马氏珠母贝丝裂原活化蛋白激酶p38的克隆与表达

Cloning and expression analysis of mitogen-activated protein kinase(MAPK)p38 in pearl oyster Pinctada fucata martensii

丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是一种丝氨酸/苏氨酸蛋白激酶,该蛋白激酶通过磷酸化级联介导的信号通路在细胞对细胞外刺激的反应中起着重要作用。本研究利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术克隆获得马氏珠母贝MAPK p38(PmMAPK p38)cDNA全长序列并对其序列进行生物信息学分析;利用实时荧光定量PCR(qPCR)技术分析了PmMAPK p38在马氏珠母贝不同组织以及不同免疫刺激后的表达水平。结果显示,PmMAPK p38 cDNA全长为1516 bp,开放阅读框长度为1071 bp,共编码356个氨基酸,理论分子量为40.88 ku;结构域预测结果表明PmMAPK p38含有MAPK家族典型的S_TKc结构域;多序列比对、进化树构建以及MatGAT计算结果显示PmMAPK p38与其他物种的相似度、保守程度较高;荧光定量分析结果表明,该基因在马氏珠母贝中存在广泛表达,在肝胰腺中表达量最高,其次为外套膜,最低是闭壳肌。马氏珠母贝在受到LPS刺激后,PmMAPK p38的相对表达量在刺激后2 h达到最高,12 h降到最低,最高约为最低的5倍;而在哈维氏弧菌刺激后,PmMAPK p38的相对表达量在2 h达到最高,8 h降到最低,最高约为最低的4倍。研究表明,PmMAPK p38可能在马氏珠母贝的免疫反应,尤其是在抵御外部细菌侵入中起着重要的作用。本研究为贝类免疫防御系统的研究提供了基础资料。

The mitogen-activated protein kinase(MAPK)signaling pathway is crucial in cellular response to extracellular stimuli.This pathway utilizes serine/threonine-protein kinases that transmit extracellular signals through a phosphorylation cascade to cells.Rapid-amplification of cDNA ends(RACE)was utilized for cloning and quantitative PCR(qPCR)used for expression profiling of p38 MAPK in this study.Our findings reveal that the Pinctada fucata martensii(PmMAPK p38)has a full-length cDNA of 1516 bp,an open reading frame(ORF)of 1071 bp,and has an estimated molecular mass of 40.88 ku which is encoded 356 amino acids.Domain prediction analysis indicates that PmMAPK p38 has the typical MAPK family S_TKc domain and sequence alignment,tree construction,and MatGAT calculation demonstrate its high similarity and conservation to MAPK genes in other species.Our qPCR results show that PmMAPK p38 is extensively expressed in various P.fucata martensii tissues,with the highest levels in hepatopancreas,followed by mantle,and the lowest levels in adductor muscle.Stimulation with LPS resulted in relative expression peaking at 2 h,decreasing to the least at 12 h.The greatest expression was roughly 5 times higher than the lowest.After stimulation with Vibrio harveyi,relative expression peaked at 2 h,decreased to the lowest at 8 h,with the highest expression approximately 4 times greater than the lowest.Our findings suggest that PmMAPK p38 may be a crucial component of the immune response in P.fucata martensii,and this study provides essential data for further investigation on the immune defense system of shellfish.