La(III)通过衔接蛋白基因异常甲基化激活植物胞吞作用探究

It is incontrovertible that the accumulation of rare earth elements (REEs) in edible plants increases the REEs content in human body through food chain, and finally influences human health. Therefore, the limit standard of REEs in edible plants is of urgent need, however, the primary premise, the mechanism of REEs acting plants, is still not clear. It was reported that La(III) can break the sluggishness of endocytosis in plant leaf cells, which is the result of long-time evolution, and activate the endocytosis of plant leaf cells. However, the mechanism of the activation is unknown. Adapter protein can start endocytosis by binding clathrin, and influence the gene express and the content of adaptin through the methylation of DNA promoter. In this project, Arabidopsis thaliana is chosen as the research object. By using bisulfite sequencing PCR and a newly developed capillary electrophoresis-mass spectrum system, we will study the relationship between the La(III)-induced DNA methylation of adapter protein promoter and the variety of metabolism molecules. Moreover, by using real-time fluorescence quantification, Western blotting and transmission electron microscope, we will explore the effects of the aberrant methylation of adapter protein on the gene expression, synthetic amount and recruitment of itself. Furthermore, using fluorescence resonance energy transfer, yeast two-hybrid, computational chemistry and laser scanning confocal microscope, we will investigate the effects of La(III) on the mode of interaction between adapter protein and clathrin, as well as its effect on the activation of endocytosis. This project is expected for illuminating the mechanism of La(III) activating the endocytosis in plant leaf cells via aberrant DNA methylation of adapter protein.

稀土在可食用植物中累积并通过食物链蓄积于人体，影响人类健康，已成不争事实。亟需建立可食用植物中稀土限量标准，其首要问题是明晰稀土作用于植物机理。报道显示，La(III)可打破植物叶片长期进化形成的胞吞惰性，激活胞吞作用，然而激活机制尚不明确。衔接蛋白可绑定网格蛋白而启动胞吞作用，通过DNA启动区甲基化影响其基因表达及含量。鉴此，本项目以模式植物拟南芥为代表，采用亚硫氢酸盐测序和独创毛细管电泳-质谱，探讨La(III)诱导衔接蛋白DNA启动区甲基化与代谢分子种类间关系；利用实时荧光定量PCR、Western杂交、透射电镜，探究衔接蛋白异常甲基化对衔接蛋白基因表达、合成量及招募的影响；使用荧光共振能量转移、酵母双杂交、化学计算、激光共聚焦显微镜技术，研究La(III)对衔接蛋白与网格蛋白相互作用方式及其激活胞吞作用的影响。以期阐明La(III)通过衔接蛋白基因异常甲基化激活植物胞吞作用的机制。

稀土元素(REEs)在可食用植物中累积并通过食物链蓄积于人体，影响人类健康，已成不争事实，亟需建立可食用植物中REEs限量标准，其首要问题是明晰稀土作用于植物机理。项目以环境中累积量高的La(III)和模式植物拟南芥为代表，以稀土生物无机化学、环境科学、植物生物化学、细胞和分子生物学交叉学科技术的优化组合为研究手段，获得重要结果：(1) 低剂量短时间La(III)暴露，4个DNA甲基化酶基因(MET1、CMT3、DRM2、DNMT2)和2个脱甲基化酶基因(DML2、DML5a)表达量升高，4种甲基化产物(S-腺苷高半胱氨酸、胆碱、甘氨酸和蛋氨酸)含量增加，导致甲基化和脱甲基化代谢增强—AP2总甲基化水平降低，继而引发AP2基因表达量上调、AP2合成量增加，大量招募网格蛋白至质膜与之复合，增强根细胞网格蛋白介导的胞吞作用；伴随根细胞胞吞作用的增强—光形态建成正调控因子表达量上升，负调控因子表达量下降—光形态建成被促进—叶片叶绿素荧光反应、Rubisco基因RBCL和RBCS1A表达量增加，光合作用增强；同时气孔导度增加，蒸腾作用增强—终呈现植物整体正效应响应的复杂机制。(2)随暴露剂量增强和暴露时间延长—所有甲基化酶基因(MET1、CMT1、CMT2、CMT3、DRM2、DNMT2)和脱甲基化酶基因(ROS1、DME、DML2、DML5a、DML5b)表达量和甲基化代谢产物增加—AP2基因表达量和合成量进一步增加—根细胞胞吞作用急剧增强—叶片光合作用降低—终呈现植物整体负效应响应的复杂机制。在Chemosphere (SCI-top)等期刊上发表论文8篇，他引28次。这些研究结果，可为从细胞及分子层次揭示REE(III)作用于植物的机制，REE(III)在植物(尤其食品)中限量标准的建立，提供借鉴。

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