三疣梭子蟹C-JUN氨基末端激酶基因克隆及在病原胁迫后的表达特征分析

Cloning and Expression Analysis of c-Jun N-Terminal Kinase Gene in Portunus trituberculatus after Pathogenic Stress

中文关键词: 三疣梭子蟹  PtJNK  基因克隆  病原感染  实时荧光定量PCR

英文关键词: Portunus trituberculatus  PtJNK  Gene cloning  Pathogenic infection  Quantitative Real-time PCR

中文摘要:

      C-JUN氨基末端激酶(c-Jun N-terminal kinase, JNK)作为丝裂原活化蛋白激酶(MAPK)超家族的重要一员，在细胞增殖、凋亡和免疫应激等过程中发挥着重要作用。为深入研究JNK基因的免疫防御机制，本研究从三疣梭子蟹(Portunus trituberculatus)转录组数据库中筛选到JNK基因的EST序列，利用RACE扩增技术克隆得到该基因全长序列，命名为PtJNK，cDNA全长为3240 bp，开放阅读框(ORF)为1380 bp，编码459个氨基酸，具有TPY磷酸化位点的S_TKC保守结构域，是JNK基因家族典型特征。组织表达分布结果显示，PtJNK基因在所有组织中均有表达；Real-time PCR检测不同病原刺激下PtJNK基因的表达水平，结果显示，注射副溶血弧菌(Vibrio parahaemolyticus)后，该基因显著下调表达(P<0.05)；而注射白斑综合征病毒(WSSV)后，该基因显著上调表达(P<0.05)。综上所述，PtJNK基因是一种广泛表达基因，且在不同的病原感染情况下，该基因的表达模式存在差异，在免疫防御过程中具有重要作用。

英文摘要:

      The c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase superfamily, which plays an important role in cell proliferation, apoptosis, and immune stress. To further explore the immune defense mechanism of JNK in Portunus trituberculatus, the EST sequence of PtJNK was isolated from the transcriptome database of P. trituberculatus. In this study, the JNK gene of P. trituberculatus (PtJNK) was successfully cloned by RACE; measurements showed a cDNA length of 3240 bp and an open reading frame of 1380 bp. PtJNK consists of 459 amino acids, including a serine/threonine protein kinase (S-TKc) domain with a conserved Thr-Pro-Tyr (TPY) motif, which is a typical feature of the JNK gene family. The results of tissue expression and distribution show that the PtJNK gene is expressed in all tissues. Real-time PCR was used to detect the expression of PtJNK under different pathogenic stimuli. The results show that expression of the gene is significantly down-regulated after injection of Vibrio parahaemolyticus (P<0.05), while expression is significantly up-regulated when white spot syndrome virus is injected (P<0.05). To sum up, PtJNK gene is a widely expressed gene, and expression of the gene differs according to pathogen.