菠萝MYB转录因子AcMYB1的抗寒功能鉴定及调控机理解析

Cold injury is an important limiting factor for the development of pineapple industry in China. Mining the key regulatory factors in cold resistance and exploring related regulatory mechanism are important prerequisites for the genetic improvement of pineapple cold resistance traits. In our prophase research, the pineapple cold-resistant somaclonal mutant was created by applicant’s group. A MYB transcription factor, AcMYB1, was selected from genes which were significant differentially expressed between the control and mutant under cold stress treatment, and AcMYB1 was probably to be tightly correlated to the pineapple cold resistance. In this proposal, we plan to (i) suppress AcMYB1 gene expression by RNA interference in cold-resistant mutant and overexpress it in control plants, to validate the effects of AcMYB1 on pineapple cold resistance, (ii) mine the biological processes regulated by AcMYB1 through transcriptome analysis of the transgenic lines, further screen and identify the downstream target genes and interaction proteins, to understand the molecular mechanisms of AcMYB1 in the pineapple cold resistance regulatory network. The implementation of this proposal will clarify the cold resistant function and transcriptional regulation mechanisms of AcMYB1, and lay the foundation for the genetic improvement of pineapple cold resistance traits.

低温寒害是限制我国菠萝产业发展的重要因素。挖掘影响抗寒性状的关键调控因子并探究相关调控机理，是对菠萝抗寒性状进行遗传改良的重要前提。申请人前期结合所在课题组创制的菠萝抗寒体细胞突变体材料，筛选到了一个低温胁迫下在突变体与对照中显著差异表达的MYB转录因子AcMYB1，该基因很可能与菠萝的抗寒性状有密切的关联。本项目拟通过在突变体中RNA干涉、在对照中超量表达AcMYB1基因的方法，以验证目的基因在菠萝抗寒过程中的功能；通过转基因系的转录组分析挖掘AcMYB1调控的生物学途径，并进一步筛选鉴定其下游靶基因及互作蛋白，以探明AcMYB1在菠萝抗寒调控网络中的分子作用机制。本项目的实施将明确AcMYB1基因的抗寒功能及转录调控机理，为菠萝抗寒性状的遗传改良奠定基础。

深入开展菠萝抗寒分子机制的研究，挖掘影响抗寒性状的关键调控因子并探究相关作用机理，是对菠萝抗寒性状进行遗传改良的重要前提。本项目旨在解析关键差异基因AcMYB1在菠萝抗寒过程中的功能及相关分子调控机制，为菠萝抗寒性状的遗传改良提供理论依据和重要的候选功能基因。本研究围绕AcMYB1基因，从基因的特性、生物学功能及调控机制等方面展开了全面的分析与研究。主要取得以下方面的结果：.基因表达模式分析表明，AcMYB1基因除了响应外源低温胁迫应答外，同时还参与到了其他逆境胁迫及激素信号通路中，在不同抗逆信号转导途径的交叉调控机制中起到了重要的作用。菠萝叶肉原生质体中的亚细胞定位实验证实了AcMYB1蛋白定位于细胞核中。菠萝中的遗传转化实验初步证实了AcMYB1提升植株抗寒能力的生物学功能；烟草中的异源表达说明AcMYB1在影响植株成花等方面也有一定的作用。综合菠萝转基因系的转录组分析及DAP-seq分析结果，共筛选鉴定出包括糖酵解途径中的关键基因AcPGK在内的90个下游候选靶基因，利用酵母单杂交实验进一步验证了其AcPGK启动子与AcMYB1蛋白的互作关系。酵母双杂交文库筛选共获得12个候选互作蛋白，进一步的实验证明了AcMYB1蛋白与糖酵解路径中的另一个关键蛋白FBA存在着互作关系。目的蛋白调控机制的分析表明AcMYB1蛋白通过直接结合下游基因启动子及与关键蛋白互作的方式，参与调控糖酵解代谢路径，影响植株的抗寒性能。