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青蛤(Cyclina sinensis)IKK基因的克隆及其在鳗弧菌(Vibrio anguillarum)刺激下的表达分析
侯梓园1, 高姗1,2, 潘宝平1, 闫春财1
1.天津师范大学生命科学学院 天津市动植物抗性重点实验室 天津 300387;2.天津市兴南中学 天津 300021
摘要:
利用构建的青蛤(Cyclina sinensis)转录组文库,筛选到青蛤IKK基因的类似序列。经设计引物克隆比对后确认为CsIKK基因。利用生物信息学软件在线对该基因进行结构分析。采用PCR技术克隆基因,并使用实时荧光定量PCR技术克隆得到CsIKK基因在青蛤五个不同组织中的表达情况及在鳗弧菌(Vibrio anguillarum)的刺激下IKK基因在青蛤血淋巴中的时序性表达情况。综合结果得到,CsIKK基因序列开放阅读框长2298bp,编码765个氨基酸。IKK基因在青蛤的血淋巴、外套膜、闭壳肌、肝脏、性腺和鳃六个组织中均表达,在血淋巴中表达量最高。青蛤IKK基因在鳗弧菌胁迫下表达量在6h时达到最大值,与对照组相比差异极显著(P<0.01),表明该基因所指导的蛋白是青蛤重要的免疫信号通路蛋白。
关键词:  青蛤  CsIKK基因  鳗弧菌  荧光定量PCR
DOI:10.11693/hyhz20181100267
分类号:Q789;S968.3
基金项目:天津市自然科学基金项目,18JCYBJC96100号;天津市科学技术普及项目,17KPXMSF00040号;天津市高校“中青年骨干创新人才培养计划”,135305JF79号;天津师范大学基金项目,043135202-XK1706号,043135202-XB1715号;天津市高等学校科技发展基金项目,20090608号;天津师范大学引进人才基金项目,5RL104号。
CLONING AND EXPRESSION OF IKK GENE IN CYCLINA SINENSIS AND ITS EXPRESSTON UNDER VIBRIO ANGUILLARUM STRESS
HOU Zi-Yuan1, GAO Shan1,2, PAN Bao-Ping1, YAN Chun-Cai1
1.College of Life Sciences, Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University, Tianjin 300387, China;2.Tianjin Xingnan Middle School, Tianjin 300021, China
Abstract:
We obtained the sequence of an inhibitor of nuclear factor kappa-B kinase (IKK) in Cyclina sinensis (termed CsIKK gene) by building a transcriptome library and online analyzed and compared its gene structure in bioinformatics. In addition, we analyzed the expression of some tissues of C. sinensis by PCR and RT-PCR, and the expression of CsIKK gene in hemolymph after stimulated by Vibrio anguillarum. The result shows that the open reading frame of CsIKK consists of 2298bp encoding 765 amino acids. The CsIKK gene was expressed in the hemolymph, liver, coat film, closed shell, gonad, and gill, and the expression level was the highest in its hemolymph. After stimulated by V. anguillarum, the expression level increased obviously and peaked in 6h, which is significantly different from that of the control group (P<0.01). Therefore, we believe that the CsIKK-guided protein is an important immune signaling pathway protein of C. sinensis.
Key words:  Cyclina sinensis  CsIKK gene  Vibrio anguillarum  real-time quantitative PCR
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