杂色鲍紫色酸性磷酸酯酶基因克隆及应激下的表达  

Molecular cloning and expression of purple acid phosphatase in Haliotis diversicolor under stress

作　　者：黄贻涛[1] 蔡秀红[1] 张子平 王国栋[1] 邹志华[1] 王淑红[1] 王艺磊[1] 

机构地区：[1]集美大学水产学院,农业部东海海水健康养殖重点实验室,福建厦门361021 [2]西东大学生物系

出　　处：《中国水产科学》2013年第5期939-949,共11页Journal of Fishery Sciences of China

基　　金：国家自然科学基金项目(41006105;41176152);福建省科技重点项目(2011N0022);集美大学创新团队基金项目(2010A001)

摘　　要：利用本实验室获得的杂色鲍(Haliotis diversicolor)转录组测序数据库,筛选出杂色鲍紫色酸性磷酸酯酶(Purple acid phosphatase,PAP)同源片段,进而克隆获得PAP的cDNA全序列,命名为HdPAP,并进行了HdPAP蛋白的结构分析和功能预测,同时利用实时定量PCR(qRT-PCR)技术分析HdPAP基因的组织表达谱和应激条件下的HdPAP表达变化。HdPAP基因cDNA全长1 215 bp,含有1个编码322个氨基酸的969 bp的开放阅读框。经Blast比对,与无脊椎动物半索动物门囊舌虫(Saccoglossus kowalevskii)的PAP氨基酸序列一致性最高,达59%。通过实时定量PCR检测,HdPAP在检测的杂色鲍各组织中均有表达,其中在血细胞和肝胰腺的表达量显著高于其他各组织(P<0.05)。高温应激下,在检测的6个时相中,HdPAP在血细胞、肝胰腺和鳃中均出现不同程度的表达抑制。缺氧应激后,4 h血细胞中HdPAP显著低于对照组,24 h恢复到正常水平,96 h显著高于对照组(P<0.05),到192 h又恢复到正常水平。副溶血弧菌感染实验中检测到HdPAP在血细胞中同样出现表达抑制现象,3 h和6 h均检测到感染组HdPAP表达量显著低于对照组(P<0.05)。PAP在高温、缺氧及弧菌感染下显著的表达抑制从转录水平揭示了其作为表达下调的酯酶可能参与胁迫下的生物代谢和免疫反应。The small abalone Haliotis diversicolor is one of the most commercially important cultured abalone in southern coastal areas in China. However, the frequent occurrence of infectious disease, especially during hot summers, is a major problem that has threatened the abalone aquaculture industry for a long time. As an invertebrate, abalone lacks an adaptive immune system and relies exclusively on innate immunity to defend against bacterial challenge. However, research on gastropod immune responses is limited. Purple acid phosphatase （PAP） belongs to a large family of dinuclear metalloenzymes and is distinguished from other acid phosphatases by its purple color, which is due to a Tyr-to-iron （III） charge transfer transition. PAPs are a group of tartrate resistant, molybdate sensitive, iron containing acid phosphatases with a molecular weight of about 35-40 kD and a high activity towards activated phosphoric acid monoesters and anhydrides. They catalyze the hydrolysis of a wide range of phosphate esters. PAPs play important roles in response to different stresses in plants and mammals. However, to date it has not been investigated in molluscs. In this study, the first molluscan PAP gene, HdPAP from H. diversicolor, was cloned by combining the expressed sequence tag （EST） and rapid amplification of cDNA end （RACE） methods. Its full length cDNA sequence is 1 215 bp, with a 969 bp open reading frame encoding a protein of 322 amino acids （GenBank： KC337074）. The 5′ and 3′ untranslated regions （UTR） of HdPAP contain 28 bp and 218 bp, respectively. Pairwise analysis results revealed that the HdPAP amino acid sequence has the highest identity, 59%, to the invertebrate Saccoglossus kowalevskii PAP. The calculated molecular mass of deduced HdPAP is 36.8 kD with a theoretical isoelectric point （PI） of 5.27. Multiple sequence alignment of the HdPAP amino acid sequence with other known vertebrate PAPs and invertebrate PAP family proteins revealed that it was conserved, while their lengths varied

关 键 词：杂色鲍 紫色酸性磷酸酯酶 高温 缺氧 副溶血弧菌 

分 类 号：S917[农业科学—水产科学]