巴西橡胶树表观遗传学相关冷胁迫应答分子机理研究

The successful extension of Hevea brasiliensis cultivation to high-latitude areas has long believed to benefit from the breeding of cold-tolerant cultivars. A puzzling incongruity is the variation in cold tolerance among the cultivated clones despite their similar genetic make-up. In previous work, we found that cold-related genes (such as HbICE1 and HbCBF2), cold-responsive (COR) genes, and DNA-methylation related genes (such as HbMET1) were induced by cold treatment. Furthermore, cold treatment not only elevated the transcriptional activities of the HbICE1, HbCBF2, and HbMET, but also induced DNA demethylation of their promoters under natural condition. The DNA demethylation induced by cold was highly correlated with low temperature, but not with the genetic backgrounds of cultivars, suggesting that cold-induced epigenetic modification might play an important role in cold tolerance of H. brasiliensis. In this work, we intend to identify the histone acetylation of HbICEs and HbCBFs using chromatine co-immunoprecipitation (CHIP) under cold acclimation. Furthermore, to identify how cold stress alters the DNA demethylation and histone acetylation of HbICEs and HbCBFs, the factors interacted with epigenetics modification related genes were screened by yeast-two hybrid and yeast-three hybrid. Using combined analysis of Digital Gene Expression (DGE) analysis and Reduced Representation Bisulfite Sequencing (RRBS) to globally reveal the epigenetic related cold responsive genes in Hevea brasiliensis. The study might help us to reveal the molecular mechanism of epigentic related cold acclimation in rubber tree.

巴西橡胶树缺少种质遗传多样性且生长周期较长，为何能培育出适应高纬度地区低温的橡胶栽培种目前仍是个未解之谜。通过前期研究发现，长时间冷处理能使橡胶冷胁迫关键基因(HbICEs, HbCBFs)，冷应答相关基因(COR)及DNA甲基化修饰等相关基因的启动子发生去甲基化。在自然条件下，低温诱导的耐寒相关基因表达水平与其启动子去甲基化水平正相关，与其栽培品种的遗传背景不相关，说明冷诱导的表观遗传学修饰是橡胶适应冷胁迫的关键因素。为深入解析橡胶表观遗传学相关的耐寒分子机理，本项目拟采用染色质共沉淀等方法鉴定HbICEs和HbCBFs等基因的组蛋白冷胁迫相关表观遗传学修饰变化，利用酵母双杂交等技术筛选哪些因子参与DNA甲基化和组蛋白乙酰基化。利用基因数字表达谱分析技术和基因组甲基化分析技术的关联分析，从全基因组水平筛选表观遗传学相关的冷胁迫应答基因，从而解析表观遗传学修饰相关的橡胶耐寒的分子机理。

我们前期的研究发现冷处理诱导橡胶冷胁迫关键基因（HbICEs, HbCBFs）、 冷应答相关基因（COR）以及DNA甲基化修饰等相关基因的启动子发生去甲基化。低温诱导的耐寒相关基因表达水平与其启动子去甲基化水平正相关，说明冷诱导的表观遗传学修饰是橡胶适应冷胁迫的关键因素。为了深入解析橡胶表观遗传学相关的耐寒分子机理，本项目利用基因数字表达谱分析技术和基因组甲基化分析技术的关联分析，全基因组简化甲基化测序分析发现低温处理后橡胶基因组的CpG岛发生普遍去甲基化现象，广泛地分布在基因的编码区及功能调控区。我们进行了个性化的RNA-seq贯穿RRBS的分析。通过分析冷胁迫差异表达基因的甲基化区域（DMR）的分布，发现在基因上游2kb序列中，距离编码区越远，DNA甲基化程度越高，而低温处理后DNA去甲基化程度明显提高，发生去甲基化的CpG岛类型主要是CHG和CHH。有趣的是，低温处理后基因组中转座子序列的甲基化程度有所提高，甲基化水平提高的CpG岛类型主要是CG型。对贯穿得到全基因组差异基因的甲基化差异分布及数据聚类分析。我们挖掘出400多个甲基化差异变化明显的基因，主要集中在甲基化转移酶一类的基因和锌指蛋白等基因家族。之后，我们比较不耐寒的RY-7-20-59和相对耐寒的RY-7-33-97橡胶树在冷胁迫下的生理生化指标、冷应答相关基因的表达及组蛋白修饰变化，发现RY-7-33-97多个冷胁迫相关基因的表达量显著高于RY-7-20-59，组蛋白H3K4me3和H3K27me3修饰与橡胶冷相关基因的表达发生协同作用,表明组蛋白修饰在橡胶树冷胁迫应答中也发挥调控作用。本项目的实施，加深了我们对巴西橡胶树耐寒的分子机理理解，为培育出适应高纬度地区的耐低温橡胶栽培种提供理论支撑。