轉酮醇酵素基因油膜蛋白基因轉移至甘藍與結球白菜之研究

Abstract

轉酮醇酵素基因(transketolase)為植物戊糖磷酸代謝途徑中的關鍵性酵 素，與植物光合作用及碳的初步固定上有重要的關係。油膜蛋白基因( oleosin)為富含於種子特殊胞器-油體(oilbody)表面的蛋白，其功能為穩 定油體，增加油體與磷脂酵素的接觸面積，與三酸甘油脂的合成與分解有 關。本試驗將分離自酵母菌的轉酮醇酵素基因(TKL)與水稻油膜蛋白基因( OLE)，構築到含有rubisco samll subunit(rbc S)及CaMV 35S啟動子的轉 殖載體，並利用農桿菌直接基因轉移的方式，將帶有目標基因的pKctTt、 pKcTt、pKrtTt、pKrTt、pKcOn、pKrOn質體分別送入甘藍(新豐、初秋、 高峰)、結球白菜(夏陽、玉冠、新38號)的子葉或下胚軸。本試驗的目的 為：(1)建立甘藍、結球白菜組織培養以及基因轉移的再生系統，(2)研究 不同啟動子組合對轉酮醇酵素基因、油膜蛋白基因在轉殖甘藍及結球白菜 表現的影響，(3)探討轉移轉酮醇酵素基因、油膜蛋白基因到甘藍及結球 白菜，及培育成耐逆境與高品質之蔬菜的可行性。 研究結果顯示，經農 桿菌感染供試之三個品種的甘藍下胚軸之轉殖再生率為2.3%~9.0%，三個 品種的結球白菜下胚軸之轉殖再生率為0.17%~1.68%。子葉的轉殖再生率 幾乎為零，以“夏陽”結球白菜子葉再生率0.35%為最高。再生植株經PCR 、南方Slot 轉漬雜交做初步篩選後，以南方電泳墨點雜交法分析檢驗， 均可在轉殖植株的DNA上偵測到雜交訊號。北方雜交墨點分析的結果顯示 ，存在轉殖植株體內的轉酮醇酵素基因、油膜蛋白基因皆可轉錄出RNA。 轉殖攜帶有rbc S啟動子之轉酮醇酵素基因或油膜蛋白基因的再生植株， 其基因的表現較以CaMV 35S為啟動子者為佳，其中又以rbc S啟動子及TKL 啟動子的表現較好。轉殖轉酮醇酵素基因(pKcTt)的再生甘藍較未轉殖者 ，有較高的轉酮醇酵素活性，且有顯著的耐高溫的能力。

In most photosynthetic and chemoautotrophic organisms, CO2 is fixed into organic carbon by means of the reductive pentose phosphate pathway. Transketolase is the key enzyme of the non- oxidative part of the pentose phosphate pathway. Oleosin are a group of Mr 15~26 kD amphipathic proteins which are associated with the surface of oil bodies in lipid-storing tissues such as seeds and pollen. The role of oleosin in oil body is biogenesis and structural stabilization. This research focuses on the use of cabbage (Brassica oleracea L. var. capitata L.) and Chinese cabbage (Brassica campestris L. ssp. pekinensis) as a model system to establish the gene transfer technology, and possibility for improvement of cabbage and Chinese cabbage with stress resistance and nutrient quality, through the art of genetic engineering. Transketolase (TKL) and Oleosin (OLE) genes were isolated from yeast and rice, respectively, constructed into plant transformation vectors drived by CaMV 35S or rubisco small subunit (rbc S) promoter, and introduced into the hypocotyl and cotyledon of cabbage and Chinese cabbage using Agrobacterium-mediated transformation. Regenerated plants of cabbage and Chinese cabbage were obtained after transformation with six kinds of plasmids. The regeneration rates from cabbage and Chinese cabbage hypocotyl were 2.3% ~ 9.0% and 0.2 ~ 2% respectively. Only few plants were regenerated from the cotyledon of cabbage and Chinese cabbage. The regenerated plants were examined by PCR, southern, and Northern hybridization.The results indicated that the expression of constructed genes was higher in transgenic plants drived by the rbc S promoter than by CaMV 35S promoter. The TKL-transformed "New Top" cabbage had higher TKL activity and heat resistance than the controls.