臺灣博碩士論文加值系統

革蘭氏陰性植物病原菌 Xanthomonas campestris pv. campestris (Xcc)，主要感染十字花科植物造成黑腐病 (black rot)，導致農業上的重大損失。本菌尚能分泌大量胞外多醣以及多種胞外酵素，如蛋白酶、纖維酶、聚甘露糖酶和果膠酶等，為本菌重要的致病因子。這些胞外產物的產生，由訊號擴散因子 (diffusible signal factor, DSF)，進行細胞與細胞間訊息的傳遞調控。DSF 的合成需要 RpfF (putative enoyl-CoA hydratase)，而訊號的感應與傳遞則是由 RpfC 及 RpfG 組成之双成員 (two component system) 負責。最近研究也指出多元轉錄因子 Clp (cAMP receptor protein-like protein) 蛋白，對於 DSF 調控毒力因子產生是必要的。
本研究主要探討 Xcc 果膠酶中之果膠甲基酯酶 (pectin methyl esterase, PME) 基因 pmeA。首先構築 pmeA 突變株 YL17 及其互補株 YL17(pRKpmeA)，並與不同 Xcc 菌株比較胞外 PME 活性。結果顯示 AU56E (clp 基因缺損突變株)、RM17F (rpfF 基因缺損突變株) 及 YL17 之 PME 活性輕微下降，而 YL17(pRKpmeA) 可回復 YL17 產生 PME 之能力。由此推測 Clp 及 RpfF 均參與 PME 的表現，而 pmeA 主導 Xcc 之次要 PME。在致病性測試上，YL17 病癥出現的速度與 Xc17 相比只具輕微差異，推測 PmeA 在 Xcc 致病性方面可能扮演次要的角色。其次，利用 5’-RACE (rapid amplication of cDNA ends) 技術來訂定 pmeA 之轉錄起始點。結果得知，pmeA 之轉錄起始點為核苷酸“G”，位於起始密碼上游 54 nt 處。最後，將不同長度的 pmeA 上游片段，個別選殖於 pFY13-9 (為 promoter-probing vector) 中 lacZ 之上游，獲得之 PpmeA-lacZ transcriptional fusion constructs 再經電孔法分別送入 Xc17、AU56E 與 RM17F。啟動子活性分析指出 pmeA 之轉錄起始點上游 -359 至 +46 的區域，為 pmeA 完全表現所必需。轉錄融合分析也顯示 pmeA 啟動子活性，不論在 clp 突變株或是 rpfF 突變株，其活性均比野生株低，因此推測 Clp 與 RpfF 正調控 pmeA 之轉錄。另外，pmeA 基因之轉錄也受到代謝抑制 (catabolite repression) 作用、高滲透度 (osmolarity)、限氧 (oxygen limitation) 及缺氮 (nitrogen starvation) 所影響。

Abstract
Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative plant-pathogenic bacterium that causes black rot in crucifers, resulting in tremendous losses in agriculture. This organism is capable of producing large amounts of exopolysaccharides and secreting several extracellular enzymes (such as protease, endoglucanase, mannanase, and pectinase), which have been considered to be important virulence determinants. The production of these extracellular products is coordinated by a cell-cell communication mechanism through diffusible signal factor (DSF), which is dependent on RpfF, a putative enoyl-CoA hydratase, for synthesis and the RpfC/RpfG two component system for perception and signal transduction. A recent study also indicated that global transcription factor cAMP receptor protein-like protein (Clp) is essential for DSF regulation of virulence factor production.
The aim of this study was to characterize the pmeA gene encoding one of the pectinases, pectin methyl esterase (PME), in Xcc. First, the pmeA mutant YL17 and its complementary strain YL17(pRKpmeA) were constructed and the extracellular PME activity was compared with different Xcc strains. The result showed that the PME activity of AU56E (clp mutant), RM17F (rpfF mutant), and YL17 were slightly reduced, whereas the activity was restored in YL17(pRKpmeA). It was suggested that Clp and RpfF are involved in PME expression and pmeA gene might code for a secondary PME in Xcc. In pathogenicity test, the rate of symptoms development in YL17 was slightly slower than Xc17. These findings deduced that PmeA of Xcc plays a minor role in pathogenesis. Second, nucleotide G at 54 nt upstream of the pmeA start codon was mapped as the pmeA transcriptional initiation site by using the 5’-RACE (rapid amplication of cDNA ends) technique. Third, seven PpmeA-lacZ transcriptional fusion constructs were generated by cloning PCR fragments into the broad-host-range-promoter-probing vector pFY13-9, which uses lacZ as the reporter. The resultant PpmeA-lacZ transcriptional fusion constructs were introduced into Xc17, AU56E and RM17F by electroporation, respectively. Reporter assay indicated that the -359/+46 region contains the complete promoter and is capable of maximal-level expression. Transcriptional fusion analysis also revealed that the promoter activity of pmeA was reduced in either AU56E or RM17F, suggested that Clp and RpfF positively regulate transcription of the pmeA gene. In addition, the pmeA expression was affected by catabolite repression, osmolarity, oxygen limitation, and nitrogen starvation.

目錄
中文摘要------------------------------------------------------------------------------- 1
英文摘要------------------------------------------------------------------------------- 3
第一章 前言---------------------------------------------------------------------------- 5
一、 果膠與果膠甲基酯酶相關研究----------------------------------- 5
二、 Xanthomonas 之特性----------------------------------------------- 6
三、 Xcc 之致病機轉----------------------------------------------------- 7
四、 Xcc 胞外酵素之基因調控----------------------------------------- 8
第二章 文獻回顧---------------------------------------------------------------------- 9
一、 果膠甲基酯酶相關研究-------------------------------------------- 9
1. E. chrysanthemi PME-------------------------------------------- 9
(1) PemA----------------------------------------------------------- 10
(2) PemB----------------------------------------------------------- 14
2. R. solanacearum PME------------------------------------------- 15
3. D. carota PME---------------------------------------------------- 17
4. L. esculentum PME----------------------------------------------- 19
二、 Xcc 之胞外酵素調控相關研究----------------------------------- 19
1. rpf 基因串之相關研究------------------------------------------ 19
2. clp 基因之相關研究--------------------------------------------- 21
第三章 材料與方法-------------------------------------------------------------------- 23
一、 材料--------------------------------------------------------------------- 23
1. 菌種及質體-------------------------------------------------------- 23
2. 一般藥品----------------------------------------------------------- 23
3. 酵素----------------------------------------------------------------- 23
4. 培養基-------------------------------------------------------------- 24
(1) 液體培養基---------------------------------------------------- 24
(2) 固體培養基---------------------------------------------------- 25
5. 引子----------------------------------------------------------------- 25
二、 儀器--------------------------------------------------------------------- 25
1. 光譜儀-------------------------------------------------------------- 25
2. 桌上型微量離心機----------------------------------------------- 25
3. 桌上型低溫冷凍離心機----------------------------------------- 25
4. 電孔儀-------------------------------------------------------------- 25
5. 聚合酶連鎖反應儀----------------------------------------------- 25
6. 低溫迴轉式振盪培養箱----------------------------------------- 25
7. 紫外光照相系統-------------------------------------------------- 26
8. 電泳設備----------------------------------------------------------- 26
(1) 洋菜凝膠電泳------------------------------------------------- 26
(2) 聚丙烯醯胺凝膠電泳---------------------------------------- 26
9. 低溫反應器-------------------------------------------------------- 26
10. 其他----------------------------------------------------------------- 26
(1) 電子天秤------------------------------------------------------- 26
(2) 攪拌器---------------------------------------------------------- 26
(3) 酸鹼測定儀---------------------------------------------------- 26
(4) 振盪器---------------------------------------------------------- 26
(5) 乾浴機---------------------------------------------------------- 26
(6) 水浴機--------------------------------------------------------- 26
三、 方法-------------------------------------------------------------------- 26
1. 細菌之培養與保存---------------------------------------------- 26
2. 胞外果膠甲基酯酶之活性分析------------------------------- 27
3. DNA 的製備----------------------------------------------------- 27
(1) 小量質體 DNA 抽取法----------------------------------- 27
(2) 染色體 DNA 抽取法-------------------------------------- 28
4. 聚合酶連鎖反應 (Polymerase Chain Reaction，PCR)-- 29
5. 洋菜凝膠製備與電泳分析-------------------------------------- 30
(1) 凝膠製備------------------------------------------------------- 30
(2) 電泳分析------------------------------------------------------- 30
6. 質體的選殖-------------------------------------------------------- 31
(1) PCR 增幅片段之選殖--------------------------------------- 31
(2) 限制酶的切割分析------------------------------------------- 31
(3) DNA 回收----------------------------------------------------- 31
(4) DNA 的黏接-------------------------------------------------- 32
7. 轉形作用----------------------------------------------------------- 32
(1)E. coli 之轉形------------------------------------------------- 32
(2)Xcc 之轉形---------------------------------------------------- 34
8. 致病性測試-------------------------------------------------------- 35
9. 重組蛋白之大量表現與誘導----------------------------------- 35
10. SDS-聚丙烯醯胺凝膠電泳------------------------------------- 36
(1) 10% 聚丙烯醯胺之解析凝膠 (resolving gel) 溶液--- 36
(2) 4% 聚丙烯醯胺之集體凝膠 (stacking gel) 溶液----- 36
(3) 凝膠製備------------------------------------------------------- 37
(4) 電泳分析------------------------------------------------------- 37
11. Xcc total RNA 的抽取------------------------------------------ 37
12. cDNA 末端的快速增幅 (Rapid Amplification of
cDNA Ends，RACE)-------------------------------------------- 38
(1) 引子設計------------------------------------------------------- 38
(2) 第一股互補 DNA (complementary DNA，cDNA)
合成------------------------------------------------------------- 39
(3) 第一股 cDNA 的純化-------------------------------------- 40
(4) cDNA 的 tailing 反應-------------------------------------- 40
(5) dC-tailed cDNA 的 PCR 反應與選殖------------------- 40
(6) 巢氏 PCR 增幅---------------------------------------------- 41
13. 啟動子活性分析-------------------------------------------------- 41
(1) 培養方式------------------------------------------------------- 41
(2)

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