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青蛤(Cyclina sinensis)AP-1基因的克隆及在鳗弧菌(Vibrio anguillarum)侵染下的表达分析
丁丹, 潘宝平, 王玉梅, 侯梓园, 闫春财
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
摘要:
为了探究青蛤(Cyclina sinensis)转录因子(transcription factor gene,AP-1)在病原微生物侵染下的免疫防御应答过程中的潜在作用,本研究采用转录组测序筛选、设计特异性引物并成功克隆得到青蛤AP-1基因的cDNA序列(GenBank注册号:KX840340),利用生物信息学在线软件进行了生物信息学分析,结果显示:AP-1基因全长1914bp,结构域全长195bp,编码65个氨基酸,开放阅读框全长825bp,编码274个氨基酸,存在一个保守的bZIP功能域;是重要的转录因子。运用实时荧光定量PCR(qPCR)的方法分析了Ap-1基因在青蛤5个组织中表达过程变化,结果表明:Ap-1基因在血淋巴、外套膜、肝脏、闭壳肌、腮等组织中广泛表达;在血淋巴中表达量最高,在闭壳肌中次之,然后为鳃和外套膜,在肝脏中表达量最低,约为血淋巴的1/7。另外,我们还分析了鳗弧菌(Vibrio anguillarum)侵染青蛤血淋巴后的变化情况,结果显示:在鳗弧菌侵染后,与对照组相比,青蛤血淋巴中的AP-1表达量明显升高,在24h时Ap-1基因表达量最大,与对照组差异极显著(P<0.01)。说明Ap-1基因在青蛤的免疫防御反应中具有重要作用,同时也为进一步研究AP-1在病原微生物侵染青蛤过程中的功能和作用机制奠定了基础。
关键词:  青蛤  转录因子(AP-1)  定量PCR  鳗弧菌  免疫应答
DOI:10.11693/hyhz20170700192
分类号:Q789
基金项目:天津市科委应用基础与前沿技术重点项目资助,12JCZDJC22800号,09JCZDJC19300号;天津市科学技术普及项目,17KPXMSF00040号;天津师范大学基金项目,043135205-GC54号,043135202-XK1706号。
CLONING AND EXPRESSION OF AP-1 GENE FROM CYCLINA SINENSIS INFECTION BY VIBRIO ANGUILLARUM
DING Dan, PAN Bao-Ping, WANG Yu-Mei, HOU Zi-Yuan, YAN Chun-Cai
Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University, Tianjin 300387, China
Abstract:
To understand the roles of transcription factor AP-1 in immune response triggered by pathogenic microbes, we used clam Cyclina sinensis transcriptome library information and designed primers to clone the cDNA sequence of C. sinensis transcription factor gene AP-1 (GenBank Accession Number:KX840340). Online bio-information software analysis shows that the full length of the AP-1 is 1914bp, with a 195bp domain encoding 65 amino acids and an 825bp ORF encoding 274 amino acids. Tissue expression analysis by fluorescent quantitative PCR (qPCR) revealed that the AP-1 gene was expressed in haemolymph, mantle, liver, adductor muscle, and gill, and the expression level of AP-1 was the highest in haemolymph and lowest in liver. The sequence and expression of Vibrio vulnificus infecting hemolymph were also analyzed. After stimulated by V. anguillarum, the expression level increased and reached peak point in 24h, showing significant difference from that of the control (P<0.01). Therefore, the Ap-1 gene plays an important immune role in C. sinensis. Meanwhile, for study basis function and mechanism of AP-1 gene was response of V. anguillarum infecting in C. sinensis.
Key words:  Cyclina sinensis  transcription factor gene (AP-1)  quantitative PCR  Vibrio vulnificus  immune response
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