Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92106
標題: 分析大量表現McSnRK1對冰花培養細胞之影響暨分析Agrobacterium rhizogenes感染冰花之效率
Analysis of expressing McSnRK1 in cultured ice plant cells and infection efficacy of Agrobacterium rhizogenes in ice plant seedlings
作者: Hsiao-Wei Huang
黃筱薇
關鍵字: 冰花
農桿菌
ice plant
McSnRK1
Agrobacterium
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摘要: Ice plant (Mesembryanthemum crystallinum L.) can grow in salinity and drought environments. It has been used as a model plant for studying salt-tolerant mechanisms. Previous studies showed ice plant SNF1-related protein kinase1 (McSnRK1) interacted with suppressor of K+ transport growth defect 1 (McSKD1) that has been proposed to play a role in mechanisms of salt-tolerance. In order to investigate the effect of McSnRK1 on the mechanism of salt-tolerance in ice plant, this thesis used full-length McSnRK1, partial sequence containing UBA and KA1 domain of McSnRK1 (UK) in forward orientation (McSnRK1-UK-F) and reversed orientation (McSnRK1-UK-R). These sequences were introduced separately into cultured ice plant cells using Agrobacterium tumefaciens-mediated transformation. The hygromycin-resistant cells were treated with 0 or 200 mM NaCl and the growths, Na+ and K+ contents of cultured cell were measured. The results showed the packed cell volumes of WT, McSnRK1, McSnRK1-UK-F and McSnRK1-UK-R transgenic cells cultured in 0 mM NaCl was higher than cells cultured in 200 mM NaCl. The growth of transgenic cells were slower than WT cells in the beginning, but the cell volume of transgenic cells continued to increase at the end of 14 days. The highest cell volume was observed in SnRK1-transformed cells when cells were cultured in 200 mM NaCl. Under salt treatment, the cell growth rate of McSnRK1-UK-F or McSnRK1-UK-R-transformed cells were much slower suggesting that expression of McSnRK1-UK may interfere with the action of endogenous SnRK1. The sodium and potassium contents of McSnRK1 and McSnRK1-UK-F-transformed cells were measured. After salt stress for 14 days, Na/K ratio of McSnRK1-transformed cells was lower than that of SnRK1-UK-F-transformed cells, suggesting expression of functional McSnRK1 increased long-term salt tolerance of cultured cells, while expression of dominant negative form of McSnRK1 interfered with the cellular Na-K homeostasis. When the culture cells was salt-shocked in 200 mM NaCl for 6 hours, McSnRK1-expressed cells had reduced Na/K ratio. The distribution of Na+ in McSnRK1-UK-F-transformed cells changed from distributed evenly in 6 hours to punctate distribution under salt treatment for 24 hours. The results indicated Na+ compartmentation in the specific vesicles of McSnRK1-UK-F-transformed cells under salt treatment. Based on the results, expressing McSnRK1 would activate McSKD1 to increase K+ uptake and salt-tolerance of culture cells, while interfering McSnRK1 function would cause abnormal accumulation of Na+. In addition to use transgenic cultured cells for functional studies, attempt has been made in this thesis to establish root culture using A. rhizogenes-mediated transformation for functional studies at tissue and organ levels. Ice plants seedlings were infected with A. rhizogenes strains A8196, A136, A4 and 15834 separately and the numbers of lateral root were observed. Seedlings infected with A. rhizogenes strain A4 generated the highest numbers of lateral root. Further, I used A. rhizogenes strain A4 carrying GUS construct to infect intact ice plants seedlings and cut roots. The results showed no blue color was detected in all the tissues tested indicating A. rhizogenes strain A4 was unable to transform ice plants seedlings effectively. Ice plants seedlings and cut roots were transformed by A. rhizogenes strain 15834 carrying GUS construct, the transformation rate of cotyledon in 14-day-old seedlings was 100%, seedling roots was 67%, cut roots was 10%. The infected roots were further cultured in kanamycin-containing selection medium for 2 weeks. Trypan blue staining of roots transformed by A. rhizogenes strain 15834 showed similar patterns to dead roots, indicating the infected roots were sensitive to kanamycin leading to cell death. In conclusion, A. rhizogenes strain 15834 is able to transiently express target gene in ice plants seedling but the subsequent processes of screening and culturing of transformed roots are still needed to establish. Mass production of transformed hairy roots is expected to provide a useful tool for studying the mechanisms of salt-tolerance in ice plant.
冰花(Mesembryanthemum crystallinum L.)為一可於高鹽和乾旱環境生長的耐鹽模式植物,研究發現在鹽逆境下冰花SNF1-related protein kinase1 (McSnRK1)會與suppressor of K+ transport growth defect 1 (McSKD1)進行交互作用,參與冰花的耐鹽機制,為了探討McSnRK1對冰花耐鹽機制的影響,本論文將全長的McSnRK1以及僅含有UBA和KA1序列的McSnRK1-UK-F (正向)及McSnRK1-UK-R (反向)的構築以Agrobacterium tumefaciens-mediated轉殖到冰花培養細胞中,分別以0和200 mM NaCl處理後觀察培養細胞的生長及細胞內鈉鉀離子含量。結果發現WT、McSnRK1、McSnRK1-UK-F及McSnRK1-UK-R轉殖細胞在0 mM NaCl培養下細胞累積量會高於處理200 mM NaCl的培養細胞;而轉殖細胞經鹽處理後生長曲線與WT培養細胞相比皆較為平緩,細胞增加速率較慢,但在14天的觀察中細胞量持續增加,並且以SnRK1轉殖細胞在鹽處理下細胞累積量最高;其中在大量表現McSnRK1-UK-F及McSnRK1-UK-R的轉殖細胞經鹽處理後細胞累積速率緩慢,推測大量表現McSnRK1-UK-F可能會擾亂內生McSnRK1表現。進一步測試轉殖McSnRK1或McSnRK1-UK-F對培養細胞鈉鉀含量的影響,經14天培養後發現在鹽逆境下,表現McSnRK1可降低冰花培養細胞的鈉鉀比,而表現SnRK1-UK-F則會造成細胞內鈉鉀比值升高,顯示McSnRK1可增加長時間鹽逆境下培養細胞的鹽容忍度,而McSnRK1-UK-F可能會干擾正常的McSnRK1活性,導致鈉鉀離子調節機制失靈。培養細胞處理6小時200 mM NaCl後,大量表現McSnRK1可幫助降低冰花培養細胞在鹽處理下的鈉鉀比。觀察McSnRK1-UK-F轉殖細胞鈉離子的累積情形,發現McSnRK1-UK-F轉殖細胞在6小時鹽處理後細胞內鈉離子會均勻的分佈在細胞中,而增加鹽處理培養時間至24小時後,則會發現McSnRK1-UK-F轉殖細胞內鈉離子以點狀分布的形式累積在細胞質,顯示McSnRK1-UK-F會隨著鹽處理時間的增加,將鈉離子累積於細胞質特定囊泡中。綜合以上結果推論,大量表現McSnRK1會促使McSKD1活化進而增加細胞內鉀離子吸收,來提高冰花培養細胞的短時間鹽逆境容忍度;而大量表現McSnRK1-UK-F可能會干擾正常McSnRK1活性,改變鈉離子在細胞內的分佈,進而影響鈉鉀離子的調節。除了觀察細胞層次的變化,本論文亦利用A. rhizogenes來進行冰花根部的轉殖,嘗試觀察組織器官層次的變化。分別以A. rhizogenes菌株A8196、A136、A4和15834感染冰花幼苗,4週後發現以A. rhizogenes A4感染的冰花植株側根生長數量最高,進一步使用A. rhizogenes A4轉殖GUS報導基因於冰花幼苗及切離根中,結果顯示在冰花幼苗及切離根上皆無法觀察到代表GUS活性的藍色區域,因此認為A. rhizogenes A4無法對冰花幼苗及切離根進行有效的轉殖;以A. rhizogenes 15834轉殖GUS報導基因於冰花幼苗及切離根,發現在子葉的轉殖率可達到100%,而根部則以14天大幼苗最佳轉殖率約為67%,在切離根方面也有10%轉殖率。進一步將受感染的冰花根系,放入抗生素篩選培養液中2週後以trypan blue染劑進行根部染色,結果顯示15834所轉殖的冰花根系細胞染色情形與死亡的根部細胞相似,認為此感染根系不具有抗kanamycin能力,造成死亡。以上結果得知,A. rhizogenes 15834對14天大冰花幼苗有較高的轉殖能力,在冰花植株轉殖研究中可利用A. rhizogenes 15834做為轉殖菌株以提高冰花幼苗轉殖效率,但後續篩選培養流程尚需建立,期能建立大量培養的基因轉殖毛狀根來研究冰花生理耐鹽的相關機制。
URI: http://hdl.handle.net/11455/92106
文章公開時間: 2018-02-06
Appears in Collections:生命科學系所

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