低浓度微囊藻毒素干扰营养生长期水稻光合作用特性及机制研究

In recent years, cyanobacterial blooming has occurred frequently in many lakes in China, rising the concern about the potential risks of microcystins entering the terrestrial ecosystem along with the agricultural irrigation water. So far the mechanisms of the inhibition effects of these toxins on photosynthesis of terrestrial higher plants are not yet fully known. We choose the rice (Oryza sativa L. japonica) as testing organisms and carry out the following studies: 1) to investigate the long-term effects of microcystin-LR at low concentrations on the basic growth indexes, photosynthetic pigment content and pattern, photosynthetic rate, chlorophyll fluorescence induction kinetics, electron transport activity of thylakoid membrane, activities of key enzymes involved in photosynthetic reaction, thylakoid membrane protein complexes, and the chloroplast ultrastructure of rice during the vegetative growth stage; 2) to investigate the dynamic changes of differentially expressed functional genes and related proteins in rice leaves after microcystin-LR treatment using gene chip and other modern molecular and cellular biology technologies, and their respective dose-effect relationships during this process; 3) to identify the characteristics of resistant reaction of rice to microcystin-LR at the enzyme, protein and gene levels, respectively; 4) to determine the free radicals contents and distribution using the electron paramagnetic resonance spin trapping and in situ coloration technology, and therefore explore the molecular mechanisms of interference of microcystin-LR to rice photosynthesis based on the analysis the relationship between the oxidative stress, differentially expressed genes and related proteins, resistant reaction, photosynthetic parameters. This study will be of great help in elucidation of the toxic effects of microcystin at low concentrations on the photosynthesis of terrestrial plants and the related toxicity mechanisms.

近年来，我国蓝藻水华频发，灌溉农田有使水体中微囊藻毒素进入陆生生态系统的潜在风险，目前对该过程中微囊藻毒素抑制陆生高等植物光合作用的机制尚未明确。本项目以水稻为受试生物，开展以下研究：1）低浓度MC-LR长期胁迫对营养生长期水稻的基本生长指标、光合色素、光合速率、叶绿体荧光动力学参数、类囊体膜电子传递活性、光合作用关键酶活性、类囊体膜蛋白复合体及叶绿体生理形态的影响；2）利用表达谱基因芯片等现代分子生物学技术考察水稻光合相关基因差异表达和相关编码蛋白表达动态变化，并研究这其中的剂量效应关系；3）从酶学、蛋白水平和基因水平研究水稻对毒素抗性反应特性；4）采用电子顺磁共振自旋捕集和原位显色技术研究自由基含量和分布，联合植株体内氧化应激状况，相关基因和蛋白表达、抗性反应和植株光合特性变化，探明MC-LR干扰水稻光合作用机制。该研究有助于阐明低浓度微囊藻毒素干扰陆生高等植物光合作用特性和作用机制。

微囊藻毒素MC-LR可以随着灌溉用水进入到农田中，对农作物尤其是水稻的影响不可忽视。本研究着重于研究低浓度MC-LR干扰营养生长期水稻光合作用特性及机制，并应用基因组和蛋白组学分析方法，从分子水平研究了MC-LR对水稻的毒性机制，主要进展如下：1）0.05 μg·L-1MC-LR对水稻悬浮细胞的影响可以随着暴露时间的延长而恢复。0.5 μg·L-1 MC-LR暴露处理能诱导水稻悬浮细胞产生大量MDA。POD在清除ROS的过程中发挥着积极作用，在暴露后期（6 d），T-SOD在清除ROS的过程中占主导作用。2）MC-LR可以诱导水稻悬浮细胞发生程序性细胞死亡（PCD），MC-LR诱导水稻悬浮细胞OsPDCD5基因和OsMC-6基因显著表达，而抑制VPE家族基因OsVPE-1和OsVPE-2基因表达量，同时细胞跨膜蛋白OsOATPM的表达，加速MC-LR排出细胞外。3）不同浓度微囊藻毒素长期胁迫影响营养生长期水稻叶和根的生长发育、光合作用系统指标以及叶片特定基因表达的变化。MC-LR对水稻光合作用的影响主要通过净光合速率和蒸腾速率这两个因子来反馈。MC-LR可以显著抑制ATP酶的调控基因AK105925的表达量，还通过刺激基因COMBINER_EST的表达对PSⅡ产生影。4）基因组学研究表明MC-LR暴露后水稻叶片下调基因功能集中在萜类骨干生物合成、光合生物固碳作用、果糖和甘露糖代谢、光合作用和氮代谢。表明MC-LR暴露对水稻的光合作用和营养生长均造成了干扰；蛋白组学研究表明，50.0 μg·L-1 MC-LR对水稻的毒性效应主要表现为抑制核糖体、抑制卟啉叶绿素代谢和光合系统通路、抑制萜类骨干生物合成通路，促进硫胺、硫酸肌醇代谢、维他命B6代谢及类黄酮生物合成等。