镉胁迫下DNA MMR系统对拟南芥G2期阻滞的调控机制

Both in Europe and, more recently in China, Cd has been used extensively in the chemical industry and agriculture (e.g. in electroplating, metallurgy, painting, plastic generation) and has also been released as a by product of mining. Cd has thus been released into the biosphere with consequent contamination of water and soil. Cd is a non-essential metal with genotoxic effects and can move rapidly through the food chain; thus Cd accumulation can pose a direct threat to human health. Biomarkers for detecting bioavailable Cd are therefore important for assessing the level of contamination and the success of efforts in land remediation. Plants provide excellent biomarkers as they are sedentary and can be used for in situ monitoring. Many results have demonstrated that the DNA MMR system in organisms plays important roles in activation of checkpoints in cell cycles, maintaining the stability of genomic DNA, and fidelity of DNA replication etc. In this project, zeocin will be used as a positive control and Cu as a reference pollutant. Specific primers for MMR-related PCR have been designed previously, and siRNA techniques will be used for infection of specific small interfering RNAs (siRNA) against MSH2, MSH6 and MLH1 in MMR-positive Arabidopsis thaliana cells. At the same time, gene knock out mutants (KO) for the same genes will also be chosen as experimental materials. Changes in mitotic index, G2 phase arrest, gene expression of regulatory proteins such as WEE1, Knolle and CYCB1;1, critical for controlling the cell cycle transition from the G2 phase to M phase, will be analysed in Arabidopsis under Cd stress. Effects of gene knock out mutants (MLH1-KO, MHS2-KO, MHS6-KO), and transformation of siRNA (MLH1-siRNA，MHS2-siRNA，MHS6-siRNA) on the above processes will be explored in the DNA MMR system of Arabidopsis under Cd stress. Furthermore, mechanisms of molecular behavior of the MLH1, MSH6 and MSH2 genes (i.e. levels of gene transcript and expression levels of their proteins) and of DNA damage checkpoint of G2/M phase arrest during mitotic cell cycle will be elucidated in response to DNA damage induced by Cd, which will verify whether Cd stress can result in escape from the DNA damage checkpoint of G2/M phase arrest during the mitotic cell cycle in MLH1-deficient, MSH6-deficient or MSH2-deficient cells of Arabidopsis under Cd stress and whether MLH1，MSH6 and MSH2 genes are the key genes of controlling cell cycle G2 phase arrest. In addition, cell cycle-related indices/DNA damage/molecular behavior of MLH1, MSH6 and MSH2 genes and their dose-effect relationship will be intensively researched and compared according to the content of Cd in plant cells and culture medium under Cd stress. Our long-term objective is to find sensitive/specific biomarkers for exposure and effects of Cd from the MMR family and/or cell cycle related indices in Arabidopsis. The above results may help to unravel the mechanism of Cd toxicity and provide a scientific basis for molecular marker techniques.

本项目以铜为参照污染物，应用基因敲除突变体(KO)和siRNA等技术，研究镉胁迫下野生型拟南芥有丝分裂指数，G2期阻止，G2/M期关键调控基因WEE1、Knolle和CYCB1;1表达的变化趋势；探讨基因敲除（MLH1KO、MSH2KO、MSH6KO）和siRNA转染（MLH1siRNA、MSH2siRNA、MSH6siRNA）对上述过程的影响；阐明镉胁迫诱导拟南芥DNA MMR系统中关键基因MLH1、MSH2和MSH6的分子行为和细胞周期DNA损伤检验点对DNA损伤的响应机制；明确镉胁迫是否导致MLH1、MSH2和MSH6功能缺失拟南芥可以绕开细胞周期G2/M期DNA损伤检验点，证明3种基因是否调控植株细胞周期G2期阻滞。分析上述指标的分子行为及其与镉暴露之间的剂量-效应关系，从拟南芥DNA MMR家族与细胞周期相关指标中筛选镉暴露的分子生物标志物，丰富对镉毒性机理的认识并提供分子标记。

DNA错配修复系统（MMR）是一种DNA复制后修复，在激活细胞周期检验点、确保基因组的稳定性和DNA复制的精确性等方面发挥关键作用。但是Cd胁迫下，拟南芥DNA MMR系统中MSH2、MSH6和MLH1基因是否调控DNA损伤诱导的细胞周期G2期阻滞，目前国内外尚未见报道。本项目研究结果表明，Cd（0.125～2.5 mg•L-1）胁迫5 d后，对Cd胁迫敏感度上，拟南芥DNA损伤修复系统总体表现为DNA MMR＞HR（同源重组, RAD51, BRCA1）＞NHEJ（非同源末端连接修复, KU70, MRE11, GR1）；Cd抑制了拟南芥幼苗根尖细胞周期进程；MSH2和MSH6是Cd诱导 DNA 损伤的关键感受器，直接参与DNA损伤的识别/传递与 G2/M 期阻滞,而MLH1不参与此过程：Cd胁迫诱导拟南芥WT、mlh1-、msh2-和msh6-KO幼苗根尖细胞周期阻滞在G2/M期，与G2/M期调控相关的基因表达异常有关，其中DNA错配修复基因MLH1、MSH2和MSH6转录水平显著降低，DNA损伤检验点基因ATM、ATR、SOG1和 DNA损伤修复基因BRCA1、RAD51、KU70和MRE11的表达量呈典型的倒U型，G2/M期标志性基因WEE1、CYCD4;1、MAD2、CDKA;1、CYCB1;2 和 CYCB1;1的表达量呈现下降或者倒U型变化趋势。DNA MMR基因调控大豆的耐Cd性能，具体表现于以下几方面：Cd胁迫下，Cd敏感品种LD10细胞内的DNA损伤明显小于Cd耐性品种SN20；Cd诱导的DNA损伤导致LD10阻滞在G1/S期，SN20而阻滞在G2/M期；MSH2和MSH6是调控大豆的耐Cd性的关键基因。上述结果可以丰富对镉毒性机理的认识并提供分子标记。