黄姑鱼转录因子激活蛋白AP2α的克隆和特征分析

cDNA cloning and characterization of transcription factor activating protein AP2α from yellow drum, Nibea albiflora

中文关键词: 黄姑鱼  转录因子激活蛋白AP2α  哈维氏弧菌  实时荧光定量PCR  亚细胞定位  蛋白表达

英文关键词: Nibea albiflora  Transcription factor activating protein 2α  Vibrio harveyi  Real-time quantitative PCR  Subcellular localization  Protein expression

中文摘要:

      转录因子激活蛋白AP2α是一类能特异地结合DNA的核转录因子，参与动物胚胎发育的调节、细胞生长、细胞凋亡、肿瘤发生以及免疫反应等多种生物过程。课题组前期通过基因组关联分析(GWAS)发现，AP2α是黄姑鱼(Nibea albiflora)抗哈维氏弧菌(Vibrio harveyi)的候选基因。本研究克隆了黄姑鱼转录因子AP2α，其开放阅读框(ORF)为1 275 bp，编码424个氨基酸；所编码的AP2α蛋白质N端是富含脯氨酸和谷氨酰胺(P/Q-rich domain)的反式激活结构域，中间是中心基本结构(central basic region)，C端为高度保守的helix-span-helix基序，负责结合DNA和蛋白质二聚化。氨基酸序列多重比对表明，AP2α保守性强，与所检测的鱼类、两栖类、鸟类和哺乳动物同源性在84.63%以上。实时荧光定量PCR检测结果显示，AP2α基因的mRNA广泛分布于所检测的黄姑鱼9 种组织样品中，其中血液中的表达量最高；受哈维氏弧菌攻毒感染后，肝脏、脾脏和头肾中AP2α表达量均明显升高，特别是在肝脏中，AP2α mRNA表达水平在24 h时达到攻毒前的39倍。通过构建重组真核表达质粒pGEFP-AP2α并转染HEK293T细胞进行亚细胞定位研究的结果显示，AP2α分布于HEK293T细胞的细胞核中。进一步通过原核克隆表达获得了可溶性的GST-AP2α融合蛋白。以上结果表明，AP2α在黄姑鱼抵御哈维氏弧菌感染过程中发挥重要作用。本研究对AP2α在硬骨鱼类先天免疫中的重要功能提供了新的见解，为深入研究黄姑鱼的分子免疫机理和抗病分子育种奠定了基础。

英文摘要:

      The transcription factor activating protein 2α (AP2α) is a nuclear transcription factor that specifically binds to DNA and is involved in the regulation of animal embryonic development, cell growth, apoptosis, tumorigenesis, immunity, and other biological processes. In our previous study, the transcription factor AP2α was discovered as a key disease-resistance candidate gene in the yellow drum, Nibea albiflora, in response to a Vibrio harveyi infection through genome-wide association analysis. In the present study, the AP2α gene, which encodes a protein of 424 amino acids, was cloned from a yellow drum. The N-terminal is a trans-activation domain rich in proline and glutamine (P/Q-rich domain), and the middle is a central basic structure (central basic region), which is a highly conserved helix-span-helix motif responsible for DNA binding and protein dimerization. Multiple alignments of amino acid sequences showed that AP2α was highly conserved, with a homology of more than 84.63% among the detected fish, amphibians, birds, and mammals. Quantitative RT-PCR demonstrated that AP2α was widely distributed in the nine tested tissues, with the highest expression in the blood. Moreover, its transcription was significantly activated in the liver, spleen, and head kidney by V. harveyi infection, especially in the liver wherein the transcript level of AP2α reached a peak at 24 h post infection. Subcellular localization by constructing the recombinant eukaryotic expression plasmid pGEFP-AP2α and transfection into HEK293T cells revealed that AP2α was localized in the nucleus. In addition, the soluble GST-AP2α fusion protein was expressed in Escherichia coli BL21. These results demonstrate that AP2α plays an important role in the immune response against V. harveyi in N. albiflora. We provide new insights into the role of AP2α in the innate immunity of teleost fishes and provide a basis for studies on immune mechanisms and disease-resistant breeding in N. albiflora and other marine fish.