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研究生:程穎賢
研究生(外文):Ying-Hsien Cheng
論文名稱:擬南芥對青枯病菌的系統性抗病之探討
論文名稱(外文):Resistance of Arabidopsis thaliana to bacterial wilt caused by Ralstonia solanacearum through systemic
指導教授:楊長賢楊長賢引用關係
指導教授(外文):Chang-Hsien Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:68
中文關鍵詞:系統性抗病擬南芥青枯病菌
外文關鍵詞:systematic requared resistanceArabidopsis thalianaRalstonia solanacearum
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青枯病菌(Ralstonia solanacerarum)會引起擬南芥(Arabidopsis thaliana)部分生態型之明顯的細菌性萎凋病產生病徵,為一個可行性高的模式系統來探討寄主植物與其病原菌的相互關係。以致病性較弱的青枯病菌Ps95品系穿刺擬南芥S96生態型的葉片後,在穿刺點周圍會出現壞死現象,但植株不會產生更嚴重的病徵;但以致病性較強的青枯病菌Rd15品系穿刺S96生態型的葉片,則會在穿刺十天後造成葉片萎凋,且其病徵會有系統性的在兩星期內蔓延到整株植株,甚至造成植株死亡。先以致病性較弱的青枯病菌Ps95穿刺S96生態型,再以致病性較強的Rd15進行挑戰穿刺,則植株所產生的病徵有降低的情況產生。本研究藉用擬南芥為實驗材料,進行擬南芥對青枯病菌感染後所引發的系統性抗病現象的探討。根據之前所觀察到的初步現象,設計在不同時間點進行二次穿刺,若以致病性較弱的青枯病菌Ps95先穿刺S96生態型,再於穿刺後一到五天以致病性較強的Rd15進行穿刺,則植株所產生的病徵會有明顯的延遲與減緩現象產生。由此結果推論在穿刺較弱的Ps95之後,在整株擬南芥S96植株中均會引起類似系統性抗病機制(Systematic Acquired Resistance)的反應。進一步分析與抗病性相關基因PR-1(pathogen-related gene)和GST1(glutathione S-transferase)的表現,證實這些基因不只在穿刺葉中被誘導表現,也於穿刺Ps95之後6-12小時後,在非穿刺葉中有被誘導的反應表現。由此結果推論擬南芥抵抗青枯病菌的機制,與其他植物疾病中所觀察到的系統性抗病反應非常相似。進一步以Rd15接種擬南芥NPR1(Non-pathogen- related gene;系統性抗病的一個關鍵調控基因)轉基因植株,結果發現其抗病性明顯加強,更得到進一步的結果支持以上的推論。

Rastonia solanacearum strains have been shown to cause bacterial wilt in certain ecotypes of Arabidopsis thaliana. After inoculating the leaves of S96 ecotype with less aggressive R. solanacearum strain Ps95, necrosis around the inoculation site rapidly appeared and no symptoms were developed further. Leaves of S96 ecotype completely wilted 10 days after inoculation with aggressive R. solanacearum strain Rd15, and symptoms spread systematically throughout the whole plant within two weeks after inoculation. Interestingly, the disease symptom was significantly delayed in Ed15-inoculated S96 plants one to five days after inoculation with strain Ps95. This result suggested that a mechanism similar to systemic acquired resistance (SAR) was triggered in whole plant of Arabidopsis S96 after inoculation with Ps95. Further analyzes on the expression of defense-related genes, PR-1(pathogen-related gene 1)and GST1(glutathione S-transferase) indicated that expression of these genes was not only up-regulated in inoculated leaves but also in non-inoculated leaves 6-12 hours after inoculation with Ps95. The result suggested that the resistance of Arabidopsis to R. solanacearum is due to a response similar to the SAR observed in other plant disease. Analyzing transgenic Arabidopsis plants ectopically expressed NPR1 gene, a key regulator of SAR, further supported this assumption. And showed significant enhancement of the resistance to severe strain Rd15

I. Abstract(English)……………………………………3
II. Abstract(Chinese)……………………………………4
III.Introduction ………………………………………….. 6
IV. Materials and Methods ………………………………. 14
A. Bacterial strains and cultivations
B. Arabidopsis thaliana resource and cultivation
C. Bacterial inoculation
D. Disease assessment
E. Induction treatment and symptoms observation
F. Analysis of defense gene ( PR-1 and GST1 ) expression in
infected plants
G. Disease assessment contrast to NPR1 gene expression in
infected plants
H. Purification of DNA fragment
I. Preparation of DNA probe
J. RNA extraction
K. RNA gel blot analysis
L. Quantitative reverse transcriptase-polymerase chain
reaction (RT-PCR)
M. Southern blot analysis
V. Results ...……………………………………………… 23
A. The mechanism of resistance of Arabidopsis to Ralstonia
solanacearum is similar to systematic acquired rsistance.
B. Similar expression of PR-1 and GST1 mRNAs was induced in
susceptible Arabidopsis leaves after inoculation with
Ps95.
C. NPR1 over-expression plants display a significant
enhancement of the resistance to R. solanacearum.
D. NPR1 over-expressing transgenic plants are more
resistant to bacterial pathogen.
E. Increased PR-1 mRNA in NPR1-overexpression transgenic
plants is associated with increased NPR1 gene expression.
F. GST1 and Cu, Zn SOD mRNAs were elevated in NPR1-H
transgenic plants associated with different resistant
abilities.
VI. Discussion …………………………………………… 30
VII.Reference ………………………………………..… 36
VIII.List of Figures ……………………………………… 46
IX. List of Tables …… ……………………………………57
X. Appendix ……………………………………………59
XI. Abbreviations ……………………………………… 68

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