卵叶牡丹花色苷合成相关基因bHLH的克隆与功能分析

Cloning and Functional Analysis of bHLH Gene Related to Anthocyanin Synthesis in Paeonia qiui

【目的】以卵叶牡丹(Paeonia qiui)为试验材料,研究PqbHLH1基因在叶片发育过程中对花色苷合成的调控机制。【方法】基于前期获得的卵叶牡丹叶片转录组数据设计引物,利用PCR技术从卵叶牡丹叶片中克隆得到bHLH1基因,采用生物信息学方法分析PqbHLH1基因的理化性质、亲疏水性、理论等电点、蛋白二级结构以及物种的进化关系。通过荧光定量PCR技术检测其在不同组织、不同时期的表达量。利用农杆菌介导法使其在拟南芥(Arabidopsis thaliana)中过表达,检测过表达PqbHLH1对卵叶牡丹叶片花色苷的影响。【结果】PqbHLH1基因的编码区全长为1920 bp,编码639个氨基酸。PqbHLH1蛋白属于亲水性蛋白,同时不含有信号肽和跨膜结构,且定位在细胞核上。系统进化分析表明卵叶牡丹PqbHLH1蛋白与葡萄VvMYCA1的亲缘关系最近。荧光定量PCR分析表明PqbHLH1基因在卵叶牡丹叶片中的表达量呈先降低后增高的趋势,在叶片S6时期表达量显著,且花色苷合成结构基因PqCHS、PqF3'H、PqDFR和PqANS随着叶片发育其表达水平呈现出先上升后下降的趋势。PqbHLH1基因过表达的拟南芥株系中,与花色苷合成相关结构基因的表达量显著高于野生型拟南芥,且花色苷含量与结构基因AtCHI、AtF3'H和AtUFGT的表达趋势一致,黄酮醇含量与结构基因AtFLS的表达趋势一致。【结论】PqbHLH1基因通过调控花色苷合成相关基因的表达,从而正向调控卵叶牡丹叶片中花色苷和黄酮醇的合成。

【Objective】Using Paeonia qiui as the experimental material,the regulatory mechanism of PqbHLH1 gene on anthocyanin synthesis during leaf development was studied.【Method】Based on the transcriptome data obtained in the early stage,primers were designed and the bHLH1 gene was cloned from the leaves of Paeonia qiui using PCR technology.Bioinformatics methods were used to analyze the physicochemical properties,hydrophobicity,theoretical isoelectric points,protein secondary structure,and evolutionary relationships of the PqbHLH1 gene.Fluorescence quantitative PCR technology was used to detect its expressions in different tissues and stages.Agrobacterium mediated method was employed to overexpress it in Arabidopsis thaliana and detect the effect of overexpressing PqbHLH1 on the anthocyanins of peony leaves.【Result】The total length of the coding region of PqbHLH1 gene was 1920 bp,encoding 639 amino acids.The PqbHLH1 protein was hydrophilic and does not contain signal peptides or transmembrane structures,and was localized in the nucleus.Phylogenetic analysis showed that the PqbHLH1 protein in peony ovale had the closest genetic relationship with VvMYCA1 in grapes.Fluorescence quantitative PCR analysis showed that the expression of PqbHLH1 gene in peony leaves had a trend of first decreasing and then increasing,and the expression was significant in the S6 stage of leaves.The expressions of anthocyanin synthesis structural genes PqCHS,PqF3'H,PqDFR,and PqANS showed a trend of first increasing and then decreasing with leaf development.In A.thaliana strains overexpressing the PqbHLH1 gene,the expressions of structural genes related to anthocyanin synthesis was significantly higher than that of wild-type Arabidopsis,and the expression trend of anthocyanin content was consistent with that of structural genes AtCHI,AtF3'H,and AtUFGT,while the expression trend of flavonol content was consistent with that of structural gene AtFLS.【Conclusion】The PqbHLH1 gene positively regulates the synthesis of anthocyanins and flavonols in peony leaves by regulating the expressions of genes related to anthocyanin synthesis.