合浦珠母贝表皮生长因子样(EGF-like)基因的克隆与表达分析

Cloning and expression of an novel EGF-like gene from Pinctada martensii

为探究表皮生长因子(epidermal growth factor,EGF)在合浦珠母贝幼体发育以及组织生长过程中的作用,实验通过RACE技术克隆了合浦珠母贝EGF-like基因并做了相应的表达分析。实验结果获得c DNA全长序列4 107 bp,命名为Pf-egf1,开放阅读框(ORF)702 bp,编码234个氨基酸,包含一个信号肽序列和一个跨膜结构域,功能结构域分析发现Pf-egf1具有一个EGF-like结构域,有6个半胱氨酸残基,由3个二硫键维持,是表皮生长因子家族及其相关蛋白的特征结构域,但其余序列与现有相关基因序列差异较大,推测可能是一个新的EGF-like基因。表达结果显示,Pf-egf1 mRNA在合浦珠母贝外套膜、闭壳肌、鳃、肝胰腺、珍珠囊、肠和性腺中均有表达,在肠中的表达显著高于其他组织;在合浦珠母贝幼体发育的担轮期、D型期、壳顶期、眼点期和变态期的表达呈现逐渐升高的趋势,并且其在变态期的表达量极显著地高于其他时期。上述结果表明,Pf-egf1基因可能在合浦珠母贝肠道修复和幼虫变态发育阶段起着重要作用,为进一步开展育珠与生长调控奠定了基础。

Epidermal grow th factor（ EGF） plays important roles in grow th,wound healing and metamorphosis,etc. The pearl oyster Pinctada martensii is a commercially important species cultured for pearl production,in which a series of developmental transformation occur in young stage and an injury will happen during nuclear insertion. In order to evaluate the function of EGF gene during P. martensii larval development,tissue grow th and nuclear insertion. In this study,cloning and expression of an EGF-like gene was carried out to provide some background for further study on grow th development and pearl culture improvement. As a result,a full length c DNA of 4 107 bp was obtained and named Pf-egf1. The complete ORF encoded 234 amino acid residues,including a signal peptide,a transmembrane domain and an EGF-like domain. The EGF-like domain contained six cysteines that can form three disulfide bonds,consistent with the characteristics of Epidermal Growth Factor protein family. How ever,the remaining Pf-egf1 sequences were quite different from other EGF family members. So we speculated that Pf-egf1 is probably a novel EGF-like gene. The tissue expression profile show ed that Pf-egf1 expressed in all the six tissues tested（ mantle,muscle,gill,hepatopancreas,pearl sac,intestines and gonad）,with significantly higher expression in intestines than in other tissues. The expression of Pf-egf1 presented an increasing trend from trochophore stage to metamorphosis stage in larval development,with significantly higher expression in metamorphosis stage than in other stages. The observations above indicated that Pf-egf1 gene may play important roles in intestine repairing and larval metamorphosis in P. martensii,possibly through promotion of cell proliferation,and thus can benefit to some extent further studies on modulation of growth and pearl culture.