臺灣博碩士論文加值系統

本研究應用六個隨機引子以RAPD分析20個葉表與根圈綠膿桿菌菌株間之差異性，結果顯示葉表與根圈綠膿桿菌菌株間具差異性，又根圈之綠膿桿菌菌株間之差異性較葉表之綠膿桿菌菌株間為大，除葉表分離的W135菌株外，其餘葉表分離之綠膿桿菌菌株其相似度為0.73，而與大部分根圈分離菌株較近，由根圈分離的S2菌株與葉表菌群之相似度則較其與其他根圈菌株為高。測試此些綠膿桿菌對Biolog 95種碳素源之利用情形，其中根圈菌株大部分皆會利用Tween 80及Pyruvic acid methyl ester此兩種碳素源，而大部分葉表菌株則會利用Succinamic acid、Hydroxy-L-proline及L-serine三種碳素源，然對於碳素源利用與RAPD之分析結果其間未具明顯之相關性。以隨機引子OPC11對供試之綠膿桿菌可增幅出一專一性DNA片段 (約700 bp)，此片段經選殖及進行核甘酸序列分析，其核酸序列與綠膿桿菌PAO1菌株之核酸序列高達99％相同度，依其序列所設計出之引子對PaC11-F1 / PaC11-R1，以聚合酶連鎖反應對綠膿桿菌菌株進行測試，結果皆能增幅出556 bp之DNA專一性片段。然對於17個產氰之P. fluorescens及P. putida菌株，此引子對可對其中之六個菌株增幅出556 bp之條帶；對21個不產氰之P. fluorescens及P. putida菌株，則有三個菌株能增幅出此片段。由茄科作物根圈土壤所分離之十一株綠膿桿菌菌株皆具有氰酸產生之能力，其對番茄種子發芽及生長之影響，結果顯示綠膿桿菌於菌量108 CFU/g 泥炭土其發芽率可由對照組之93％降至37％ ~ 63％間，且大部分綠膿桿菌菌株皆明顯抑制番茄幼苗根部之生長，可由對照組之8.31 cm降至4.56 ~7.01 cm間，而對植物莖部生長則無明顯之抑制現象。

壹、前言………………………………………………………………………………1
貳、材料與方法……………………………………………………………………… 5
一、供試菌株……………………………………………………………………….5
二、細菌全DNA的抽取與濃度測定……………………………………………..5
(一) 細菌全DNA的抽取……………………………………………………….5
(二) DNA濃度及純度測定……………………………………………………...6
三、隨機增幅核酸多型性分析 (Random amplified polymorphic DNA, RAPD).. .6
四、綠膿桿菌專一性DNA片段之回收及純化…………………………………..7
五、南方雜合法 (Southern hybridization)…………………………………………7
(一) 轉漬 (transfer) 及聯結 (UV-crosslinking)………………………………..7
(二) 核酸探針之製備…………………………………………………………...8
(三) 核酸雜合反應 (hybridization) 及偵測反應 (detection)…………………8
六、綠膿桿菌專一性DNA片段之選殖……………………………………………9
七、小量質體DNA (plasmid DNA) 的製備及選殖株篩選……………………..10
八、選殖株重組質體DNA嵌入片段之核甘酸定序及專一性引子對之設計…10
九、綠膿桿菌引子對專一性之測定………………………………………………11
(一) 對綠膿桿菌菌株專一性片段之增幅……………………………………..11
(二) 引子對專一性之測試……………………………………………………..11
十、測試綠膿桿菌對碳素源之利用………………………………………………12
十一、樹狀圖分析…………………………………………………………………12
十二、根圈綠膿桿菌對植物生長之影響…………………………………………12
(一) 供試植物及土壤………………………………………………………….12
(二) 接種源的製備…………………………………………………………….13
(三) 對種子發芽之影響……………………………………………………….13
(四) 對植物生長之影響……………………………………………………….13
參、結果……………………………………………………………………………..14
一、綠膿桿菌之RAPD分析…………………………………………………….14
二、綠膿桿菌RAPD分析之樹狀圖……………………………………………..16
三、綠膿桿菌對碳素源之利用…………………………………………………..16
四、綠膿桿菌對碳素源利用之樹狀圖…………………………………………..17
五、綠膿桿菌專一性DNA片段之篩選…………………………………………18
六、綠膿桿菌專一性DNA片段之選殖…………………………………………18
七、綠膿桿菌專一性引子對之設計………………………………………………18
八、引子對之專一性測定…………………………………………………………19
九、根圈綠膿桿菌對番茄生長之影響……………………………………………19
(一) 種子發芽之影響………………………………………………………...20
(二) 幼苗生長之影響………………………………………………………...20
肆、討論…………………………………………………………………………….21
伍、引用文獻……………………………………………………………………….24
陸、摘要…………………………………………………………………………….29
柒、Abstract…………………………………………………………………………30
捌、圖表…………………………………………………………………………….32
玖、附錄…………………………………………………………………………….52

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