臺灣博碩士論文加值系統

蝴蝶蘭軟腐病菌Erwinia chrysanthemi會產生多種果膠質分解酵素，其中pectate lyases (Pels)是最主要分解植物細胞壁內果膠質的酵素，其分解產物為unsaturated oligogalacturonates被認為會引起植物的防禦作用。因此若能在健康植物以Pels或是oligogalacturonates處理，使植物事先產生防禦反應，或可產生抗軟腐病菌的效果。本實驗室已建構可表現E. chrysanthemi PelE和PelZ蛋白質之E. coli BL21 (DE3)和E. coli Rosetta (DE3) pLysS菌株，純化得到47.4 kDa PelE和48.5 kDa PelZ蛋白質。蝴蝶蘭軟腐病葉汁液具有Pel酵素活性，利用所得到PelE和PelZ蛋白抗體，對蝴蝶蘭軟腐病葉汁液進行Western blot，可偵測到PelE和PelZ，證明E. chrysanthemi感染蝴蝶蘭時，PelE和PelZ蛋白質有表現。所構築的E. coli BL21 (DE3) (pET29a-pelE)和E. coli Rosetta (DE3) pLysS (pET29a-pelE)，可將所產生PelE蛋白質分泌至細胞外，並可分解polygalacturonic acid，其分子量為40 kDa，較原本估計的PelE蛋白質小，因此經由軟體分析及蛋白質定序發現PelE N-端的胺基酸序列含有一段signal peptide和其切點。利用PCR得到此段signal peptide DNA片段，以構築新的表現載體，將用於其他蛋白質的表現。使用分泌至細胞外的PelE蛋白質，純化得到具活性PelE酵素，得知PelE酵素置於室溫七天後，酵素活性才會開始明顯下降，並且需以100℃高溫處理30分鐘才能完全去除酵素活性，PelE酵素之Km為0.46而Vmax為1.75。PelE酵素分解polygalacturonic acid所得產物進行HPLC，偵測波長230 nm處之吸光值，可以在5.3 min時產生一個明顯的波峰，與digalacturonic acid產生明顯波峰在5.2 min時間相近。將可分泌PelE蛋白質之E. coli接種菸草會引發過敏性反應，接種至蝴蝶蘭則都沒有。將純化得到具活性PelE酵素(40 kDa)接種菸草與蝴蝶蘭都會引發過敏性反應，以100℃高溫加熱30分鐘後所得不具活性PelE酵素(40 kDa)則不會引發過敏性反應。將具活性PelE酵素(40 kDa)水解PGA之產物接種菸草與蝴蝶蘭目前都沒有引發過敏性反應。

Phalaenopsis soft rot bacterium, Erwinia chrysanthemi produces several pectic enzymes. Pectate lyases (Pels) the main E. chrysanthemi enzyme, depolymerize pectic substances of plant cell walls. The degradation products, unsaturated oligogalacturonates, have been proposed to induce plant defence reactions. If healthy plants are treated with Pels or oligosaccharides to make them produce defence reactions or resist soft rot pathogen. Constructed E. coli BL21 (DE3) and E. coli Rosetta (DE3) pLysS E.coli strains could express PelE and PelZ. Purified 47.4 kDa PelE and 48.5 kDa PelZ were used to produce PelE and PelZ antibodies. Soft rot leaves filtering solution had Pel activity. PelE and PelZ antibodies were used in Western blot for detecting soft rot leaves filtering solution. There had detected PelE and PelZ expression, the results indicated that PelE and PelZ express in soft rot pathogenesis. E. coli BL21 (DE3) (pET29a-pelE) and E. coli Rosetta (DE3) pLysS (pET29a-pelE) could secrete PelE protein out of the cell and was also able to degrade polygalacturonic acid. Its molecular weight of 40 kDa was smaller than we calculated. The N-terminal amino acid sequence of PelE had a signal peptide and its cleavage site based on sequence analysis and N-terminal amino acid sequencing of PelE. Signal peptide DNA sequence amplification by PCR was used to construct a new expression vector for other protein expression. PelE enzyme was purified from culture supernatant. PelE enzyme activity decreased significantly after seven days at room temperature. A complete removal of PelE enzyme activity required 100 ℃ for 30 minutes. The Km of PelE enzyme was 0.46 and the Vmax of PelE enzyme was 1.75. Analysis the products of PelE degraded polygalacturonic acid by HPLC. A maximum absorption was found at 230 nm, and the HPLC separation test showed a clear peak at 5.3 minute after sample injection. The time was similar to digalacturonic acid showed a clear peak at 5.2 minute after sample injection. The E.coli strains which could secrete PelE protein were inoculated into tobacco and triggered hypersensitive reaction but not seen in phalaenopsis. Purified active PelE enzyme was inoculated into tobacco and phalaenopsis both triggered hypersensitive reaction. The products of PelE degraded polygalacturonic acid were inoculated into tobacco and phalaenopsis both could not trigger hypersensitive reaction.

論文目錄
中文摘要
英文摘要
前言
材料與方法
菌種的取得、培養條件、儲存的方法
實驗用植株
序列分析
將pelE、pelZ基因接入pET29a載體中
蛋白質大量表現
分析蛋白質產物是可溶性或不可溶性
目標蛋白質為可溶性
目標蛋白質為不可溶性
以His TrapTM套組純化大量表現之蛋白質產物
BCA蛋白質定量
抗體效價測試
Enzyme–linkedimmunosorbentassay (ELISA)
Western blot
墨點法的準備與蛋白質轉印
色度偵測
1.E. chrysanthemi感染蝴蝶蘭之PelE表現情形
(1)製作葉片過濾液
(2)葉片過濾液進行Pel酵素活性測試
(3)葉片過濾液進行Western blot測試

2.pET29apelE在E. coli BL21 (DE3)和E. coli Rosetta (DE3) pLysS的表現情形
(1)測試含pET29apelE之E. coli BL21 (DE3)和E. coli Rosetta (DE3) pLysS分解polygalacturonic acid能力
(2)由E. coli分泌(secretion)出的PelE的大小
(3)分泌出PelE N端的胺基酸序列分析
(4)PelE之signal peptide及切點(cleavage site)分析
(5)構築好的pET29apelE質體pelE與其他E. chrysanthemi pelE之序列比對 －不同E. chrysanthemi pelE之signal peptide的序列差異
(6)PelE之signal peptide的核苷酸序列選殖
(7)將pelEsp基因接入pET29a載體中
3.PelE酵素活性及其產物測定
(1)PelE酵素之生產及純化
誘導PelE蛋白質大量表現
蛋白質沉澱(鹽析)及透析
以His TrapTM套組純化大量表現之蛋白質產物
Optimisation Protocol (找尋最佳化流洗濃度)
蛋白質透析去鹽(恢復活性)
PelE酵素保存方式
(2)Pel酵素活性測定
利用分光光度計測試Pel酵素活性
(3)PelE酵素活性、Km、及Vmax測定
(4)PelE酵素之穩定性測定
(5)PelE水解PGA (polygalacturonic acid)之產物用HPLC分析
4.PelE誘導煙草及蝴蝶蘭之過敏性反應(hypersensitive reaction)
(1)E. coli BL21 (DE3) (pET29apelE)和E. coli Rosetta (DE3) pLysS (pET29apelE)誘發煙草及蝴蝶蘭之過敏性反應測試
(2)純化之PelE酵素誘發煙草及蝴蝶蘭之過敏性反應測試
(3)PelE水解PGA之產物誘發煙草及蝴蝶蘭之過敏性反應
基礎分生技術
實驗結果
自Erwinia chrysanthemi選殖得到部分pectinase基因
PelE和PelZ蛋白質表現結果
PelE和PelZ蛋白質純化結果
PelE和PelZ蛋白質抗體測試結果
Enzyme –linked immunosorbent assay (ELISA)
Western blot
1.E. chrysanthemi感染蝴蝶蘭之PelE表現情形
(1)葉片過濾液進行Pel酵素活性測試
(2)葉片過濾液進行Western blot測試
2.pET29apelE在E. coli BL21 (DE3)和E. coli Rosetta (DE3) pLysS的表現情形
(1)測試含pET29apelE之E. coli BL21 (DE3)和E. coli Rosetta (DE3) pLysS分解polygalacturonic acid能力
(2)由E. coli分泌(secretion)出的PelE的大小
(3)分泌出PelE的胺基酸序列分析
(4)PelE之signal peptide及切點(cleavage site)分析
(5)構築好的pET29apelE質體pelE與其他E. chrysanthemi pelE之序列比對－不同E. chrysanthemi pelE之signal peptide的序列差異
(6)PelE之signal peptide的核苷酸序列選殖
3.PelE酵素活性及其產物測定
(1)PelE酵素之生產及純化
(2)具活性PelE蛋白質Pel酵素活性測試
(3)具活性PelE酵素之酵素反應速率、活性unit、比活性、km、Vmax
(4)PelE酵素之穩定性測定
(5)PelE水解PGA (polygalacturonic acid)之產物用HPLC分析
4.PelE誘導煙草及蝴蝶蘭之過敏性反應(hypersensitive reaction)
(1)E. coli BL21 (DE3) (pET29apelE)和E. coli Rosetta (DE3) pLysS (pET29apelE)誘發煙草及蝴蝶蘭之過敏性反應測試
(2)純化之PelE酵素(40 kDa)誘發煙草及蝴蝶蘭之過敏性反應測試
(3)PelE水解PGA之產物誘發煙草及蝴蝶蘭之過敏性反應
討論
參考文獻
圖表

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