马氏珠母贝TRACP基因的克隆及组织表达  

Cloning and Tissue Expression Analysis of TRACP Gene in Pinctada martensii

作　　者：郑哲[1] 吴宣瑾 焦钰[1] 黄荣莲[1] 杜晓东[1,2] ZHENG Zhe;WU Xuan-jin;JIAO Yu;HUANG Rong-lian;DU Xiao-dong(Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;Pearl Breeding and Processing Engineering Technology Research Center of Guangdong Province, Zhanjiang 524088, China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省珍珠养殖与加工工程技术研究中心,广东湛江524088

出　　处：《广东海洋大学学报》2019年第3期24-29,共6页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(31672626)

摘　　要：【目的】克隆马氏珠母贝(Pinctada martensii)抗酒石酸酸性磷酸酶(Tartrate resistant acid phosphatase,TRACP)基因,分析该基因在不同组织中的表达模式。【方法】用RACE技术克隆得马氏珠母贝TRACP基因(PmTRACP),用实时荧光定量PCR分析该基因在外套膜、闭壳肌、足、性腺、珍珠囊、肝胰腺和鳃中的表达。【结果与结论】PmTRACP基因长度为2 034 bp,开放式阅读框972 bp,编码323个氨基酸,5′UTR长度为27 bp,3′UTR长度为1 035 bp。预测PmTRACP分子质量约为36.39 ku,理论等电点为5.97。该基因含有一个钙调神经磷酸酶样磷酸酯酶结构域。PmTRACP与其他物种TRACP的同源性为48%～65%,与长牡蛎(Crassostrea gigas)TRACP的同源性最高,同时D^(32)、D^(70)、Y^(73)、N^(108)、H^(203)、H^(212)、H^(237)、H^(239)等8个氨基酸活性位点和N^(114)糖基化位点在不同物种TRACP中高度保守。PmTRACP与长牡蛎TRACP的亲缘关系最近。PmTRACP在马氏珠母贝各个组织均有表达,且在肝胰腺和珍珠囊中高表达。【Objective】 To clone the full length of tartrate resistant acid phosphatase(TRACP) gene in the pearl oyster Pinctada martensii and analyze the expression profile of TRACP at different tissues.【Method】The TRACP gene in pearl oyster Pinctada martensii(designated as PmTRACP) was obtained by RACE, and quantitative real-time PCR was used to detecte the expression level of PmTRACP in different tissues including the mantle, adduct muscle, foot, gonad, pearl sac, hepatopancreas and gill.【Result and conclusion】The length of PmTRACP is 2 034 bp, with an open reading frame of 972 bp that encodes 323 amino acids, 5′ untranslated region(UTR) was 27 bp, and 3′ UTR is 1 035 bp. The predicted molecular weight of PmTRACP was 36.39 ku and the theoretical isoelectric point was 5.97.PmTRACP contains a calcineurin-like phosphatase domain. Multiple sequence alignment showed that the TRACP homology between P. martensii and other species ranged from 48% to 65%, with the highest homology to Crassostrea gigas. The 8 amino acid active sites(D^32, D^70, Y^73, N^108, H^203, H^212, H^237, H^239)and the N-glycosylation site(N^114) are highly conserved in different species of TRACP. Phylogenetic analysis showed that PmTRACP was mostly close to TRACP of C. gigas. Quantitative real-time PCR analysis showed that PmTRACP m RNA was constitutively expressed in different tissues, with significantly high expression in the hepatopancreas and pearl sac.

关 键 词：马氏珠母贝 抗酒石酸酸性磷酸酶 基因克隆 基因表达 实时荧光定量PCR 

分 类 号：Q78[生物学—分子生物学] Q959.21543