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研究生: 黃蕙蘭
Huei-Lan Huang
論文名稱: 水稻胞外擬抗菌蛋白的生化分析與生理表現之研究
Biochemical and physiological characterizations of a rice (Oryza sativa L.) apoplastic thaumatin-like protein
指導教授: 王玉麒
Wang, Yu-Chie
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2001
畢業學年度: 89
語文別: 中文
論文頁數: 93
中文關鍵詞: PR蛋白質擬甜蛋白陽離子交換管柱水楊酸等焦電泳
英文關鍵詞: pathogenesis-related protein, thaumatin-like protein, Mono S, salicylic acid, IEF
論文種類: 學術論文
相關次數: 點閱:172下載:7
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  • 摘要
    在正常的生長條件下,懸浮培養的水稻細胞分泌多種蛋白質至液態Murshige-Skoog培養基中,其中,有一分子量約為29 kDa的蛋白質(簡稱Osm29蛋白質),其N端胺基酸序列與植物PR蛋白質( pathogenesis-related protein )中第五群 thaumatin-like protein具高度相似性。 本論文的研究目的即在於分離純化該蛋白質以進行其生化與抗菌特性的分析,同時也用以生產抗體探針,以探測該蛋白質的生理表現情形。
    在蛋白質純化方面,本研究者先利用硫酸銨鹽析(60-80%飽和度)以局部提高Osm29蛋白質的相對含量,再經Mono S陽離子交換管柱的分離過程後,可將Osm29蛋白質純化至幾近均質狀態。 經此一純化過程Osm29蛋白質的產量約佔培養液蛋白質的0.64%,而其純化倍數約為39倍。
    在生化特性分析方面,由於Osm29蛋白質可吸附於陽離子交換管柱,因此其結構表面可能帶有許多正電荷,而IEF電泳也顯示Osm29蛋白質多為pI>7.1的鹼性蛋白質,並具有五種等電點相異的同功蛋白,經peptide mapping分析的結果顯示,此五種同功蛋白的胺基酸序列因極為相近或完全相同。 而醣蛋白分析的結果則說明Osm29蛋白質並非為醣化的分泌性蛋白質。
    在抗菌分析方面,Osm29蛋白質對所測試的紋枯病菌、胡麻葉枯病菌以及葉鞘腐敗病菌並不具有明顯抑制菌絲生長的作用,不過在150 μg劑量下,Osm29蛋白質似乎對胡麻葉枯病菌有減緩其菌絲生長的現象。
    在生理表現分析方面,Osm29蛋白質會受到植物生長調節劑salicylic acid的處理以及受真菌感染而誘導分泌。 另外在水稻胚,及水稻幼苗的各器官內並無發現Osm29蛋白質的存在,推測Osm29蛋白質在植株體內的表現可能受病原菌感染誘導表現。
    在病原菌感染方面,Osm29蛋白質在水稻懸浮細胞受到葉鞘腐敗病菌感染時,會有大量被誘導分泌的現象。 不過,當以紋枯病菌感染時,Osm29蛋白質卻反而被抑制分泌,因此Osm29蛋白質似乎只能針對特定幾種真菌產生反應。

    ABSTRACT
    Under conditions of normal growth, suspension-cultured rice cells secreted some different proteins into liquid Murashige-Skoog medium. Among them, a protein with a monomeric mass of 29 kDa (designated Osm29 protein) has a N-termintal amino acid sequence homologous to some thaumatin-like proteins in plants. The purposes of this study were to purify the Osm29 protein and to produce antibodies of it . In addition, biochemical and physiological characterizations of the Osm29 protein were also conducted.
    With regard to purification of the Osm29 protein, a scheme using in combination of ammonium sulfate fractionation (60-80% saturation) and Mono S chromatography were developed. By using this protocal the Osm29 protein was purified to near homogeneity with a purification factor of 39-fold and the protein yield was about 0.64% of the total secreted proteins.
    With regard to the biochemical properties of Osm29 protein, it appears to be a non-glycosylated basic protein with 5 isomers varying in pI value. According to the results of peptide mapping, the amino acid sequence of the five isoforms were highly comparable between each other.
    With regard to the physiological properties of the Osm29 protein, it is present in the medium of cell culture, but not detectable in extracts of rice embryo and various organs of rice young seedlings. The secretion of Osm29 protein in culture medium can be rapidly induced by treatment of salicylic acid and by Sarocladium oryzae infection, but not by treatment of methyl jasmonic acid and Rhizoctonia solani infection.
    In an antifungal bioassay, Osm29 protein did not inhibit the growth of Rhizoctonia solani, Bipolaris oryzae, and Sarocladium oryzae, but at the dose of 150 μg, it exhibited growth-retarding effects on Bipolaris oryzae.

    目 錄 壹、緒論---------------------------------------------- 1 貳、研究材料與方法------------------------------------ 16 一、研究材料與細胞培養及水耕栽培---------------------- 16 A. 材料----------------------------------------------- 16 B. 水稻細胞懸浮培養及生長調節劑methyl jasmonate (MeJA) 與 salicylic acid (SA)的處理-------------------------- 16 1. 水稻細胞懸浮培養----------------------------------- 16 2. 植物生長調節劑的處理------------------------------- 16 C. 懸浮培養細胞的真菌感染----------------------------- 17 D. 水稻植株的水耕栽培--------------------------------- 17 二、細胞培養液內蛋白質之收集及Osm 29 分泌性蛋白質之分離及純化 -------------------------------------------------------18 A. 培養液之收集--------------------------------------- 18 B. 培養液蛋白質之初步分離----------------------------- 18 1. 硫酸銨沈澱(鹽析)--------------------------------- 18 2. 透析----------------------------------------------- 19 C. 蛋白質之純化--------------------------------------- 19 1. FPLC之操作流程------------------------------------- 20 2. 純化後Osm 29蛋白質之收集--------------------------- 21 三、蛋白質的分析-------------------------------------- 21 A. 蛋白質的萃取--------------------------------------- 21 B. 蛋白質的電泳分析----------------------------------- 22 C. 蛋白質的銀染--------------------------------------- 22 D. Coomassie brilliant blue 的染色-------------------- 23 E. 蛋白質的定量--------------------------------------- 23 F. 醣蛋白的膠體染色----------------------------------- 24 G. Osm29蛋白質的電泳釋出 (electroelution)------------- 25 H. 等焦電泳 ( isoelectric focusing gel electrophoresis ) 分析 -------------------------------------------------------26 I. 免疫轉漬 (immunoblotting) 試驗--------------------- 27 四、Osm 29 蛋白質的peptide mapping 分析--------------- 28 A. 蛋白質切割藥劑的種類------------------------------- 28 B. Peptide mapping 分析------------------------------- 29 五、Osm 29 蛋白質微定序分析 (Microsequencing)--------- 29 A. 微定序Mono S管柱所純化之Osm29蛋白質---------------- 29 B. 微定序經切割處理後之Osm29蛋白質-------------------- 30 六、Osm29 蛋白質多株抗體之製備------------------------ 31 A. 免疫注射------------------------------------------- 31 1. 施打Mono S管柱所純化之Osm29蛋白質------------------ 31 2. 施打電泳分離所純化之Osm29 蛋白質------------------- 31 B. 血清之分離----------------------------------------- 32 七、Osm29 蛋白質之抗菌效能分析------------------------ 32 A. PDA (Potato dextrose agar) 培養基的製備------------ 32 B. 菌株的種類(為台中霧峰農試所所提供)--------------- 33 C. 抗菌效能分析--------------------------------------- 33 參、研究結果------------------------------------------ 34 Osm29蛋白質的鑑定------------------------------------- 34 Osm29蛋白質的差異性硫酸銨鹽析------------------------- 35 Osm29蛋白質的離子交換管柱純化------------------------- 36 Osm29蛋白質的純化係數--------------------------------- 37 Osm29蛋白質於培養液中的累積測定----------------------- 38 Osm29蛋白質之多株抗體生產與其效價測定(titer determination) -------------------------------------------------------38 醣蛋白的的測定---------------------------------------- 39 Osm29蛋白質的等焦電泳分析----------------------------- 40 Peptide mapping分析----------------------------------- 40 植物生長調節劑的影響---------------------------------- 41 —MeJA的處理------------------------------------------ 41 -SA處理---------------------------------------------- 41 -Osm29蛋白質於水稻植株內的表現情形------------------- 43 -Osm29蛋白質於真菌感染水稻細胞時的表現情形----------- 43 Osm29蛋白質抗菌活性的測定----------------------------- 44 肆、討論 ----------------------------------------------45 Osm29蛋白質的鑑定------------------------------------- 45 Osm29蛋白質的分離與純化------------------------------- 44 Osm29蛋白質之多株抗體生產與其效價測定----------------- 48 Osm29蛋白質之物化性質分析----------------------------- 50 Osm29蛋白質受植物生長調節劑處理的表現分析------------- 52 Osm29蛋白質於水稻不同發育時期的器官部位之表現分析----- 54 Osm29蛋白質受病原菌誘導的表現分析--------------------- 55 Osm29蛋白質的抗菌活性測定----------------------------- 55 總結-------------------------------------------------- 56 伍、參考文獻------------------------------------------ 57 附錄一、 Murashige-Skoog medium----------------------- 91 附錄二、Kimura B solution----------------------------- 92 附錄三、FPLC program---------------------------------- 93 附錄四、V-7 valves------------------------------------ 93 圖 表 次 圖一、硫酸銨 (0-80%) 沈澱的培養液蛋白質經Mono S管柱分離的結果---------------------------------70 圖二、Rice Osm29 蛋白質與四種植物的thaumatin-like protein之N端序列 比對情形-------------------71 圖三、培養液蛋白質的硫酸銨鹽析沈澱結果---------------------------------72 圖四、硫酸銨 (60-80%) 沈澱的培養液蛋白質經Mono S管柱純化的結果---------------------------------73 圖五、水稻細胞培養液蛋白質經Mono Q管柱純化的結果---------------------------------74 圖六、硫酸銨(60-80%)沈澱的培養液蛋白質在pH 6.8的條件下,以Mono S管柱分離純化的結果-----------75 圖七、硫酸銨(60-80%)沈澱的培養液蛋白質在pH 4.6的條件下,以Mono S管柱分離純化的結果-----------76 圖八、將局部純化之培養液蛋白質 [圖四(b), fr. 28, 29] 再經Mono S管柱(pH 4.6)純化之結果----------77 圖九、Osm29蛋白質在培養液中的累積變化情形------------78 圖十、Osm29蛋白質多株抗體的製作流程圖----------------79 圖十一、第一次Osm29蛋白質之抗血清的效價與專一性分析的結果----80 圖十二、第二次Osm29蛋白質之抗血清的效價與專一性分析的結果----81 圖十三、Osm29蛋白質的醣化分析結果--------------------82 圖十四、Osm29蛋白質的等焦電泳分析--------------------83 圖十五、Osm29蛋白質的peptide mapping分析-------------84 圖十六、MeJA處理對培養液中Osm29蛋白質含量的影響------85 圖十七、SA處理(1 ml細胞)對培養液中Osm29蛋白質含量的影響----86 圖十八、SA處理對培養液中Osm29蛋白質含量的影響--------87 圖十九、SA處理 (5 ml細胞) 對培養液中Osm29蛋白質含量於短時間內的 影響-------------------------------------------------88 圖二十、Osm29蛋白質於水稻植株內的表現情形------------89 圖二十一、懸浮培養細胞受葉鞘腐敗病菌感染的Osm29蛋白質之表現--90 圖二十二、懸浮培養細胞受紋枯病菌感染的Osm29蛋白質之表現------91 表一、Osm29蛋白質的純化結果--------------------------77

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