连作胁迫下地黄DNA甲基化谱及其与连作障碍的关系

Rhemannia glutinosa is a major chinese medicinal plant. The continuous cropping obstacle is the main problem in Rhemannia glutinosa's production. Multiple adverse stresses including from diseases and plant autotoxic allelochemicals occuring in the continuously cropped soil influence normal expression of its genes and inhibit plant's normal growth and development, thus decreasing greatly yield and quality of Rhemannia glutinosina. As a main epigenetic modification, DNA methylation plays an important role in regualtion of plant gene expression, growth and development, and adverse stress responsiveness. However, It remains unknow that how DNA methylation in Rhemannian glutinosa is altered under continuous cropping condition and related with the occurrence of continuous cropping obstacle . So we aim at studying the epigenetic mechanism of DNA methylation of Rhemannia glutinosa during the occurence of continuous cropping obstacle by: 1) analyzing levels and statuses of DNA methylation in various developing stages of tuberous roots and leaves of Rhemannia glutinosina germplasms in first year of cropping and second year of cropping using HPLC and high methylation sensitive amplified polymorphism (MSAP) and comparing the temporal and tissue differences of DNA methylation responding to contintuous cropping stress; 2) cloning, sequencing differentially methylated DNA fragments induced by the continuous cropping stress and bioninformatically predicting their functions ; 3) analyzing expression profilings of candidate genes , furthe revealing the relationship between alterations of DNA methylation under continuous cropping and formation of continous cropping obstacle. These studies in this proposal will provide the basis for further study on molecular mechanisms of the ocurrence of continuous cropping obstalces and for improvement of tolerance of Rhemannia glutinosina to continuous cropping via genetic modifications.

连作障碍是我国大宗药材地黄生产上的主要问题。连作土壤中的化感物质、病害等多种逆境胁迫影响地黄的基因表达和生长发育，最终严重降低地黄的产量和品质。DNA 甲基化是植物调控其基因表达、生长发育和逆境胁迫响应最重要的表观遗传修饰机制。目前还未有连作胁迫下植物DNA甲基化变化及其与连作障碍形成关系的研究和报道。本课题拟 1）利用高效液相层析(HPLC)法和甲基敏感扩增多态性（MSAP）法首次研究正茬和重茬条件下地黄DNA 甲基化水平和状态的时空模式差异；2）克隆测序连作诱导的甲基化差异片段，并利用生物信息学手段分析其功能；3）结合候选基因的差异表达模式，分析连作下DNA甲基化与连作障碍形成的关系。所得结果将有助于深入研究连作障碍形成的分子机理和耐连作的遗传改良。

地黄的生产具有明显的连作障碍， DNA甲基化是调控植物基因表达和生长发育最重要的一种表观遗传修饰机制。本项目首次利用甲基敏感扩增多态性（MSAP）法研究正茬和重茬种植下地黄发育过程中块根和叶片DNA甲基化的水平、状态，分析连作对地黄DNA甲基化的影响。对240对MSAP选择性引物中18对多态性较好及能较好揭示甲基化状态的引物进行了2个栽培种（温85-5、北京1号）3个组织器官（叶、新生块根、母根）3个时期（60、80和120DAP）的MSAP，初步揭示了连作对地黄基因组甲基化的影响。分析结果显示，连作对地黄叶片的甲基化水平及状态几乎没有影响；地黄块根甲基化位点略低于叶片中，新生根为21-23%左右，半甲基化位点数比例3-6%；全甲基化位点数比例为17%；母根半甲基化位点数比例低于相应的新生根半甲基化水平。18对引物中9对引物检测到地黄块根甲基化状态在连作下发生改变；这些甲基化状态变化主要表现为去甲基化。本项目克隆到4条甲基化差异片段，为以后研究深入研究DNA甲基化与地黄块根形成和连作障碍奠定了基础。对地黄母根和新生根的转录子表达谱进行了分析，首次揭示了连作对两种不同生长状态的地黄块根的基因表达调控机制；对DNA/组蛋白甲基化相关的候选基因进行了分析，在99个候选基因中2个DNA甲基化转移酶和1个组蛋白去甲基化酶在新生根中的表达受连作影响。