R2R3-MYB转录因子调控薄壳山核桃嫁接愈合的分子机制研究

Grafting is a key technique to promote the development of pecan industry, it can remain the good characteristic of its parents, and reduce the juvenile stage, thus accelerating the process of fruiting. Successful grafting is a complex biochemical and structural process beginning with the adhesion of the two grafted partners, followed by callus formation and the establishment of a functional vascular system. Graft union development is an intricate process which involves cell dedifferentiation, cell differentiation and stress-resistance cause by mechanical damage during graft process.The formation of vascular, as a mark of successful grafting, needs the involvement of lignin. The metabolic pathway of ‘Phenylpropanoid biosynthesis’ is important for lignin production, which is regulated by R2R3-MYB transcription factors. In this research, based on our former transcriptome database, the full length cDNA and promoter of lignin related R2R3-MYB would be cloned. The expression profile of R2R3-MYB would be tested by real-time PCR. By using yeast two-hybrid, the transactivation R2R3-MYB would be analyzed. The down-stream regulators would be characterized by electrophoretic mobility shift and yeast one-hybrid assay. Transient expression analyses were performed in tobacco to study the function of R2R3-MYB. Promoter + GUS vectors of R2R3-MYB would be constructed and transformed into Arabidopsis thaliana. The transformed plants would be grafted for further evaluation of the regulatory elements of R2R3-MYB. Combination of the above analysis, the functions and mechanism of R2R3-MYB in the regulation graft union formation will be disclosed.This investigation will provide an insight into the molecular mechanism of graft process in pecan and lay a theoretical basis for better guiding the production practice.

嫁接是薄壳山核桃产业发展的关键技术，它具有维持亲本优良性状、促进提早结果、增强适应性等功能。嫁接成活是一个复杂的生物学过程，涉及到细胞的脱分化、再分化、维管系统形成及各种生理生化过程。嫁接成活的标志是维管组织的形成，该过程需要木质素的参与。“苯丙烷合成代谢”是产生木质素的重要途径，该代谢通路受到R2R3-MYB转录因子的调控。本项目拟以先前构建的转录组测序数据为基础，克隆木质素生物合成相关的R2R3-MYB基因及其启动子；利用荧光定量PCR分析其表达模式；通过酵母双杂试验分析其转录激活特性；利用酵母单杂、凝胶阻滞技术分析其下游调控基因；在烟草中瞬时过表达分析其功能；构建启动子融合载体转化拟南芥，进行拟南芥嫁接实验，分析R2R3-MYB基因受诱导的条件，揭示R2R3-MYB基因在嫁接愈合过程中的功能及其分子调控机制，这对于嫁接技术改进及促进薄壳山核桃产业发展均具有重要的理论与实践意义。

项目首先以薄壳山核桃（Carya illinoinensis）基因组为基础，从薄壳山核桃基因组中共鉴定到153个R2R3-MYB基因家族成员，构建了薄壳山核桃、拟南芥、杨树和水稻R2R3-MYB系统进化树，分析成员之间的进化关系，并利用RNA-seq分析了薄壳山核桃R2R3-MYB基因在嫁接后0、8、15、30d 4个不同时间点中的表达水平，共鉴定到64个差异表达的R2R3-MYB基因，并在嫁接后表现出不同的表达模式，构建其共表达网络，挖掘这些基因之间的关系；其次从转录组数据中鉴定到CiMYB46基因并进行克隆，分析其表达模式及启动子区的诱导元件，以薄壳山核桃基因组DNA为模板，并使用PCR方法克隆得到CiMYB46基因的启动子区域，最终克隆得到CiMYB46序列，实时定量PCR分析显示，CiMYB46在嫁接体发育的维管组织形成期具有高表达量，并与部分次生壁合成相关功能基因具有共表达趋势，研究结果表明CiMYB46可能与嫁接体发育的维管形成期有关，并受激素诱导表达；最后通过酵母双杂试验分析其转录激活特性，在烟草中瞬时过表达分析其功能，构建启动子融合载体转化拟南芥，进行拟南芥嫁接实验，分析CiMYB46基因受诱导的条件，揭示了CiMYB46基因在嫁接愈合过程中的功能及其分子调控机制，研究结果对于嫁接技术改进及促进薄壳山核桃产业发展均具有重要的理论与实践意义。本项目共发表研究论文5篇（其中SCI论文2篇，中文核心期刊论文3篇），授权国家发明专利1件，出版专著1部，培养博士、硕士研究生各1人，利用该技术培育的薄壳山核桃优良品种嫁接苗在江苏及安徽、浙江等南方省份累计推广面积达到50余万亩，新增产值近5.6亿元，产生了显著的经济、社会和生态效益。项目组完成的《薄壳山核桃产业化开发关键技术创新与应用》成果获2020年梁希林业科技进步奖二等奖。