β-glucuronidase（GUS）基因轉移至馬鈴薯之研究

Studies on the β-glucuronidase (GUS) Gene Transfer to Potato (Solanum tuberosum L. cv. Russet Burbank)

馬鈴薯為世界性最重要之非榖類糧食作物，提供人類大量的能量及營 養來源，較其他作物而言更有增加產量及品質之潛力。新的基因轉移及細胞培養技術，使外來基因導入植物細胞並再生成轉殖株成為可能，這也提供馬鈴薯品種改良之可能新途徑。本研究之目的為探討使用PEG直接基因轉移法，將GUS報導基因轉移進入栽培種馬鈴薯（Solanum tuberosum L. cv. Russet Burbank）葉肉細胞原生質體中，並培養成為再生轉殖植株之可行性。由試驗結果發現，以15%PEG濃度轉移GUS基因到馬鈴薯葉肉細胞原生質體時GUS基因的transient expression最高。轉殖原生質體，經4至6個月的培養可得再生植株，由癒傷組織分化出芽梢之再生率為41%。以組織化學染色法篩選三次基因轉移試驗產生之再生植株所得之相對再生植株轉移率分別為17.7%、2.7%、4.6%。CaMV 35S啟動子在轉殖馬鈴薯之不同組織的表現有差異性存在，但都以在維管束韌皮部表現最強。篩選所得之轉殖馬鈴薯經DNA雜交、RNA雜交、原位雜交及SDS-PAGE蛋白質電泳分離等分析，証實GUS基因之存在及表現。轉殖植株之間，GUS基因表現之差異，乃因為在轉譯層次上GUS RNA產生量不同所致。由轉殖馬鈴薯薯球及長出之芽及根，經分析也有GUS基因之表現，顯示轉移進入馬鈴薯之外來基因可經由無性繁殖而遺傳，並且固定下來。

The potato (Solanum tuberosum L.) is the most important non-cereal world food crop. It has the capacity to produce more energy and protein than any other single food crop. Breeding for better clones with high yield and good quality is promising. Genetic manipulation through cell culture and recombinant DNA technology would certainly facilitate the progress. The objectives of this study are to establish the system of PEG- mediated direct gene transfer and protoplast regeneration system of potato and to examine the tissue-specific expression of CaMV 35S promoter in transgenic potato plants via β -glucuronidase (GUS) gene. Results show that the suitable concentration of PEG (MW. 4000) for transferring pBI221 into mesophyll protoplasts of potato (S. tuberosum L, cv. Russet Burbank) is 15% to 20%. The regenerated transgenic plants were obtained from mesophyll protoplasts after 4 to 6 months of culture. The transfer efficiency is about 5% as evaluated from the percentages of total regenerated plants.Transformation was confirmed by Southern, northern and in situ hybridization, protein gel electrophoresis, histochemical staining and fluorometric assay. The results of Southern blot hybridization indicate that more than one copy of GUS gene was integrated into the genome of potato. Although GUS activity was detected in roots, stems, leaves, and tubers, tissue-specific expression was observed in the tubers and roots. This is the first report in regard to transfer foreign gene into cultivated potato protoplasts and regenerate into plants via PEG-mediated transformation.