三角帆蚌EFCB1基因cDNA序列克隆及表达研究

为了发掘更多三角帆蚌具有EF-hand结构域的功能基因及其蛋白质,本研究运用RACE-PCR技术,克隆得到了三角帆蚌包含EF-hand结构域钙结合蛋白1基因(EF-hand calcium-binding domain-containing protein 1,EFCB1)的cDNA全长并进行了生物信息学分析;通过real-time Q-PCR技术,分析了EFCB1基因在三角帆蚌10个组织,以及内脏团、外套膜插核后不同时间点的时空表达特点。结果表明三角帆蚌EFCB1基因cDNA序列全长981 bp,ORF为531 bp,编码176个氨基酸残基,5'-UTR 239 bp和3'-UTR 211 bp。EFCB1分子式为C₈₇₇H₁₃₄₈N₂₃₈O₂₇₀S₁₀,分子量约19.9 ku,等电点为4.70,不稳定系数为62.65,属亲水蛋白。其序列无信号肽序列,存在1个跨膜区域和2个EF-hand结构域,EF-hand模块分别为DLNDDKLISPEE(98-109)和DTNGDDKLDGEE(129-140)。荧光定量结果显示三角帆蚌EFCB1基因在各组织中均有表达,其中在肠和鳃中表达量最高(P<0.05),外套膜中表达量显著高于内脏团(P<0.05)。EFCB1基因在插核后不同时期的外套膜和内脏团育珠部位组织中表达具有显著差异(P<0.05),在外套膜中的表达量均显著高于内脏团(P<0.05),在插核后第20 天时表达量显著高于各时期(P<0.05)。研究表明,EFCB1在三角帆蚌Ca²⁺的吸收过程中发挥调节作用,在珍珠囊形成过程中以及珍珠形成初期具有重要功能。

Molecular cloning and expression research of EFCB1 cDNA in Hyriopsis cumingii

In order to find more functional genes with EF-hand domain in Hyriopsis cumingii,we cloned the full-length cDNA of EF-hand calcium-binding domain-containing protein 1(EFCB1)by using RACE-PCR technique,and performed bioinformatics analysis;EFCB1 gene expression levels were analyzed in different tissues from Hyriopsis cumingii(mantle pallial,gill,visceral mass,adductor muscle,axe foot,gonad,liver,gut,haemolymph and kidney)and in different periods of the mantle and visceral mass after inserting nucleus.The results showed that the EFCB1 cDNA sequence length was 981 bp,containing a 531 bp open reading frame(ORF)encoding 176 amino acid residues,239 bp 5'-untranslated region(UTR)and 211 bp 3'-UTR.The molecular weight of the peptide was predicted 19.9 ku,with an isoelectric point of 4.70.Amino acid sequence analysis showed that there was a transmembrane region.Results of the ProtScale online analysis showed that the protein was hydrophilic protein.There were two typical EF-hand calcium binding domains,respectively DLNDDKLISPEE(98-109)and DTNGDDKLDGEE(129-140).Phylogenetic analysis indicated that the EFCB1 of H.cumingii was closely related to seawater bivalve Crassostrea gigas,followed by that of fish and then insect.Real-time Q-PCR results showed that the EFCB1 mRNA is ubiquitously expressed in various tissues(mantle paillial,gill,visceral mass,adductor muscle,axe foot,gonad,liver and gut),with the highest level in the gut and gill(P<0.05),and it is also highly expressed in the pearl culture tissues(mantle and visceral mass).These data suggested that EFCB1 plays an important role in the absorption process of Ca²⁺,and there might be an intrinsical relationship between EFCB1 and pearl formation of H.cumingii.Furthemore,expression differences were found among different periods of the mantle and visceral mass after inserting nucleus in the H.cumingii,and the expression level of EFCB1 in the mantle was significantly higher than that in visceral mass(P<0.05),after nucleus inserted 20 d it was significantly higher than other periods(P<0.05),suggesting EFCB1 has an important function in the formation process of pearl sac and nacre secretion,indicating EFCB1 might be an important regulator of pearl formation.The results of present study may provide useful information for further studies on regulation mechanism of calcium metabolism and pearl formation.