建立穩定螢光標記轉殖內生菌株及其於植物組織內之定位研究

Development of Stable Fluorescent Markers in Bacterial Endophytic Transformants and Their Localization in Plant Tissue

黃偉哲(Wei-Che Huang)

指導教授 ： 楊禮亘

國立虎尾科技大學/文理學院/生物科技系碩士班/碩士(2017年)

摘要

近年來有許多文獻指出由植體內分離的植物內生菌株回感染到植株能使植株有顯著的生長促進或抗病蟲害效果，然而菌株是否進入植株體內且在植物體內之期程卻鮮少人探討。為了確認菌株是否有進入植體內，本研究將有益內生菌編號LS004 (Rhizobium daejeonense)、LS077 (Curtobacteriumcitreum)、LS079 (Rhizobium radiobacter)、與LS183 (Rhizobium leguminosarum)藉由電穿孔技術轉殖入帶有綠色螢光基因(Green Fluorescent Protein，GFP)的質體，使內生菌株表現螢光，再將具有螢光的內生菌感染植株，輔以螢光顯微鏡觀察以研究植物與微生物之共生關係。結果發現GFP標記的內生菌株LS004與LS079在感染水稻種子之後，在螢光顯微鏡下可在水稻植株根部的木質部與葉部的葉肉組織觀察到LS004，而LS079在水稻植株根部的內皮層觀察到，這意謂著雖然內生菌是分離自水稻植株的根部，不僅只生存於根部，也可以存活於葉部，也代表著內生菌可經由根部移動到地上部。同時，我們也將此兩種水稻內生菌感染至水稻植株體內，發現植株15天齡與30天齡實驗組葉綠素含量低於控制組，60天齡葉綠素含量與實驗組相比沒有顯著差異，然而90天齡實驗組葉綠素含量與控制組相比增加11 %。我們於120 天收取成熟的水稻種子，實驗組與控制組相比，稔實率降低了1.6 %，百粒重增加了1.6 % ，穗長增加了17 %，穗重增加了4.7%。將收取成熟的水稻種子，運用萃取內生菌與基因鑑定的方式，驗證感染過具有GFP內生菌的植株是否會遺傳到下一代，結果在本實驗中無法偵測到種子內有GFP基因。此外，為了確定分離自水稻植株的內生菌是否具有跨物種生存及應用的能力，我們將轉殖GFP的內生菌感染至小白菜以觀察在不同作物的結果，結果顯示LS004、LS077、LS079、與LS183可分別存活於7天齡小白菜的根部與葉部，意味著這些內生菌具有跨物種生存的潛力，在未來要開發生物性肥料將可為一項有利的依據。在未來我們可以將這些結果應用於研究內生菌在植物體內的位置，藉此以研究與探討內生菌在植物體內如何促進植物生長或是如何抵抗外來病原菌的相關作用機制。

關鍵字

綠色螢光蛋白 ； 內生菌 ； 水稻 ； 電穿孔 ； 定位 ； 根瘤菌(Rhizobium)

並列摘要

There are many recent reports on the beneficial effects of plant bacterial endophytes on growth promotion and disease resistance. However, very little attention has been focused on the actual colonization of endophytes in planta through inoculation. In order to confirm whether endophytes could colonize plant tissues, we have selected beneficial endophytic bacterial strains LS004 (Rhizobium daejeonense), LS077 (Curtobacteriumcitreum), LS079 (Rhizobium radiobacter) and LS183 (Rhizobium leguminosarum) and introduced the Green-Fluorescent Protein (GFP) plasmid into these bacteria using an electroporation approach. Transformants were confirmed by PCR and fluorescent microscopy and used to understand endophytic relationship between plant and microbes. Rice seeds were inoculated with GFP-tagged strains LS004 and LS079. LS004-GFP was present in rice root xylem and the leaf mesophyll cells, while LS079-GFP was present in the root endodermis. These results suggest that although these endophytic bacteria were originally isolated from rice roots, they can translocate and survive in the leaf as well. Meanwhile, we inoculated two beneficial endophytic bacterial strains LS004 and LS079 to inoculate into rice plant and test their growth promoting properies. We found 15-day old and 30-day old experimental group chlorophyll content were lower than the control group, 60-day old chlorophyll content had no significant difference as compared to the control group, but 90-day old experimental group chlorophyll content were significantly higher than the control group. We harvested matured rice and measured ear of mass, ear of length, hundred grain weight and fertility rate. We found the experiential group compared with control group have increased ear of mass 4.7%, ear of length 17% and hundred grain weight 1.6%, decrease fertility rate 1.6%.We used matured rice seeds to confirm whether the GFP endophytic bacteria could be passed to the next generation by extracting endophytic bacteria and PCR the GFP gene. In result, we cannot detect GFP gene in the rice seed. In addition, we want to examine whether endophytic bacteria from rice have the ability to survive in other crops, we used GFP endophytic bacteria to inoculate into Brassica rapa chinensis (bok choy). Results showed that we can observe LS004 (Rhizobium daejeonense), LS077 (Curtobacterium citreum), LS079 (Rhizobium radiobacter) and LS183 (Rhizobium leguminosarum) in the root and leaf of 7-day old bok choy. The result indicates that these endophytes exhibit cross species potential for developing of bio-fertilizer. This result could provide us a platform for future research in understanding localization of endophytic bacteria in the plant to research interaction mechanism of plant growth or disease resistance in plants.

並列關鍵字

Green-Fluorescent Protein ； Endophyte ； Rice ； Electroporation ； Localization ； Rhizobium

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