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標題: | FIN219及FIN219-FIP1蛋白複合體的純化、結晶及生化分析 Purification, crystallization and biochemical assay of FIN219 and the complex of FIN219-FIP1 |
作者: | Sih-Syun Ho 何思勳 |
指導教授: | 鄭貽生 |
關鍵字: | FIN219,JAR1,茉莉酸,JA-Ile,FIP1, FIN219,JAR1,Jasmonate,JA-Ile,FIP1, |
出版年 : | 2009 |
學位: | 碩士 |
摘要: | FIN219(Far-red insensitive 219)又被稱為JAR1 (Jasmonate Resistant 1),屬於GH3-like基因家族成員之一,受生長素Auxin誘導而快速地表現。目前已知除了與光型態發生有關外,並且與植物賀爾蒙茉莉酸(Jasmonic acid, JA)調節有關。在遠紅外光的照射之下,fin219的突變株會出現長下胚軸的表現型。在胺基酸的序列初步分析上,FIN219蛋白的N端和C端皆有捲曲的螺旋區域(coiled-coil domain)。此外,FIP1(FIN219- interacting protein 1)基因,它所產生的蛋白質被證實可與FIN219的C端作用,並參與光訊息調控。FIN219具有將茉莉酸(JA)和異白胺酸(Ile)結合成JA-Ile的酵素活性,JA-Ile可與泛素連結酶COI1作用並促使26S蛋白酶體將轉錄抑制體JAZ1分解,這顯示了FIN219在茉莉酸訊息傳遞路徑上擔任了活化JA的角色。
由於FIN219的蛋白質晶體結構尚未被解出,且FIN219-FIP1複合體在植物體內的功能尚不清楚。本論文的研究目的即是針對FIN219蛋白以及FIN219-FIP1複合體,建立可取得高均質性與穩定性佳的蛋白之表現與純化流程,並藉由酵素動力學,希望能了解FIN219和FIN219-FIP1複合體,在調控光訊息傳遞和茉莉酸傳遞途徑所扮演的角色。 本實驗採用大腸桿菌表現系統,分別表現FIN219及FIP1,並以膠體過濾層析管柱純化FIN219-FIP1複合體。由酵素活性分析發現,JAR1-1蛋白不具酵素活性;JAR1-3蛋白的酵素活性,則介於JAR1-1和FIN219蛋白之間;結果證實,第101個胺基酸絲胺酸和第334個胺基酸谷胺酸在FIN219蛋白進行茉莉酸腺苷酸化作用時的重要性。將FIN219-FIP1複合體進行活性測試,發現FIN219在與FIP1作用後其酵素活性會提升。綜合蛋白質交互作用的實驗結果,推測FIP1可能藉由和FIN219 C端的捲曲螺旋區域作用,促使FIN219的活性上升。FIN219-FIP1複合體在經過晶體條件篩選後,可找到數個結晶條件,這些晶體的結晶條件,將再進一步調整後,取得較佳結晶條件,進行後續收集X射線繞射分析。 Arabidopsis FIN219 (far-red insensitive 219), also called JAR1(Jasmonate Resistant 1), is a member of GH3-like gene family and induced rapidly by auxin. Under far-red light, fin219 mutant exhibited a long hypocotyl phenotype. Amino acid sequence analysis revealed FIN219 contained two putative coiled-coil domains in both N- and C-terminal regions. Moreover, a gene fip1 (FIN219-interacting protein 1) encoding a glutathione S-transferase (GST) was demonstrated to interact with FIN219 in vitro and in vivo. FIN219 showed the ability to conjugate Jasmonic acid (JA) and amino acids Ile for biological activity. JA-Ile could bind to COI1, ubiquitin ligase, and cause the transcriptional repressor, JAZs, to be digested by 26S proteasome. It showed JA can be activated by FIN219 in JA signal pathway. Since the structure of FIN219 has not been resolved and the biological function of FIN219-FIP1 complex is still unknown. The proteins of FIN219 and FIP1 in E. coli were expressed then FIN219-FIP1 complex was purified by gel filtration. In biochemical analysis, JAR1-1 didn’t show any enzymatic activity and JAR1-3 showed the activity between JAR1-1 and FIN219. The results revealed S101 and E334 play a role of adenylation of JA by FIN219. In conclusion, FIP1 interacts with C-terminal coiled-coil of FIN219 and increases the enzymatic activity of FIN219. Through this interaction, FIP1 increases the enzymatic activity of FIN219. Several crystals of FIN219-FIP1 complex can be found after crystal screening. The crystallization will be further optimized for data collection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43521 |
全文授權: | 有償授權 |
顯示於系所單位: | 植物科學研究所 |
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