臺灣博碩士論文加值系統

革蘭氏陰性菌 Xanthomonas campestris pv. campestris (簡稱Xcc) 為造成十字花科植物黑腐病之病原菌。 目前已知 Xcc 產生的一些物質如: 胞外多醣 (exopolysacchride) 及胞外酵素與致病性有關。 這些胞外酵素如 protease, pectate lyase, alpha-amylase 及 cellulase 等係以第二型分泌途徑將蛋白分泌出去。 胞外蛋白質分泌, 經由 pilD 所主導的signal peptidase (PilD) 將欲分泌出蛋白之信號胜肽 (signal peptide) 切除再釋放成熟的蛋白於細胞間質, 然後經由其他輔助蛋白送出胞外。 　
過去本實驗室曾將 Xcc strain P20H 之 pilD 基因, 以染色體上同質性片段, 進行 single crossover, 得到突變株 MC1220(pilD::pSMP104)。 從測試胞外酵素分泌的活性染結果顯示, pilD 突變株同樣具有與 P20H相同的活性。 為了比較 P20H 及 MC1220(pilD::pSMP104) 的胞外蛋白 N 端序列, 收集兩者的胞外蛋白以 SDS-PAGE 電泳分離, 轉漬到 Nylon membrane後, 回收較清楚且再現性一致的三個蛋白色帶 (24-kDa, 28-kDa, 35-kDa) 進行 N 端定序。 結果顯示 P20H 及 MC1220(pilD::pSMP104) 的三個胞外蛋白之 N 端序列相同。 N 端定序的三個胞外蛋白24-kDa 、28-kDa 及 35-kDa, 經 Xcc strain ATCC33913 genome比對的結果分別為 conserved hypothetical protein (XCC0694)、 cellulase S (XCC3381) 及 pectate lyase II (XCC2815)。 此一結果顯示至少在此三個胞外蛋白, PilD 應該不是負責胞外蛋白分泌的 signal peptidase。
已知在 Xcc, 當 clp 基因破壞後, 多種胞外酵素包括 pectate lyase 的產量會減少。 收取等菌量的 P20H 及 clp 突變株之胞外蛋白, 經 SDS-PAGE 電泳分離, 發現 pectate lyase II (PelB) 於 clp 突變株中的蛋白產量較 P20H少, 推測 PelB 的蛋白表現也受 Clp 調控。 本實驗另外將 pelB 基因予以 insertional mutation 突變後, 進行胞外果膠酶的活性染, 分析得知 pelB 缺失並不影響果膠酶的活性 (平板上分解果膠形成澄清暈圈的能力), 推測 PelB 應非 Xcc 的主要果膠酶。 在致病性測試的結果顯示, pelB 突變後, 有延緩病徵出現的現象。 另外, 將 pelB 基因可能包含啟動子的區域, 選殖到泛寄主載體 pFY13-9 (為一 promoter-probing vector), 結果顯示 pelB-lacZ transcriptional fusion 之啟動子活性於 clp 突變株及 rpfF 突變株都很低, 亦即 Clp 及RpfF 為 pelB 表現所需之轉錄調控因子。

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium causing black rot in cruciferous plants. The virulence of Xcc depends on a number of pathogenic genes and virulent factors including the exopolysaccharide and extracellular enzymes such as proteases, endoglucannases, and pectinases. It is known that extracellular enzymes in various Gram-negative bacteria are secreted by type II secretion pathway using PilD as the signal pepetidase for N-terminus processing.
A pilD mutant, MC1220 (pilD::pSMP104), has previously been constructed from Xcc strain P20H in our laboratory. Plate assays for the extracellualr enzymes showed that pilD mutant exhibited the same levels of activity as those in the wild-type cells. To compare the N-terminal sequence, extracellular proteins from the culture supernatants of P20H and MC1220 (pilD::pSMP104) were separated in SDS-polyacrylamide gel electrophoresis, transblotted onto Nylon membrane, then three of the proteins (24, 28 and 35-kDa) were subjected to chemical determination of the N-terminal sequences. The results showed that each of the protein pairs has the same terminal ends. N-terminal sequencing also identified the 24, 28, and 35-kDa proteins as the conserved hypothetical protein (XCC0694 in the genome of Xcc strain ATCC33913), cellulose S (XCC3381), and pectate lyase II (XCC2815, designated as PelB herein), respectively. These results indicate that PilD is not the signal peptidase responsible for the N-terminus processing during secretion of these extracellular proteins.
In a separate experiment, SDS-PAGE showed that the amounts of extracellular PelB were significantly reduced in a mutant deficient in Clp, a transcription factor homologous to CRP (cyclic AMP-receptor protein), suggesting that expression of pelB is regulated by Clp. Because of the latter finding, pelB was further studied. The results indicate that 1) the pelB mutant of Xcc acquired by insertional mutation showed no change in plate assays for pectinolytic activity, indicating that PelB is not the major pectate lyase in Xcc, 2) in pathogenicity testing using pelB mutant, appearance of symptoms was delayed compared with that of the wild-type virulent strain, and 3) in transcriptional fusion (pelB-lacZ) assays, the promoter activities were found to be greatly reduced in clp and rpfF mutant; suggesting that transcription of the pelB gene is positively regulated by Clp as well as RpfF.

中文摘要 -------------------------------------------------- 1
英文摘要 -------------------------------------------------- 3
縮寫字對照表 ------------------------------------------------ 5
壹. 前言----------------------------------------------------- 8
貳. 材料---------------------------------------------------- 14
参. 方法 --------------------------------------------------- 18
一. 細菌的培養和保存 -------------------------------------- 18
二. DNA之製備---------------------------------------------- 18
1. 小量質體之製備質體 ------------------------------------ 18
2. 細菌染色體 DNA 之抽取---------------------------------- 18
三. 聚合酶連鎖反應 (polymerase chain reaction, PCR)-------- 19
四. 洋菜膠電泳分析 (agarose gel electrophoresis)----------- 19
五 質體選殖 (clonig)--------------------------------------- 20
1. PCR 增幅片段之選殖------------------------------------- 20
2. 限制酶切割 ( restriction enzyme digestion)------------- 20
3. DNA 片段回收------------------------------------------- 20
4. DNA 黏接反應(ligation)-------------------------------- 20
六. 細菌之轉形作用(transformation)------------------------- 21
1. Escherichia coli (大腸桿菌) 之轉形--------------------- 21
(1) 勝任細胞 (competent cells) 之製備------------------- 21
(2) 轉形作用 ------------------------------------------ 21
2. X. campestris pv. campestris 之轉形-------------------- 22
(1) X. campestris pv. campestris 勝任細胞之製備 -------- 22
(2) 轉形作用 ------------------------------------------ 22
七. 點測試法 (spot test) ---------------------------------- 22
八. 平版測試胞外果膠酶之活性------------------------------- 23
九. 以 TCA 沉澱的方式收取胞外蛋白-------------------------- 23
十. 以硫酸銨將胞外蛋白進行分割試驗------------------------- 23
1. 收取X. campestris pv. campestris 之胞外蛋白------------ 23
2. 以硫酸銨做蛋白分割------------------------------------- 24
十一 供 N 端蛋白定序之前處理------------------------------- 24
1. SDS-polyacrylamide gel 之電泳分析---------------------- 24
(1) SDS-聚丙烯醯胺凝膠之配製---------------------------- 24
(2) 電泳分析-------------------------------------------- 25
2. 蛋白質轉漬及 N 端定序前之處理-------------------------- 25
十二. 啟動子之活性分析------------------------------------- 25
1. 培養方式----------------------------------------------- 25
2. -galactosidase 之活性測試---------------------------- 25
十三. 致病性測試------------------------------------------- 26
肆. 結果
第一部份: X. campestris pv. campestris 內 signal peptidase 之 探討 ------------------------------------------------------- 27
一. 野生株 P20H及 MC1220(pilD::pSMP104) 之胞外蛋白的分析--- 27
1. 以TCA 沉澱野生株 P20H所產生之胞外蛋白------------------ 27
2. 以硫酸銨將 P20H 及MC1220(pilD::pSMP104) 所產生的胞外蛋白進行分割------------------------------------------------------ 27
二. 野生株 P20H 及 MC1220(pilD::pSMP104)之胞外蛋白 N 端定序結果及分析---------------------------------------------------- 28
三. 預測signal peptidase 的切位---------------------------- 29
第二部份: 胞外酵素 PelB 之探討----------------------------- 29
一. 野生株P20H 和TC820之 PelB的表現------------------------ 30
二. PelB 於蛋白表現載體之蛋白表現-------------------------- 30
1.質體 pETpelB 之構築------------------------------------- 30
2. BL21(DE3)(pETpelB) 之蛋白表現-------------------------- 31
第三部份: pelB 基因之探討--------------------------------- 31
一. pelB 之轉錄調控分析------------------------------------ 31
1. pelB 上游片段之選殖與質體構築-------------------------- 31
2. 轉型株之篩選------------------------------------------- 32
3. pelB 基因啟動子之表現情形------------------------------ 32
二. pelB 突變株之特性分析---------------------------------- 33
1. pelB 突變株之構築------------------------------------- 33
2. 以平板測試分解果膠酶之能力----------------------------- 34
3. pelB 突變株之致病性測試 (pathogenicity assay)---------- 35
伍. 討論---------------------------------------------------- 36
陸. 參考文獻------------------------------------------------ 41
柒. 表------------------------------------------------------ 48
捌. 圖------------------------------------------------------ 53

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