茄SmCDF2在花青素生物合成和开花中的功能分析

Functional characterization of SmCDF2 in anthocyanin biosynthesis and flowering in Solanum melongena L.

[目的]本文拟通过验证茄单锌指(DNA binding with one finger,Dof)家族转录因子SmCDF2的功能,探索其对茄花青素生物合成及开花响应调控的作用。[方法]采取同源克隆方法从茄中分离SmCDF2基因,利用生物信息学分析其氨基酸序列;采用实时荧光定量PCR技术(RT-qPCR)检测其组织特异性表达,在烟草中进行亚细胞定位研究其表达位置;利用双荧光素酶报告试验验证其对花青素合成相关基因的转录活性;通过转基因拟南芥研究其对花青素合成和开花调控的作用。[结果]SmCDF2蛋白N端具有1个保守结构域zf-Dof,茄SmCDF2蛋白序列与同科的蔬菜作物马铃薯、辣椒和番茄中的同源蛋白具有较高的同源性。亚细胞定位试验显示SmCDF2定位于细胞核中。双荧光素酶报告试验结果表明,SmCDF2可显著上调花青素合成相关结构基因SmF3H、SmCHS的表达。拟南芥遗传转化试验结果显示,SmCDF2转基因植株具有开花延迟、花青素累积增加的表型。RT-qPCR和花青素含量测定结果表明,转基因拟南芥中的AtF3H、AtCHS的表达量显著上升,且花青素的含量也明显增加,而AtFT的表达量显著下降。[结论]SmCDF2通过正向调控SmF3H、SmCHS的表达,促进茄花青素的生物合成,同时还通过调控FT的转录,参与调控开花时间。

[Objectives]The purpose of this study was to verify the function of SmCDF2 in Dof(DNA binding with one finger)transcription factor family and explore its effect on anthocyanin biosynthesis and flowering response.[Methods]SmCDF2 was isolated from eggplant(Solanum melongena L.)by homologous cloning.Bioinformatics method was used to analyze the amino acid sequence.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect gene expression level in different organs.Subcellular localization was analyzed by transient expression system in tobacco.Dual luciferase reporter assay was used to verify the transcriptional activities of anthocyanin biosynthesis genes.Transgenic Arabidopsis thaliana plants were used to study the function of SmCDF2 in anthocyanin biosynthesis and flowering.[Results]SmCDF2 protein had a conserved domain called zf-Dof.The sequence of eggplant SmCDF2 protein had high homology with the homologous proteins in Solanaceae vegetables,including S.tuberosum,Capsicum annuum and S.lycopersicum.Subcellular localization assay showed that SmCDF2 was located in the nucleus.Dual luciferase reporter assay revealed that SmCDF2 significantly upregulated the expression of SmF3H and SmCHS structural genes in the anthocyanin biosynthesis pathway.The transgenic A.thaliana plants of SmCDF2 resulted in delayed flowering and increased anthocyanin accumulation.The results of RT-qPCR and anthocyanins content determination indicated that the expression levels of AtF3H and AtCHS in transgenic A.thaliana plants significantly increased and the content of anthocyanins was raised.However,the expression level of AtFT significantly decreased.[Conclusions]SmCDF2 promoted the biosynthesis of anthocyanins through positively regulating SmF3H and SmCHS.SmCDF2 also influenced the flowering time by negatively regulating the transcription of SmFT.