利用農桿菌轉殖冰花培養細胞與植株系統之改良

Abstract

冰花(Mesembryanthemum crystallinum L.)為耐鹽模式植物，利用分生與生化的策略，已鑑定出許多耐鹽相關基因，但因冰花的轉殖與再生不易，使得目前尚無高效率的轉殖系統，故目前冰花耐鹽基因的功能性分析，不易在冰花體內進行須利用其它模式植物，因此本論文利用農桿菌轉殖法，嘗試建立冰花的穩定轉殖系統。依先前建立的冰花培養細胞轉殖條件，使用β-glucuronidase (GUS)報導基因，進行農桿菌轉殖冰花培養細胞系統的改良。欲加速轉殖冰花培養細胞的增殖，將農桿菌與冰花培養細胞於全光照下共培養；而為了降低實驗成本，在去除農桿菌過程中以無菌水進行清洗，僅在最後一次時加入timentin，結果轉殖的冰花培養細胞經過一年多的繼代培養，仍能表現GUS基因，顯示T-DNA可穩定的存在冰花的基因體中。進一步嘗試建立冰花植株的轉殖系統，以完整或不同部位的冰花小苗進行感染，結果以3天大剪去根尖的冰花小苗轉殖率最高為90%，GUS染色多分布於子葉和下胚軸。由於GUS染色無法觀測活細胞的動態變化，進一步選用黃色螢光蛋白(yellow fluorescence protein, YFP)報導基因，進行冰花轉殖並觀察螢光表現，結果顯示感染3天的培養細胞無法分辨YFP所釋放的螢光或是冰花細胞的自體螢光，持續將轉殖的培養細胞進行篩選培養，至長出新的癒傷組織，首先確認轉殖培養細胞的基因體DNA含有YFP基因，進一步觀察螢光的釋放，發現轉殖YFP培養細胞的細胞核、細胞質、細胞膜和細胞壁皆有黃色螢光分布，且在細胞質、細胞膜和細胞壁上有明顯的黃色亮點，顯示轉殖冰花培養細胞能穩定表現YFP基因。為了以冰花進行基因功能分析，嘗試將冰花培養細胞轉入逆境相關蛋白激酶sucrose non-fermenting 1 (SNF1)基因，並以誘導型啟動子驅動，由於轉殖的培養細胞生長緩慢，目前細胞量不足進行大規模耐鹽性測試。預備實驗則是進行未轉殖冰花培養細胞生長的耐鹽性測試，結果發現在適當的鹽分(100 mM NaCl)環境中，冰花培養細胞的生長狀況良好，而200 mM以上的鹽濃度會延緩冰花培養細胞的生長速度。綜合以上結果得知，利用農桿菌轉殖法，可成功地穩定轉殖冰花培養細胞和小苗，未來可利用冰花轉殖株，進行耐鹽基因功能的分析，以進一步了解高等植物的耐鹽機制。

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for halophytes. Many salt tolerant-related genes have been identified using molecular and biochemical approaches. Because ice plant is difficult to transform and regenerate, it lacks efficient transformation system and needs to use other model plants to analyze salt tolerant-related genes. In this thesis, I used Agrobacterium-mediated transformation to establish a stable transformation system of ice plant. In order to refine Agrobacterium-mediated stable transformation system in cultured ice plant cells, I used β-glucuronidase (GUS) reporter system previously established in the lab. For increasing the growth of transformed cultured cells, Agrobacterium and cultured ice plant cells were co-cultured under continuous light. For reducing the cost of antibiotics, Agrobacterium was washed with sterile water, and timentin was added in the final round of washing. As a result, transformed cultured cells continued to express GUS gene after being subcultured over one year indicating that T-DNA was stably integrated into ice plant genome. Furthermore, I established an Agrobacterium-mediated stable transformation system in seedlings of ice plant using intact or parts of seedlings for infection. Three-day-old cut root tip seedlings of ice plant yielded the best transformation rate of 90% with GUS staining distributed in cotyledon and hypocotyl. Yellow fluorescence protein (YFP) reporter gene was transformed into ice plant to observe fluorescence in lived cells. The result revealed that YFP fluorescence and autofluorescence of ice plant were hard to distinguish in cells infected with Agrobacterium for 3 days. Newly emerged callus transformed with YFP was obtained and the integration of YFP gene into ice plant genome was confirmed. When these cells were used to observe the fluorescence, yellow fluorescence was detected in the nucleus, cytosol, cell membrane and cell wall of YFP-transformed cultured cells with punctate spots found in cytosol, cell membrane and cell wall. The result showed that cultured ice plant cells were able to express YFP gene stably. Furthermore, in order to establish a system to study gene function in ice plant, a stress-related protein kinase sucrose non-fermenting 1 (SNF1) gene were transformed into cultured ice plant cells under the control of an inducing promoter. Because transformed cultured cells grew slowly, it was unable to perform a large-scaled salt tolerance test due to insufficient amount of cells on hand. The preliminary experiment for salt tolerance assay was done in non-transformed cultured cells. Cultured cells grew well in 100 mM NaCl condition, and the growth of cultured cells started to decrease in 200 mM NaCl or higher. In conclusion, I have successfully established an Agrobacterium-mediated transformation procedure for cultured cells and intact plants in ice plant. This is one step towards making transgenic ice plant for analyses the functions of salt-tolerance genes.