珍稀濒危植物珙桐自噬相关基因DiATG3在高温胁迫响应中的功能研究

Davidia involucrata Baill. is a Tertiary relict tree species endemic to China and is reputed to be a ‘living fossil’ in the plant kingdom. It is also a rare and endangered species that has been listed as a first-grade nationally protected plant of China. High temperature is a major environmental stress limiting the distribution of natural D. involucrata population and ex situ cultivation. However, the molecular mechanisms underlying the response of D. involucrata to heat stress are poorly understood. Previously, we performed de novo transcriptome sequencing of D. involucrata leaves exposed to heat stress, identified differentially expressed genes, and found that DiATG3 was highly induced upon heat treatment. In this project, we aim to clone and characterize the DiATG3 gene promoter, and identify the upstream regulators of DiATG3. Furthermore, we will establish the expression pattern at the protein level, investigate the subcellular localization of the DiATG3 protein, analyze the interactions between DiATG3 and DiATG8, and perform functional complementation in the yeast atg3 mutant. Importantly, Arabidopsis lines overexpressing DiATG3 will be generated. Subsequently, we will determine autophagic activities, observe the growth phenotype, measure physiological and biochemical indexes of heat tolerance, and analyze the transcriptional profiles in DiATG3-overexpressing Arabidopsis plants. In addition, we will assess the impact of pretreatment with autophagy modulators on stress tolerance of D. involucrata. The expected results of this project will help to unravel the molecular basis of heat stress response in D. involucrata, and also provide new strategies for effective conservation and utilization of rare and endangered plant resources.

珙桐是我国特有的第三纪孑遗树种，被誉为植物界的“活化石”，属国家一级重点保护珍稀濒危植物。高温是限制珙桐种群分布以及引种栽培的主要环境因素，但珙桐响应高温胁迫的分子机制目前尚不清楚。在前期研究中，我们对高温胁迫下的珙桐叶片转录组进行分析，得到差异表达基因，筛选出响应高温胁迫的自噬相关基因DiATG3。在此基础上，本项目拟克隆DiATG3基因启动子序列，分析启动子功能，并分离上游调控因子；建立其蛋白水平的表达模式，研究蛋白亚细胞定位，分析DiATG3与DiATG8的相互作用，进行酵母异源功能互补；创建过表达DiATG3的转基因拟南芥，分析转基因拟南芥的自噬活性、生长表型以及耐热性生理生化指标，并进行转录水平的分析。此外，将分析自噬调节剂预处理对珙桐逆境耐受性的影响。本研究结果不仅有助于揭示珙桐高温逆境应答的分子机理，而且可以为珍稀濒危植物资源的有效保护及开发利用提供新的思路。

高温是珙桐引种栽培的主要限制因子，严重影响了对珙桐种质资源的有效保护。细胞自噬作为维系细胞稳态的关键机制，在植物响应高温胁迫中发挥重要作用。本研究从珙桐中克隆出一个受高温胁迫显著诱导的自噬相关基因DiATG3，并对其进行一系列生物学功能研究，解析了DiATG3调控高温胁迫应答的作用机制。基于植物稳定转化体系的亚细胞定位分析表明DiATG3主要定位于细胞质。研究发现拟南芥中过表达DiATG3基因提高了对高温胁迫的耐受性。进一步研究表明，DiATG3过表达株系的光合能力增强，抗氧化酶活性升高。同时，DiATG3过表达促进了高温胁迫下多胺的积累，并提高了自噬活性。通过转录组分析，发现DiATG3过表达在高温胁迫下激活了钙信号通路和MAPK信号通路，并诱导了AtHSFA3、AtDREB2C、AtWRKY26和AtWRKY33等重要逆境相关基因的表达。此外，发现施用自噬调节剂AZD8055可以有效缓解高温对珙桐幼苗的伤害。本项目的实施进一步阐明了自噬途径在植物逆境胁迫响应中的作用，同时为全球气候变化背景下濒危植物的引种栽培提供了新的策略。

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