香蕉轉殖細胞利用葡萄糖苷酸酶活性組識化學染色之改進

Improvement of Histochemical Staining for β-glucuronidase (GUS) Activity in Transformed Banana Cells

在基因轉殖試驗中，β-葡萄糖苷酸酶（β-glucuronidase; GUS）基因是應用最廣泛的報導基因（reporter gene）。本試驗針對香蕉轉殖細胞及非轉殖細胞，試驗五種CUS作用基質配方，以檢視GUS組織化學活性。結果顯示，最適用於香蕉轉殖細胞的GUS組織化學染色法，乃是先將組織以含有1% Triton X-100的磷酸根之磷酸鈉系列緩衝液（50mM sodium phosphate buffer, pH 7.0）前處理，置於37℃下二小時後，再以不含Triton X-100之緩衝液清洗兩次，而後加入含有1.0nM 5-bromo-4-chloro-indolyl-β-glucuronic acid X-gluc的磷酸緩衝液及20%甲醇，可以得到最好的染色效果。

The bacterial β-glucuronidase (GUS) gene is advantageous over other genes when introduced into plants as a reporter gene. The histochemical assay of β-glucuronidase activity, as described by Jefferson (1987), is excellent for many dicotyledonous plants. However, some researchers encountered difficulties with monocotyledons. Therefore, we examined a series of histochemical β-glucuronidase assays for transformed banana cells. The best method was that time banana calli were incubated, first, in phosphate buffer (50 mM Sodium phosphate buffer, pH 7.0) containing 1% Triton X-100 at 37℃ for 2 hours. The buffer, was then removed and fresh phosphate buffer containing 1.0 nM 5-brosno-4-chloro-indolyl-β-glucuronic acid (X-gluc) and 20% methanol was added to the calli. This method enabled us to determine β-glucuronidase gene expression more effectively for transformed banana cells.