| 摘要: |
| JNKs (c-Jun氨基末端激酶)具有调节细胞凋亡的功能，可对多种细胞外信号刺激产生反应。为研究高温对小黄鱼(Larimichthys polyactis)肝脏细胞凋亡的调控机制，对细胞凋亡相关基因jnk进行研究。以NCBI中大黄鱼(Larimichthys crocea) jnk1和jnk2的CDS序列作为参照，克隆获得小黄鱼这两个基因的CDS序列。通过生物信息学分析，发现小黄鱼jnk1 ORF (开放阅读框)序列共1155 bp，编码384个氨基酸；jnk2 ORF序列共1263 bp，编码420个氨基酸。通过对结构域进行预测，发现jnk1与jnk2均有一个保守的STKc结构域和一个双磷酸化TPY催化位点。序列比对和系统进化结果分析表明，小黄鱼jnk1与jnk2均与大黄鱼亲缘关系较为接近。利用三级结构分析，发现jnk1与jnk2结构均比较保守。利用实时荧光定量方法检测jnk1与jnk2在组织中的分布情况，结果表明小黄鱼jnk1与jnk2在脑中的表达量最高。进一步分析jnk1与jnk2在高温处理不同时间的小黄鱼肝脏中的表达模式，发现随着高温处理时间的增加，jnk1表达量未发生显著变化；而jnk2在水温达到32 ℃时，其表达量即显著高于对照组，随着高温处理时间的增加，基因表达量逐渐下降，说明jnk2在高温胁迫过程中发挥了重要的调节作用。研究结果为探究小黄鱼肝脏响应高温胁迫及细胞凋亡的分子机制提供了分析基础。 |
| 关键词: 小黄鱼 高温胁迫 肝脏 jnk1 jnk2 基因表达 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金项目, 32102765号; 浙江省重点研发计划项目, 2021C02055号; 南通市科技计划重点项目，MS12022029号 |
|
| Cloning of jnk1 and jnk2 and expression analysis in response to high temperature stress in Larimichthys polyactis |
|
ZHANG Tian-Le1, LIU Hao-Wen1, LIU Si-Fang1, LI Qian2, ZHU Jia-Jie3, ZHU Fei4, LIU Feng5, LOU Bao5
|
|
1.College of Life Sciences, China Jiliang University;2.Zhejiang Ocean University;3.Ningbo University;4.Jiangsu Marine Fisheries Research Institute;5.Institute of Hydrobiology, Zhejiang Academy of Agricultural Sciences
|
| Abstract: |
| JNKs (c-Jun amino-terminal kinases) has the function of regulating apoptosis and can respond to various extracellular signal stimuli. In order to study the regulatory mechanism of high temperature on liver cell apoptosis in Larimichthys polyactis, the apoptosis related gene jnk was investigated. The CDS sequences of jnk1 and jnk2 in Larimichthys crocea from NCBI were used as reference, the CDS sequences of these two genes in L. polyactis were cloned. Through bioinformatics analysis, it was found that the ORF (Open Reading frame) sequence of jnk1 in L. polyactis is 1155 bp, encoding 384 amino acids, while the ORF sequence of jnk2 is 1263 bp, encoding 420 amino acids. By predicting their structural domains, it was found that both jnk1 and jnk2 have a conserved STKc domain and a dual phosphorylation TPY catalytic site. Sequence alignment and phylogenetic analysis showed that both jnk1 and jnk2 in L. polyactis are closely related to those in L. crocea. By tertiary structure analysis, it is found that both jnk1 and jnk2 structures are conserved. Real-time fluorescence quantitative PCR method was used to detect the distribution of jnk1 and jnk2 in tissues, which showed that the expression levels of jnk1 and jnk2 in the brain of L. polyactis were the highest. Further analysis on the expression patterns of these two genes at different times under high temperature treatment revealed that there was no significant change in expression level for jnk1 with increasing treatment time. However, jnk2 exhibited significantly higher expression level than control group when the water temperature reached 32 ℃, but its expression gradually decreased with increasing treatment time, indicating an important regulatory role played by jnk2 during high temperature stress response process. These results provide a basis for exploring molecular mechanisms underlying liver response to high temperature stress and cell apoptosis regulation in L. polyactis. |
| Key words: Larimichthys polyactis high temperature stress liver jnk1 jnk2 gene expression |