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

黃蕙蘭(Huei-Lan Huang)

指導教授 ： 王玉麒

國立臺灣師範大學/理學院/生物學系/碩士(2000年)

摘要

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。

關鍵字

PR蛋白質 ； 擬甜蛋白 ； 陽離子交換管柱 ； 水楊酸 ； 等焦電泳

並列摘要

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.

並列關鍵字

pathogenesis-related protein ； thaumatin-like protein ； Mono S ； salicylic acid ； IEF