杨树转录因子MYB50在调控苯丙烷代谢和胁迫防御中的机制研究

As a class of secondary metabolites ubiquitously found in plants, phenylpropanoids play important roles in plant growth, development and defense against the stress. To identify candidate MYB regulators of stress-induced phenylpropanoid metabolism in poplar, transcriptome analysis was performed using the poplar leaves treated with fungi and high-salt stresses. To verify the biological function of MYB50 which is strongly responsive to various stresses, the following experiments will be conducted: Over-expression vector of MYB50 will be constructed and transformed into wild Populus tomentosa, and the myb50 mutants will be generated by the CRISPR/Cas9 system. The contents of phenylpropanoids will be determined, and the expression levels of genes involved in phenylpropanoid biosynthesis will be detected by quantitative real-time PCR (qRT-PCR). Chromatin immunoprecipitation followed sequencing (ChIP-Seq) will be used to explore the binding of MYB50 to the promoters of downstream genes and yeast two-hybrid library will further be utilized to screen functional proteins interacting with MYB50. We will further use transient transcription activation assay to identify the activation of in the phenylpropanoid biosynthetic genes by MYB50. Furthermore, the relationship between phenylpropanoids and stress resistance will be clarified by comparing the resistance of transgenic poplars to the fungal pathogen and high salt treatments. Our study will not only figure out the mechanism of MYB50 in regulating the phenylpropanoid pathway and stress assistance in poplars, but also provide molecular evidence for breeding trees to improve stress resistance and wood properties.

苯丙烷类化合物是植物中广泛存在的一类次生代谢产物，在植物生长发育和胁迫防御中扮演重要角色，但其在木本植物中生物合成的调控机制尚不清楚。本课题组前期分析了真菌病和高盐等胁迫处理毛白杨的转录组测序数据，筛选出受胁迫诱导并参与苯丙烷类代谢调控的转录因子MYB50。本项目将克隆杨树MYB50基因，通过载体构建和遗传转化获得过表达和Cas9基因敲除MYB50的转基因杨树，并测定苯丙烷类化合物含量，qRT-PCR分析转基因杨树中该代谢途径相关基因的表达变化;ChIP-seq筛选MYB50下游靶标基因，酵母双杂交文库筛选与MYB50相互作用的功能蛋白，瞬时表达实验分析MYB50对下游基因的转录调控作用，解析MYB50调控苯丙烷代谢的机制；比较转基因毛白杨对真菌病和高盐等胁迫的抗性，搞清苯丙烷代谢和胁迫防御的关系。最终阐明MYB50在调控苯丙烷代谢和胁迫防御中的机制，为林木抗逆性与木材品质育种提供参考。

苯丙烷类化合物是植物中广泛存在的一类次生代谢产物，在植物生长发育和胁迫防御中扮演重要角色。本项目通过真菌病、伤害、冷和高盐胁迫处理毛白杨的转录组测序数据筛选并克隆了受胁迫诱导并参与苯丙烷类代谢调控的转录因子编码基因MYB50和MYB61，在毛白杨中对其进行功能解析，取得如下研究结果：进化树及同源比对分析显示，MYB50和MYB61的序列相似性为61.84%，与调控杨树苯丙烷代谢的转录因子MYB6亲缘关系最近；亚细胞定位分析显示MYB50和MYB61均定位于细胞核；酵母转录激活实验证实，MYB50具有转录激活活性，且激活结构域位于蛋白质C端，而MYB61在酵母细胞中没有自激活活性；利用转基因技术分别获得MYB50和MYB61的超量表达及Cas9基因敲除的转基因杨树，分析其生长表型、苯丙烷类代谢通路基因表达及组织切片等发现MYB50对毛白杨苯丙烷类代谢通路没有明显调控作用；荧光定量PCR分析发现，MYB61基因的表达受到盐、ABA和真菌病的诱导表达，表明其可能参与胁迫防御；在杨树中过表达和敲除MYB61基因发现，与野生型相比，转基因植株的生长表型没有明显变化，但其茎中的木质部发育受到影响，表现为木质部细胞层数均减少；对野生型和MYB61过表达及敲除株系进行盐处理发现，敲除MYB61基因后，毛白杨对盐更为敏感；进一步通过转录组测序并进行KEGG富集分析发现，差异基因主要富集在类黄酮代谢通路、植物病原菌互作通路和苯丙烷代谢通路，表明MYB61可能通过调控苯丙烷代谢通路来影响植物对盐及病原菌的抵御能力。本研究通过解析MYB61在调控苯丙烷代谢和胁迫防御中的作用机制，为林木抗逆性与木材品质育种提供参考。