臺灣博碩士論文加值系統

西瓜為台灣地區重要的葫蘆科作物。Fusarium oxysporum f. sp. niveum (Fon)所引起的西瓜蔓割病是西瓜生長期影響最大的真菌病害之一，對西瓜產量造成極大的損失。西瓜蔓割病(Fusarium wilt of watermelon)為土壤傳播型病害，西瓜蔓割病菌可經由種子帶菌或土壤中殘存的孢子發芽侵入植株根莖組織，切開罹病植株的維管束可見明顯褐化病徵，致使植株萎凋死亡，其厚膜孢子可在土壤中殘存數年仍可發芽，嚴重影響西瓜連作地區的產量。本研究將西瓜蔓割病菌Fon H0103 菌株以農桿菌轉殖法(Agrobacterium tumefaciens-mediated transformation)獲得帶有綠螢光蛋白(green fluorescent protein)基因插入的Fon轉殖菌株共30株，在經過接種測試後，得知轉殖菌株Fon-Y1對藍寶(Grand Baby)西瓜的致病能力幾乎喪失，其他轉殖菌株的致病能力則與H0103野生型菌株相近(共25株轉殖菌株)或毒力稍微降低(共四株轉殖菌株)或上升(僅有一株轉殖菌株)。所有轉殖菌株在產孢能力上與H0103野生型菌株並無明顯差異，惟Fon-Y1轉殖菌株在半糖馬鈴薯葡萄糖瓊脂平板(potato dextrose agar, PDA)培養基上之氣生菌絲生長減少且色素沉積減少。利用共軛焦顯微鏡觀察極低毒力的Fon-Y1轉殖菌株和與H0103野生型菌株毒力相近之Fon-X3轉殖菌株於感病品種蜜寶西瓜(Sugar Baby, SB)與抗病品系JSB西瓜根部之侵入感染情形，得知兩轉殖菌株在接種後第一天的發芽與侵入能力在SB與JSB西瓜根部皆無明顯差異，接種後第三天即可見到Fon-X3菌絲進入SB維管束中柱進行快速纏據，而Fon-Y1菌絲侵入速度較慢；Fon-X3菌株於接種後第八天即可在SB西瓜根部觀察到產孢構造形成，但是Fon-Y1菌株在接種後第十二天仍無法觀察到產孢現象，也無法如同Fon-X3轉殖菌株一般形成濃密的菌絲網，不過，在SB西瓜的莖基部都有觀察到此二轉殖菌株的纏據。此外，Fon-X3與Fon-Y1轉殖菌株在侵入感染JSB西瓜根部的速度較感染SB西瓜根部為慢，二轉殖菌株在JSB西瓜根部只會沿著維管束方向生長，並且都不會形成濃密的菌絲網，在接種後第十二天也無法觀察到產孢現象，更不會感染到JSB西瓜的莖基部。以南方墨點法確認Fon-Y1轉殖菌株只有一個T-DNA插入位點，進一步利用熱不對稱交錯聚合酶連鎖反應(thermal asymmetric interlaced PCR, TAIL-PCR)分析Fon-Y1轉殖菌株的T-DNA插入點之鄰近序列，經核酸序列比對後，得知其與番茄萎凋病菌F. oxysporum f. sp. lycopersici 4287 菌株的一個未定性蛋白(hypothetical protein) 之基因序列相似，其DNA序列相同度為99%，而此F. oxysporum f. sp. lycopersici 4287 菌株的未定性蛋白序列與Purpureocillium lilacinum 真菌之MFS maltose permease partial mRNA (XM_018323113.1)的DNA序列相同度為67%，又與Metarhizium majus ARSEF 297 菌株之MFS maltose permease partial mRNA (XM_014724167.1)的DNA序列相同度為65%，因此推測此未定性蛋白可能是麥芽糖通透酶。由碳氮素源生長測試結果顯示，轉殖菌株Fon-Y1在分別含有半乳糖和尿素的Czapek氏培養基上的生長勢較差，推論其可能是T-DNA插入使其無法有效利用半乳糖和尿素作為其碳氮素營養源。病原菌和寄主植物之間的交互作用機制複雜，未來可透過基因剔除的轉殖菌株深入研究此基因的功能。

中文摘要……………………………………………………………………………...i
Abstract…………………………………………………………………………….. iii
目次…………………………………………………………………………………...v
表次索引…………………………………………………………………………….vii
圖次索引…………………………………………………………………………….viii
附錄索引……………………………………………………………………………..ix
前言………………………………………………………………………………..…1
材料與方法…………………………………………………………………………..4
供試菌株來源與培養…………………………………………………..………4
以帶綠螢光蛋白基因之農桿菌轉殖Fon..……………………………………..4
真菌基因組(genomic) DNA之萃取……………………………………………4
以聚合酶連鎖反應(polymerase chain reaction, PCR)增幅轉殖菌株中之綠螢光 蛋白基因…………………………………………………………...…………....5
DNA探針製作………………………………………………………………….5
南方雜合法……………………………………………………………………...6
分生孢子產量測試……………………………………………………………...7
轉殖菌株對培養條件酸鹼值需求測試………………………………………...7
轉殖菌株於PCNB選擇性培養基上之生長測試………………………….….7
以帶綠螢光蛋白基因之Fon轉殖菌株接種西瓜………………………….…..7
以共軛焦顯微鏡觀察轉殖菌株侵入感染與纏據抗感病西瓜根部之情形…..8
TAIL PCR (thermal asymmetric interlaced PCR)及其產物序列分析...………..8
轉殖菌株對碳素源需求測試………………………………………………..…9
轉殖菌株對氮素源需求測試…………………………………………………..9
結果…………………………………………………………………………………..10
以農桿菌轉殖綠螢光蛋白基因………………………………………………..10
以聚合酶連鎖反應增幅轉殖菌株中之綠螢光蛋白基因……………………..10
以帶綠螢光蛋白基因之Fon轉殖菌株接種西瓜………………………………10
轉殖菌株之菌落型態觀察……………………………………………………..10
轉殖菌株之分生孢子產量測試…………………………………………….….11
轉殖菌株在不同酸鹼值下之生長情形………………………………………..11
轉殖菌株於PCNB選擇性培養基之生長與菌落型態……………………….11
以共軛焦顯微鏡觀察轉殖菌株侵入感染與纏據抗感病西瓜根部之情形….11
Fon-Y1轉殖菌株T-DNA插入點鄰近序列之解序與NCBI序列比對結果..12
轉殖菌株對不同碳素源之需求測試………………………………………......13
轉殖菌株對不同氮素源之需求測試………………………………………......13
轉殖株T-DNA插入情形………………………………………………………13
討論…………………………………………………………………………………..15
參考文獻……………………………………………………………………………..20
圖表………………………………………………………………………………......24
附錄……………………………………………………………………………...…...51

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