马尾松衰亡胚性愈伤复生调控的甲基化分子作用机理研究

项目名称： 马尾松衰亡胚性愈伤复生调控的甲基化分子作用机理研究

项目编号： No.31200481

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 林学

项目作者： 杨模华

作者单位： 中南林业科技大学

项目金额： 23万元

中文摘要： 体胚发育是马尾松良种苗木规模化快繁的最佳途径，但胚性愈伤在增殖培养中容易出现衰亡老化，严重限制马尾松体胚发育植株再生的实践应用。项目组已研发建立马尾松衰亡胚性愈伤复生调控技术，但该技术调控衰亡胚性愈伤复生的甲基化分子作用机理不清楚。本项目在复生技术调控马尾松衰亡胚性愈伤复生增殖的基础上，以复生调控前后同一细胞系胚性愈伤为材料，用HPLC和MSAP技术研究复生调控前后胚性愈伤的甲基化多态性，对差异性片段进行生物信息学功能注释，筛选出调控区（或编码区）甲基化/去甲基化的关键基因DNA片段，经TAIL-PCR与巢式PCR扩增获得目的基因基因组全长DNA克隆，解析目的基因在复生调控前后胚性愈伤中的差异表达谱式，揭示复生技术调控衰亡胚性愈伤复生的部分甲基化分子作用机理。研究结果将为马尾松胚性愈伤增殖与胚性保持提供DNA甲基化分子作用依据，并开发出马尾松高效体胚发生技术体系，丰富林木体胚发育理论基础。

中文关键词： 马尾松；老化胚性细胞系；复生调控；增殖；DNA甲基化

英文摘要： Masson pine (Pinus massoniana.Lamb.) is one of the economic important plants in southern China. Masson pine somatic embryogenesis (SE) had been developed and it is a powerful tool for clonal propagation of superior clones. One phenomenon, which had been observed in embryogenic tissue, was the loss of ability to proliferation after the tissue had been cultured for several months.We had established a procedure for recovering the aged embryonal mass (EM) recently. To further investigate mechanism of recovering tissue proliferation, we will analyze DNA methylation pattern (content and its distribution) between aged and recoveryed EM using HPLC and MSAP techniques. The focus of our research is to screen the segregation and annotation of different MSAP bands bioinformatics function according to methylation/demethylation in the contral region or coding region with the target gene fragment, clone the target gene's genomic DNA by chromosome walking of TAIL-PCR and Nested PCR , and obtain the target gene express pattern by quantitative PCR analysis in the same line between aged and recoveryed EM.The results should be able to reveal the DNA methylation/demethylation pattern on the target genes involved in the recovery procedure on aged EM and better explain and adjust DNA methylation of EM in masson pine related to aged EM

英文关键词： Pinus massoniana Lamb；ageing embryogenic cultures；recovering；proliferation；DNA methylation