生物資源暨農學院: 農藝學研究所指導教授: 蔡育彰侯雅文Hou, Ya-WenYa-WenHou2017-03-062018-07-112017-03-062018-07-112016http://ntur.lib.ntu.edu.tw//handle/246246/275987細胞分裂素 (cytokinin, CK) 為重要的植物荷爾蒙之一，在調節植物生長發育上具有重要功能，而細胞分裂素訊息傳遞為一重要調控機制。細胞分裂素之訊息傳遞為雙分子訊息傳遞系統 (two-component signaling system, TCS)，藉由磷酸轉移傳遞訊息，其中Type-A RRs能被細胞分裂素所誘導，為細胞分裂素訊息中之原初反應 (primary response) 分子，前人研究顯示在阿拉伯芥及部分水稻 Type-A Response regulators (RRs) 具有負向調控細胞分裂素訊息傳導的功能。目前已知在水稻中有 13 個 Type-A OsRRs，預期在細胞分裂素生理功能上會有補償性，根據親緣關係分群與表現組織，本研究對細胞分裂素訊息反應調節子 OsRR6 與 OsRR11 進行生理功能探討。一般生長狀況下，OsRR6 具有較高的表現量且全株皆有表現；OsRR11 表現量很低且具有地上部的組織專一性。檢測 OsRR6 及 OsRR11 在不同非生物逆境下的基因表現，發現 OsRR6 在高溫逆境與乾旱逆境下能被誘導基因表現，OsRR11 基因表現會受到鹽逆境與乾旱逆境抑制，顯示 OsRR6 與 OsRR11 調控水稻遭受非生物逆境下之反應並且可能具有不同的生理功能。為了解 OsRR6 與 OsRR11 在細胞分裂素訊息之功能，以 CRISPR/Cas9 基因編輯系統建立 Osrr6/11 雙重突變株，在 OsRR6 與 OsRR11 突變效率皆達 80% 以上，突變型態以缺失 1bp 最多，進行與細胞分裂素相關反應實驗，結果顯示相較於野生型 Osrr6/11 突變株之不定根數目較多、能夠延緩葉綠素降解且促進水稻癒傷組織地上部再生，皆支持 OsRR6 與 OsRR11 在細胞分裂素訊息傳遞系統中扮演負向調控的角色。Cytokinins are plant hormones that play important regulatory roles in plant developmental and physiological processes. Cytokinin signaling pathway is a multistep His-Asp phosphorelay system known as the two-component system. The type-A response regulator genes in Arabidopsis and rice are rapidly induced by exogenous cytokinin and have been characterized as cytokinin primary response genes. Some of type-A RRs in Arabidopsis and rice act as negative regulators of cytokinin responses by a feedback mechanism. It has been known that there are thirteen type-A RRs in rice and may perform partially redundant functions. Based on the phylogenetic relationship and tissue specificity, in this study, I characterized the functions of OsRR6 and OsRR11, using CRISPR/Cas9 gene-editing approach to establish Osrr6/11 mutants. Mutation rate of Osrr6/11 mutants are over 80% and mainly occurring in 1 bp deletion. Real-time qPCR analysis indicated that OsRR6 exhibits high expression and is expressed in all organs; while OsRR11 exhibits very low expression and is expressed only in the shoot. Besides, OsRR6 can be up-regulated by high temperature and drought stresses; while OsRR11 can be down-regulated by salinity and drought stresses. These results illustrated that OsRR6 and OsRR11 participate in plant responses to different stresses and may have different functions under abiotic stresses. In order to gain insight into their functions, we conduct cytokinin response analysis by using Osrr6/11 mutants. Compared to WT, Osrr6/11 mutants have more adventitious roots, delay leaf senescence and promote callus shoot regeneration. Taken together, these data suggested that OsRR6 and OsRR11 play negative role in cytokinin signaling transduction.論文使用權限: 不同意授權雙分子訊息傳遞系統細胞分裂素訊息OsRR6OsRR11CRISPR/Cas9基因編輯系統two-component systemcytokinin signalingCRISPR/Cas9thesis10.6342/NTU201602905