miR5149介导GTL1调控气孔密度提高文冠果幼苗抗旱性的机制研究

Xanthoceras sorbifolium, a tree species endemic to China, has high oil content in its seeds and is recognized as an economically and ecologically biodiesel crop. X. sorbifolium is characterized by high tolerance to poor soil and drought stress. Stomatal characteristics can reveal the adaptation strategies of plants under drought stress. GTL1 transcription factor regulates drought tolerance by modulating stomatal density via transrepression of SDD1. MicroRNA (miRNA) are involved in plant response to stresses, whether the regulation of GTL1 expression has not been elucidated. On the basis of our previous work, the targeted regulating relationships between miR5149 and GTL1 have been confirmed by RLM-5’RACE (RNA ligase mediated 5’rapid amplification of cDNA ends) assay. So we propose the following hypothesis that miR5149 can mediates drought tolerance in X. sorbifolium seedlings by modulating stomatal density via targeting regulate the expression of GTL1. In this project, we will adopt ultrastructural observations、dual-luciferase reporter (DLR) assay systems and qRT-PCR(real-time quantitative PCR) to comparative analysis of expression patterns of miR5149, GTL1 and SDD1, regulatory relationships and the effect on stomatal density in different X. sorbifolium germplasms with drought tolerance. The project intends to study the functions of miR5149 and GTL1 in drought-stress tolerance in X. sorbifolium by reverse genetics methods. We also intend to observe the changes of stomatal density and expression of miR5149, GTL1 and SDD1 in wild-type (WT) and transgenic plants before and after drought treatment. This project will obtain a new evidence for miR5149 mediate GTL1 tuning stomatal density. We expect to reveal the molecular mechanisms of miR5149-GTL1-SDD1 functional modules in X. sorbifolium seedlings, and provide a theoretical basis for miRNA regulatory mechanisms involved in plants response to drought stress.

文冠果是我国特有的优良木本油料树种，抗旱耐瘠薄。转录因子GTL1下游靶基因SDD1可调控气孔密度从而影响植物抗旱性，但其miRNA调控机制仍不清楚。前期发现文冠果miR5149靶向GTL1，本项目以干旱胁迫下抗性存在明显差异的不同种质为材料，利用超微结构观测、双荧光素酶报告基因检测和qRT-PCR等手段，通过对叶片气孔密度的观察及miR5149、GTL1和SDD1基因表达的分析，明确三者间的调控关系及其对气孔密度的影响；结合过表达及沉默的方法，比较转化miR5149和GTL1植株及对照叶片中SDD1基因表达量、气孔密度及抗性生理指标的差异，揭示miR5149介导GTL1调控气孔密度提高文冠果幼苗抗旱性的生理及分子机制。这将首次阐明“miR5149-GTL1-SDD1”模块在文冠果抗旱中的作用，可为植物miR5149调控GTL1提供直接例证，对深入理解miRNA响应干旱胁迫机制具有重要意义。

文冠果是我国特有的优良木本油料树种，干旱是影响文冠果成活与生长的主要逆境之一。探明文冠果抗旱的分子机制，有助于培育优良品种，增产增收。miRNA强大的调控作用近年来倍受关注，但专注于干旱胁迫相关miRNA的研究仍未见报道。我们发现miR5149可通过靶向转录因子GTL1调控文冠果的抗旱性。利用生物信息学预测、qRT-PCR及RLM-5’RACE等技术手段，验证了miR5149对GTL1转录因子具有切割和转录调控作用。亚细胞定位实验证实GTL1定位在细胞核中，干旱胁迫后miR5149与GTL1表达呈负相关。利用STTM技术沉默miR5149的文冠果植株生长势较弱、叶片窄小、叶缘消失、叶片面积减小，而气孔密度显著增大，表明miR5149在文冠果叶片形态建成和气孔密度调控中发挥着重要的作用。干旱处理后，转化植株抗性降低，叶片相对含水量和水分利用率都显著低于野生型，而气孔导度和叶片失水率显著高于野生型。对miR5149-STTM材料进行转录组分析发现，miR5149能影响389个转录因子基因的表达。过表达GTL1基因的文冠果植株表现出叶片狭小、叶面积降低和气孔密度增大等特征。干旱处理后，转化植株抗性降低，叶片相对含水量和水分利用率均显著低于野生型，其靶基因SDD1表达量显著下降，而叶片失水率高于野生型。利用RNAi技术沉默GTL1基因的文冠果效果正好相反，叶片变宽大，叶面积增加，气孔密度减少，抗旱性增强。对过表达和沉默GTL1转录因子基因的材料进行转录组分析，GTL1能影响1710个基因的表达，这些基因主要包括在淀粉与蔗糖代谢、植物激素信号转导和MAPK相关的信号通路中。本项目较好地阐明了miR5149-GTL1模块在文冠果响应干旱胁迫中的作用，既有利于揭示文冠果抗旱的分子机制，有可为文冠果基因资源的有效利用和品种改良提供重要信息，同时还可以为全面系统地研究miRNA提供新的思路。

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