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|標題:||Studies on Transformation of Heat Shock Protein Gene and Catalase Gene into the Chloroplast of Cabbage (Brassica oleracea L. var. capitata L.) and Chinese Cabbage (Brassica campestris L. ssp. Pekinensis)
Taiwan is located at the subtropical zone with high temperatures and rich rainfall, especially in the summer. Vegetable productivity could be subjected to great loss due to the environmental stresses and disease resulted form high temperature and humidity. Cabbage and Chinese cabbage are two of the most widely grown leafy vegetable crops in the Taiwan. Therefore, developing summer cultivars of Cabbage and Chinese cabbage with high-temperature and stress-tolerance is one of the most important researches in Taiwan. The important roles of heat shock protein and catalase in protection of plants from heat stress and oxidation had been well documented. We attempt to transfer the heat shock protein and catalase genes into the chloroplast of cabbage and Chinese cabbage. The objective of this study is to explore the possibility for improvement of Brassica vegetables with high-temperature and stresses resistance by chloroplast gene transformation.
In this study, hsc70-2 gene of tomato and catalase 78 gene (cat78) of Chinese cabbage driven by prrn promoter were constructed into the Brassica-specific-plastid vector, pASCC201, to form the pASCCHSC and pASCCCAT, respectively. The hsc70-2, and cat78 genes were transferred into the cabbage and Chinese cabbage chloroplast via particle gun mediated transformation. Regenerated plantlets were conferred by resistance to spectinomycin antibiotics. The results of PCR and Southern hybridization analysis indicated that the aadA, hsc70-2, and cat78 genes were integrated into the chloroplast genome of cabbage and Chinese cabbage. Furthermore, the expression of hsc70-2 mRNA and Hsc70 protein were detected in the hsc70-2 gene transformed cabbages by Northern blot analysis and Western blot analysis, respectively. After 3 days of 42℃ treatments, hsc70-2 gene transformed cabbage showed high degree of high-temperature resistance than the un-transformed plants.
臺灣地處亞熱帶，夏季氣候高溫多濕，常有溫度逆境的發生，影響夏季蔬菜的生產甚钜。甘藍及結球白菜是台灣栽培面積最大的葉菜類蔬菜，因此培育耐高溫及逆境的甘藍及結球白菜品種，一直是專家學者改善臺灣夏季蔬菜生產的努力目標。過氧化氫酵素 (catalase, CAT) 及熱休克蛋白 (heat shock protein, HSP) 是植物內生抗氧化及抗高溫逆境之的酵素蛋白，其能提昇作物抗氧化逆境及耐高溫的能力，已有許多研究報導。本研究分別將以prrn為啟動子的番茄hsc 70 與結球白菜cat 78 基因構築至蕓苔屬葉綠體基因轉殖之通用轉殖載體 (pASCC201)，成為pASCCHSC及pASCCCAT之蕓苔屬葉綠體基因轉殖載體。再以基因槍法將其分別轉移到結球白菜與甘藍葉片、子葉及下胚軸，再經誘導再生植株。本研究的目的為探討以葉綠體基因轉移方式培育出耐高溫、逆境及低基因污染之甘藍及結球白菜蔬菜的可行性。
以基因槍法轉殖之培殖體，以低濃度的抗生素 (spectinomycin) 持續篩選4週後，可獲得轉殖的再生植株。以PCR及南方墨點分析hsc70、cat 78之再生甘藍及結球白菜的植株之葉片結果均可偵測到轉殖之 aadA、hsc70與cat 78基因的存在，顯示hsc70、 cat 78基因已轉移到結球白菜與甘藍之葉綠體基因組。以北方墨點分析轉殖甘藍植株可偵測到轉移之hsc 70 mRNA的表現。以西方墨點分析Hsc 70之結果顯示在轉殖甘藍有較強的Hsc70表現。轉殖甘藍植株經由42℃高溫處理三天後，較未轉殖對照組呈現極耐高溫之效果。
|Appears in Collections:||分子生物學研究所|
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