文蛤粪卟啉原Ⅲ氧化酶基因克隆及与壳色性状的相关性分析

粪卟啉原Ⅲ氧化酶(coproporphyrinogen-III oxidase,CPOX)是卟啉类化合物合成过程中的关键酶,而卟啉是形成机体色素的重要化合物。采用RACE方法克隆获得文蛤粪卟啉原Ⅲ氧化酶基因(MmCPOX)的cDNA序列,该序列全长1490 bp,其中开放阅读框1173 bp,编码390个氨基酸,预测分子量为12.04 ku,理论等电点为5.05;预测该酶含有跨膜结构域和Coprogen-oxidas结构域;从构建的系统进化树来看,软体动物门的文蛤、加州海兔和太平洋牡蛎首先聚在一起,显示出较近的亲缘关系。qRT-PCR结果显示,MmCPOX基因在文蛤早期发育的各个时期均有表达,但在D形幼虫期以后表达量显著升高;在文蛤成贝的7个组织中,外套膜和血液中表达量显著高于其他组织,表明其与血卟啉合成和壳色卟啉形成相关;在不同壳色文蛤中,红壳文蛤、暗纹文蛤、细纹文蛤的外套膜中表达量显著高于黑斑文蛤和白壳文蛤,表明其参与形成红色和褐色壳色。

Gene cloning of CPOX and correlation analysis of shell colors in Meretrix meretrix

Coproporphyrinogen-Ⅲ oxidase (CPOX) is a key enzyme in the synthesis of porphyrins, which plays an important role in the formation of pigment. cDNA of Meretrix meretrix CPOX (MmCPOX) was cloned by SMART RACE techniques, then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed. The results indicated that the full length cDNA of MmCPOX gene was 1490 bp, containing a 1173 bp opening reading fame(ORF) encoding 390 amino acids. The molecular weight of MmCPOX was 12.04 ku and pI was 5.05. It was also predicted that protein MmCPOX had a transmembrance region and a Coprogen-oxidas region. Comparisons of amino acid sequence, MmCPOX was highly homologous withAplysia californica and shares 64.5% similarity. The result of qRT-PCR showed that MmCPOX gene was expressed in all stages, and significantly increased from D-shaped larva stage (P<0.01).MmCPOX was expressed in all seven tissues, in which the expressions in mantle and blood were significantly higher than those of other tissues (P<0.01), indicating that it was related to synthesis of hematoporphyrin and formation of shell porphyrin. Considering different shell colors, the relative expressions of mantle in red color, dark fringe and thin checkered shells, were significantly different from the black and white shells (P<0.05), suggesting that MmCPOX gene was responsible for the formation of red and brown shell color.