UV-B激活BpMPK6催化BplMYB46磷酸化调控白桦细胞壁发育及木质素合成的分子机制研究

Enhanced UV-B radiation has become a serious global environmental problem. Our previous studies have shown that UV-B can activate the BpMPK6 enzyme in 5 min. Then BpMPK6 interacted and phosphorylated the key transcriptional factor BplMYB46, which regulates secondary wall development and lignin synthesis. Based on the results, MPK6 RNAI plant, BplMYB46 RNAI and overexpression plants and wild birch (Betula platyphylla) are used as research materials. We will analyze the phosphorylation sites of MYB46 gene, and regulatory mode of phosphorylation on the function of the target protein, which include stability, subcellular localization, binding activity with DNA and identification sequence. Using the chromatin immunoprecipitation sequencing (CHIP-Seq) and transcriptome sequencing (RNA-Seq), we will determine the target gene of BplMYB46 and regulation mode of UV-B induced BpMPK6 to catalyze the phosphorylation of BplMYB46.The study will reveal the molecular mechanism of secondary cell wall synthesis regulated by UV-B induced “MPK6-MYB46-target genes” pathway in Betula platyphylla. The study can explain the signaling pathway that UV-B mediated by MAPK pathway effects on tree cell wall development, but also Lay the foundation for improving the adaptability of trees to environmental changes and improvement of wood properties.

紫外线UV-B辐射增强已成为重大的全球环境问题，本项目前期研究表明增强UV-B辐射5分钟即可激活激酶BpMPK6，进而催化调控白桦细胞壁发育和木质素合成的关键转录因子BplMYB46磷酸化修饰。基于此，本项目以已经获得的BpMPK6抑制表达株系、BplMYB46抑制表达及过表达株系和野生型白桦为材料，解析BpMPK6催化BplMYB46磷酸化的修饰位点，揭示磷酸化修饰对BplMYB46转录因子的功能活性（稳定性、亚细胞定位、与DNA结合活性及识别序列）的影响。通过CHIP-Seq和RNA-Seq确定UV-B激活BpMPK6催化BplMYB46磷酸化后调控的靶基因及表达模式，揭示“BpMPK6-BplMYB46-靶基因”通路介导UV-B辐射对白桦细胞壁发育和木质素合成的调控机制。本研究能够阐释UV-B调控树木细胞壁发育和木质素合成的分子机理，为增强树木适应环境变化及定向改良木材性状奠定基础。

UV-B辐射增强严重影响树木的生长，尤其是细胞壁的发育和木材形成。使用UV-B辐射处理野生型白桦，五分钟即可激活MAPK级联途径。通过分析UV-B处理和对照条件下的野生型白桦的转录组，代谢组和磷酸化蛋白组，发现BpMPK6蛋白的磷酸化水平受UV-B辐射上调。本研究鉴定出三个潜在的UV-B诱导的BpMPK6关键底物，分别为BplMYB46、BpMYB4和BpTT2-like，通过酵母双杂交、双分子荧光素酶系统、Pull-down等实验验证了其互作，并且通过体外磷酸化证实BpMPK6能够在体外磷酸化BplMYB46、BpMYB4和BpTT2-like。此外，酵母双杂交证实BpMPK3也可以与BplMYB46、BpMYB4和BpTT2-like互作。BpMYB4受到BpMPK6磷酸化后降解，而BpTT2-like被BpMPK6磷酸化后稳定性无明显变化。BpANR, Bp4CL2, BpCCR, BpCHI3, BpCHS2, BpDFR在UV-B处理后表达量明显上调，通过EMSA和酵母单杂交，本研究证实BplMYB46能够和BpCCR和Bp4CL2的启动子结合，BpTT2-like能够结合BpDFR和BpANS的启动子。综上研究揭示了UV-B胁迫下白桦中MAPK级联途径调控机制，即UV-B诱导BpMAPK3/6-BplMYB46/BpMYB4/BpTT2-like-靶基因来调节白桦的UV-B响应，为增强树木适应环境变化及定向改良木材性状奠定基础。