Effects of Promoters on the Expression of Antifreeze Protein Genes in the Transformed Broccoli (Brassica oleracea L. var. italica Plenk.) and Cabbage (Brassica oleracea L. var. capitata)

Abstract

Environmental factors are the most important matters affecting the growth and distribution of the crope and in which temperature is especial importance. Low temperature affects a wide range of plant species when they are subjected to low temperature stress, and also causes plant loss and reduced yield. The purposes of this research are to study the effects of promoters on antifreezing protein gene (AF) expression and to study the possibility for improvement of cruciferous vegetable with frost resitance, through the art of genetic engineering. The AF gene of winter flounder was constructed with the combinations of CaMV 35S or rubisco small subunit (rbcS) promoters as plant transfer vectors. The constructed plasmids were transferred into hypocotyls and cotyledon of broccoli (Green King) , and cabbage (K-Y cross and Tropical Delight) via Agrobacterium mediated transformation. Regenerated plants of Brassica vegetables were obtained after transformation with six kinds of plasmids. The regenerated plants were examined by Southern and Northern blot hybridization. The results indicated that the expression of constructed genes was higher in transgenic plants driven by one or two of the rbcS promoter than by CaMV 35S promoter. Increasing in freezinf tolerance was also founded in AF transformed plants.環境因子是影響作物生長與分布最重要的因素，其中溫度的影響佔著重要的地位。過低的溫度，常造成幼苗或植株產生霜害或凍害。本試驗將分離自比目魚的抗凍蛋白基因(AF)構築到攜帶有不同組合的CaMV 35S及rubisco small subunit(rbcS)啟動子的轉殖載體，並利用農桿菌將其轉移到青花菜(綠王)及甘藍(初秋、和風)的子葉或下胚軸。再生植株經南方墨點法分析檢驗，可在轉殖植株得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AF基因之表現量大都較接近構造基因的啟動子為CaMV 35S啟動子之質體(pKcAn, pKccAn, pKrcAn)之再生植株高。轉殖AF基因之再生植株進行生物檢定，可提高轉殖植物2~5℃之抗凍能力