金花茶CnbHLH79转录因子的克隆、亚细胞定位及表达分析

Cloning,Subcellular Localization and Expression Analysis of CnbHLH79 Transcription Factor from Camellia nitidissima

为探究CnbHLH79转录因子在金花茶花色形成中的作用机理,克隆了金花茶CnbHLH79转录因子的编码序列,对其进行生物信息学和亚细胞定位分析,利用荧光定量PCR技术分析了CnbHLH79转录因子在金花茶不同组织,以及不同发育阶段的花瓣中的表达模式,并分析CnbHLH79转录因子的表达量与色相b^(*)、类黄酮物质含量的相关关系。研究结果表明,CnbHLH79转录因子的开放阅读框长度为855 bp,共编码284个氨基酸,具有bHLH_AtBPE_like保守结构域。系统进化分析发现,金花茶CnbHLH79蛋白与茶树bHLH79蛋白的亲缘关系最近。亚细胞定位分析显示,CnbHLH79转录因子主要在细胞核中发挥功能。对CnbHLH79转录因子进行实时荧光定量PCR分析发现,该转录因子在金花茶的根、花等组织中表达量较高;还发现在花朵开放过程中,CnbHLH79转录因子的表达量总体呈先高后低,逐步下降的趋势;此外,CnbHLH79的表达量与色相b^(*)值、槲皮素-7-O-葡糖苷的相关性系数分别为-0.92、-0.7,它们之间的负相关性较高。本研究将为阐明CnbHLH79转录因子在金花茶中调控类黄酮的合成机制,以及调控花瓣显黄色的作用机理奠定基础。

In order to explore the regulation mechanism of CnbHLH79 in the flower color formation of Camellia nitidissima,the coding sequence of CnbHLH79 was cloned and analyzed by bioinformatics,and the subcellular localization of CnbHLH79 protein was performed.Subsequently the expression pattern of CnbHLH79 was analyzed by fluorescence quantitative PCR(RT-qPCR)in different tissues and petals of different flowering stages,and the correlation between the expression of CnbHLH79 and value of chromaticity b^(*)and the content of flavonoid compounds was analyzed.It was found that the coding sequence of CnbHLH79,with bHLH_AtBPE_like conserved domain,was 855 bp encoding 284 amino acids.CnbHLH79 had the closest relationship with bHLH79 protein of Camellia sinensis.Subcellular localization analysis showed that CnbHLH79 mainly had functions in the nucleus.CnbHLH79 highly expressed in the roots and flowers of C.nitidissima and gradually decreased at flowering stages by RT-qPCR.In addition,correlation coefficient between the expression of CnbHLH79 and the value of chromaticity b^(*)and content of Qu7G was-0.92 and-0.70 respectively,which had negative relationship.This study lays a foundation for elucidating the mechanism of CnbHLH79 transcription factor regulating the synthesis of flavonoids in C.nitidissima and the mechanism of regulating the yellow color of petals.