大青杨 CCCH 型锌指蛋白PuC3H35调控干旱胁迫下根发育的分子机制研究

ABA is an important hormone that plants response to soil drought stress and regulate root growth. However, the molecular mechanism on root growth by which ABA signals regulate in the forest tree is not yet clear. The growth of root is mainly determined by the regulation of division and differentiation of root meristematic cells. In our previous study, the root zone-specific expressed and ABA-induced IX subgroup CCCH-type zinc finger protein PuC3H35 was identified from Populus ussuriensis Kom., overexpression of PuC3H35 gene significantly promote the root development under drought stress and ABA treatment. In order to understand the physiological function and the mechanism on the PuC3H35 gene in response to drought stress, genetic transformation with inhibited expression and promoter driven GUS gene experiments will further be performed to compare with root growth in the root meristem at the morphological, developmental and cellular levels. Meanwhile, integrative analysis of RNA-seq and ChIP-seq data will be performed to identification of target genes related to root development and ABA signal transduction, with functional identification of the key target gene through genetic transformation experiments. In addition, yeast one-hybrid system and electrophoretic mobility shift assay will be applied for identification of upstream regulatory factors, and yeast two-hybrid system and bimolecular fluorescence complementation assay will be performed for identification of interacting proteins related to root development and ABA signal transduction. It is expected to elucidate the molecular regulation network that PuC3H35 involved in the ABA signaling pathway in response to drought stress and regulation of root growth in P. ussuriensis.

ABA是植物响应土壤干旱胁迫、调控根系生长的重要激素。但在林木中，ABA信号调控根生长的分子机制尚不清楚。根的生长主要由根分生区细胞的分裂与分化的调控决定的。在我们前期研究中，挖掘到了在根分生区特异性表达和ABA诱导表达的大青杨CCCH型锌指蛋白转录因子基因PuC3H35，能显著促进干旱胁迫下的根系生长。本项目拟继续从形态、发育和细胞水平研究PuC3H35基因超表达、基因敲除和启动子驱动GUS转基因大青杨在根分生区响应干旱胁迫、调控根生长的途径，明确其功能。同时，综合运用转录组测序和染色质免疫共沉淀测序、酵母单杂交和酵母双杂交等技术筛选出与根发育和ABA相关的下游靶基因、上游调控因子和互作蛋白，解析PuC3H35参与的ABA信号途径在大青杨根分生区响应干旱胁迫、调控根生长的分子调控网络。

树木在森林生态系统中具有重要的经济和生态价值。干旱是影响树木生长和发育较严重的逆境因子之一。了解树木如何响应干旱，分析树木抗旱分子机制，培育抗旱新品种对未来林业的发展具有重要意义。亚细胞定位结果发现 PuC3H35 蛋白定位于细胞核中；转录自激活实验发现 PuC3H35的转录激活区位于 390-449 aa 和 615-710 aa 处；RT-QPCR结果表明，PuC3H35 基因在大青杨根中表达量最高，同时对 PEG6000 胁迫具有响应；分别获得 了 PuC3H35 过 表 达 （ PuC3H35-OE ） 和 抑 制 表 达 （ PuC3H35- SRDX）大青杨转基因株系。对组培苗进行 6% PEG6000 渗透胁迫后发现，与野生型大 青杨（WT）相比，PuC3H35-OE 株系的根系更发达，根部电导率（EL）、丙二醛 （MDA）和活性氧（ROS）含量少，相反，与 WT 相比，PuC3H35-SRDX 株系根部生 物量小、EL、MDA 和 ROS 含量高。对土培苗的干旱胁迫实验后发现，在长期干旱胁迫 后，与 WT 相比，过表达株系的根部发达，木质化程度提高，而抑制表达株系根部生长受到抑制，木质化程度低。这些结果表明干旱胁迫时，PuC3H35 锌指蛋白转录因子能够 通过提高根部清除 ROS 的能力和提高根部木质化程度来抵御干旱胁迫；对正常生长和 6% PEG6000 胁迫 7 天的 WT 和 PuC3H35-OE 株系根部进行转 录组测序，GO功能注释发现，这些差异基因很多与代谢途径相关如类黄酮代谢过程、次生代谢过程、原花青素生物合成途径、木质素生物途径；染色质免疫共沉淀、酵母单杂交和凝胶迁移实验实验验证了 PuC3H35 蛋白可以与原花青素合成途径中的花青素还原酶（PuANR）基因和木质素生物合成调控基因 PuEARLI1 的启动子区相结合，从而直接调控这两个基因的表达综上所述，在干旱胁迫下，PuC3H35 能够通过促进根部原花青素的合成，降低胁迫后根部产生的 ROS，另一方面通过促进根部木质素合成来提高根部木质化程度，两种机制相辅相成，共同作用最终提高大青杨的抗旱性。本研究为理解杨树根系抗旱响应的分子机制提供了新的思路，为杨树遗传改良奠定了理论基础。