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研究生:陳懷如
論文名稱:甘藷sporamin基因於轉殖芥藍及阿拉伯芥內之表現及分析
論文名稱(外文):Characterization of sporamin gene in transgenic Brassica oleracea var. alboglabra and Arabidopsis thaliana
指導教授:葉開溫葉開溫引用關係
指導教授(外文):Kai-Wun Yeh
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:胰蛋白抑制因子
外文關鍵詞:trypsin inhibitor
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Sporamin是甘藷塊根中含量非常豐富的儲存性蛋白質,過去對sporamin的研究方向著重於其運送過程及被蔗糖誘導的表現機制,直到1997年,Yeh等人才證明其具有胰蛋白抑制因子的活性,並在轉殖菸草中發現對斜紋夜盜蛾等幼蟲的啃食具有抗性。
在本篇論文中,我們期望得到具有抗蟲能力的十字花科作物,但基於CaMV 35S啟動子所驅動的基因轉殖很容易引起基因靜默的現象,故採用sporamin基因本身的prospoA啟動子來驅動sporamin基因的表現,prospoA是一具有受傷誘導的特性啟動子。經前人的研究得知,影響一基因的表現不止轉錄區的部分,在非轉錄區的地方也可能會影響基因的表現,因此我們也比較sporamin cDNA及genomic sporamin在prospoA驅動下的表現。我們首先構築了35S/sporamin、prospoA/sporamin、prospoA/sporamin —3’flanking region三種質體,利用農桿菌(Agrobacterium tumefaciens)轉殖進入阿拉伯芥及芥藍植物中。
利用阿拉伯芥及芥藍兩種植物來表現sporamin基因,經抗生素及聚合連鎖反應來初步篩選轉殖成功的植株,在阿拉伯芥的轉殖上相當有2∼5%的轉殖率,而芥藍植株也只有5%左右的成功率。轉殖成功的植株經西方點墨法分析後,證實植株均表現sporamin蛋白質,但各轉殖株之間的表現量均稍有差異。利用胰蛋白抑制因子膠體活性分析轉殖株,並沒有偵測到sporamin蛋白質表現胰蛋白抑制因子的活性,但經過胰蛋白親和性管柱純化轉殖株的粗萃取蛋白質,會得到具有活性的sporamin蛋白質。故採用敏感度較高的胰蛋白脢抑制因子生化活性分析轉殖株之間的活性,比較三種載體之間的活性,發現具有3’flanking region的sporamin基因在prospoA啟動子的驅動下表現了較高的胰蛋白抑制因子的活性。
利用芥藍轉殖株餵食二∼三齡的玉米穗蟲幼蟲,來分析轉殖株的抗蟲能力,經過2次的重複測試後,測量葉片啃食重量、蟲子平均重量級蟲子存活率,以野生型植株作為對照組,結果顯示有表現胰蛋白脢抑制因子活性的植株,都有抵抗玉米穗蟲幼蟲的啃食的能力。
Sporamin is an abundant storage protein in tuberous roots of sweet potato. It accounts for about 80% of total soluble proteins in sweet potato tubers, and no expression in leaves and a little expression in stems at the growing stage.
In 1997’s, Yeh et al., constructed a recombinant plasmid with sporamin cDNA and expressed it’s in Escherichia coli cells as fusion proteins. This protein was identified by strong inhibitory activity to trypsin in vitro and was proved to be pest-resistant in transgenic tobacco plants.
The transgenes driven by CaMV 35S promoter usually have gene silencing. To compare the gene expression driven by CcaMV 35S promoter and sporamin promoter, we selected prospoA promoter to drive it’s own cDNA sporamin and genomic sporamin, then established three different constructs: 35S::sporamin, prospoA::sporamin and prospoA::sporamin 3’-flanking region. They were introduced into Brassica oleracea var. alboglabra and Arabidopsis thaliana plants by Agrobacterium tumefaciens-mediated transformation. Genomic sporamin has longer 3’-flank region than cDNA sporamin, and contains one MAR T-box, three E-box, one SEF4 box. All transformants were screened for kanamycin resistance and confirmed by PCR. Arabidopsis thaliana transformants showed 2~5 % transformation frequency, and Brassica oleracea var. alboglabra expressed 5 % transformation ration. We used western analysis, trypsin inhibitory gel activity analysis and biochemical analysis to detect sporamin protein expression level in the transformants. Among the three-constructs, prospoA::sporamin 3’-flanking region shows better trypsin inhibitory activity than other two constructs.
We also tested insecticidal activity of these transformants by the feeding test of Helicoverpa armigera Hubner. It was demonstrated the genomic transgenic plants, with a genomic construct expressing better trypsin inhibitory activity, had better pest-resistant than wild type plants.
目錄
檢索表…………………………………………………………………………..3
中文摘要………………………………………………………………………..5
英文摘要………………………………………………………………………..7
第一章 前言
第一節 植物的防禦機制…………………………………………………9
第二節 防禦性蛋白質……………………………………………………11
第三節 基因靜默…………………………………………………………15
第四節 Sporamin蛋白質………………………………………………..17
第五節 芥藍的轉殖………………………………………………………19
第二章 材料與方法
實驗材料…………………………………………………………………….21
實驗方法…………………………………………………………………….21
第一節 載體的構築………………………………………………………21
第二節 阿拉伯芥的轉殖…………………………………………………28
第三節 芥藍的轉殖………………………………………………………32
第四節 轉殖植物之鑑定與分析…………………………………………38
第五節 抗蟲能力測試……………………………………………………47
第三章 結果
第一節 構築載體…………………………………………………………48
第二節 轉型至大腸桿菌XL1-blue strain及鑑定……………………...49
第三節 阿拉伯芥植物之轉殖及篩選……………………………………50
第四節 轉殖阿拉伯芥之鑑定……………………………………………51
第五節 芥藍植物之轉殖及篩選…………………………………………51
第六節 轉殖芥藍之鑑定…………………………………………………52
第七節 西方點墨法分析sporamin蛋白質……………………………..53
第八節 分析芥藍及阿拉伯芥轉殖株胰蛋白抑制因子活性…………53
第九節 胰蛋白抑制因子生化活性分析……………………………...54
第十節 抗蟲測試…………………………………………………………55
第四章 討論
第一節 載體的構築目的……………………………………………….…56
第二節 利用農桿菌進行基因轉殖……………………………………….58
第三節 胰蛋白抑制因子活性分析…………………………………….61
第四節 未來展望………………………………………………………….61
參考文獻…………………………………………………………………………63
附圖………………………………………………………………………………70
附錄………………………………………………………………………………86
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