抗凍蛋白基因轉移到青花菜、花椰菜及小白菜之影響

Abstract

This research focused on the use of cruciferous vegetables as a model system to establish the gene transfer technology, and to study the possibility for improvement of cruciferous vegetables with frost resistance. Antifreeze protein gene (AF) of winter flounder driven by the promoter of CaMV 35S or rubisco small subunit (rbcS) was constructed into plant transfer vectors. The constructed plasmids were transferred into hypocotyls and cotyledon of Broccoli (Green King), Cauliflower (Chiu-Wang), and Pak-Choi (Tainung No. 1) via Agrobacterium-mediated transformation. Regenerated plants of three Brassica vegetables were obtained after transformation with two kinds of plasmids. The regeneration rate of transformants was 1％ to 3％. The regenerated plants were examined by GUS, PCR, Southern, and northern analysis. The results of genetics analysis indicated that the transgenes were inserted into the chromosome of transgenic plants and expressed. The expression of the AF gene was higher in transgenic plants driven by the rbcS promoter than by CaMV 35S promoter. Increasing in 2~3℃ of freezing tolerance was found in AF-transformed plants.本試驗將分離自比目魚抗凍蛋白基因(AF)，構築到攜帶有rubisco small subunit(rbcS) 或CaMV 35S啟動子的轉殖載體，並利用農桿菌將其轉移到‵綠王′青花菜、‵秋王′花椰菜及‵臺農一號′小白菜的子葉或下胚軸。本試驗的目的建立青花菜、花椰菜及小白菜基因轉移及植株再生系統，研究不同啟動子對表現此種基因的影響，並探討培育成抗凍之蔬菜的可行性。 本研究的結果顯示，以農桿菌轉殖供試之三種蕓薹屬蔬菜之再生率在1~3%之間。檢測轉殖植株之GUS基因，有95%以上成正反應。以南方墨點法分析PCR反應呈正反應之轉殖植株，均可偵測到轉移AF基因存在於轉殖的DNA。北方墨點法分析之結果顯示，轉移之AF基因在轉殖植物體內均有轉錄。轉殖以rbcS啟動子為啟動子之AF基因的再生植株，其AF mRNA表現量，較轉殖以CaMV 35S啟動子為啟動子者為高。經AF基因轉移之植株較未轉殖之對照組植株，可增加耐2~3℃的低溫