水稻細胞分裂素訊息反應調節蛋白 OsRR9 和OsRR10 之生理功能探討

Abstract

細胞分裂素 (Cytokinin, CK) 訊息在調節植物生理功能扮演重要角色。CK之訊號傳導屬於磷酸傳遞雙分子訊號系統 (two-component signal system, TCS)，其中水稻與阿拉伯芥經基因序列比對顯示目前水稻中有相似的磷酸接受A型反應調節蛋白 (Type-A Response regulator, Type-A RR)。本次研究主要探討A型RR中的OsRR9與其高相似度之OsRR10在水稻生理及逆境過程中可能參與之調節反應，OsRR9/10主要表現時期為幼苗及孕穗期，表現部位主要於地上部，在CK處理高溫逆境下則會顯著受到誘導表現。本研究利用Tos17插入型水稻突變株以及CRISPR/Cas9基因編輯植株作為研究OsRR9和OsRR10功能之研究材料，OsRR9轉錄降低表現突變水稻在幼苗期生長較野生型緩慢。外施細胞分裂素時，受細胞分裂素誘導之Type-A OsRRs表現與野生型有相似趨勢，突變株會加速葉綠素降解。另有較低之癒傷組織再生率相對於對照組。表示OsRR9降低表現突變株對細胞分裂素訊號傳導之敏感度下降。在Osrr9/10基因編輯株中，OsRR9和OsRR10之突變率超過80%，突變類型以1 bp缺失之頻率最高，並仍有轉錄表現。然而轉譯之胺基酸序列會造成截斷及功能性區域消失，於細胞分裂素反應發現會延緩葉綠素降解、促進癒傷組織再生。此外，插入型與基因編輯突變株在CK反應的不同，可能為Type-A OsRRs表現不同所引起，因此認為基因編輯突變株更能夠代表OsRR9和OsRR10功能缺失的情形。綜合以上結果可以說明OsRR9/10會影響植株生長勢並在細胞分裂素訊號傳導途徑扮演負向調控之角色。

Cytokinin (CK) signaling plays an important role in regulating plant development and physiological functions. Type-A response regulators (Type-A RRs) are part of two-component signal system (TCS). Phylogenetic analysis of TCS components in rice and Arabidopsis indicates that most of rice and Arabidopsis type-A RRs are in different clades. My study focuses on whether OsRR9 and OsRR10, which has high similarity of rice. Participate in the regulation of physiological and stress responses. Both OsRR9 and OsRR10 are highly expressed in seedlings and booting stages, mainly in the shoot. OsRR9/10 are significantly up-regulated in response to heat stress. To understand the function of OsRR9 and OsRR10, we used an insertion mutant and generated CRISPR/Cas9-edited mutants to evaluate the role of OsRR9 and OsRR10. OsRR9 knockdown mutant shows delayed growth during seedlings stage. By conducting dark-induced chlorophyll degradation and callus regeneration experiments, we found that OsRR9 knockdown mutant is more sensitive to CK than WT. Besides, additional CRISPR/Cas9 mutants were generated, mutation rates of CRISPR/Cas9-edited mutants in OsRR9 and OsRR10 are over 80%, mainly occuring in 1 bp deletions but the transcription still can be detected. Our results show that CRISPR/Cas9-edited mutants were more sensitivity to BA, as indicated by the delay of leaf senescence, and the induction of callus regeneration. Moreover, the opposite response between insertion and CRISPR/Cas9-edited mutant may be caused by the different transcription levels of type-A other OsRRs family. Above all, OsRR9/10 regulates plant growth and plays a role as a negative regulator in CK signaling transduction.