细胞周期及MMR系统对Cd胁迫下拟南芥DNA损伤的响应机制

Many results have demonstrated that the DNA MMR system in organisms plays important roles in maintaining the stability of genomic DNA, fidelity of DNA replication, and activation of checkpoints in cell cycles etc. In this project, Arabidopsis thaliana wild type, MLH1 Knockout (KO)，MSH6-KO and MSH2-KO lines will be selected as experimental materials, Zeocin will be used as positive control and Cu as a reference pollutant. Change in trends of DNA damage, cell cycle component phases and WEE1 gene expression and protein response, a sensitive marker of the DNA damage checkpoint during G2/M phase, will be studied in the four lines of Arabidopsis seedlings under Cd/Zeocin/Cu stress. Correlation of molecular behavior of the MLH1, MSH6 and MSH2 genes (i.e.promoter activity, methylation level of CG sites in promoter region, levels of gene transcript) with cell cycle component phases and sensitive indexes of the DNA damage checkpoint during G2/M phase will be elucidated in the four lines of Arabidopsis seedlings, which will verify whether MLH1，MSH6 and MSH2 genes are the key genes of controlling cell cycle progress and that Cd can result in escape from the DNA damage checkpoint in Arabidopsis. In addition, cell cycle-related indexes/the DNA damage/molecular behavior of MLH1, MSH6 and MSH2 genes and their dose-effect relationship will be extensively researched and compared according to the content of Cd and Cu in plant cells and culture medium under Cd stress. Our long-term objective is to find sensitive/specific biomarkers of exposure and effect 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.

生物体内DNA错配修复系统在确保基因组的稳定性、DNA复制的精确性和激活细胞周期检验点等方面担负主导作用。本项目以拟南芥野生型、基因敲除突变型MLH1-KO、MSH6-KO和MSH2-KO为材料，博来霉素为阳性对照，铜为参照污染物，探讨镉胁迫下拟南芥幼苗中DNA损伤、细胞周期时相及G2/M期DNA损伤检验点敏感指标WEE1基因表达及蛋白应答的变化趋势；阐明镉胁迫诱导植物细胞中MMR系统中关键基因MSH6、MSH2和MLH1的分子行为/细胞周期/DNA损伤之间的相互关系；明确上述3种基因是调控植物细胞周期G2/M期的关键基因；证明镉诱导拟南芥DNA损伤检验点的无效应答。结合植株体内镉测定结果，分析DNA损伤、细胞周期、上述3种基因的分子行为及其与镉暴露之间的剂量-效应关系，从拟南芥MMR基因家族/细胞周期相关指标中筛选和确认镉暴露的分子生物标记物，丰富对镉毒性机理的认识并提供分子标记。

研究了Cd/Cu胁迫下拟南芥幼苗基因组DNA甲基化多态性、MLH1启动子甲基化、WEE1基因表达的变化趋势。研究进展如下：..1. 进一步完善了拟南芥启动子甲基化的亚硫酸盐测序技术：为使DNA完全修饰，建议基因组DNA与亚硫酸盐修饰液修饰液的比例为500ng ：1000µL。明确了MLH1启动子-346~ 42 bp区域内CG2, CG3, CG4, CG8, CG10, CG11, CG15 位点未发生甲基化。..2. 建立了拟南芥基因组DNA甲基化的MSAP-PCR检测技术:（1）DNA模板量在150ng-250ng、4%PAGE（含50%尿素）电泳分辨PCR产物多态性最佳；（2）对Cd胁迫敏感性高；（3）AP-4对CpG位点甲基化变化敏感，AP-3在CHG位点的甲基化多态性最高。..3. SSR、RAPD和MSAP-PCR结果表明: (1) 0.25、1.0、4.0和5.0 ppm Cd胁迫15天后，拟南芥幼苗全基因组的甲基化多态性、CpG位点和CHG位点甲基化位点数均明显增加，表现为微弱的倒U字型变化趋势；而CpG和CHG位点去甲基化位点数在不同Cd处理间没有差异。(2) 4.0 ppm Cd胁迫15天后，幼苗表现为微弱的RAPD多态性; 5.0 ppm Cd胁迫15天、0.25和1.0ppm处理21天幼苗均表现为较高的RAPD多态性。(3) 0.25-5.0 ppm Cd胁迫15 天后，使用46对SSR引物没有检测出SSR变异；8.0ppm Cd胁迫15 天后产生一个重复序列的SSR变异。表明对Cd胁迫最敏感性的生物标记物是MSAP-PCR，RAPD多态性次之，SSR多态性最差。 . .4. MSAP结果表明：0，0.25，1.0，3.0 ppm Cu胁迫15 d后，幼苗基因组MSAP率分别为15.93% ，16.28% ，15.83%，14.3%；基因组DNA的甲基化多态性位点数、超甲基化（M型）位点及去甲基化（D型）位点数呈增加趋势，而且M型位点数高于D型，表明拟南芥MSAP变化可以作为Cu污染的潜在生物标记物。..5. qPCR结果表明，0.25 - 8.0 ppm Cd处理15天后，拟南芥MLH1，MSH2，MSH6，WEE1基因的转录水平降低；0.25 ppm Cu处理15天后，上述4个基因的表达水平增加，1-2.5 ppm Cu降低其表达水平。