内源活性氧引发大蒜试管苗细胞膜异变和玻璃化发生的机理解析

Hyperhydricity in plantlet in vitro, a common problem, has seriously influenced virus-free and rapid propagation in plant tissue culture,as well as gene genetic transformation. However, its mechanism remains unclear. It is shown that accumulation of reactive oxygen species（ROS）, disturbance in ROS scavenging system and damage to cell membrane were found during the process of hyperhydricity. Our previous research demonstrated that ROS was related to the hyperhydricity and oxidative damage of cell membrane. To date，the relationship between them was rarely studied at home and abroad. According to the previous reports and our studies, it would be predicted that ROS, as a signal transduction or oxidative damage substances, plays a key role in hyperhydricity, which could be the probable novel mechanism of hyperhydricity in plantlet in vitro. To prove this hypothesis, the model between the accumulation of ROS in different treatments and hyperhydricity in plantlet in vitro would be established by means of the observation of the tissue structure, the determination of physiological function and transcriptome sequencing. From the tissue, cell and molecule levels, we can analyze and discuss the effect of different ROS concentration on the hyperhydricity, the structural variation of cell membrane and function and the expression of related genes. This research will provide a new perspective of hyperhydricity mechanism and lay a foundation on controlling hyperhydricity in plantlet in vitro.

试管苗玻璃化是植物组织培养中普遍发生的现象，对植物脱毒、快繁及基因遗传转化影响严重，但其发生机制至今尚未明确。研究表明，试管苗玻璃化过程中存在活性氧物质(ROS)的产生和积累、活性氧清除系统紊乱和细胞膜损伤，我们前期试验也证实ROS与试管苗玻璃化和细胞膜损伤相关，但相互关系的机理国内外迄今尚少有报道。根据相关研究和我们前期工作，我们推测，具有信号传导和氧化伤害功能的ROS在试管苗玻璃化中有重要作用，其不同功能的表达可能是调控试管苗玻璃化的新机制。为证实这一假设，我们在不同培养条件下培养试管苗,建立内源ROS产生和积累与试管苗玻璃化发生模型，采用组织观察，生理测定，转录组测序和分析等技术，从组织-细胞-分子等不同层次，探讨内源ROS对试管苗玻璃化、细胞膜结构和功能异变、相关基因差异表达的影响。本课题将从新的角度提出试管苗玻璃化的机制，为试管苗玻璃化调控奠定基础。

玻璃化是植物组织培养过程中普遍存在的一种生理障碍，严重影响植株的离体再生和快繁，成为植物组织培养的三大难题之一。在研究了大蒜试管苗玻璃化发生规律的基础上，构建了防控试管苗玻璃化发生的综合技术体系，提出了构建了“内源ROS调控玻璃化发生”的机理模型。在胁迫条件下，质外体中NADPH氧化酶催化产生大量活性氧，发生氧化迸发，激活了植物体内的抗氧化系统发生响应，进而缓解内源ROS的过量增加。当产生的ROS超出植物活性氧调控系统的解毒能力，造成氧化胁迫，细胞膜饱和脂肪酸含量升高、磷脂含量降低、细胞膜功能蛋白活性和钙离子含量降低，细胞壁纤维素、木质素和果胶含量减少，导致细胞间隙增大、细胞壁厚度减小、细胞膜和细胞壁组成改变，细胞膜破损增强，从而诱导试管苗质膜过氧化和细胞膜损伤，导致植物细胞超度含水，最终发生试管苗玻璃化。外源ABA和AsA处理可缓解外源H2O2诱导的氧化胁迫，进而缓解试管苗玻璃化。利用miRNAs和转录组测序，得到133条上调和76条下调的与玻璃化相关的miRNAs，496个上调和239个下调的玻璃化相关基因。差异基因的功能注释发现，大多参与调控跨膜蛋白合成、能量代谢以及激素响应等过程，其中，细胞分裂素、生长素、乙烯、水杨酸、赤霉素、脱落酸合成相关的基因均参与了内源ROS的响应，氧化胁迫响应基因ACO2M、叶绿体抗逆基因Y2766、逆境蛋白基因DNJH2以及乙烯调控因子ERF82直接参与了胁迫的响应。利用RACE技术克隆3个与玻璃化相关的质膜水通道蛋白基因，qPCR发现，质膜水通道蛋白基因表达影响玻璃化的发生，并参与试管苗玻璃化的恢复过程。利用大蒜花序轴为外植体诱导出体细胞胚，为转基因功能验证奠定基础。我们的研究成果极大丰富了植物试管苗玻璃化相关知识，理论上填补和完善试管苗玻璃化机理的空白，为揭示玻璃化的科学理论问题奠定基础；在实践上，为有效控制玻璃化的发生提供依据。