REF6/JMJ705同源基因如何调控AP1基因启动麻竹开花

Bamboo flowering has been an unsolved mystery since it was found, as such, little data could be tracked in the area. One of our recently published works identified an attractive gene DlAP1 that may mark the initiation of flowering in an important bamboo species Dendrocalamus latiflorus. This gene showed a significant high expression level when flowering initiated, and then decreased furiously. Thus, identifying it’s true “marker” role in D. latiflorus is a crucial work. AP1 was reported bring Arabidopsis thaliana flowering much earlier when its H3K27me3 marker was decreased. This mission was accomplished by REF6, a Jumonji-C domain containing protein. JMJ705 is another H3K27me3 demethylase in rice which is homologous to REF6. An attractive point is that we found 4 structural counterparts of REF6 and JMJ705 from extensive transcriptome and genome sequences of D. latiflorus, named DlJMJ10, DlJMJ11, DlJMJ8 and DlJMJ9. Following this point, we will check if these four proteins possess H3K27me3 demethylase activity or they are other demethylases by using in vivo and in vitro demethylase assays and western blot analysis. We will also test their targeting capability to DlAP1 applying ChIP-Seq technique and investigate their effect on the histone methylating status of DlAP1 chromatin. To understand how DlJMJ10, DlJMJ11, DlJMJ8 and DlJMJ9 regulate DlAP1 expression, we will identify their interacting proteins first through GST pull-down experiment and then confirmed by CoIP experiment. The goal of this project is to confirm the “marker” status of DlAP1 indicating the initiation of D. latiflorus flowering, and to demonstrate if DlJMJ10, DlJMJ11, DlJMJ8 and DlJMJ9 play roles in demethylating H3K27me3 on DlAP1, and finally establish a mechanism by which DlJMJ10, DlJMJ11, DlJMJ8 and DlJMJ9 proteins modulate DlAP1 expression by interacting with co-effective factors in regulating D. latiflorus flowering. This is a new exploration and will be essential to speed up the process of uncovering mechanisms of bamboo flowering.

竹类植物的开花现象一直是植物界的“未解之谜”，相关机理研究十分欠缺。前期研究发现AP1基因在麻竹开花时表达量显著高于开花完成后，推测很可能是麻竹开花启动的标志基因。本项目拟将视野扩大到麻竹整个生命周期，细化到不同发育阶段不同组织，进一步确认AP1对麻竹开花启动的“标志”作用。AP1 H3K27me3水平的降低能使其提前表达，植株提早开花。目前仅发现REF6和JMJ705能去除H3K27me3。我们在麻竹中找到4个与REF6和JMJ705高度同源的序列。本项目将用组蛋白去甲基化酶活性实验确认这4个序列能否去除H3K27me3，用ChIP-Seq技术检测其能否靶定DlAP1并影响其组蛋白甲基化，用GST pull-down和CoIP技术寻找其互作蛋白，构建其与DlAP1在麻竹开花进程中的调控关系。本项目首次从基因marker和组蛋白修饰的表观遗传学角度为竹类植物开花机理研究提供新视角和新论据。

竹类植物的开花现象一直是植物界的“未解之谜”，相关机理研究十分欠缺。前期研究发现AP1基因在麻竹开花时表达量显著高于开花完成后，推测很可能是麻竹开花启动的标志基因。本项目研究的目标是将视野扩大到麻竹整个生命周期，细化到不同发育阶段不同组织，进一步确认AP1对麻竹开花启动的“标志”作用。AP1 H3K27me3水平的降低能使其提前表达，植株提早开花。目前仅发现REF6和JMJ705能去除H3K27me3。前期我们在麻竹中初步找到4个与REF6和JMJ705高度同源的序列。本项目拟用组蛋白去甲基化酶活性实验确认这几个序列能否去除H3K27me3，用ChIP-Seq技术检测其能否靶定DlAP1并影响其组蛋白甲基化，用EMSA技术进一步验证其互作，构建其与DlAP1在麻竹开花进程中的调控关系。. 本项目研究最终确定了基因27779是能够预示麻竹开花启动发生的“标志”基因。同时从麻竹中克隆到了33个JmjC domain containing 基因，并根据系统发育关系从中鉴定出了2个REF6/JMJ705同源基因DlJMJ7 和DlJMJ14（前期找到的4条序列后来经鉴定属于2个基因，因此最终改名为DlJMJ7 和DlJMJ14），并检测了其组蛋白去甲基化酶活性。同时，也制备了DlJMJ7 和DlJMJ14 蛋白的特异性抗体，在此基础上，完成了ChIP-seq实验，实验结果可判断DlJMJ7和DlJMJ14 蛋白能否与基因27779在体内互作而调控麻竹开花启动的发生。对27779的基因序列分析发现，其基因区含有ZnF-C2H2结构域簇的靶定序列CTCTGYTY motif（Y代表C或T）。已完成了验证DlJMJ14蛋白的4×ZnF-C2H2结构域簇能否体外结合27779基因的EMSA实验。. 本项目首次从基因marker和组蛋白修饰的表观遗传学角度为竹类植物开花机理研究提供新视角和新论据。