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

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 recovering proliferation potential. These innovation research findings will significantly enhance plant somatic embryogenesis technique, specially at the stage of EM proliferation. With better understanding and recoverying of EM, we could set up a high-efficiency masson pine somatic embryogenesis system and satisfy the commercial application in clonal propagation of superior clones.

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。

马尾松是中国南方重要的针叶树造林树种之一，马尾松体细胞胚胎发生（Somatic Embryogenesis SE）一直比较困难。应用复生调控技术处理可以使马尾松老化胚性细胞系（Embryoginic Cultures EC）恢复增殖并保持胚性，从而保障了马尾松SE的顺利进行，但该复生调控技术对老化EC恢复增殖的DNA甲基化调控机制不清楚，不利于该技术的应用推广。本项目在马尾松SE发生过程，定期观察记录EC的表型变化结构特征，获得马尾松SE过程发育模式图，丰富和发展了针叶树SE技术理论基础；为保障DNA甲基化实验材料的稳定供应，我们既探讨了不同蔗糖与肌醇浓度对老化EC结构发育的影响，同时也建立了一套马尾松胚性细胞系悬浮培养增殖体系；以此为基础，对复生调控处理前后不同表型状态的马尾松EC取样，应用MSAP（甲基化敏感多态性扩增）技术，检测到了不同EC细胞表型的DNA甲基化多态性模式，确证不同细胞表型的结构变化确实伴随着DNA甲基化多态性表现。基于MSAP差异带的多态性比对，挑选清晰可辨的多态片段，进行分离、纯化、克隆、测序、生物信息学分析，NCBI核酸数据库blast比对，结果表明，1）大多数序列没有任何比对信息；2）暂时还没有检测到与胚性发育密切相关的差异MSAP片段；3）发现了2个片段同源相似性比对于松科植物相关序列，其中1个同源相似性比对于白加云杉（Picea sitchensis）一条mRNA未知功能片段中，另1个高度同源相似性比对于松科植物叶绿体基因组DNA（cpDNA）中petA基因的部分区段。这两个特定片段的发现，说明复生调控处理使得EC表型恢复增殖，不仅受到了核基因组DNA甲基化的调控，同时也受到了cpDNA基因组甲基化的调控。片段序列测序分析结果比对于petA基因的表现，可以推测，发生在petA上的甲基化在复生调控老化EC复生增殖的细胞表型变化中发挥了一定的作用；但由于老化EC在RNA提取质量方面的限制和困难，对该基因的表达分析，还需继续探讨。本项目研究成果一方面揭示了马尾松体胚发育模式以及复生调控技术对EC细胞结构发育的影响，另一方面也让我们可从甲基化调控方面理解和把握马尾松体细胞胚胎发生过程，认识到老化EC恢复增殖并保持胚性是一个全局性基因表达调控的结果；本研究成果为加速马尾松体细胞胚胎发生技术的推广应用，提供了坚实的研究基础和科学依据。