三角帆蚌钙调蛋白(CaM)基因的cDNA序列克隆与表达分析

[目的]研究三角帆蚌钙调蛋白(CaM)基因在育珠和非育珠蚌各组织中的表达和不同Ca2+浓度下外套膜中的表达变化。[方法]通过RACE技术,以我国重要的淡水珍珠贝——三角帆蚌为研究客体,克隆了CaM的cDNA全长,并通过Real-Time Q-PCR技术分析了该基因在育珠和非育珠蚌各组织中的表达和不同Ca2+浓度下外套膜中的表达变化。[结果]三角帆蚌CaM基因cDNA全长659bp,包括70 bp的5'UTR,299 bp的3'UTR和447 bp的ORF,编码149个氨基酸,预测其分子量为16.8 kDa,等电点为4.14。cDNA序列比对信息表明三角帆蚌与池蝶蚌、合浦珠母贝间均具有57%的相似度,而其蛋白具有高度的保守性,除斑马鱼外,各生物CaM相似率达97%。定量数据表明,CaM基因广泛表达于三角帆蚌各组织,外套膜内膜与腮中表达量显著升高(P0.05),其次是外套膜外膜和内脏团组织。插核育珠能增加该基因在各组织的表达,特别是在外套膜外膜和鳃组织中,育珠蚌该基因的表达显著高于非育珠蚌(P0.05)。此外,水体Ca2+浓度的增大对CaM基因表达有显著的影响,外套膜内膜该基因的表达随Ca2+浓度升高而增加,而外套膜外膜1.25 mmol/L Ca2+浓度下该基因的表达水平显著高于0.50 mmol/L、3.00 mmol/L组(P0.05)。[结论]为进一步深入研究钙调蛋白基因的在珍珠生长过程中的功能及其调控机理奠定了分子基础。

Sequence Clone of cDNA and Gene Expression of Calmodulin in Hyriopsis cumingii

[Objective] The aim was to study CaM gene expressing levels at different tissue and Ca2＋ concentrations in inserting-nucleus or non-inserting-nucleus mussel.[Method] The full-length cDNA sequence of calmodulin （CaM） gene was C from the Hyriopsis cumingii by using RACE technique.CaM gene expressing levels were analyzed at different tissue and Ca2＋ concentrations in inserting-nucleus or non-inserting-nucleus mussel.[Result] The results indicated that the entire CaM cDNA was 659 bp,containing a 70 bp 5＇UTR,a 299 bp 3＇UTR,and a 447 bp complete open reading frame which encoding a protein with 149 amino acids.Bioinformatics analysis indicated the CaM gene had 57％ similarity to Hyriopsis schlegelii or Pinctadafucata,while the protein had high conservatism （97％） to listed life except for zebra fish.Real-time PCR revealed that CaM gene was ubiquitously expressed in all tested tissues,but far more abundant in outer mantle and gill （P ＜0.05）,followed by in inner mantle and visceral mass.The CaM gene expression levels were increased by inserting nucleus to cultivated pearl,especially in outer mantle and gill tissue （P ＜ 0.05）.Moreover,Ca2＋ concentration of medium had significant effect to CaM gene expression.CaM gene expression levels were increasing with rising Ca2＋ concentration in inner mantle （P ＜ 0.05）.While,the CaM gene expression levels of 1.25 mmol/L group was significantly higher compared with 0.50 and 3.00 mmol/L groups （P ＜ 0.05）.[Conclusion] The results of present study will provide useful information for further studies on function and regulation mechanism of CaM gene.