马氏珠母贝TNFR27基因克隆与功能初探

Cloning and preliminary study on the functions of PmTNFR27 gene in Pinctada fucata martensii

为了探究肿瘤坏死因子受体(tumor necrosis factor receptor,TNFR)基因在马氏珠母贝中的免疫应答机制,本实验采用cDNA末端快速扩增(RACE)技术克隆了马氏珠母贝TNFR27(PmTNFR27)基因cDNA全长并进行生物信息学分析,运用实时荧光定量PCR(qRT-PCR)技术检测了马氏珠母贝在脂多糖(LPS)、聚肌胞苷酸[Poly(I:C)]及镉胁迫后,PmTNFR27 mRNA在其不同组织中的表达模式。结果显示,PmTNFR27 cDNA全长为1524 bp,5′UTR长为186 bp,3′UTR长为248 bp,包含28 bp的poly(A)尾巴,开放阅读框(ORF)为1062 bp,编码353个氨基酸;结构域预测表明PmTNFR27具有一个典型的CRD结构域和一个跨膜蛋白结构域,符合肿瘤坏死因子受体超家族特征;多序列比对结果显示,贝类种间的相似性不高,但功能结构域位置较保守。系统进化树结果显示,马氏珠母贝与其他贝类聚为一支。qRT-PCR结果显示,PmTNFR27 mRNA在马氏珠母贝各组织中均有表达,在鳃中相对表达量最高。LPS刺激后,PmTNFR27基因在鰓中的相对表达量于3 h显著上升并达到最高值,于72 h降到最低值,最高值约为最低值的9.67倍;Poly(I:C)刺激后,PmTNFR27基因在鰓中的相对表达量在6、12 h显著上升并达到最高值,至96 h时降到最低值,最高值约为最低值的13.16倍。镉胁迫后,3 h相对表达量达到最高,24、48 h相对表达量显著下降。研究表明,PmTNFR27可能参与了马氏珠母贝的免疫应答反应。本研究可为进一步探究TNFR在贝类中的生物学功能提供重要的理论基础和参考价值。

Tumor necrosis factor receptor(TNFR)is an important cytokine receptor,mainly involved in biological processes such as apoptosis,host immune defense,and inflammation.In order to reveal the role of TNFR gene in the immune process of Pinctada fucata martensii,a full length of PmTNFR27 was obtained using rapid amplification of cDNA ends technology,and the expression levels of different tissues,LPS and Poly(I:C)immune stimulation and cadmium stress were analyzed by quantitative real-time PCR(qRT-PCR).Results showed that the total length of cDNA was 1,524 bp,including a 5′UTR of 186 bp,a 3′UTR of 248 bp and an open reading frame(ORF)of 1,062 bp encoding 353 amino acids.Domain prediction showed that PmTNFR27 contained a transmembrane domain and a CRD domain which is typical of the TNFR superfamily.Multiple sequence alignment indicated that PmTNFR27 has low similarity compared with other bivalve species,but its domain regions were highly conservative.Phylogenetic analysis showed that it clustered with other bivalves.In addition,qRT-PCR data indicated that PmTNFR27 was expressed in all tested tissues,with the highest expression in gill.After LPS stimulation,the relative expression of PmTNFR27 in gills reached the maximum at 3 h and decreased to the minimum at 72 h,and the maximum was 9.67 times of the minimum.After Poly(I:C)stimulation,the relative expressin of PmTNFR27 in gills was significantly up-regulated,reached the maximum at 6 h and 12 h,and decreased to the minimum at 96 h,the maximum being13.16 times of the minimum.After cadmium stress,the relative expression of PmTNFR27 reached the maximum at 3 h,and was significantly down-regulated at 24 h and 48 h.This study suggested that PmTNFR27 may be involved in the immune defense response of P.fucata martensii and could provide basis for the further study of the biological functions of TNFR in the shellfish.