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題名 | 葡萄糖苷酸酶活性組織化學染色法分析蝴蝶蘭轉殖事件之改進=Improvement of Histochemical Staining for ß-glucuronidase Activity in Transformed Phalaenopsis |
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作者 | 徐善德; 黃鵬林; | 書刊名 | 臺灣園藝 |
卷期 | 53:3 2007.09[民96.09] |
頁次 | 頁301-312 |
分類號 | 435.432 |
關鍵詞 | 植物基因轉殖; 報導基因; 葡萄糖苷酸酶; 組織化學染色法; Plant transformation; Reporter gene; ß-glucuronidase; GUS; Histochemical staining; |
語文 | 中文(Chinese) |
中文摘要 | 在基因轉殖試驗中,β-葡萄糖苷酸酶(β-glucuronidase; GUS)基因是應用最為廣泛的報導基因(reporter gene)。然而,用以偵測GUS活性的組織化學染色法,一般較適用於雙子葉植物,用於單子葉植物則會遭遇部分困擾。因此,本試驗針對癒合組織和葉圖片進行參試,以評估適用於轉殖蝴蝶蘭癒合組織和葉片的GUS組織化學染色方法與流程。結果顯示,最適用於蝴蝶蘭轉殖癒合組織的GUS祖織化學染色法,乃是先將組織以含有1%Triton X-100的磷酸緩衝溶液(50 M NPO4, pH 7.0)於37℃下前處理l或2小時,再以不合Triton X-100的緩衝溶液清洗,最後加入含有1.0 mM 5-bromo- 4-chloro-indolyl-ßglucuronic acid的磷酸緩衝溶液,可以得到最好的染色效果。而一般GUS染色分析所採用的真空處理步驟,則可以省略不做,此種作法可簡化轉殖蝴蝶蘭癒合組織的染色分析流程,同時不會影響染色的結果。至於,甲醇、95%乙醇、20%Clorox和Triton X-100 (1 %和2%)等前處理藥劑與處理時間,對蝴蝶蘭轉殖植株葉圖片採用GUS染色法分析效果的評估結果顯示,以95%乙醇進行10分鐘的前處理,再予以GUS染色處理,可得相對較慢的轉殖評估效能。 |
英文摘要 | The bacterial ß-glucuronidase (GUS) gene is advantageous over other genes when introduced into plants as a reporter gene. The general histochemical assay of ß-glucuronidase activity is suitable for many dicotyledonous plants. However, some researchers have encountered difficulties with monocotyledons. We 1hereforehave examined a series of histochemical ß-glucuronidase assays for transformed phalaenopsis callus and leaf disc. Our results indicated that the best method was achieved by first incubating phalaenopsis calli in phosphate buffer (50 mM NaP04, pH 7.0) containing 1 % Triton X-100 at 37℃ for 1 or 2 hours. The buffer was 1henremoved and fresh phosphate buffer containing 1.0 mM 5-brom04-chloro-indolyl -ß-glucuronicacid (X-gluc)was added to the calli. In the meantime, the vacuum step that usually adopted for histochemical ß-glucuronidase assays has been omitted from our optima1 protocol without any effect on staining. This method enabled us to determine ß -glucuronidase gene expression more effectively for transformed phalaenopsis calli. For pha1aenopsis leaf disc, the optimal procedure was achieved by incubating leaf discs in 90 % ethanol at room temperature for 10 minutes, followed by addition of X-gluc solution immediately after e1hanol removal. |
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