马氏珠母贝(Pinctada fucata martensii)细胞粘附分子3(Pm-CAM3)基因的克隆与表达分析

细胞粘附分子3 (cell adhesion molecule 3, CAM3)是免疫球蛋白家族(immunoglobulin family, IgSF)的一员,在细胞黏连、外源病原体的识别等方面具有重要作用。本实验利用末端快速克隆(RACE)技术,克隆获得马氏珠母贝(Pinctada facata martensii)细胞粘附分子3的全长序列(Pm-CAM3),并使用荧光定量PCR(Real-time PCR, q RT-PCR)技术检测了Pm-CAM3在马氏珠母贝不同组织中的表达模式。结果显示,Pm-CAM3基因全长2 245 bp。其中5'UTR 335 bp,3'UTR 166 bp,开放阅读框(ORF)长度为1 744 bp,共编码581个氨基酸;预测其相对分子量为63.21 kD,等电点为5.07,脂溶指数(aliphatic index)为67.21;总平均亲水性(grand averageofhydropathy, GRAVY)为-0.549,属于亲水性蛋白;Pm-CAM3具有跨膜结构域,其胞外区域具有一个Ig SF家族典型的Ig结构域以及一个Ig-like结构域。多序列比对结果显示,Pm-CAM3在物种间的保守性较低,其中Pm-CAM3与太平洋牡蛎的CAM3 (Crassostrea gigas, Cg-CAM3)的氨基酸序列相似性最高,但仅为37%。qRT-PCR分析表明Pm-CAM3在马氏珠母贝的9个组织中均有表达,其中在鳃中的表达量最高(p<0.05)。

Cloning and Expression Analysis of Cell Adhesion Molecule 3(Pm-CAM3) Gene from Pinctada fucata martensii

Cell adhesion molecule 3(CAM 3) is a member of the immunoglobulin family(IgSF), which plays an important role in cell adhesion and recognition of exogenous pathogens. In this study, we used the terminal rapid cloning(RACE) technique to clone the full length sequence of P. f. martensii cell adhesion molecule 3(Pm-CAM3).And the expression patterns of Pm-CAM3 in different tissues of P. f. martensii were detected by real-time PCR(qRT-PCR). The results showed that the full length of Pm-CAM3 gene was 2 245 bp, containing a 5’ UTR of 335 bp and a 3’ UTR of 166 bp. The length of open reading frame(ORF) was 1 744 bp, encoding 581 amino acids. The predicted relative molecular weight was 63.21 kD, isoelectric point was 5.07, aliphatic index was 67.21. The grand average of hydropathicity(GRAVY) was-0.549, belonging to hydrophilic proteins. Pm-CAM3 had a transmembrane domain and its extracellular domain had a typical Ig domain of Ig SF family and an Ig-like domain. The results of multiple sequence alignment represented that the conservation of Pm-CAM3 among species was low. The amino acid sequence similarity of Pm-CAM3 to CAM3(Crassostrea gigas, Cg-CAM3) in Pacific oyster was the highest,but only 37%. q RT-PCR analysis showed that Pm-CAM3 was expressed in nine tissues of P. f. martensii with the highest expression in the gill(p<0.05). This study would provide basic materials for further elucidating the role of Pm-CAM3 in the innate immunity of P. f. martensii.