植物再生过程中细胞重编程的表观遗传调控机制研究

Directed by phytohormones, plant tissues and organs are easily regenerated to form a whole plant through reprogramming of plant cells, showing an ability that most animals do not possess. During plant regeneration, the cultured plant tissues and organs usually first produce a kind of pluripotent cell mass, called callus. Although this process have been known for a long time, the underlying mechanism that guides the callus formation is still unclear. Because the genome-wide changes of gene expression should occur due to dramatic morphologic changes of the cultured tissues during callus formation, it is reasonable to speculate that epigenetic regulations, which are usually responsible for large-scale control of gene expression, must be involved in this process. Our previous study revealed that the Polycomb and ISWI epigenetic pathways are important for the callus formation and act in different steps of transdiffentiation of stem cells. In this current research, we plann to explore the molecular mechanisms of how the Polycomb and ISWI pathways regulate the callus regeneration, using approaches of molecular genetics, biochemistry, live imaging and deep sequencing. It is important to conduct studies on plant regeneration at present, not only because it links to the plant basic life activity, but also its perspective of wide applications in the future. As only a few reports were seen in recent year in terms of the epigenetic regulations of plant regeneration, it seems of great significance to initiate the research addressing the mechanisms for plant regeneration.

植物细胞能在合适的条件下通过激素诱导再生成愈伤组织，并进一步再生为完整植株。虽然愈伤组织再生早已被广泛运用于农业生产中，但是控制这一过程的分子机理却未深入了解。愈伤组织的形成是一个细胞重编程过程，并伴随着大规模基因表达的变化；而我们的前期工作已表明，表观遗传机制是有效控制这一过程中大规模基因表达变化的基本机制。我们已发现，Polycomb和ISWI是两条控制再生过程关键的表观遗传调控途径，并作用在植物干细胞转分化的不同阶段。本申请项目将从分子遗传、生化、转录与表观遗传组学和实时荧光分析等多角度，系统分析Polycomb和ISWI表观遗传途径在愈伤组织再生过程中特异性和分子机理。表观遗传因子调控植物再生的研究尚处于起步阶段，且在植物细胞重编程领域具有重要理论价值，也可能给其他真核系统的表观遗传学研究提供借鉴；同时，这一理论基础也为改造植物细胞和农艺生产提供广阔的应用前景。

植物组织培养是广泛使用的农艺技术。组织培养的第一步就是将植物外植体培养在富含生长素的愈伤组织诱导培养基上，使其再生出愈伤组织。此项目集中研究了表观遗传调控机制在愈伤组织再生过程中的功能。表观遗传调控机制是基因表达的重要控制途径。我们的研究集中在Polycomb和ISWI这两类表观因子上。研究结果显示，Polycomb和ISWI都具有控制愈伤组织再生的能力。通过LC-MS/MS技术发现了与Polycomb和ISWI形成蛋白复合体的多个组分。结果发现在Polycomb途径中的PRC2蛋白复合体还包括了LHP1和EMF1，这一复合体与PRC1的功能有分工。另外，激素通路（尤其是生长素通路）的多个因子也在PRC2和ISWI复合体中被发现。BiFC和Co-IP实验证实了蛋白相互作用。利用RNA-seq配合ChIP/MNase-seq技术发现Polycomb和ISWI在控制下游基因表达中的规律。我们的研究结果证实，Polycomb主要贡献于外植体特征基因的关闭，而ISWI主要贡献于愈伤组织特征基因的开启。这些研究结果不仅揭示了Polycomb和ISWI是植物发育与愈伤组织再生中重要的调控因子，也为将来继续深入研究植物再生的表观遗传调控机制奠定了基础。在项目执行过程中还发现了表观因子调控再生的一些新现象，也为今后了解植物再生能力提供了新的线索。