【摘要】 c-Jun氨基端激酶(c-JunN-terminalkinases,JNKs)是丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)蛋白超家族成员之一,参与细胞骨架构建、细胞凋亡等多种细胞活动。本研究克隆了香鱼(Plecoglossusaltivelis)JNK1基因cDNA序列,并检测了其成熟卵在排入体腔保存不同时间(0—96h)的过熟过程中JNK1基因的表达变化。结果显示,香鱼JNK1基因cDNA序列全长1670bp,含一个长度1155bp的开放读码框,编码384个氨基酸,预测蛋白分子质量约44.2kDa、理论等电点6.61。氨基酸序列多重比对显示硬骨鱼类中JNK1氨基酸序列高度保守,香鱼与黄鳝的JNK1序列相似性最高(97.6%)。系统进化树分析显示各种脊椎动物的JNK1、JNK2、JNK3分别聚为一簇;本研究获得的香鱼JNK1位于JNK1大簇中并与黄鳝JNK1优先相聚,表明二者进化关系较近。RT-PCR检测结果显示健康香鱼JNK1基因在脑和性腺中高表达,肝和鳃中无表达。实时荧光定量PCR检测显示香鱼成熟卵JNK1基因的表达变化与卵的受精率、孵化率随保存时间延长而降低之间存在相关性:成熟卵随保存时间的延长JNK1表达量逐渐升高、在48h时达到峰值(24h、48h时的表达量分别为0h时的1.49倍和2.55倍),在此期间卵有较高的受精率和孵化率(48h时分别为88.99%和67.32%);保存时间继续延长时卵内JNK1基因都处于高表达水平,72h和96h时的表达量分别为0h时的2.32倍和1.53倍,而卵的质量也在保存超过48h后急剧下降(72h时受精率和孵化率分别为50.2%和25.54%)直至基本失去受精、孵化能力(96h时受精率和孵化率分别为10.83%和0.54%)。综上,香鱼JNK1基因表达上调与香鱼成熟卵的过熟凋亡过程密切相关,为深入研究香鱼JNK1基因的功能及卵过熟机制提供了基础资料。更多还原

【Abstract】 The c-Jun N-terminal kinases(JNKs) are one subgroup of the mitogen-activated protein kinases(MAPLs)and play crucial roles in cytoskeleton and apoptosis. Here, JNK1 cDNA was cloned and characterized from sweetfish Plecoglossus altivelis, and the expression changes of JNK1 gene were detected in eggs which were held in the body cavity for 0—96 h. The results showed that P. altivelis JNK1 cDNA contained 1670 nucleotides with an open reading frame of1155 nucleotides and encoding 384 amino acids, the 5’-and 3’-untranslated region contained 45 nucleotides and 469 nucleotides, respectively. The molecular mass of predicted JNK1 protein was 44.2 kDa and its theoretical isoeletric point was 6.61. The multiple sequence alignment of amino acid sequences indicated that the JNK1 of different vertebrates was highly conserved, P. altivelis JNK1 shared the highest amino acid similarity(97.6%) with JNK1 of swamp ell Monopterus albus. Phylogenetic tree analysis also confirmed that P. altivelis JNK1 fell into the JNK1 cluster and was most closely relation to M. albus JNK1. Reverse transcription PCR(RT-PCR) analysis showed that P. altivelis JNK1 mRNA had high expression in brain and gonad, but under-detectable in liver and gill. Quantitative real-time PCR(qRT-PCR) analysis showed there was a correlation between the expression of JNK1 and the quality of the eggs during the process of egg post-ovulatory ageing. Compared to freshly ovulated eggs(0 h), the expression of JNK1 increased 1.49 fold when the eggs were held in the body cavity for 24 h, and the expression peak appeared at 48 h(2.55 fold). In this period(0—48 h), the eggs had high fertilization rate(88.99% at 48 h) and hatching rate(67.32% at 48 h). The expression level of JNK1 maintained the higher level at 72 h and 96 h(2.32 and 1.53 folds compared to 0 h, respectively). At this period, the quality of the eggs sharply decreased(50.2% fertilization rate and 25.54% hatching rate), and was almost lose their developmental capacities(10.83% fertilization rate and 0.54% hatching rate at 96 h). Therefore, the increased expression of JNK1 is closely related to the apoptosis of egg post-ovulatory ageing, and these data could be helpful to understand both the biological function of JNK1 gene and the mechanism of egg post-ovulatory ageing.更多还原