臺灣博碩士論文加值系統

瓜類細菌性果斑病 (Bacterial fruit blotch disease, BFB) 是由Acidovorax avenae subsp. citrulli所引起的細菌性病害，可藉由hrp/hrc基因組所組成的第三型分泌系統分泌致病相關的蛋白質至植物細胞內，以干擾植物的生理生化反應和防禦機制並引起寄主病害，及引起非寄主植物的過敏性反應。而分離自不同寄主的A. avenae菌株，在hrp/hrc基因組於GALA和hrcT之間約4 kb的區域具有變異性，例如：苦瓜果斑病菌Aac156含有hrpW和transposase兩個基因；西瓜果斑病菌Aac148及Aac31則含有兩個PE_PGRS基因，分別命名為Aave_0456和Aave_0457。本研究利用E. coli BL21 (λDE3) 大量表現Aac148之Aave_0457蛋白質以製備其多株抗體，並以此抗體進行西方墨點法分析，顯示於XVM2培養基誘導下可發現Aave_0457蛋白質藉由第三型分泌系統分泌至胞外。經胺基酸序列比對後，發現Aave_0457蛋白質N端不具有分泌訊號序列，與Aac156之HrpW harpin domain具有高度相似性，且於E. coli BL21 (λDE3) 中所誘導表現的Aave_0457蛋白質具有熱穩定性，於非寄主菸草可誘發過敏性反應，推測Aave_0457蛋白質為一個harpin類蛋白。進一步構築Aac31之Aave_0457非極性突變株，此Aave_0457基因的缺失會些微降低病原菌於菸草誘發過敏性反應的能力，以及降低寄主植株的罹病度和發病率，但缺失株於寄主西瓜葉片內的族群菌量卻與野生株無顯著差異。另外本研究利用gfp作為報導基因 (reporter gene)，證實Aave_0457基因上游的非轉譯區域不具有啟動子活性，因此推測hrcT、Aave_0456和Aave_0457同屬於一個操縱子 (operon)。

Acidovorax avenae subsp. citrulli (Aac), which causes bacterial fruit blotch disease (BFB) in Cucurbitaceae, is a gram-negative, rod-shaped plant pathogen. The ability of Aac to grow and cause diseases in plants is dependent on the injection of multiple effector proteins into plant cells via the type III secretion system (T3SS), encoded by hrp/hrc genes cluster. Effectors contribute to pathogenesis by suppressing plant defenses and promoting disease symptom in host plants. In nonhost plants, it can elicit the hypersensitive response (HR). However, the DNA sequence between GALA and hrcT in hrp/hrc genes cluster of Aac strains from different hosts are variable. For example, Aac156 isolated from bitter gourd contains hrpW and transposase in this region, but Aac148 and Aac31 isolated from watermelon contain two putative PE_PGRS genes, named Aave_0456 and Aave_0457. The recombinant Aave_0457 protein was overexpressed in E. coli BL21 (λDE3) and applied to raise polyclonal antibody in rabbit. According to western blotting results, Aave_0457 was induced in XVM2 minimal medium and secreted into medium. The amino acid sequence of Aave_0457, which has no N-terminal signal peptide, is homologous to the harpin domain of HrpW in Aac156. The recombinant Aave_0457 protein overexpressed in E. coli BL21 (λDE3) is heat-stable and can elicit the HR in nonhost plant after 24 hours post inoculation, suggesting that Aave_0457 is a harpin-like protein. Aave_0457 mutant elicited the delayed HR on nonhost tobacco leaves, and reduced the disease incidence and disease severity in host plant, but the growth of Aave0457 mutant and wild type was no significant difference in Cucurbitaceae. Furthermore, the non-coding region between Aave_0457 and Aave_0456 might be promoter-less, suggesting that hrcT, Aave_0456 and Aave_0457 belong to an operon.

中文摘要...................................................i
Abstract..................................................ii
縮寫字對照表.............................................iii
壹、前言...................................................1
貳、材料與方法.............................................9
一. 供試的菌株與質體及菌體生長環境...................9
二. 重組DNA技術......................................9
三. 聚合酶連鎖反應...................................9
四. 勝任細胞的製備..................................10
五. 核酸序列的定序與分析............................10
六. 構築西瓜果斑病菌Aac31 Aave_0457非極性突變株.....11
七. 三親本接合作用與標記置換突變....................11
八. 西瓜果斑病菌染色體DNA的製備.....................12
九. 南方雜合反應....................................13
十. 構築西瓜果斑病菌Aac31 Aave_0457基因互補株及
Aave_0457蛋白質過表現株.........................14
十一. 利用T7 RNA polymerase-dependent system於E. coli
BL21 (λDE3) 中大量表現Aave_0457蛋白質..........15
十二. 製備Aave_0457多株抗體...........................16
十三. 西方墨點法......................................17
十四. 誘導與偵測Aave_0457蛋白質於西瓜果斑病菌內的表現.18
十五. 非寄主菸草之過敏性反應測定......................18
十六. 寄主西瓜之病原性測定............................19
十七. 西瓜果斑病菌於寄主植株葉片內的生長測定..........20
十八. 啟動子活性測試..................................20
參、結果.................................................22
一. 西瓜果斑病菌Aac148 Aave_0457 (PE_PGRS) 基因序列之
分析............................................22
二. 選殖西瓜果斑病菌Aac31位於GALA (aac1) 和hrcT區域間
的基因片段......................................22
三. 利用T7 RNA polymerase-dependent system於E. coli
BL21 (λDE3) 菌株中誘導表現Aac148 Aave_0457蛋白
質..............................................23
四. 誘導表現Aave_0457蛋白質以製備Aave_0457多株抗體..23
五. 偵測西瓜果斑病菌Aac148 Aave_0457蛋白質，以及苦瓜果
斑病菌Aac156 HrpW蛋白質於E. coli BL21 (λDE3) 菌株
中的表現........................................24
六. Aave_0457蛋白質於菸草葉片上引起過敏性反應的情形.24
七. 西瓜果斑病菌Aac31 Aave_0457::nptII非極性突變株的構
築與確認........................................25
八. 西瓜果斑病菌Aac31 Aave_0457基因互補株及Aave_0457蛋
白質過表現株的構築與確認........................26
九. 偵測Aave_0457蛋白質於西瓜果斑菌中的表現.........26
十. Aac31野生株、Aave_0457基因缺失株、Aave_0457基因互
補株及Aave_0457蛋白質過表現株的病原性分析.......27
十一. 啟動子活性測試..................................29
肆、討論.................................................30
伍、參考文獻.............................................37
陸、圖表.................................................48

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