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,通过IPTG诱导,Ni2+-NTA亲和层析柱纯化,成功获得预期大小的融合蛋白。凝胶电泳迁移率的变化证明PMMG1蛋白具有结合Ca2+/Mg2+的活性,组织特异性表达表明PMMG1基因在外套膜的表达量远高于其他组织。大珠母贝外套膜基因PMMG1的克隆与表达研究为进一步研究该蛋白在珍珠质矿化中的作用、探讨珍珠质形成的分子生物学机制奠定了一定基础。
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.
出处
《海洋学报》
CAS
CSCD
北大核心
2010年第3期121-128,共8页
基金
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)
