Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/23058
標題: 利用農桿菌轉殖冰花培養細胞系統之建立
Establishment of Agrobacterium-mediated transformation system in Mesembryanthemum crystallinum L.
作者: 陳曉慧
Chen, Hsiao-Huei
關鍵字: 冰花;Agrobacterium-mediated transformation;農桿菌轉殖;Mesembryanthemum crystallinum L.
出版社: 生命科學系所
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摘要: 
冰花為耐鹽模式植物,是一種研究滲透性逆境之生理生化特性之模式植物,因冰花的轉殖與再生不易,使探討耐鹽相關分子機制受到侷限,故本論文以農桿菌轉殖技術,建立冰花培養細胞之轉殖系統。本論文以農桿菌轉殖之影響因子,農桿菌濃度、共培養時間以及冰花培養細胞生長階段此三因子來進行探討。
為了測得冰花培養細胞轉殖之最佳條件,使用農桿菌菌系EHA105內含GUS (β-glucuronidase)報導基因與nptII (neomycin phosphotransferase II)基因之pBISN1質體感染冰花培養細胞,透過PCR與GUS組織化學染色法分析轉殖細胞。分別選用農桿菌濃度為1.25 x109、2.5x 109或5 x 109 cells mL-1和共培養24或48小時進行測試,發現農桿菌濃度2.5x 109 cells mL-1和共培養48小時可得2.4%的轉殖率。進而測試冰花培養細胞生長階段的影響,以生長5天大的培養細胞可獲得轉殖效率3%最高。且以懸浮培養的方式增加篩選轉殖冰花的能力,使原本只有3%的轉殖細胞提升為70%,並以PCR的方式證實轉基因成功插入基因組,說明此農桿菌系統可使GUS報導基因在冰花細胞內穩定表現。再利用農桿菌GV3101體內含有GFP (green fluorescence protein)報導基因與hpt (hygromycin phosphotransferase)篩選基因之pCAMBIA1302質體感染冰花培養細胞,以PCR方式確認GFP報導基因已插入植物基因組,進一步利用免疫標定方式偵測GFP蛋白的累積,證實GFP可累積在冰花細胞內。當以共軛焦螢光顯微鏡觀察轉殖GFP細胞,發現其螢光散發量微弱,與未轉殖細胞無法區分,故使用共軛焦螢光顯微鏡之全波段掃描技術,發現冰花原生質體的細胞膜與細胞質自體螢光波段分佈在520-540 nm,說明冰花細胞自體螢光與GFP綠色螢光之emisson重疊。為了證實已轉殖之冰花細胞可重覆進行農桿菌轉殖,將已轉殖的冰花利用農桿菌再次感染,以PCR的方式得知二次轉殖細胞同時有GUS和GFP報導基因存在,且觀察GUS染色與GFP綠色螢光,可見細胞質區域有亮點的分布,計算二次轉殖效率為11%,表示冰花細胞可以進行二次轉殖。
綜合以上結果可知,本論文已成功的使用農桿菌針對冰花細胞進行轉殖,也證實轉殖的冰花可以重覆進行農桿菌轉殖,對未來冰花耐鹽相關的研究有所幫助。

Halophyte Mesembryanthemum crystallinum L. (ice plant) is used as a model to study the physiology of osmotic-related abiotic stresses. The molecular studies are hampered by lack of efficient transformation and regeneration procedures in ice plant. To develop an efficient protocol for Agrobacterium-mediated transformation in cultured ice plant cells, the effects of three factors were examined. Factors which significantly affect the transformation frequency were the concentrations of Agrobacteria, the durations of co-culture time, and the growth stages of cells.
To determine the suitable transformation conditions of cultured ice plant cells, Agrobacterium tumefaciens strain EHA105 was used. The EHA105 harbors the pBISN1 plasmid containing a GUS (β-glucuronidase) reporter gene and an nptII (neomycin phosphotransferase II) gene. Cultured ice plant cells were incubated with Agrobacterium concentration of 2.5×109 cells mL-1 and co-cultured for 48 hours, β-glucuronidase assays showed transformation efficiency of 2.4%. As for the growth stages of cultured cells, 5-day-old cultured cells had the highest transformation efficiency (3%). Transformed cells were transferred into liquid culture media to increase the antibiotic selection efficiency and the percentage of transformed cells increased dramatically from 3% to 70%. GUS gene integration was confirmed by polymerase chain reaction (PCR). This result showed that the GUS reporter gene was stably integrated into ice plant genome. Another Argobacterium tumefaciens strain GV3101 harboring pCAMBIA1302 plasmid was also used to confirm the transformation procedures. The pCAMBIA1302 contains a GFP (green fluorescence protein) reporter gene and an hpt (hygromycin phosphotransferase) selection marker gene. The integration of GFP gene into ice plant genome was confirmed by PCR, and accumulation of GFP protein was detected by immunolabeling. Although the accumulation of GFP was found in transformed ice plant cells, the emission of green fluorescence was weak, indistinguishable from the untransformed cells. It is due to the strong autofluorescence emitted from cultured cells. The autofluorescence of cultured ice plant cells was still strong even when cell wall has been removed. Wavelength scanning of fluorescence emitted from plasma membrane and cytoplasm revealed that the wavelengths of autofluorescence and GFP emission were overlapped. Cultured ice plant cells were further tested whether cells can be transformed repeatedly. The double-transformed cultured cells containing both GUS and GFP reporter genes were confirmed by genomic PCR. Blue GUS staining and punctuate yellow-green GFP fluorescence were overlapped in cytosol, and the double transformation efficiency was estimated as 11%. The result indicated that cultured ice plant cells can be transformed repeatedly.
Based on the abovementioned results, I successfully established an efficient Agrobacterium-mediated transformation procedure for cultured ice plant cells, and also confirmed the cultured cells can be repeatedly transformed by Agrobacterium. The developed Agrobacterium-mediated transformation protocol will be helpful for exploration of the molecular mechanism of salt tolerance in ice plant.
URI: http://hdl.handle.net/11455/23058
其他識別: U0005-1401201115073300
Appears in Collections:生命科學系所

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