg350） and 4 conserved sequences （Ile111-Val116、 ValE07-Ala215, Phe247-Va1251 、 Vala08-Asn315）. Blast analysis shows that pmAMY shares the highest identity （82%） with Pteria penguin, the lowest （27%） with Thermococcus hydrothermalis and 57%-79% with some other organisms. Two introns are obtained, being 846 bp and 162 bp, respectively. Both introns begin with GT and ended with AG, consistent with common sequences of intron splice sites. Tissue expression analysis indictes that pmAMY only exists in hepatopancreas. The results above lay a base work for further research on the function of pmAMY and discovery of SNPs as well as its relationship with growth trait of P. maxima.
出处
《热带海洋学报》
CAS
CSCD
北大核心
2013年第3期52-58,共7页
Journal of Tropical Oceanography
基金
现代农业产业技术体系建设专项资金(CARS-48)
国家高技术研究发展计划项目(2006AA10A415)
广东省海洋与渔业局科技推广项目(A200701C02
A200899C04
A200900A07)
中央级公益性科研院所基本科研业务费(2007TS07
2009YD02)
