低温胁迫下转录因子RaMYB1对花毛茛花青素苷合成的调控模式研究

The phenomenon of changes of flower colors under cold stress is prevalent, while related reports of transcription regulation are limited to few papers. In previous researches, we have cloned a transcription factor gene of Ranunculus asiaticus L. named as RaMYB1. With the aid of analysis of gene differential expression, bioinformatics, protein translation and chromatin immunoprecipitation, RaMYB1 was confirmed as a key transcription factor regulating anthocyanin biosynthesis under cold condition in Ranunculus asiaticus L.. Based on previous researches, this project will apply the technique of chromatin immunoprecipitation combined with sequencing to isolate the genome-wide target sequences specifically bound by RaMYB1 in response to cold stress in Ranunculus asiaticus L.; apply the technique of genome walking to clone the promoter sequences of differentially expressed genes of key enzymes of anthocyanin biosynthesis; identify the target genes initially by means of alignment of target sequences and promoter sequences, and apply the technique of chromatin immunoprecipitation combined with PCR to verify above target genes; draw the regulation pattern of RaMYB1 along anthocyanin biosynthesis pathway responding to cold stress in Ranunculus asiaticus L. based on the sites and features of expression differences of target genes, and use the features of anthocyanin content differences as well as flower color variation to verify above regulation pattern. This project will provide basic data for researches of molecular regulation mechanism, quality control, breeding, molecular monitoring and indicating of color characteristics, and will provide a new reference for researches of transcriptional regulation of anthocyanin biosynthesis in non-model plants.

低温胁迫下花瓣呈色变化的现象普遍存在，然而其转录调控机制鲜有报道。项目组前期克隆到花毛茛转录因子基因RaMYB1。经基因差异表达、生物信息学、蛋白表达水平和染色质免疫共沉淀分析，确定RaMYB1是低温条件下调控花毛茛花青素苷合成的主效转录因子。基于上述研究，本项目拟应用染色质免疫共沉淀结合测序技术确定RaMYB1经低温诱导后在花毛茛全基因组范围内特异性结合的靶序列；利用染色体步移技术克隆差异表达的花青素苷合成结构基因启动子序列；比对靶序列和启动子序列初步确定靶基因，应用染色质免疫共沉淀结合PCR技术验证靶基因；根据靶基因位置及其表达差异特征，在花毛茛花青素苷合成途径上绘制RaMYB1响应低温信号的调控模式图，并利用花青素苷含量和花色表型变化特征进行验证。本项目将为研究颜色性状分子调控机理、质量控制、育种改良、分子监测指示技术提供基础数据，为非模式植物花青素苷合成转录调控研究提供新思路。

花色是重要的植物品种测试性状及选育指标，常随温度变化而变化。然而，花色转录调控机制研究不多，对花青素苷代谢通路调控模式以及结合位点全局性定位的报道较少。本项目前期明确了花毛茛品种“Doppio Pandora-purple”在温度降低时花色变化，在构建花毛茛花青素苷合成通路以及确定RaMYB1是低温胁迫下花青素苷合成转录调控主效因子的基础上，本项目获得27条低温胁迫下差异表达的花青素苷合成结构基因启动子序列，最长4121bp，最短503bp；获得低温条件下RaMYB1结合的靶序列；挖掘到RaMYB1经低温诱导后在花青素苷合成通路上转录调控靶基因7条；明确RaMYB1对上述7条基因的调控属于激活增强型，其中，RaMYB1与靶基因 DFR的结合为诱导型，与其他6条基因的结合为组成型。与之对应的是，低温胁迫下外轮花瓣天竺葵素含量由正常温度条件下185.74μg/g增加为低温胁迫下的407.93μg/g，矢车菊素由140.34μg/g增加为182.52μg/g，花色亮度和蓝度减小、红度增加。基于上述研究结果，项目组阐释了 RaMYB1经低温诱导后对花青素苷合成的调控模式，即：RaMYB1在低温胁迫下，通过组成型或诱导型的方式转录激活花毛茛花青素苷合成通路上游7个结构基因表达，增强F3’H和F3H分支代谢，上调天竺葵素和矢车菊素骨架成分合成。为获得准确的花色表型测定结果，项目组构建了花色测定取样参数判定技术以及花色定量定性测定技术。本项目将为其他分子背景较少的非模式植物种属代谢通路调控模式研究提供参考，获得的调控模式可用于植物颜色育种、颜色表达环境控制，建立的花色测定技术及花色测定取样参数判定方法已用于上海、福州、北京等品种测试分中心花色测定以及测试指南研制中测试参数定义、颜色分组和表达状态分级。通过项目实施，项目组发表SCI收录论文4篇；获得专利4项；承担或参与测试指南行业标准6项；2人完成云南省技术创新人才培养及考核，1人被认定为云南省知识产权高层次人才。