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  • 學位論文

香蕉重泛素及beta-1,3-葡聚醣酶基因啟動子活性分析

Promoter Activity Analyses of Banana Polyubiquitin and beta-1,3-Glucanase genes

指導教授 : 杜宜殷
共同指導教授 : 黃鵬林(Pung-Ling Huang)

摘要


為了分析香蕉重泛素基因Mh-UBQ3、Mh-UBQ4及beta-1,3-葡聚醣酶基因Mh-BGL1啟動子之表現活性,將五個基因轉譯起始點上游長度不等的5’-flanking 區域分別構築至轉殖載體,連接報導基因GUS並以農桿菌轉殖至阿拉伯芥及菸草進行分析。在阿拉伯芥苗期發育階段方面,Mh-UBQ3基因啟動子則在各發育階段,均維持不表現之特性,此外,其在菸草轉殖植株亦不表現; Mh-UBQ4基因啟動子在子葉的表現會明顯受到不同苗期發育階段之影響,當新葉萌發時,於子葉的表現活性會明顯提升,而後其表現活性隨新生葉之展開而降低,而於新生之真葉則具有高表現活性;而Mh-UBQ4啟動子在菸草幼苗中,僅會表現於上胚軸靠近生長點之周圍,且不隨發育而改變;在Mh-BGL1啟動子方面,在苗期時為專一地表現的於子葉與上胚軸相連處,且隨子葉展開角度之增加,其表現活性亦隨之提升,在菸草幼苗中,Mh-BGL1啟動子亦有相同之表現。在成熟植株中的組織特異性方面, Mh-UBQ4動子在阿拉伯芥方面,主要在發育早期之花序葉及花苞,或老化之蔟生葉及發育後期之花苞有較強之表現活性,而於成熟之蔟生葉的表現活性則相對較低;而Mh-UBQ4啟動子在菸草中,仍表現於莖頂靠近生長點處,手切片顯示,其表現分布位置與木質部類似,此外,於根尖及側根尖端均有表達之活性。Mh-BGL1啟動子在阿拉伯芥中,主要表現於器官與器官相連接處、成熟果莢之胎做框(replum)及葉面及花序莖表面的毛狀體;而在菸草中,Mh-BGL1基因啟動子表現於老化之葉片。   植物生長調節劑誘導試驗方面,Mh-UBQ1啟動子會受IAA、BA、ABA、GA3、2,4-D、SA、Me-JA及ethylene之誘導而促進表現活性; Mh-UBQ2啟動子同樣會受IAA、BA、ABA、GA3、2,4-D、SA、Me-JA及ethylene之誘導而促進表現活性;Mh-UBQ3在本試驗中則不受任何處理之誘導;Mh-UBQ4啟動子方面,IAA、BA、GA3及2,4-D不影響啟動子之表現活性,ethylene及SA處理則會進啟動子的表現活性,而ABA及Me-JA則會抑制啟動子的表現活性;Mh-BGL1啟動子方面,IAA、BA、ABA、2,4-D、SA及Me-JA會抑制啟動子的表現活性,GA3及ethylene則會促進啟動子的表現活性。 非生物性誘導物誘導試驗方面,Mh-UBQ1啟動子會受乾旱及高鹽處理之誘導表現,創傷、低溫、暗及低溫及暗處理則不影響表現活性,高溫及淹水處理則會抑制表現活性。Mh-UBQ2啟動子則會受高鹽、創傷、低溫及暗處理及高溫處理誘導表現活性,乾旱、暗及低溫則不影響其表現活性,淹水處理則抑制其表現。Mh-UBQ3啟動子仍不表現。Mh-UBQ4啟動子方面,創傷、暗及高溫處理會促進其表現活性,乾旱、高鹽及低溫則不影響其表現活性。

並列摘要


To analyze promoter activity of banana polyubiquitin Mh-UBQ3, Mh-UBQ4 and beta-1,3-glucanase Mh-BGL1 genes, 5’-flanking region upstream translation start codon of each gene was constructed into stable transformation vector to drive reporter gene β-glucuronidase (GUS). Then, these expression vectors were transformed into Arabidopsis and tobacco. Results of GUS histochemical analysis for both of Arabidopsis and tobacco transgenic plants reveals that each promoter expressed differentially in tissues and development stages. No detactable expression activity was observed in Mh-UBQ3::GUS transgenic plants for both Arabidopsis and tobacco. In Mh-UBQ4::GUS transgenic plants, expression pattern in cotyledon was greatly affected during seedling development, while it always had strong expression activity in the newly born leaves. GUS expression in Mh-BGL1::GUS transgenic plants was tissue and developmental specific. When cotyledons was encloed, expression in the junction site of cotyledons and hypocotyls was weak. As it expanded, expression activity was gradually eleveated. Result of treatment analysis indicated that expression activity of Mh-UBQ1 gene promoter could be induced by plant growth regulators IAA, BA, ABA, GA3, 2,4-D, SA, and Me-JA and by abiotic stimuli such as drought and high salt. Expression activity of Mh-UBQ2 gene promoter could be induced by palnt growth regulators such as IAA, BA, ABA, GA3, 2,4-D, SA, and Me-JA and by abiotic stimuli like high salt, wounding, cold and dark, and heat. Expression activity of Mh-UBQ3 gene promoter was not induced by any treatment in the experiment. Expression activity of Mh-UBQ4 gene promoter could be induced by plant growth regulators SA and ethylene and by abiotic stimule like dark and heat while repressed by ABA and Me-JA. Promoter activity of Mh-BGL1could be indeuced by GA3 and ethylene but repressed by IAA, BA, ABA, 2,4-D, SA, and Me-JA.

並列關鍵字

banana ubiquiti beta-1,3-glucanas promter

參考文獻


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