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 β-glucuronidase Activity in Transformed Phalaenopsis

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圓片進行參試，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組織化學染色法，乃是先將組織以含有1% Triton X-100的磷酸緩衝溶液（50 mM NaPO4, pH 7.0）於37℃下前處理1或2小時，再以不含Triton X-100的緩衝溶液清洗，最後加入含有1.0 mM 5-bromo-4-chloro-indolyl-β-glucuronic 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圓片採用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，可得相對較優的轉殖評估效能。 The bacterial β-glucuronidase (GUS) gene is advantageous over other genes when introduced into plants as a reporter gene. The general histochemical assay of β-glucuronidase activity is suitable for many dicotyledonous plants. However, some researchers have encountered difficulties with monocotyledons. We therefore have examined a series of histochemical β-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 NaPO 4 , pH 7.0) containing 1% Triton X-100 at 37℃ for 1 or 2 hours. The buffer was then removed and fresh phosphate buffer containing 1.0 mM 5-bromo-4-chloro-indolyl-β-glucuronic acid (X-gluc) was added to the calli. In the meantime, the vacuum-step that usually adopted for histochemical β-glucuronidase assays has been omitted from our optimal protocol without any effect on staining. This method enabled us to determine β-glucuronidase gene expression more effectively for transformed phalaenopsis calli. For phalaenopsis 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 ethanol removal.