- 學位論文
植物 RNase L inhibitors 之功能研究
Functional study of plant RNase L inhibitors
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摘要
RNase L inhibitor proteins (RLIs) 為真核生物與古細菌共有之高度保守蛋白質，最早被發現調控動物 RNase L 相關之免疫反應，然而在植物中並無 RNase L 之同源基因，故除了已知 RLIs 是植物生長發育必需基因外，在植物之其他功能尚待研究。本研究室先前的研究指向茄科植物之 RLIs 在青枯病、細菌性軟腐病及氧化逆境之防禦系統為負調控者，但尚需更多研究與證據方能有定論。本研究旨在進一步探討阿拉伯芥 AtRLI2 與番茄 SlRLI 在植物生長發育、逆境防禦反應及基因靜默之功能，以驗證先前研究之推測。首先，結果顯示 AtRLI2 在植物各組織中皆有表現，且其蛋白質主要分佈於細胞核，而 SlRLI 則在細胞質呈現點狀分佈但卻非座落於粒線體、過氧化體或細胞核。先前研究發現 RLIs 表現異常會影響植物生長發育，進一步短暫全株過表現 SlRLI 會造成番茄與菸草生長異常，然而 atrli2-1 knock-down 突變株卻無明顯的生長異常，推測 AtRLIs 有功能冗餘特性 (gene functional redundancy)，但因多次嘗試仍無法成功培育AtRLI1與AtRLI2單一基因與雙基因基因靜默之轉殖株，故推測 RLIs之表現量可能必須受嚴格管控。此外，植物RLIs 在對抗病原菌與氧化逆境防禦反應中為負向調控者，且經比較 atrli2-1 與野生株中防禦賀爾蒙標誌基因表現後發現 AtRLI2 可能透過抑制水楊酸、茉莉酸、乙烯及抗氧化逆境訊息傳遞路徑而調控植物抗病與防禦氧化逆境反應。另外，雖然先前研究指出 AtRLI2 具有抑制基因靜默之功能，然而本研究所得結果卻不支持 AtRLI2 與 SlRLI 有抑制基因靜默之能力。綜合本研究室所得結果指向 RLIs 為植物逆境防禦反應之關鍵負向調控者，且可能為植物調控生長發育與逆境反應間之樞紐。
並列摘要
RNase L inhibitor proteins (RLIs) are highly conserved in eukaryotes and archaea. RLIs are involved in regulating mammal defense against viruses by modulating RNase L activity. However, no typical RNase L proteins can be found in plants, and despite of an essential role in plant growth and development, additional functions of plant RLIs remain largely unknown. Our previous studies suggested a negative role of RLIs in the defense responses of tobacco/tomato against bacterial wilt, bacterial soft rot and oxidative stress. This study aimed to further investigate the roles of Arabidopsis RLI2 (AtRLI2) and tomato RLI (SlRLI) in plant defense against pathogens and oxidative stress. The results revealed that AtRLI2 is ubiquitously expressed in all organs. AtRLI2 predominantly localized in nucleus, while SlRLI distributed as cytosolic foci but not localized in mitochondria, peroxisome or nucleus. In addition, although overexpression of SlRLI in tomato and tobacco caused defective plant growth, an atrli2-1-knockdown line did not display defects in growth and development, proposing possible gene functional redundancy. However, generation of single and double knock-down lines of AtRLI1 and AtRLI2 failed despite several attempts, implying a tight regulation of RLI expression. Furthermore, results of functional genetic studies using atrli2-1 and pAtRLI2::AtRLI2-FLAG transgenic Arabidopsis plants further revealed a negative role of AtRLI2 in defense against pathogens and oxidative stress via repressing SA, JA, ET and ROS scavenging signaling pathways. Moreover, although AtRLI2 was previously reported to be an endogenous suppressor of RNA silencing, our results did not support a role of AtRLI2 and SlRLI in suppressing RNA silencing. These studies and our previous studies together evidence that plants RLIs are important negative regulator in stress defense responses, and may be the hub of plant growth and development and stress responses.
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