一种三角帆蚌肌浆网Ca2+-ATP酶基因cDNA的全长克隆与表达分析

doi:10.3969/j.issn.1004-1524.2019.04.06

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (4): 545-555.DOI: 10.3969/j.issn.1004-1524.2019.04.06

一种三角帆蚌肌浆网Ca²⁺-ATP酶基因cDNA的全长克隆与表达分析

张爱菊¹, 刘士力¹, 刘金殿¹, 张根芳², 周志明^1,*

1.浙江省淡水水产研究所,中国水产科学研究院东海水产研究所浙江研究中心,农业农村部淡水渔业健康养殖重点实验室,浙江省淡水水产遗传育种重点实验室,浙江湖州 313001;
2. 金华职业技术学院,浙江金华 321000

收稿日期:2018-10-25 出版日期:2019-04-25 发布日期:2019-04-19
通讯作者: *周志明,E-mail: zjzzm@163.com
作者简介:张爱菊(1981-),女,浙江湖州人,博士,副研究员,从事水生生物与水污染生态修复。E-mail:zhangaiju2013@163.com
基金资助:
浙江省科技厅重大科技专项(2012C12907-5)

Cloning, characterization, and expression patterns of one sarco/endoplasmic reticulum calcium ATPase isoform from freshwater mussel Hyriopsis cumingii

ZHANG Aiju¹, LIU Shili¹, LIU Jindian¹, ZHANG Genfang², ZHOU Zhiming^1,*

1. Key Laboratory of Healthy Freshwater Aquaculture of Ministry of Agriculture and Rural Affairs, Key Laboratory of Freshwater Aquaculture Genetic and Breeding of Zhejiang Province, Zhejiang Research Center of East China Sea Fishery Research Institute, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;
2. Jinhua Polytechnic, Jinhua 321000, China

Received:2018-10-25 Online:2019-04-25 Published:2019-04-19

摘要/Abstract

摘要： 采用RACE技术,从三角帆蚌(Hyriopsis cumingii)鳃组织中成功克隆得到一种肌浆网Ca²⁺-ATP酶(sarco/endoplasmic reticulum calcium ATPase,SERCA)基因的全长cDNA序列,共3 326 bp,包含201-bp 5’-UTR区域、3 060-bp编码框(ORF)和65-bp 3’-UTR。ORF共编码1 019个氨基酸,预测无信号肽。该基因氨基酸序列呈现出典型的Ca²⁺-ATP酶特征,由Cation_ATPase_N、E1-E2_ATPase、Hydrolase、Cation_ATPase_C四种类型结构域组成,其内含SERCAs的常见结构组成包括磷酸化区域、异硫氰酸荧光素位点、FSBA结合位点、受磷蛋白结合区以及毒胡萝卜素位点。分析显示,该基因序列高度保守且与海洋软体动物具有最高同源性。荧光定量PCR检测,该基因在三角帆蚌外套膜、斧足、鳃、肝胰腺、性腺等5个组织中均有表达,且在鳃、外套膜、肝胰腺组织中表达较高。不同Ca²⁺浓度处理试验的结果表明,随水体中Ca²⁺浓度逐渐升高,该基因在外套膜中的表达水平呈先下降后上升趋势,并在Ca²⁺浓度为60 mg·L^-1时达到最低值,80 mg·L^-1时达到最高值。同时在60 mg·L^-1 Ca²⁺浓度条件下,外套膜中SERCA基因的表达量随时间推移先上升,并于48 h时达到最高,而后逐渐下降。上述结果为进一步深入研究SERCA基因的功能及其调控机理奠定了基础。

关键词: 三角帆蚌, SERCA, 克隆, 基因表达, 钙刺激

Abstract: The full-length cDNA sequence of sarco/endoplasmic reticulum calcium ATPase isoform gene was isolated from the gill of Hyriopsis cumingii by using SMART RACE technique. The entire SERCA cDNA was 3 326 bp, including a 201-bp 5'-UTR, a 3 060-bp ORF and a 65-bp 3'-UTR, encoding a 1 019-amino acid protein, and no putative signal peptide was predicted. Four types of domains were found and the general structures of SERCA gene, containing phosphorylation region, fluorescein isothiocyanate site, 5'-(p-fluorosulfonyl) benzoyladenosinebinding site, phospholamban-binding motif and thapsigargin sites were also identified. Compared with SERCA homologs from seawater mollusks and other animals, the SERCA of H. cumingii had high similarity with them in both sequence and structure. Tissue-specific expression analysis revealed that SERCA mRNA was detected in all the sampled tissues, but was prominently expressed in the gill, mantle and hepatopancreas. When exposed to a series of increasing Ca²⁺ that lasted 7 days, the mRNA expression of SERCA gene in mantle was shown to be cove-shaped, ascending when Ca²⁺ concentration was more than 60 mg·L^-1 and peaking at 80 mg·L^-1. Moreover, the temporal expression of SERCA transcripts in mantle after 60 mg·L^-1 Ca²⁺ exposure was slightly down-regulated at 24 h, but up-regulated from 24 h to 48 h post-treatment, peaking at 48 h. The results of present study will provide useful information for further studies on function and regulation mechanism of SERCA gene.

Key words: Hyriopsis cumingii, SERCA, cloning, gene expression, calcium stimulation

中图分类号:

S966.22⁺1

张爱菊, 刘士力, 刘金殿, 张根芳, 周志明. 一种三角帆蚌肌浆网Ca²⁺-ATP酶基因cDNA的全长克隆与表达分析[J]. 浙江农业学报, 2019, 31(4): 545-555.

ZHANG Aiju, LIU Shili, LIU Jindian, ZHANG Genfang, ZHOU Zhiming. Cloning, characterization, and expression patterns of one sarco/endoplasmic reticulum calcium ATPase isoform from freshwater mussel Hyriopsis cumingii[J]. , 2019, 31(4): 545-555.

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一种三角帆蚌肌浆网Ca²⁺-ATP酶基因cDNA的全长克隆与表达分析

Cloning, characterization, and expression patterns of one sarco/endoplasmic reticulum calcium ATPase isoform from freshwater mussel Hyriopsis cumingii

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