毛叶杜鹃热激转录因子Hsfs高温应答的表达模式与功能解析

Rhododendron (Rhododendron L.) has a very high ornamental value and extensive application in landscape, while high-temperature is an important environmental factor limits its introduction and cultivation. Previous study shows Hsfs(Heat stress transcription factors) play a pivotal role in gene expression regulatory network in thermotolerance. This program takes Rhododendron championiae as material, which is a wild thermo-tolerant rhododendron originated from South China. Through detecting transcriptome expressional profile of leaves after high-temperature treatment and/or control, transcript sequence and specific gene expression profile of Hsfs will be identified. Then we conduct RACE cloning, quantitative Real-time PCR and GUS fusion expression experiment, in order to clarify the expression pattern of a given Hsf under heat stress and space-time distribution. In addition, Arabidopsis transformation, microarray and yeast one-hybrid system will be applied to characterize their molecular function and signaling pathway involved in thermotolerance. The execution of this program would be helpful to understand the signaling response and transcriptional regulatory networks of R. championiae under high temperature stress, as to achieve novel genes involved in thermotolerance from elite Rhododendron germplasm for molecular marker-assisted breeding, in order to provide a theoretical basis for improved themotolerance by genetic engineering techniques.

杜鹃（Rhododendron L.）具有极高的观赏价值与丰富的园林应用，高温是限制其引种栽培的重要环境因素。前人的研究表明Hsfs（热激转录因子）是耐热表达调控的关键。本项目以我国原产的耐热毛叶杜鹃（Rhododendron championiae）为试材，通过比较对照与高温胁迫处理下的杜鹃叶片表达谱，鉴定出杜鹃转录组中Hsfs的序列信息与动态变化特点，结合RACE克隆、荧光定量PCR与GUS融合表达等手段，弄清Hsfs家族基因高温应答的表达模式。利用拟南芥转基因、微阵列芯片与酵母单杂交系统，验证差异表达的Hsfs在高温胁迫下的功能及其作用途径。本项目的实施有助于理解毛叶杜鹃高温胁迫下的信号应答与转录调控网络，从而挖掘出耐热杜鹃种质资源中的优异耐热基因，为开发杜鹃耐热分子标记用于辅助育种或者通过基因工程技术进行杜鹃耐热改良提供理论基础。

杜鹃是原产我国的重要木本花卉，高温是限制其引种栽培的重要环境因素。前人的研究表明，Hsfs（热激转录因子）是植物耐热基因表达调控的关键。本项目筛选到耐热杜鹃材料，以此建立了一套高效繁育的体系，通过比较对照与高温胁迫处理下的杜鹃叶片表达谱，鉴定出杜鹃转录组中Hsfs的序列信息与动态变化特点，结合RACE克隆、荧光定量PCR与GUS融合表达等手段，弄清Hsfs家族基因高温应答的表达模式。利用拟南芥转基因、RNA-seq与酵母单杂交系统，验证差异表达的Hsfs在高温胁迫下的功能及其作用途径。.我们的研究从表型层面筛选到了耐热性和观赏性兼具的杜鹃优质高抗材料，并对它们进行了高效繁育；从生理层面解释了杜鹃属植物不耐热的重要原因在于光合系统II对高温胁迫极度敏感，证实它可能是导致叶肉细胞叶绿体降解及随后细胞死亡的重要成因；从分子水平上发现了杜鹃热激转录因子RoHSFA2、RoHSFA3、RoHSFA6和RoDREB2C是杜鹃响应高温胁迫的关键基因元件。它们均定位于细胞核，作为转录激活子参与到高温适应性基因调控过程中。其中，RoHSFA2、RoHSFA3、RoHSFA6可以结合靶基因HSP101、CAT1等下游基因，而RoDREB2C 可以作用于RoHSFA2的上游，且这种作用受到ABA的调控。.本项目的实施有助于理解杜鹃高温胁迫下的信号应答与转录调控网络，为深入阐明杜鹃属植物高温适应性的形成和分化成因奠定了理论基础，同时，获得的数个基因元件，有望直接应用于杜鹃的定向育种中，作为宝贵的基因资源进行开发利用，极大加快育种进程。