马氏珠母贝Bcl-2-like基因克隆及其功能初探

B淋巴细胞瘤-2(B-cell lymphoma, Bcl-2)基因作为一种重要的细胞凋亡调控基因,在内源性细胞凋亡通路中发挥着重要的调控作用。实验利用c DNA末端快速扩增(RACE)技术克隆获得PmBcl-2-like基因cDNA全长序列,并对其序列进行生物信息学分析;利用实时定量PCR(qPCR)技术分析了PmBcl-2-like在马氏珠母贝不同组织、不同发育时期以及不同免疫刺激后的表达水平。结果显示,PmBcl-2-like cDNA全长为2 180 bp,开放阅读框长度为1 650 bp,共编码549个氨基酸,分子量为21.62 ku;结构域预测分析表明PmBcl-2-like含有Bcl-2家族典型的BH1-4结构域;多序列比对以及进化树构建结果表明,PmBcl-2-like与其他物种的相似度较高,保守性较强;荧光定量结果表明PmBcl-2-like在马氏珠母贝8个组织中均有表达,在鳃中表达量最高,其次为性腺,表达量最低为中央膜区;在胚胎期表达量较高,受精卵时期表达量最高。机体受到脂多糖(LPS)刺激后,相对表达量在24 h达到最高,72 h降到最低,最高约为最低的2.5倍;肽聚糖(PGN)刺激后,相对表达量在6 h达到最高,48 h降到最低,最高约为最低的7.28倍;聚肌胞苷酸(Poly:IC)刺激后,相对表达量在3 h达到最高,12 h降到最低,最高约为最低的6.49倍。研究表明,PmBcl-2-like可能在马氏珠母贝发育和免疫防御反应中担任着重要的角色。

Cloning and preliminary study on the function of Bcl-2-like gene in Pinctada fucata martensii

As an important apoptosis-regulating gene, Bcl-2 gene plays an important role in endogenous apoptotic pathways. In this study, Pinctada fucata martensii Bcl-2 gene (Pm Bcl-2-like) was cloned by rapid-amplification of cDNA ends (RACE) technology and its characteristics was analyzed by bioinformatics methods. The expression levels of PmBcl-2 -like in different tissues, development stages and immune stimulation were analyzed by real-time fluorescence quantitative PCR (qPCR). The results showed that the full length of PmBcl-2-like cDNA was 2180 bp, the length of open reading frame was 1650 bp, encoded a total of 549 amino acids and the molecular weight was 21.62 kDa. Domain prediction showed that PmBcl-2 -like contained BH1-4 domains typical of bcl-2 family. Multiple sequence alignment and phylogenetic tree analysis showed that PmBcl-2-like had high similarity with other species and was highly conservative in phylogeny. Fluorescence quantitative results showed PmBcl-2-like was expressed in all the tested eight tissues of P. f. martensii, with the highest expression in the gill, followed by the gonad, and the lowest expression in the mantle central; At different developmental stages, the expression level of PmBcl-2-like was higher in the embryonic stage and highest in the fertilized egg stage. After stimulated by LPS, the relative expression reached the maximum at 24 h, and then decreased to the minimum at 72 h, with the maximum being about 2.5 times of the minimum; After PGN stimulation, the relative expression reached the highest at 6 h and the lowest at 48 h, and the highest was about 7.28 times of the lowest; After Poly: IC stimulation, the relative expression reached its maximum at 3 h, and decreased to its minimum at 12 h, and the maximum was 6.49 times of the minimum. This study suggests that PmBcl-2 -like may play an important role in the development and immune defense response of Pinctada fucata martensii.