DNA甲基化在杜梨耐盐过程中对CBL-CIPK信号通路的调控研究

Under the salt stress, the changes of methylation level in genomic DNA and the transcriptional responses of gene signal network are the common phenomena among different plants. Until now, the relation between methylation pattern and gene transcript regulation in specific signal pathway needs to be clarified. Our preliminary study showed that the CBL-CIPK signal pathway was involved in the salt tolerance process through upstream and downstream gene cascade response, which worked for the temporal and spatial distribution of Na+ and K+ ions in Pyrus betulaefolia Bunge once the seedlings suffered in salt stress. At the same time, the changes of transcription level of methyltransferase and demethylation enzyme genes were detected and the demethylation of PbCBL1 was observed. However, the specific mechanism on DNA methylation regulates CBL-CIPK signaling pathways during salt stress is unclear. Firstly, methylation specific PCR (MSP) technology is adopted to evaluate DNA methylation level under different salt stresses in P. betulaefolia. Then, the methylation map of CBL-CIPK signaling pathways will draw. Simultaneously, gene expression profile of the CBL-CIPK signal pathway will be analyzed. Furthermore, methylation and transcriptome analysis is carried out together to identify the main methylation sites which maybe control the salt tolerant traits. And the effect of these sites on gene structure and function will be explored. Next, the relationship between gene methylation pattern and expression level in above pathway would be analyzed once the 5-azacytidine, a DNA methylation inhibitor, is used in P. betulaefolia under slat stress. Finally, the methyltransferase/demethylase genes will be transformed to tobacco and P. betulaefolia in order to change their DNA methylation levels. After that, the salt tolerance, the gene transcription and DNA methylation of CBL-CIPK pathway in transgenic plants will be explored. Therefore, the effect of methylation on the function of key genes in CBL-CIPK signaling pathway could be investigated.. Based on above results, CBL-CIPK signaling pathway will be used as an example to elucidate the mechanism of DNA methylation in regulating the salt tolerance of P. betulaefolia. This research will reveal the molecular mechanism about P. betulaefolia against salt stress on the epigenetic aspect and provide the theoretical guidance for pear resistant saline-alkali breeding in the future.

盐胁迫下基因组DNA甲基化变化和信号网络基因转录响应是植物界的普遍现象，但特定信号通路中甲基化模式与基因表达调控间的对应关系有待阐明。我们前期研究发现盐胁迫下杜梨CBL-CIPK信号通路通过上下游基因级联反应，调控Na+、K+时空分布，同时甲基转移酶/去甲基化酶基因转录水平改变，PbCBL1发生去甲基化。然而，DNA甲基化如何调控CBL-CIPK信号通路的具体作用机理尚不清楚。本项目拟在绘制杜梨盐胁迫下CBL-CIPK信号通路甲基化图谱的基础上，联合分析甲基化图谱和基因表达谱，揭示甲基化模式与基因转录间的关系；甲基化抑制剂处理或过量表达甲基转移酶/去甲基化酶基因，定向调节DNA甲基化水平，评价植株耐盐能力，探讨甲基化对CBL-CIPK信号通路关键基因功能的影响。期望以CBL-CIPK信号通路为例，从表观遗传层面阐明DNA甲基化调控杜梨耐盐碱的内在机理，为梨抗盐碱育种提供深层次理论依据。

土壤盐渍化是现代农业生产中频繁发生的土地退化现象，严重影响生态环境质量，制约农业可持续健康发展。果树是重要的经济树种，大多为多年生木本植物，不可避免受到土壤盐碱化的危害。利用农艺措施提高果树耐盐碱能力、培育耐盐碱果树新品种符合国家经济发展需求，具有广阔的应用前景。梨是我国第三大果树，采用杜梨（Pyrus betulifolia Bunge）为砧木嫁接能有效提高梨树的耐盐性。在国家自然科学基金（编号31772287）的支持下，我们筛选获得可在含6‰NaCl土壤中正常生长的杜梨耐盐单株，并发现不同耐盐单株Na+转运与DNA甲基化程度相关。. 本研究以耐盐性差异显著杜梨株系为研究对象，绘制杜梨全基因组DNA甲基化图谱，通过不同株系盐胁迫下的全基因组DNA甲基化和转录组联合分析，筛选获得受DNA甲基化调控的CBL-CIPK信号通路成员，明确其盐胁迫相关甲基化位点和模式。DNA甲基化抑制剂处理,结合荧光定量PCR、甲基化特异性PCR、耐盐性鉴定和原子吸收元素分析，证实改变CBL-CIPK 信号通路关键基因的DNA甲基化状况能够调节基因转录水平。过量表达杜梨DNA甲基转移酶和去甲基化酶，会改变转基因株系的盐胁迫表型、Na+/K+离子分布、基因表达量及DNA 甲基化状况，明确DNA 甲基化在杜梨耐盐过程对 CBL-CIPK 信号通路的调控作用。. 本项目的研究以CBL-CIPK信号通路为切入点，从表观遗传水平上尝试阐明DNA甲基化在杜梨Na+转运调控网络中的作用机制，可为梨的耐盐育种提供范例。本项目的研究结果将搭建梨耐盐砧木从实验室培育到果园生产的有效桥梁，为果树抗逆新种质创制及及实际应用作出有益尝试。