不同敢動子對鎘結合蛋白基因在轉殖青花菜中表現的影響

Effects of Promoters on the Expression of Cd-binding Protein Genes in the Transformed Broccoli (Brassica oleracea L. var. italica Plenck)

鎘在工業上的應用廣泛，導致鎘污染問題日益擴大，不僅危害生態環境且鎘污染農產品亦直接影響人體健康。本試驗將分離自天竺鼠的鎘結合蛋白基因（MT II），構築到攜帶有不同組合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）的子葉或下胚軸。本試驗的目的為探討不同啟動子組合對鎘結合蛋白基因在轉殖青花菜內表現之影響。研究結果顯示，經農桿菌轉殖青花菜（綠王）子葉之轉殖再生率為零，下胚軸之轉殖再生率為4.0%～7.2%。轉殖再生植株經PCR及南方墨點雜交分析，均可偵測到MT II基因存在於轉殖植株的DNA。北方雜交墨點分析的結果顯示，轉移六種不同構築的MT II基因在轉殖植株體內均可轉錄出RNA。轉殖較接近MT II基因的啟動子為rbcS啟動子之質體（pKrMn, pKrrMn, pKcrMn）的轉殖植株之MT II基因表現量及耐鎘能力，均較轉殖接近MT II基因的啟動子為CaMV 35S啟動子之質體（pKcMn, pKccMn, pKrcMn）的再生植株為高的趨勢。

Heavy metal pollution is the major problem that has been discussed in a few years. High heavy metal concentration in soil will affect the growth and product of crops, and the health of human body. The purposes of this research are to study the effects of promoters on cadmium-binding protein gene (MT II) expression and to study the possibility for improvement of broccoli (B. oleracea L. var. italica cv. Green King) with heavy metal resistance. through the art of genetic engineering. We had constructed the MT II gene of Chinese hamster with the combinations of CaMV 35S or rubisco small subunit (rbcS) promoters as plant transfer vectors. The constructed plasmids were transferred into hypocotyl and cotyledon of broccoli (Green King) via Agrobacterium mediated transformation. Regenerated plants of broccoli (Green King) were obtained after transformation with six kinds of plasmids. The regeneration rate of transformants was 4 to 11% from broccoli hypocotyl. No plants were regenerated from broccoli cotyledon. The regenerated plants were examined by PCR, Southern and Northern blot hybridization. The results indicated that the expression of MT-Il genes and increase in Cd resistance were higher in transgenic plants driven by one or two of the rbcS promoter than by CaMV 35S promoter.