大珠母贝外套膜基因PMMG1的克隆、 表达及其特征分析

王玉梅; 夏建红; 黄桂菊; 喻达辉

大珠母贝外套膜基因PMMG1的克隆、表达及其特征分析

1.
中国水产科学研究院南海水产研究所海水养殖生态与质量控制农业部重点开放实验室,广东广州 510300;上海海洋大学水产与生命学院,上海 201306
2.
中国水产科学研究院南海水产研究所海水养殖生态与质量控制农业部重点开放实验室,广东广州 510300

基金项目: 国家高技术研究发展计划"八六三"项目(2006AA10A415);广东省科技推广项目(A200701C02;A200899C04;A200900A07);中央级公益性科研院所基本科研业务费专项资金项目(2007TS07;2009YD02)。

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出版历程
- 收稿日期: 2009-04-13
- 修回日期: 2010-03-10

Cloning, expression and characterization of Pinctada maxima mantle gene 1

1.
Key Laboratory of Ministry of Agriculture for Mariculture, Ecology and Quality Control, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China;College of Fisheries and Life Science, Shanghai Ocean Univer
2.
Key Laboratory of Ministry of Agriculture for Mariculture, Ecology and Quality Control, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510300, China

摘要

摘要: EF-hand结构域在真核生物细胞内钙离子吸收和运输中发挥重要作用,在贝类中可能参与贝壳和珍珠质的形成。根据已报道的贝类中具有EF-hand结构域的碱基序列设计简并引物,从大珠母贝外套膜cDNA文库中筛选得到PMMG1基因(大珠母贝外套膜基因1)。PMMG1基因全长618 bp,开放读码框编码140个氨基酸,N-端的22个氨基酸肽段为信号肽。PMMG1氨基酸序列与合浦珠母贝PFMG1的一致性为56％,预测有两个EF-hand结构域。选取编码PMMG1成熟蛋白的cDNA序列插入pET-32a质粒构建表达载体
- 大珠母贝 /
- PMMG1 /
- EF-hand结构域 /
- 融合蛋白 /
- 珍珠质
Abstract: EF-hand motif plays essential roles in the absorption and transportation of calcium ion in eukaryotic cells, and is possibly involved in the formation of shell and nacre in oysters. Degenerated primers were designed according to the conserved sequences of reported EF-hand proteins in oysters, and a novel gene PMMG1(Pinctada maxima mantle gene 1) was screened from the mantle cDNA library of P. maxima. The PMMG1 cDNA contained 618 nucleotides and encoded 140 amino acids (aa), including a putative signal peptide of 22 aa. PMMG1 protein shared an identity of 56％ with PFMG1 from P. fucata and had two putative EF-hand motifs. The cDNA fragment encoding mature protein of PMMG1 was cloned and integrated into prokaryotic expression vector pET32-a. A recombinant protein of expected size was induced by IPTG and then purified by Ni²⁺-NTA resin. Electrophoretic shift experiment revealed that the PMMG1 protein could bind both Ca²⁺ and Mg²⁺ion, and the tissue-specific expression level of PMMG1 was much higher in mantle than in other tissues, which may be involved in the formation of calcite while binding Ca²⁺ or the formation of aragonite while binding Mg²⁺. This work can benefit the further investigation into the roles of EF-hand proteins in the biomineralization of pearl oysters.
- Pinctada maxima /
- PMMG1 /
- EF-hand domain /
- recombinant protein /
- nacre

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