草地早熟禾水分胁迫下MYB家族基因调控机制的研究

Kentucky bluegrass is commonly used in lawn planting of Northern cities in China due to its good cold, trampling and trimming tolerance, but the poor drought tolerance affects its growth and ornamental value. So it is necessary to explore the mechanism of drought tolerance and response to water stress of Kentucky bluegrass, in this research, RNA-seq transcriptome sequencing was applied to establish a library for further analysis, explore the gene expression pattern of Kentucky bluegrass under water stress, and preliminarily establish the drought tolerance signal transduction pathway. Botanic MYB transcription factors compose a huge family with various functions and play an important role in metabolic regulation, growth and development, and stress tolerance. Molecular biological techniques and botanic physiological assay were combined in this research to preliminarily reveal the regulative role of MYB family under water stress through expression regulation analysis of Kentucky bluegrass MYB genes under water stress. Their functions were verified by MYB gene mapping, transcriptional activity analysis and transgenic over expression. The expression mechanism of MYB genes in response to water stress was analyzed comprehensively to explore the function variation of each MYB transcription factor. The results were significant for the revelation of the role MYB genes played in the drought tolerance of Kentucky bluegrass, the perfection of water regulation response mechanism of Kentucky bluegrass and improvement of water use efficiency.

草地早熟禾耐寒性耐践踏耐修剪多用于我国北方城市草坪建植中，但耐旱性较差影响其生长与观赏质量。探究草地早熟禾的抗旱原理和其响应水分胁迫的信号机理十分必要，本研究通过RNA-seq转录组测序方法，构建文库进行深入分析，探究草地早熟禾水分胁迫下基因表达规律，初步构建抗旱信号转导通路。植物MYB转录因子是一个超大家族，功能多样，在代谢调控、生长发育、抗逆响应中发挥重要作用。本研究采用分子生物学技术与植物生理测定相结合，通过草地早熟禾MYB家族基因在水分胁迫的表达调控分析，初步揭示水分胁迫下的MYB家族调节作用；通过MYB家族基因定位、转录激活活性分析、转基因超量表达进行功能验证；综合分析MYB家族基因响应水分胁迫的表达机制及探究各MYB转录因子的功能差异。研究结果对揭示草地早熟禾MYB家族基因在抗旱过程中的作用，为完善草地早熟禾水分调节应答机制，提高水分利用效率有重要意义。

草地早熟禾(Poa pratensis)是应用广泛的冷季草坪草之一，但它对水分较敏感，水分胁迫直接影响其生长发育，影响草坪质量。由于草地早熟禾其庞大且复杂的基因组结构，不利于其分子生物学研究。在本研究中，针对不同缺水时段下草地早熟禾叶片的转录组学的进行了研究。利用PEG6000模拟干旱处理后0、2和16 h的植物叶片RNA样本构建了3个转录组文库，并进行了测序，发现大量的MYB转录因子响应干旱诱导，其中多个R2R3-MYB类转录因子尤为活跃。基于转录组数据及qRT-PCR数据，筛选并成功获得多个草地早熟禾MYB转录因子，如MYB44、MYB30、MYB39、MYB3R-2、MYB1R1等，进一步进行相应的生物信息学分析。再利用实时荧光定量PCR技术手段进行了不同组织、不同干旱胁迫下草地早熟禾MYB转录因子的表达水平研究。其中MYB44、MYB4随着干旱程度的增加其表达量显著下调，而MYB30、MYB39等则显著上调。草地早熟禾R2R3-MYB、MYB1R1、MYB3R转录因子在结构域、亲缘关系及表达水平上均存在一定差异。干旱胁迫显著影响草地早熟禾叶片中的内源激素含量，尤其是ABA、JA、BR。同时，干旱对“植物激素信号传导”通路影响显著，R2R3-MYB和ERF2可能是介导“植物激素信号转导”和“MAPK信号通路-植物”在干旱响应中的相互作用的重要转录因子。本研究建立了草地早熟禾再生体系，并构建多个草地早熟禾MYB转录因子的过表达载体，后续农杆菌转化草地早熟禾中。基于以上实验，本研究获得多个草地早熟禾MYB家族基因，观察其在水分胁迫下的调控机制，为其应用提供理论基础，并丰富了草地早熟禾抗逆资源，为草地早熟禾耐旱品种的培养奠定基础。