臺灣博碩士論文加值系統

中文摘要
十字花科黑腐病菌 (Xanthomonas campestris pv. campestris，簡稱 Xcc) 是一種革蘭氏陰性菌，感染十字花科作物造成黑腐病 (black rot)。 此菌所分泌的胞外黏多醣類 [exopolysaccharide, 簡稱 EPS， 又名黃原膠 (xanthan gum) ]，被認為是和本菌的致病力有關。此外，有一些基因如avr (avirulence) 以及參與第三型分泌系統之 hrp 基因為致病必需，而胞外酵素包括蛋白酶 （proteases）、果膠酶（pectinases）與纖維酶（endoglucanases，亦稱cellulases）等也是重要的致病因子。上述致病因子的基因之調控所知不多，但已知 rpf gene cluster (regulation of pathogenicity factors)會對胞外酵素和胞外多醣合成的轉錄加以調控。將rpf 基因突變後，即造成致病性力降低。其次，由 clp 所主導之 Clp 蛋白 （cAMP receptor protein-like protein） 為一 global transcription factor ，也調控胞外酵素以及胞外多醣之產生與分泌。

Xcc 的基因體序列上有多個果膠酶基因，而主導胞外聚半乳糖醛酸酶 (polygalacturonase) 的 pglA為其中之一。此基因之上游片段具有可能的 Clp-binding site 與 HrpX-binding 之保守性motif (稱做 plant-inducible promoter〔PIP〕box)，且有文獻指出 HrpX 與 pglA 基因調控有關。因此，本研究之目的即在於探討 pglA基因如何受Clp 、Rpf 系統、HrpX蛋白之調控。
序列分析的結果顯示， PglA 屬於 polygalacturonase 之 glycosyl hydrolase 中的 family 28 ，具有四個保留性的 domain (NTD、DD、HG、RIK)。致病性試驗結果顯示 pglA 突變株的致病力與野生株 Xc17無差異。平板試驗的結果顯示二者分解果膠的能力也相近，顯示 pglA 並不是Xcc 的主要果膠酶。為了了解 pglA的轉錄調控，本研究以 5''-RACE 之方式訂出的 pglA 轉錄起始點為位於其轉譯起始點 GTG上游 -102的 A。經由 gel retardation 實驗證實 pglA 基因的啟動子與 Clp 確實能直接結合。將不同段落的 pglA 上游區域選殖於 pFY13-9 ，構築成一系列的 transcriptional fusion constructs ，並將之送入 野生株 Xc17、 Xc17(clp::Gm)、Xc17(rpfF::Gm) 及 Xc17(hrpX::Gm) ，供 pglA 啟動子活性試驗。分析結果顯示，當菌生長於含有 0.5% PGA 的 modified XVM2 培養基時，pglA 的啟動子 (pFY-402+162)除了在hrpX 基因突變株中活性偏低之外，在 clp突變株與 rpfF突變株的活性分別約為野生株 2.27 與 1.76倍。

Abstract
The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris (Xcc) causes black rot in crucifers. This bacterium produces large mounts of an exopolysaccharide (EPS), xanthan gum, and an array of extracellular enzymes, including proteases, pectinases and cellulases. These substances are collectively required for pathogenicity. In addition to these substances, Avr (avirulence) proteins and the hrp genes encoded Type III secretory pathway are also essential for virulence. Regulation of the virulence factors in Xcc is not fully understood, but it is known that rpf (regulation of pathogenicity factors) gene cluster and the global transcription factor Clp（cAMP receptor protein-like protein）are involved in regulation of EPS and the extracellular enzymes.

In Xcc, pglA is a pectinase gene encoding one of the polygalacturonases. Sequence analysis performed previously indicated that the upstream region of this gene has a region conserved to a Clp-binding site and a putative HrpX-binding site (plant-inducible promoter, also called PIP box). The purposes of this study were to investigate the regulation of the pglA gene by Clp, rpf system and HrpX protein.

My sequence analysis revealed that PglA belongs to family 28 of glycosyl hydrolases, which typically have four conserved domains (NTD, DD, HG, and RIK). In plate assays, the pglA mutant constructed by marker exchange exhibited extracellular polygalacturonase at a level similar that of the wild-type, indicating that pglA is not the major pectinase in Xcc. In pathogenicity tests, the symptoms caused by the pglA mutant appeared slightly slower than Xc17. Using 5’RACE technique, nucleotide A at 102 nt upstream of the pglA translation initiation codon GTG was mapped as the 5’ end of pglA mRNA, the transcription star site. Gel retardation assays revealed that Clp can directly bind to the pglA promoter. Nested fragments of the pglA upstream region were separately cloned into the broad host range promoter-probing vector pFY13-9 to form transcriptional fusion constructs, each of which was then introduced by electroporation into the wild-type Xc17, clp mutant Xc17(clp::Gm), rpfF mutant Xc17(rpfF::Gm), and hrp mutant Xc17(hrpX::Gm). Results of reporter assays indicated that when the cells were grown in the modified XVM2 medium supplemented with 0.5% polygalacturonic acid, the pglA promoter activity was greatly reduced in hrpX mutant compared to that in than Xc17, but increased by 2.27 and 1.76 times in clp and rpfF mutant, respectively.

目 錄
中文摘要.................................................................................................................................i
英文摘要................................................................................................................................ii
目錄........................................................................................................................................iii
縮寫字對照表.......................................................................................................................vii
壹、前言................................................................................................................................1
貳、材料與方法....................................................................................................................7
I. 材料....................................................................................................................................7
一、菌種及質體...........................................................................................................7
二、藥品.......................................................................................................................7
三、酵素.......................................................................................................................7
四、引子.......................................................................................................................7
五、抗生素濃度...........................................................................................................7
六、培養基...................................................................................................................7
七、試劑與緩衝溶液...................................................................................................8
II. 方法.................................................................................................................................12
一、細菌之培養與保存...............................................................................................12
二、小量質體 DNA 之抽取........................................................................................12
三、染色體 DNA 抽取法............................................................................................12
四、.質體之選殖..........................................................................................................13
五、凝膠電泳分析.......................................................................................................14
六、聚合酶連鎖反應 (PCR).......................................................................................14
七、Rapid amplification of cDNA ends (RACE).........................................................15
八、轉形作用 (transformation)...................................................................................16
九、質體快速篩檢法...................................................................................................17
十、β-galactosidase 活性測定....................................................................................17
十一、南方轉漬法.......................................................................................................18
十二、平版測試胞外果膠酶之活性...........................................................................19
十三、Xc17 total RNA 的純化...................................................................................19
十四、致病性測試.......................................................................................................20
十五、gel retardation..................................................................................................20
十六、PglAhis 蛋白之純化.......................................................................................21
十七、胞內蛋白之收取..............................................................................................22
十八、西方墨點法分析 (Western blot).....................................................................22
參、結果..............................................................................................................................24
一、Xcc2266 (pglA) 基因之生物資訊探討.............................................................24
二、pglA 突變株之構築...........................................................................................24
(1).質體 pOKpglAGm 之構築.........................................................................24
2. 突變株之初步篩選.......................................................................................25
3. 以 Southern hybridization 確認突變株......................................................25
三、 病原性測試 (Pathogenicity assay)..................................................................25
四、以平板測試胞外果膠酶分解果膠能力............................................................26
五、pglA 轉錄起始點之訂定...................................................................................26
六、pglA 上游片段 gel retardation..........................................................................27
七、pglA 上游片段之選殖.......................................................................................27
八、Clp 對pg1A 基因之啟動子調控.....................................................................28
1. 含 pFY-402+162 之轉殖株的 pg1A 啟動性.............................................28
(1) 以 XOLN 為培養基的情形………………………….........................28
(2) 以 XOLN 為培養基外加 0.5 % polygalacturonic acid的情形….....28
(3) 以 XVM2 為培養基的情形………………………….........................29
(4) 以 XVM2 為培養基外加 0.5 % polygalacturonic acid的情形…….29
九、PglAhis 蛋白質大量表現...................................................................................30
十、蛋白的抗體製備.................................................................................................31
十一、PglA 之胞內蛋白表現...................................................................................31
肆、討論...............................................................................................................................32
伍、參考文獻.......................................................................................................................37
陸、表與圖...........................................................................................................................46
表. 1. 本試驗中所使用之菌種..........................................................................................46
表. 2. 本試驗中所使用之質體..........................................................................................47
表. 3. (續).本試驗中所使用之質體................................................................................48
表. 4. 在 X. campestris pv. campestris ATCC33913 基因體序列中被註解為主
導果膠酶相關活性之基因.....................................................................................49
表. 5. 在 X.campestris pv.campestris 之啟動子區域中具有完整 PIP box
的基因……………………………..…………………………...............................50
表. 6. 在 X.campestris pv.campestris 基因之中，其啓動子區域的 PIP
box 與 pglA之PIP box ......................................……………..........................51
圖. 1. PglA 及其他菌種之 polygalacturonase 活性位置比較.....................................52
圖. 2 Xc17 的 pglA 上、下游基因位置簡單示意圖..............................................53
圖. 3. 質體 pOK-pglAGM 之構築…………………….................................................54
圖. 4. 突變株 XGpglA 之構築.......................................................................................55
圖. 5. 以 partial pglA 基因為探針，利用 Southern hybridization 方法
確認突變 XGpglA ………………………………………………………….......56
圖. 6. 以 GMr cartridge 片段為探針，利用 Southern hybridization
方法確認突變 XGpglA………………………………..........................................57
圖. 7. 突變株 XMpglA 致病性之偵測………….......................................................58
圖. 8. 野生株 Xc17 、突變株 XMpglA 、Xc17(clp::Gm) 、
Xc17(hrpX::Gm) 與 Xc17(rpfF::Gm) 胞外果膠酶分泌能力之測定................59
圖. 9. pglA 轉錄起始點………………………………………......................................60
圖. 10. 以不同濃度之 Clp 與 生物素標定之 pglA 上游片段 (probe)
進行之 gel retardation 試驗.................................................................................61
圖. 11. PpglA-lacZ fusion constructs 所含 pglA 上游示意圖........................................62
圖. 12. PpglA-lacZ fusion constructs 之構築………………...........................................63
圖. 13. PpglA-lacZ fusion constructs pFY-402+162 在 Xc17、Xc17(clp::Gm)
Xc17(rpfF::Gm) Xc17(hrpX::Gm)中啟動子活性分析………………...............64
圖. 14. PpglA-lacZ fusion constructs pFY-402+162 在 Xc17、Xc17(clp::Gm) Xc17(rpfF::Gm) Xc17(hrpX::Gm) 中啟動子活性分析…….............................65
圖. 15. pET30-pglAhis 蛋白質表現載體之構築……………………………………..........66
圖. 16. 以不同濃度之 IPTG 誘導 PglAhis 蛋白質表現................................................67
圖. 17. 以 0.5 Mm IPTG 誘導不同時間後 ， PglAhis 蛋白質表現情形…….........68
圖. 18. 以 SDS-PAGE 分析經 His-binding 管柱純化的 PglA his 蛋白質…..............69
圖. 19. 野生株、 clp 、 rpfF 及 hrpX 突變株胞內蛋白之SDS-PAGE
電泳分離…………………….………………………………………………...........70
圖. 20. X. campestris pv. Campestris ATCC33913 中 PglA 以 SignalP 3.0
Server 程式預測 signal peptidase 切位 ................................................................71
圖. 21 Xc17中PglA 以 SignalP 3.0 Server 程式預測 signal peptidase 切位……...…72
陸、 附錄………………………………………………………………………………....73

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