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標題: | 水稻含錳超氧歧化?基因轉殖之研究 The Transformation of Manganese-Superoxide Dismutase gene into Rice |
作者: | Chiung-Chih Chu 朱瓊枝 |
出版年 : | 1997 |
學位: | 碩士 |
摘要: | 水稻台農67號及矮南早39號兩種栽培品種之幼胚,利用粒子槍方式進行兩種含水稻mnsod或其反義基因之轉殖。質體之一具ABRC調控序列、Amy64最小啟動子及mnsod;另一質體含CaMV 35S啟動子及mnsod反義基因。轉殖後以hygromycin進行篩選並誘導癒傷組織形成,一部分作懸浮細胞培養,餘者則誘導植株再生。mnsod轉殖方面,經抗生素篩選後,選取台農67號5個細胞系,矮南早39號23個細胞系,經南方氏轉印分析,選出臺農67號之轉殖懸浮細胞系2個。北方點墨轉印分析結果顯示,在非轉殖細胞系中,mnsod表現有兩個訊號,主要訊號為0.95 kb約佔95%,另一2.6 kb訊號約佔5%,而轉殖細胞系的主要訊號為小於0.5 kb,此訊號表現會隨ABA處理而增強,0.95 kb的訊號變為次要,2.6 kb的訊號則消失。mnsod反義基因轉殖方面,台農67號有4個細胞系,作南方氏轉印分析,選出確定之4個轉殖細胞系。由北方點墨轉印分析結果顯示,這些細胞系皆多出0.62 kb左右的訊號。此外,轉殖之水稻植株則正在培養中。 台農67號之懸浮細胞以ABA處理,觀察抗氧化酵素的變化。懸浮細胞主要的SOD活性為粒腺體型MnSOD;ABA處理後,一細胞質型CuZnSOD蛋白質含量及酵素活性均會增加,MnSOD活性雖沒有明顯變化,但其基因表現,隨著ABA的處理而增加,過氧化?則於ABA處理24小時後,活性有減少之現象。 台農67號之懸浮細胞以低溫處理,觀察抗氧化酵素的變化,發現低溫處理後,mnsod mRNA量減少,但MnSOD和其他SOD同功?及過氧化?之活性則無明顯變化。 The immature embryos of two rice cultivars, TNG67 and ANT39 were transformed with plasmids containing mnsod or antisense mnsod by biolistic method. One plasmid contained three copies of ABRC, Amy64 minimal promoter and a full length cDNA of rice mature MnSOD and the other plasmid contained CaMV 35S promoter and antisense mnsod. The calli induced from rice immature embryos after bombardment were selected by 50 μg/ml hygromycin. Parts of calli were used for suspension cell culture and the others were used for plant regeneration. In the mnsod transformation experiments, 5 cell lines of TNG67 and 23 cell lines of ANT39 were analyzed by Southern blot. The sequence containing ABRC element, Amy64 minimal promoter, mnsod and HVA22 terminator was proved to be inserted into the genomic DNA of 2 cell lines of TNG67. The expression of mnsod was detected in gel blot analysis of total RNA by probing with mnsod cDNA clone of rice. Basically, two transcripts were detected from untransformed cell lines of TNG67. The size of major transcript (95%) was 0.95 kb, and the minor one (5%) was 2.6 kb. The transcripts were different in one cell line of mnsod transformants. The major one was smaller than 0.5 kb which was enhanced by ABA, and the minor one was 0.95 kb. In the antisense mnsod transformation experiments, 4 cell lines of TNG67 were analyzed by Southern blot. The sequence containing CaMV 35S, antisense mnsod and nos terminator was proved to be inserted into the genomic DNA of 4 cell lines of TNG67. From northern blot analysis, it was found that one extra transcript about 0.62 kb was detected in all cell lines of these mnsod antisense transformants. On the other hand, the rice plants of putative transformants were transplanted into soil to obtain seeds. Additionally, the effects of ABA and chilling stress on SOD in rice suspension cells were also examined. The results showed that ABA could enhance the expression of mnsod in transcription level, but had little effect on the enzyme activity of MnSOD. On the other hand, ABA could enhance cytosolic CuZnSOD both in the enzyme activity and in the protein amount. The enzyme activities of peroxidase and ascorbate peroxidase were decreased after being treated with ABA for 24 hours. When rice suspension cells were exposed to chilling treatment, the expression of mnsod in transcription level was decreased but the activities of total SOD, peroxidase and ascorbate peroxidase were no significantly changed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76276 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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