桃MYB62基因参与愈伤褐变及再生的调控及机理解析

Browning is one of the critical factors that lead to difficulties in peach regeneration. Flavonoids as the most abundant phenols, are the main substrates of tissue browning. Based on the preliminary results which showed the increased accumulation levels of flavonoid in browning callus and the differentially expressed regulation gene—PpMYB62, negative correlation between the level of auxin and flavonoid content; the present project proposes to furtherly study the molecular mechanism of PpMYB62 that involved in the regulation of peach callus browning and regeneration. Peach root genetic transformation and non-chimeric callus induction system will be applied to reveal the biological function of PpMYB62 by overexpressing and silencing. Yeast one/two hybridization, NLuc/CLuc, LUC/REN, and ChIP will be used to capture interaction proteins and downstream genes of PpMYB62, which can explore the regulatory network between and molecular mechanism crosstalk between flavonoid content and the level of auxin. This project not only provides theoretical and technological support but also creates materials for the establishment of peach tissue regeneration and genetic transformation, which will be of critical theoretical significance and practical value.

褐变是影响桃离体再生的主要障碍之一。黄酮类多酚化合物是组织褐变反应的主要底物。本项目拟在前期发现桃褐变愈伤组织中大量积累黄酮类化合物及其合成的关键调节基因PpMYB62，以及黄酮类化合物与生长素两者含量之间呈显著负相关的基础上，进一步研究PpMYB62参与桃愈伤褐变和再生调控的分子机理。采用已构建的桃根系遗传转化和非嵌合愈伤组织诱导体系，通过超表达和基因沉默等方法揭示PpMYB62基因组织再生相关的生物学功能；运用酵母单/双杂交、荧光素酶互补、双荧光素酶系统和蛋白质/染色质免疫共沉淀技术筛选PpMYB62互作蛋白和其下游基因，解析PpMYB62的基因调控网络以及黄酮类积累与生长素之间交互作用的分子机理。项目的开展不仅能为桃组培再生和遗传转化提供理论和技术支撑，而且能创制用于桃离体再生体系研究的新材料，具有重要的理论意义和应用价值。

组织褐变是植物离体培养的瓶颈问题之一，褐变会导致外植体生命力下降，失去再生能力。酚类化合物，特别是类黄酮的积累是导致组织褐变的重要底物，其中的分子调控机理尚不明确。代谢组学分析表明，黄酮是导致离体培养愈伤组织和根褐变的关键成分。外源添加黄酮(木犀草素)可导致愈伤组织和根生长迟缓和组织褐变。此外，通过比较转录组分析，挖掘到调控黄酮合成和生长素转运的候选基因PpMYB62。在桃愈伤组织中的稳定过表达PpMYB62通过刺激黄酮积累导致严重的褐变和生长迟缓。PpMYB62直接与黄酮合成酶II启动子(PpFNSII)结合，通过转录激活其表达;同时，PpMYB62在转录水平抑制参与生长素转运(PpPIN1)和侧根发育(PpLBD29)相关基因的表达。本项目研究结果拓展了黄酮类可作为内源性生长素运输抑制剂的认识，这不仅是由于黄酮类合成途径中的激活剂能够竞争PIN蛋白的生长素结合位点，而且还可以在转录水平抑制生长素转运。