請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51772
標題: | 綠竹筍乙烯受體 BoERS1 結構與功能之研究 Studies on the structure and function of ethylene response sensor 1 (BoERS1) in Bambusa oldhamii |
作者: | Yi-Lin Hsieh 謝毅霖 |
指導教授: | 楊健志 |
關鍵字: | 綠竹,乙烯受體,組胺酸激?,磷酸化, Bambusa oldhamii,ethylene receptor,histidine kinase,phosphorylation, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 本實驗室由綠竹筍 cDNA 庫中選殖出乙烯受體基因,命名為 BoERS1。其基因全長為 1899 bp。利用 PLACE (plant cis-acting regulatory DNA elements) 分析 BoERS1 5’-端上游序列,發現許多與植物荷爾蒙、光照及缺水逆境相關之 cis-acting elements。BoERS1 可轉譯出含有 632 個胺酸的蛋白質,且序列與水稻及玉米中的乙烯受體相似度達 90%。經由序列分析得知,BoERS1 主要具有三個區塊,分別為:感應功能區塊 (由三個穿膜區組成),GAF 功能區塊及 histidine kinase domain (具有保守性 H、N、G1、F 及 G2 boxes),此區塊分布與原核生物之二元訊息傳遞系統雷同。利用即時聚合酶鏈鎖反應分析 BoERS1 基因表現,田間栽種綠竹筍中 BoERS1 基因表現會隨著莖的生長而增加,且主要分布於節間及生長點,推測 BoERS1 可能參與綠竹快速生長;另外,以植物荷爾蒙處理綠竹瓶苗,發現細胞分裂素及離層酸會使 BoERS1 基因表現下降,而其他植物荷爾蒙較無影響。為瞭解 BoERS1 是否具有 histidine kinase 活性,以酵母菌 Pichia pastoris 表現 BoERS1 之 histidine kinase domain (Ala 331 至 Gly 611, BHK),經質譜儀鑑定重組蛋白質身分後,進行 in vitro 磷酸化實驗,得知 BHK 在 Mn2+ 或 Mg2+ 處理下,具有自我磷酸化活性,而在 Ca2+ 或水處理下則無活性。以質譜儀分析磷酸化反應後之 BHK,發現 Thr 442、Ser 444、Ser 489 及 Ser 503 等胺酸位置有磷酸化現象。將這些胺酸點突變後,再次進行磷酸化實驗,發現點突變並不影響磷酸化活性,說明 BHK 可能呈現多重磷酸化狀態,或具有其它磷酸化位置。以阿拉伯芥乙烯受體 AtETR1 (PDB ID: 4PL9) 及嗜熱菌 (Thermotoga maritima) 之 histidine kinase (PDB ID: 2C2A) 為模版,進行 BHK 結構模擬,發現兩個結構上具有彈性 loops,分別命名為 L1 及 L2。與原核生物比較後發現,L1 為植物 histidine kinase 所獨有,而磷酸化位置 Ser 489 及 Ser 503 正好落在 L1 兩端,推測 BHK 可能利用磷酸化調控 L1 構型,進而改變與其它蛋白質如 CTR1 或 EIN2 之交互作用,進而影響乙烯訊息傳遞;而 L2 正好位於 ATP 結合區上方,可能調控 ATP 能否進入活性區,進一步影響 BHK 磷酸化活性。BoERS1 多重磷酸化的發現,使得我們能夠更進一步地去探討乙烯受體之結構與功能關係。 An ethylene receptor gene named BoERS1 was cloned from a bamboo (Bambusa oldhamii) cDNA library. The open reading frame of BoERS1 was 1899 bp which encoded a 632-amino acid polypeptide. The encoded BoERS1 contained three domains, a sensor domain with three transmembrane regions, a GAF domain and a histidine kinase domain that contained all the conserved motifs (H, N, G1, F, and G2) that are present in the histidine kinases of the bacterial two-component systems and shared 90% sequence similarity with other ethylene receptors in plants such as rice or maize. According to real-time PCR analysis, the levels of BoERS1 mRNA in the shoots of field-grown bamboo were elevated along with the growth of the emerging shoots, especially in internodes and shoot meristems. Furthermore, the expression levels of BoERS1 were decreased under benzyladenine (BA, a cytokinin) and ABA treatments in multiple shoots of bamboo. The upstream sequences of BoERS1 were obtained using TAIL-PCR (thermal asymmetric interlaced polymerase chain reaction) and some cis-acting elements related to phytohormones, light, and dehydration were found. With in vitro kinase assay, the recombinant histidine kinase domain of BoERS1 (Ala 331 to Gly 611, BHK) showed autophosphorylation activity in the presence of Mn2+ and Mg2+, but not in the presence of Ca2+ and H2O. LC-ESI-MS/MS analysis indicated that four amino acid residues of BHK, namely Thr 442, Ser 444, Ser 489 and Ser 503, were phosphorylated by an in vitro kinase assay. Site-directed mutagenesis of these amino acids did not affect the phosphorylation activity of BHK. It indicated BHK was multiphosphorylated or had other phosphorylation residues. The model of BHK was built according to the structure of AtETR1 (PDB ID: 4PL9) and a histidine kinase of Thermotoga maritima (PDB ID: 2C2A). The three dimensional model of BHK had two flexible loops, namely L1 and L2. It is interesting to note that Ser 489 and Ser 503 were located in the both ends of L1 which was unique to the plant histidine kinase-containing enzymes and the phosphorylation may regulate the interactions between BoERS1 and other proteins; meanwhile L2 may be a gatekeeper of ATP binding pocket and regulate the entry of ATP. The identification of multiple phosphorylation sites on BoERS1 provides a new avenue for future structure–function studies of the ethylene receptor protein family. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/51772 |
全文授權: | 有償授權 |
顯示於系所單位: | 生化科技學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 7 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。