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研究生:陳冠仲
研究生(外文):KUAN-CHUNG CHEN
論文名稱:綠豆抗豆象基因VrCRP及VrArc的表現與功能分析
論文名稱(外文):Studies on the expression and functional analysis of VrCRP and VrArc bruchid resistance genes from mung bean seeds
指導教授:陳慶三陳慶三引用關係宋賢一
指導教授(外文):Ching-San ChenHsien-Yi Sung
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:172
中文關鍵詞:豆象綠豆人工種子抗菌
外文關鍵詞:bruchidmung beanartificial seedantimicrobial activity
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VrCRP (cDNA) 與VrArc (genomic DNA) 分別從具有抗豆象 (Callosobruchus chinensis) 性狀的栽培種綠豆 VC6089A 及 TC1966 中分離出來。 VrArc 基因經GCG 分析其所轉譯之胜證實其胺基酸序列與已發表的抗蟲蛋白質Arcelin-1 的 265 個胺基酸完全相同,N 端前 21 個胺基酸為信號胜。VrCRP cDNA 經 GCG分析可轉譯出 73 個胺基酸,N 端前 22 個胺基酸亦為信號胜,並擁有 8 個半胱胺酸組成二級構造 CSH motif (Cystine-stabilized -helical motif),此 motif 為許多富含硫氫基蛋白質及植物防禦素所擁有。
利用原核生物之大腸桿菌 pQE、IMPACT (intein mediated purification with an affinity chitin-binding tag) 表現系統,分別成功的將去除信號胜的 VrArcsp 及 VrCRPsp 進行表現。而含有信號胜全長的 VrArc 及 VrCRP 在表現載體誘導基因表現時,會造成寄主細胞發生溶菌現象。針對 VrArc,以此含有 His-tagged 的融合重組蛋白質為抗原對紐西蘭白兔進行免疫反應產生抗體,西方轉印法分析免疫效價高達 15 萬倍以上。就 VrCRP 而言,以此純化產物與攜帶者蛋白質KLH融合當作抗原進行免疫反應產生抗體,獲得效價達四萬倍以上的抗血清。
人工種子餵食實驗證實:含有 VrCRPsp 0.20% 以上的人工種子有能力將豆象 (Callosobruchus chinensis) 一齡幼蟲毒殺致死,而在 0.06% 即有明顯抑制幼蟲發育的跡象而導致 WSDT (Within Seed Development Time) 變長;VrCRPsp 除了對大腸桿菌有毒害外,於低濃度 ( 3.42 M) 下完全干擾並破壞昆蟲細胞株的正常生長;同時,VrCRP 蛋白質也抑制立枯絲核菌 (真菌) 的生長;試管外的蛋白質合成能力也受到 VrCRPsp 的抑制。免疫組織定位及原位雜交發現 VrCRP 主要集中在綠豆種子的種皮之中,尤其是儲積在薄壁細胞層 (parenchyma cell)。
抗蟲品系綠豆 VC6089A 中存在的天然 VrCRP 蛋白質,分別經由陰離子與陽離子層析管柱的分離,以及膠體過濾法等步驟可以獲得均質蛋白質,經西方點墨法以及蛋白質 N 端 41 個胺基酸定序的確定,證實此均質蛋白質為 VrCRP。N 端定序結果發現 N 端起始胺基酸為精胺酸,也即自 cDNA 起始密碼開始算起的第 28 個胺基酸,故斷定前 27 個胺基酸應於蛋白質成熟後被切除,天然存在的 VrCRP 應僅有 46 胺基酸,計算分子量為 4,824 Da,等電點為 8.62;進行人工種子的實驗初步也獲得具抗蟲特性的結果。
基於胺基酸序列相似性的比較、生物活性的探討以及蛋白質的分布位置,推測 VrCRP 應扮演著綠豆的重要演化屏障,來保護並阻絕植物病原及蟲害的侵襲。
VrArc genomic clone was isolated from a bruchid resistant wild mung bean, Vigna radiata TC1966, encoding a 265-amino-acid protein with a signal peptide of 21 amino acids. VrArc is completely identical to arcelin-1 which has been shown to be an insect resistant protein. A cDNA encoding a small cysteine-rich protein designated as VrCRP was isolated from a bruchid resistant nearly isogenic line of mung bean Vigna radiata VC6089A. VrArc protein was obtained by overexpression of signal peptide truncated VrArc (VrArcsp) in a pQE expression system. VrCRP (GenBank accession no. AF326687) encodes a protein of 73 amino acids, containing a predicted 22 amino acid signal peptide and 8 cysteines. VrCRP protein was obtained by overexpression of signal peptide truncated VrCRP (VrCRPsp) in an IMPACT (intein-mediated purification with an affinity chitin-binding tag) expression system. The purified VrCRPsp was identified by its molecular mass (5,944 Da) and N-terminal amino acid sequencing. Similar to a number of cysteine-rich proteins. There is a cystine stabilized -helix motif (CSH motif) in sequence of VrCRP protein that shows amino acid sequence homology to plant defensins. Artificial mung bean seeds containing 0.2% VrCRPsp killed larvae of the bruchid Callosobruchus chinensis at first instar stage. A VrCRPsp level as low as 0.06% was sufficient to delay larval development. VrCRPsp is toxic to E. coli and completely arrested growth of insect cells (Spodoptera frugiperda, Sf21) at a low concentration of 3.42 M. VrCRPsp is also a potent inhibitor of protein synthesis and arrests growth of a fungus Rhizoctonia solani. In situ hybridization revealed that VrCRP mRNA is most abundant in endosperm cells, followed by parenchyma cells. Immunolocalization indicated that VrCRP protein was predominantly present in parenchyma cells of the seed coat.
VrCRP was also purified directly from mung bean VC6089A by a procedure involving a two-column system comprising an anion-exchange (DEAE) and a cation-exchange (CM) columns, and Superdex Peptide HR10/30 gel filtration in FPLC system. The fractions containing VrCRP in the chromatography were recognized by the polyclonal anti-recombinant VrCRPsp antiserum. The first 41 amino acids of the purified VrCRP determined by N-terminal amino acid sequencing 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, and the mature VrCRP contains 46 amino acids. The calculated molecular mass and pI value of the purified VrCRP were 4,824 Da and 8.62, respectively. The anti-bruchid activity of mung bean VrCRP is similar to the recombinant VrCRPsp.
Based on the similarity of amino acid sequence with other plant defensins, biological activities and the protein localization, it is suggested that VrCRP may act as an evolutionary barrier in mung bean seeds that is important in coping with invasion of pathogens and herbivores.
頁碼
中文摘要 .......................................................................................................... I
英文摘要 .......................................................................................................... III
前言 .................................................................................................................. 1
一、綠豆種子的蟲害 ............................................................................ 1
二、抗豆象綠豆的育種與抗豆象因子的研究 .................................... 1
三、抗豆象基因的篩選 ........................................................................ 2
四、VrCRP 相似性蛋白質的分析 ...................................................... 3
五、富含硫醇基蛋白質 thionin 的生化特性 ..................................... 5
六、“Plant defensin” 植物防禦素的涵義、分類及定義 ...................... 7
七、VrArc 相似性蛋白質的分析及 arcelin 抗蟲蛋白質的介紹 ...... 9
八、外源凝集素的存在與植物防禦 .................................................... 10
九、抗豆象物質 .................................................................................... 11
十、VrCRP 抗蟲性與未來運用於農業生物科技的潛力 .................. 12
材料與方法 ...................................................................................................... 13
1.綠豆的栽培 ......................................................................................... 13
2.蛋白質定量法 ..................................................................................... 14
3.膠體電泳分析法 ................................................................................. 15
3-1. 不連續梯度SDS-聚丙烯醯胺膠片電泳檢定法 ..................... 15
3-2. 蛋白質雙向電泳分析法 .......................................................... 16
3-3. 洋菜膠體電泳分析法 .............................................................. 19
4.聚丙烯醯胺膠片染色顯影法 ............................................................. 20
4-1. CBR染色法 ............................................................................... 20
4-2. 硝酸銀染色法 .......................................................................... 20
5.總 RNA 的抽取 ................................................................................. 21
6.甲醛 RNA 洋菜膠體電泳分析法 ..................................................... 22
7.植物組織 DNA 的抽取 .................................................................... 23
8.下垂式鹼性毛細核酸轉印法 ............................................................. 25
9.Hybond N膜的雜交法 ........................................................................ 26
10.質體DNA的抽取與純化 .................................................................. 28
11.聚合鏈鎖反應 ............................................................................... 30
12.自洋菜膠中回收 DNA 片段 .......................................................... 31
13.DNA定序 .......................................................................................... 31
14.勝任細胞 (competent cell) 的製備與轉形 ..................................... 32
15.基因表現及純化系統I ~ QIAexpressionist ............................................... 33
16.基因表現及純化系統II ~ pET system ................................................. 36
17.基因表現及純化系統III ~ IMPACT ................................................ 37
18.蛋白質N端定序與 S-carboxymethylation ................................................ 39
19.抗體的製備 ....................................................................................... 41
20.蛋白質電泳轉印法及毛細轉印法 ................................................... 43
20-1 大分子以及非疏水性蛋白質轉印 ....................................... 43
20-2 小分子、鹼性以及疏水性蛋白質轉印 ~毛細轉印法 ....... 45
21.西方點墨法 (Western blotting) ........................................................ 46
22.人工種子的製備 ............................................................................... 47
23.昆蟲細胞株 (Sf 21 - AE) 的培養與毒性試驗 ............................... 48
24.轉譯抑制能力的分析 ....................................................................... 49
25.抵抗真菌正常生長能力測試 ........................................................... 50
26.組織切片與原位雜交實驗 ............................................................... 51
27.分離及純化綠豆種子中的 VrCRP 蛋白質 ..................................... 55
結果 [ I. 抗豆象基因 VrCRP 研究結果]
一、豆象飼育系統的建立與綠豆抗豆象分析 ……….......................... 57
二、溫室中綠豆開花後抗豆象性狀出現時期的決定 ........................ 58
三、VrCRP 的核酸及胺基酸序列 .................................................. 58
四、表現載體構築的鑑定與定序結果 ................................................ 59
五、VrCRPsp 基因表現與蛋白質純化 ............................................ 60
六、純化蛋白質 VrCRPsp 的 N 端序列 ....................................... 61
七、動物試驗、免疫血清的製備與西方點墨法的證據 .................... 61
八、人工種子的製作與抗豆象試驗 ….................................................. 62
九、對原核及真核細胞的影響 ..................................................…........ 63
十、VrCRPsp 對活體外轉譯作用的抑制能力 ................................ 64
十一、VrCRPsp 對真菌的拮抗能力 ................................................ 65
十二、綠豆種子內的 VrCRP 純化與鑑定 .......................................... 66
十三、VrCRP 在抗性與非抗性綠豆品系間含量的分析 ..................... 66
十四、天然綠豆 VrCRP 分子內雙硫鍵的強度分析 .......................... 67
十五、天然綠豆 VrCRP 的等電點 ..................................................… 67
十六、原位雜交及組織定位 ................................................................ 67
結果 [ II. 抗豆象基因 VrArc 研究結果]
一、VrArc 的核酸及胺基酸序列 .................................................... 69
二、表現載體的構築 ............................................................................ 69
三、VrArc 與 VrArcsp 表現對 E. coli 生長曲線影響結果 ........... 70
四、VrArcsp 基因表現與蛋白質純化 ................................................ 70
五、VrArc 抗體的製備及效價鑑定 ..................................................... 71
六、雙向電泳分析 ......................................................................…........ 71
討論
一、VrArc 與 VrCRP 的序列分析 ..................................................... 72
二、從生長曲線探討大腸菌表現系統 ................................................ 76
三、表現產物的聚集現象 (aggregation) ............................................. 77
四、表現產物的偽裝與欺騙 (trick) 誘生的手段 ~ IMPACT表現系統 78
五、IMPACT系統的優點與應用原理之研究 ..................................... 80
六、VrCRPsp 與 VrArcsp 的純化與純度分析 ............................. 81
七、VrCRPsp 與 VrArcsp 為抗原的免疫前試血與免疫 ............. 83
八、VrCRPsp 與 VrArcsp 的鑑定 ~ N-端定序 ........................... 85
九、豆象的生存及阻礙生命現象 ~ VrCRPsp 的抗豆象性質研究 87
十、VrCRPsp 對蛋白質合成的抑制性探討 .................................... 91
十一、VrCRPsp 對原核及真核細胞傷害的推論 ............................ 92
十二、VrCRPsp 對真菌的拮抗作用分析 ........................................ 94
十三、存在於抗蟲綠豆 VC6089A 的 VrCRP,分離、純化與鑑定之討論 .............................................................................................
94
十四、天然綠豆 VrCRP 與表現的重組 VrCRP 之間的差異 ........... 96
十五、非抗蟲綠豆中存在的 VrCRP 與抗蟲之間相連性探討 ........... 97
十六、組織定位的證據與其生理上的意義探討 ................................ 98
參考文獻 .......................................................................................................... 101
附錄 I ............................................................................................................... 111
附錄 II ............................................................................................................. 119
圖與表 .............................................................................................................. 125
圖一、綠豆 (Vigna radiata) 種子。 ....................................................
126
圖二、綠豆豆象 Callosobruchus chinensis 的演變及生活週期。 ........
127
圖三、VC6089A 綠豆於開花後五到二十天裡 VrCRP 的北方轉印圖。 .................................................................................................
128
圖四、VrCRP 的南方轉印圖。 ..........................................................…
129
表一、豆象的一生紀錄與觀察 .............................................................. 130
圖五、VrCRP cDNA 的核酸序列及推演的胺基酸序列。 ...............
132
圖六、VrCRPsp 的等電點演算。 .............................….......................
133
圖七、VrCRPsp的二級結構預測。 ......................................................
134
圖八、VrCRPsp 的二級結構分析。 ...................................................
135
表二、VrCRP 與相似富含硫小分子蛋白質間的比較。 .................... 136
圖九、VrCRP 與富含硫小分子蛋白質間的相似性排列。 ............... 137
圖十、pTYB4-VrCRPsp 表現載體的構築。 .......................................
138
圖十一、利用 4-12% 梯度變性膠體電泳分析 IMPACT 表現系統所生產的 VrCRPsp。....................................................................
139
圖十二、 利用FPLC系統的膠體過濾管柱 (Superdex Peptide HR 10/30) 分離並純化VrCRPsp。.................................................
140
圖十三、 VrCRPsp cDNA 推演的胺基酸序列與 VrCRPsp N 端定序序列比較。..............................…..............................…........
141
圖十四、疫前試血與免疫用兔的篩選。....................................................
142
圖十五、經三次免疫作用後的抗體效價測定。........................................
143
表三、不同含量 VrCRPsp 的人工種子餵食實驗對豆象幼蟲生長的影響。..............................................…..............................…........
144
圖十六、不同含量 VrCRPsp 的人工種子餵食實驗對豆象幼蟲生長的影響。........................................................................................
145
圖十七、VrCRP 與 VrCRPsp 對大腸桿菌的生長影響。................................................................................................
146
圖十八、VrArcDsp 與 VrCRPsp 在不同表現載體進行表現時對大腸桿菌生長態勢的影響。...........................................................
147
圖十九、VrCRPsp 對秋行軍蟲細胞株 (Spodoptera frugiperda ) 的毒害影響。....................................................................................
148
圖二十、鈣離子對 VrCRPsp 毒害秋行軍蟲細胞株 (Spodoptera frugiperda ) 的緩和作用。...........................................................
149
圖二十一、VrCRPsp 對蛋白質合成的抑制作用( I )。.......................... 150
圖二十二、VrCRPsp 對蛋白質合成的抑制作用( II ) 。~抑制 PreSS 蛋白質的合成作用。................................................................
151
圖二十三、VrCRP 重組蛋白質的抗真菌 (立枯絲核菌: Rhizoctonia solani) 實驗。.....................................................................….
152
圖二十四、光學顯微鏡下觀察 VrCRPsp 對立枯絲核菌 (Rhizoctonia solani) 菌絲的干擾現象。.................................
153
圖二十五、綠豆種子中 VrCRP 的分離,純化及 N 端胺基酸定序。............................................................................................
154
表四、綠豆種子中 VrCRP 的純化。........................................……….... 155
圖二十六、兩綠豆品系之間 VrCRP 含量差異比較。...........................
156
圖二十七、綠豆中 VrCRP 的三度空間分子模擬圖。............................
157
圖二十八、綠豆 VrCRP 分子內雙硫鍵氧化與還原態的變性電泳膠片分析。..................................................................................
158
圖二十九、綠豆 VC6089A VrCRP 的等電點演算及等焦集電泳分析。..........................................................................................
159
圖三十、利用反義 VrCRP RNA 進行綠豆 VC6089A 原位雜交圖。...
160
圖三十一、VrCRP 在綠豆 VC6089A 的免疫組織定位。.....................
161
圖三十二、VrArc 的核酸序列及推演的胺基酸序列。........................
162
圖三十三、VrArcsp 的二級結構預測。...............................................
163
圖三十四、VrArcsp 的二級結構分析。...............................................
164
圖三十五、VrArcsp二聚分子的三度空間模擬圖。..............................
165
圖三十六、VrArcsp 的等電點演算。...................................................
166
圖三十七、含 VrArc 及 VrArcsp 的表現載體構築。.........................
167
圖三十八、VrArc 的表現載體之 DNA 片段的電泳分析圖。..............
168
圖三十九、VrArc 與 VrArcsp 進行表現時對寄主細胞 (E. coli) 的影響。........................................................................................
169
圖四十、VrArcsp 的純化過程變性膠體電泳分析圖。..........................
170
圖四十一、針對 VrArcsp-6xHis 的抗血清免疫效價測定。.................
171
圖四十二、利用 2D 電泳技術鑑定 29 kDa 的 VrArcsp 重組融合蛋白質的純度。.......................................................................
172
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