水稻基因组DNA总甲基化水平测定方法

对高效液相色谱法测定DNA总甲基化水平的关键因素进行研究,即基因组DNA的提取及纯化和高效液相色谱条件的选择。结果表明:CTAB法Ⅰ提取和纯化效果优于CTAB法Ⅱ;较优高效液相色谱条件为:采用Diamonsil C18(2)(250 mm×4.6 mm,5μm)的色谱柱,以甲醇-10 mmol/L磷酸二氢钾(10-90,v/v)为流动相构成,流动相pH为4.7,流速为0.5 mL/min,柱温为30℃,紫外检测器波长为285 nm时,是分离胞嘧啶和5-甲基胞嘧啶的较优条件。以试验优化的DNA提取方法和HPLC色谱条件,基因组DNA水解液的胞嘧啶(C)和5-甲基胞嘧啶(5 mC)可得到较好的分离效果。     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

基于低氧-免疫DNA甲基化标记在肺腺癌预后中的开发

为了探究缺氧和免疫联合分析DNA甲基化位点在肺癌预后中的作用，提高肺癌免疫治疗的有效性，本研究基于甲基化和转录组二维数据，构建了一个用于预后评估的风险评分模型．从TCGA数据库中下载了247个与缺氧、免疫状态相关的去冗余肺癌样本作为训练集．然后对低缺氧-强免疫状态的样本、高缺氧-弱免疫状态的样本进行了差异基因表达、甲基化位点和临床生存率分析．生存曲线分析显示，低缺氧状态-强免疫状态组中的肺腺癌患者的1、3、5年生存率均优于高缺氧状态-弱免疫状态组（log-rank检验，P<0.05、P<0.001、P<0.001）．使用LASSO回归构建的多基因预后模型包含9个甲基化位点，在肺腺癌测试数据集和验证数据集中均显示出良好的预后预测能力（AUC=0.83,0.80,0.82）．这9个甲基化位点分别分布在PEBP4、FLI1、HLA-DMB、MYO1F、ABCC2、AKNA、ETS1、CCR7和TXNRD1基因上．此外，我们从5年生存曲线中发现，在低缺氧-强免疫状态组中高表达的PEBP4、CCR7、FLI1、AKNA、HLA-DMB、MYO1F和低表达的ABCC2基因也可作为...