SWR1染色质重塑复合体OsARP6亚基调控水稻抽穗期的分子机理

Chromatin remodeling complex SWR1 has evolutionary conserved structure and function in various organism, like Yeast, Humans, Drosophila and Arabidopsis. It can catalyzes the replacement of nucleosomal H2A with the H2A.Z variant on the gene promoter regions, to activate gene expression. However, the functions of SWR1 complex in rice have not been known. In previous study, we obtain the homologous gene of ARP6 in rice, called OsARP6, a subunit of chromatin remodeling complex SWR1. Over-expression and RNA interference (RNAi) mutant were created by DNA recombination and transgenic technology. The mutant have abnormal characteristic in heading date, plant height and leaf shape. In this study, the over-expression and RNAi mutant will be applied, to explore the regulatory mechanisms of OsARP6 on heading date control. We will use Real-time PCR, RNA high-throughput sequencing to identify the target gene through which OsARP6 regulates heading date. Furthermore, by Westen blot, cDNA library screening, chromatin immunoprecipitation technology to dissect the molecular and biochemical mechanisms on how the target genes regulated by OsARP6. These findings of the study will fully reveal the function and mechanism of OsARP6, and provide foundation for further rearch on chromatin remodeling complex SWR1 in rice.

SWR1染色质重塑复合体在酵母、人类、果蝇及拟南芥中结构和功能保守。该复合体可以催化组蛋白变体H2A.Z代替H2A，从而激活基因表达。但是，其在水稻中的功能未知。在前期的研究中，我们获得了水稻SWR1复合体ARP6亚基的同源基因，命名为OsARP6。利用DNA重组和转基因技术创制了OsARP6基因的超表达和抑制表达突变体。表型鉴定结果表明，OsARP6的抑制表达突变体抽穗期提前，剑叶伸长，超表达突变体抽穗期延迟，株高变矮。本项目拟利用已经创制的OsARP6突变体为材料，利用荧光定量PCR、RNA高通量测序等技术，寻找OsARP6调控抽穗期的靶基因；进一步通过cDNA文库筛选、Westen blot及染色质免疫共沉淀等手段，明确OsARP6调控靶基因的分子及生化机理，全面揭示OsARP6的功能及作用机制，为深入研究水稻SWR1复合体的功能及作用机理奠定理论和实践基础。

SWR1染色质重塑复合体在酵母、人类、果蝇及拟南芥中结构和功能保守。该复合体可以催化组蛋白变体H2A.Z代替H2A，从而激活基因表达，但是其在水稻中的功能未知。项目利用反向遗传的方法克隆了水稻SWR1复合体亚基OsARP6，CRISPR/Cas9基因编辑突变体展现出抽穗期延迟，株高变矮，产量降低的表型。通过表达解析分析OsARP6调控抽穗期的下游基因，发现OsARP6可能通过影响Ehd1的表达来调控成花素的积累，进而影响抽穗。Western blot检测显示OsARP6的表达能够影响染色质上组蛋白变体H2A.Z的含量。酵母双杂交检测OsARP6与其他SWR1染色质重塑复合体亚基的相互作用，发现OsARP6与OsSEF存在相互作用，OsSEF与OsPIE1存在相互作用，有可能作为复合体调控下游基因表达。项目还利用水稻cDNA文库，筛选出6个与OsARP6存在强烈相互作用的蛋白，为深入挖掘OsARP6的作用机制提供了新的方向。综上所有结果所示，OsARP6能够调控Ehd1的表达影响水稻抽穗期，还能够影响染色质上组蛋白变体H2A.Z的沉积，本项目的后续研究将重点揭示OsARP6调控Ehd1的生化机理，为水稻抽穗期的改良以及深入开展水稻SWR1染色质重塑复合体的功能研究奠定理论基础。