苦荞转录因子FtMYB22的抗逆分子机制研究

Tatary buckwheat (Fagopyrum tataricum Gaertn.) has been widely accepted as an ideal healthy coarse cereal based on the trait of containing abundance of flavonoids, and it also shows excellent resistance on biotic and abiotic stress. As one of the largest family of transcription factors (TFs), MYB TFs play a crucial role in controlling stress resistance response. Based on our previous study, a SG20 subfamily FtMYB22 TF was screened from Tartary buckwheat, because its expression could be dramatically up-regulated under the drought and salt stresses. Moreover, the transcriptomic analysis indicates it is possible to interact with MPKs family proteins. So, the main interests of this project focus on the stress resistance mechanism of FtMYB22 from Tartary Buckwheat. First, yeast model system can be used to generate a primary idea about effects of ectopic expressed FtMYB22 on the stress tolerance of yeast. Then, transgenic techniques would be applied to investigate the biological function of FtMYB22 in Arabidopsis and Tatary buckwheat, respectively. In addition, RNA-seq and CHIP-seq, together with EMSA would be conducted to identify the (cis-element) target gene and DNA binding site of FtMYB22. Moreover, function deletion analyses of the promoter of FtMYB22 can be performed to dig out the core element of stress-responsive. Furthermore, protein interaction screening and functional complementation researches are designing to explore the FtMPKs controlling the function of FtMYB22 by mediating its phosphorylation. This research would unravel the molecular mechanism of FtMYB22 on the stress resistance, and provide more theoretical bases and basic materials for molecular breeding on improving Tartary buckwheat stress tolerance traits.

苦荞是一种富含芦丁等黄酮类物质的特色小杂粮，具有优良的抗逆生产性能。作为植物中最大的转录因子家族之一，MYB转录因子在逆境胁迫响应过程中扮演着重要角色。本课题组前期从苦荞中筛选到可急剧上调响应干旱和高盐等逆境胁迫的SG20亚家族转录因子基因FtMYB22，转录组分析表明其可与MPKs家族蛋白互作。本项目拟通过转基因酵母初步分析其对酵母在多种逆境胁迫中耐受力的影响；采用转基因技术分别在拟南芥和苦荞中鉴定FtMYB22在多种逆境胁迫中的生物学效应；采用转录组和CHIP-seq高通量测序筛选并经EMSA技术鉴定FtMYB22的效应基因及其DNA结合位点；通过对FtMYB22启动子功能分析鉴定其逆境应答核心元件；通过蛋白互作和功能互补实验筛选鉴定MAPK信号通路中对FtMYB22具有磷酸化修饰和功能调节的FtMPKs。本项目将系统阐明苦荞转录因子FtMYB22 参与逆境胁迫的分子机制，将有助于加深苦荞优良抗逆性能的理解，也为苦荞抗逆分子辅助育种提供参考。

MYB转录因子家族在苦荞基因组中显著扩张，对其抗逆生产性能的形成至关重要。前期研究发现，苦荞FtMYB22基因的表达在逆境胁迫后急剧上升，且与花青素的含量呈负相关。本项目通过细胞学和生理学实验，明确了定位到细胞核且无独立转录激活活性的FtMYB22转录因子，其表达可响应ABA、SA和MeJA等激素，以及干旱和盐胁迫。生信分析结合转基因实验发现，在FtMYB22启动子序列中含有众多能够响应非生物胁迫的顺式作用元件，干旱和高盐处理能诱导ProFtMYB22驱动GUS基因的高水平表达，且MBS元件是其响应干旱和高盐胁迫的核心顺式作用元件。对过表达FtMYB22拟南芥株系分析发现，干旱和盐胁迫条件下，其抗逆生理生化指标和基因表达量均显著下调，抗旱耐盐性显著降低。ABA处理实验发现，FtMYB22可能参与了植物内源性ABA的合成调控， 过表达FtMYB22株系增强了对ABA的敏感性；同时，FtMYB22能够特异性结合ABRE顺式作用元件，通过响应ABA来调控下游基因的表达水平。利用酵母双杂交技术筛选获得了与FtMYB22互作的ABA受体蛋白RCAR1/2、ABA信号通路的蛋白激酶SnRK2.3和MPK级联途径的蛋白激酶MPK3/18/45，揭示了FtMYB22可通过ABA和MPK级联途径参与逆境应答过程。对过表达FtMYB22烟草和苦荞毛状根分析发现，FtMYB22可与FtTT8和FtTTG1形成MBW三元复合物，下调黄酮合成途径基因表达量，负控花青素的合成。过表达FtMYB22毛状根转录组数据的分析发现，FtMYB22作为一个负调控因子影响多个生物学过程，特别是对花青素合成通路基因表达有显著抑制作用。基于DAP-seq的分析和EMSA验证，FtMYB22结合的靶序列motif为“RAAGTTRGGTAA”，其中“TTRGG”为核心基序。综上可见，FtMYB22是兼具调控抗逆性和黄酮代谢的双功能转录因子，并表现出比较强烈的生物学效应，可作为苦荞生物育种的候选靶基因。