臺灣博碩士論文加值系統

摘要
本研究室利用壓制性扣除雜交技術，自近等基因品系抗蟲綠豆Vigna radiata VC 6089A (簡稱VC 6089A) 對應不抗蟲綠豆Vigna radiata VC 1973A分離到一富含半胱胺酸蛋白質的cDNA－VrCRP (cysteine-rich protein of Vigna radiata) 。經由大腸桿菌系統表現出的蛋白質VrCRPDsp (Dsp代表刪除訊號胜)，已證實具有高度的豆象抗性。為探討存在VC 6089A中VrCRP的結構、性質與功能，本研究自VC 6089A中純化出抗豆象蛋白質VrCRP。豆粉粗抽液經由CM-Sepharose 管柱層析再利用快速蛋白質液相層析系統 (FPLC systems) 進行Superdex Peptide HR 10/30膠體過濾後，可由每克綠豆中得到35微克高純度VrCRP (0.0035%, w/w)。純化過程中的主要篩選工具為大腸桿菌表現的VrCRP所製備的抗體。在西方轉印分析結果中，同時發現另一可為抗體辨識的蛋白質，經N端胺基酸定序及序列比對後，推測為一新的脂質轉運蛋白質 (lipid transfer proteins)，命名為VrLTP。對純化出的VrCRP之N端前15個胺基酸進行定序，結果與其cDNA推論出的胺基酸序列完全相符。Arg28為N端第一個胺基酸，表示在VrCRP全長73個胺基酸中，由Met1到Ala27為訊號胜。純化出的VrCRP是由46個胺基酸組成，其計算分子量及等電點分別為5123 Da及8.42。依據其胺基酸序列相似性、分子量及等電點，推測VrCRP應屬植物防禦素 (plant defensins) 中的 g-purothionin 家族，且進一步經multiple alignment 分析，VrCRP 自成一類新的植物防禦素。VrCRP可能是第一個被發現具有抗蟲活性的植物防禦素。
針對純化後的VrCRP進行胞外蛋白質合成抑制能力分析，在小麥胚芽抽出物製成的胞外轉譯系統中，5 mM VrCRP可抑制蛋白質合成達50%。對於植物病原黴菌Rhizoctonia solani的拮抗作用，隨VrCRP的含量增加而明顯。大腸桿菌在含有5 mM VrCRP的培養基中，出現明顯受抑制的生長曲線。出芽性酵母菌Pichia patoris在培養基中含有10 mM VrCRP的情況下也可觀察到類似結果。

Abstract
A cDNA named VrCRP (cysteine-rich protein of Vigna radiata) was previously isolated in our laboratory from an insect resistant nearly isogenic line of mung bean Vigna radiata VC6089A (Abbreviated as VC6089A) versus a susceptible isogenic line Vigna radiata VC1973A using suppressive substractive hybridization. The recombinant protein VrCRPDsp (Dsp represents signal peptide truncated) was shown to be highly toxic to one of the major bruchids of mung bean seeds Callosobruchus chinensis. In order to study the structure, properties and biological activities of naturally occurring VrCRP in VC6089A, VrCRP was purified directly from VC6089A by a procedure involving CM-Sepharose chromatography and Superdex Peptide HR10/30 gel filtration in FPLC system. This purification procedure was proved to be a simple and rapid method to isolate VrCRP from mung bean. Thirty-five microgram of highly purified VrCRP could be obtained from 1 g of mung bean seeds (0.0035%, w/w). The fractions containing VrCRP in the chromatography were recognized by the polyclonal anti-recombinant VrCRPDsp antiserum. A novel putative lipid transfer protein, designated as VrLTP, was also cross-reacted with the antiserum. The first 15 amino acids of the purified VrCRP determined by N-terminal Biosystems were completely consistent with the deduced amino acid sequence of VrCRP cDNA starting from Arg28. The results indicated that there is a signal peptide from Met1 to Ala27 in the full length VrCRP (73 amino acids), and the processed VrCRP contains 46 amino acids. The calculated molecular mass and pI value of the purified VrCRP were 5123 Da and 8.42, respectively. Based on amino acid sequence, molecular weight and pI value, VrCRP probably belongs to the g-purothionin-like family of plant defensins. Comparison of amino acid sequences of plant defensins and similarity dendrogram show that VrCRP appears to represent a novel subgroup of plant defensins. Plant defensins have been known as antimicrobial peptides/proteins. To our knowledge, VrCRP is the first plant defensin demonstrated to be also active against insect pests.
We also analyzed some of the biological activities of the purified VrCRP. In the in vitro translation system derived from wheat germ extract, about 50% of protein synthesis was inhibited by 5 mM VrCRP. The protein also inhibits the growth of a soil-borne pathogenic fungus, Rhizoctionia solani. Growth arrests of E. coli and a budding yeast, Pichia patoris, were also observed in media containing 5 and 10 mM VrCRP, respectively.

目 錄
頁碼
寫在前面的話
摘要
Abstract
縮寫表
第一章 緒 論
第一節 緣起：當綠豆遇到豆象 ....................1
第二節 抗豆象綠豆的育種及抗豆象因子研究 ............1
第三節 抗豆象基因的篩選 ........................3
第四節 全長VrCRP 蛋白質相似性分析 ...............4
第五節 g-Thionins、植物防禦素與植物抗微生物胜 .....5
第六節 VrCRP 的表現及蛋白質活性分析 .…………………7
第七節 自抗豆象綠豆中純化VrCRP ......………………8
第二章 材料與方法
第一節 VrCRP 純化方法............................…9
第二節 蛋白質電泳檢定法 ......................……11
第三節 蛋白質轉印及相關應用 ......................…13
第四節 蛋白質定量法 ….........………………………..15
第五節 VrCRP 蛋白質生物活性分析 .....................17
第六節 綠豆與紅豆種子的抑菌性試驗 ……....……………19
第三章 結 果21
第一節 自抗豆象品系綠豆VC 6089A中純化VrCRP ………..21
第二節 N端胺基酸定序、序列相似性分析及三級結構模擬 ...22
第三節 VrCRP生物活性分析 …...............…25
第四節 栽培種綠豆VC 1973A中VrCRP之初步純化分析 ...…26
第五節 綠豆與紅豆種子的抑菌性試驗 …...........….26
第四章 討 論
第一節 自抗豆象品系綠豆VC 6089A中純化VrCRP .........27
第二節 VrCRP之歸類與植物脂質轉運蛋白質特性的探討 ..31
第三節 VrCRP之生物活性探討 …….....…………….33
第四節 VrCRP存在栽培種綠豆VC 1973A中? ………………36
第五節 綠豆與紅豆種皮的抑菌能力 .....……………………39
未來展望 …...............………………….40
參考文獻 ...................………………….41
圖 與 表 …................……………….47
結尾的話

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