葡萄VlCKX4表达特性分析与转录调控预测

Analysis of VlCKX4 Expression Characteristics and Prediction of Transcriptional Regulation in Grape

【目的】通过克隆细胞分裂素脱氢酶/氧化酶基因VlCKX4及其启动子,进行表达特性分析和启动子活性分析并进行转录因子预测,为深入解析VlCKX4介导细胞分裂素信号途径调控葡萄坐果的分子机制提供依据。【方法】使用生物信息学方法分析‘巨峰’葡萄(Vitis vinifera×Vitis labrusca)细胞分裂素氧化酶/脱氢酶4(VlCKX4)的序列特征;利用PCR技术克隆该基因以及它的启动子,使用实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)分析VlCKX4的表达特性,GUS活性试验用于分析其启动子活性;利用PlantTFDB、CisBP等转录调控数据库对VlCKX4的转录调控关系进行预测分析,输出结果使用Gephi软件进行可视化。【结果】VlCKX4全长1 582 bp,其中包含1 566 bp的开放阅读框,编码522个氨基酸,具有家族特征的FAD结构域和细胞分裂素结合(ck-binding)结构域。qRT-PCR结果表明VlCKX4在花序中高表达,其次是叶片,在卷须中表达量最低;细胞分裂素CPPU处理后VlCKX4表达量先下降后上升,细胞分裂素抑制剂洛伐他汀(Lov)处理后VlCKX4表达量先上升后下降。顺式元件分析表明VlCKX4启动子中含有吲哚乙酸、茉莉酸甲酯等响应元件;GUS化学组织染色试验表明,VlCKX4响应这些激素的处理。VlCKX4转录调控预测分析结果显示,MYB、DOF和WRKY类转录因子对其进行转录调控,结合转录组数据和共表达关系确定WRKY20、DOF1.5和MYB59为关键候选转录因子。【结论】葡萄VlCKX4受到细胞分裂素CPPU的诱导,VlCKX4启动子受到WRKY20、DOF1.5和MYB59转录因子的调控,参与促进葡萄坐果过程。

【Objective】The cytokinin dehydrogenase/oxidase VlCKX4 and its promoter in grape(Vitis vinifera) were cloned to analyze the expression characteristics, promoter activity and transcription factor prediction, so as to provide a basis for the molecular mechanism that VlCKX4 involved in cytokinin-mediated berry set in grape. 【Method】The gene sequence of cytokinin oxidase/dehydrogenase 4(VlCKX4) in Kyoho grape(Vitis vinifera × Vitis labrusca) was studied by bioinformatics method. The gene and its promoter were cloned by PCR. The expression characteristics of VlCKX4 were analyzed by real-time quantitative PCR(qRT-PCR). GUS activity assay was used to analyze the promoter activity. The transcriptional regulatory relationship of VlCKX4was predicted by PlantTFDB, CisBP databases, and the output results were visualized by Gephi software. 【Result】The total length of VlCKX4 was 1 582 bp, which contained 1 566 bp open reading frame(ORF), encoded 522 amino acids, and had the family characteristic FAD domain and cytokinin binding(CK-binding) domain. qRT-PCR results showed that VlCKX4 was highly expressed in inflorescence and leaf, but the expression of tendril was lowest. The expression of VlCKX4 decreased first and then increased after treatment with cytokinin(CPPU), while the expression of VlCKX4 increased first and then decreased after treatment with the cytokinin inhibitor lovastatin(Lov). Cis-element analysis showed that the VlCKX4 promoter contained response elements of methyl jasmonate(MeJA) and other hormones. GUS histochemical staining showed that VlCKX4 responded to the hormones.Predictive analysis of transcriptional regulation showed that MYB, DOF and WRKY transcription factors were involved in transcriptional regulation of VlCKX4. Combined with transcriptomic expression data and coexpression relation, WRKY20, DOF1.5and MYB59 were identified as key candidate transcription factors. 【Conclusion】 VlCKX4 was induced by CPPU and participates in the process of promoting grape berry set, during which prediction was regulated by transcription factors, such as WRKY20,DOF1.5 and MYB59.