Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36067
標題: 利用T-DNA插入突變法建立Nicotiana benthamiana突變株以探討影響竹嵌紋病毒複製之寄主因子
Generation of Nicotiana benthamiana Mutant Lines by T-DNA Insertion Mutagenesis for Study of Host factors Involved in Bamboo mosaic virus Replication
作者: 蔡佳芬
Tsai, Chia-Fen
關鍵字: Bamboo mosaic virus
竹嵌紋病毒
Nicotiana benthamiana
T-DNA
Host factors
寄主因子
病毒複製
出版社: 生物科技學研究所
摘要: 病毒感染寄主的過程中,通常是由病毒及寄主所轉譯的因子經由複雜的交互作用所完成,雖然病毒因子已被研究的相當透徹,但對於寄主因子還是了解很少。愈來愈多的證據顯示寄主因子參與病毒RNA複製的過程,如與病毒RNA複製酵素有關的寄主因子、以及參與細胞蛋白轉譯之因子等,但對於實際參與病毒RNA複製所需之寄主因子仍未了解透徹,因此本實驗的目的即是利用T-DNA插入基因突變法來大量生產Nicotiana benthamiana 基因突變株,再進一步比較竹嵌紋病毒或是其衛星核酸在這些突變株的複製能力,以篩選出會影響病毒累積之株系,進而分析所插入且破壞之基因為何,以期找到可參與病毒複製所需之寄主因子。竹嵌紋病毒 (Bamboo mosaic potexvirus, BaMV) 屬於馬鈴薯病毒群 (Potexvirus group),有些BaMV分離株攜帶有衛星核酸 (satellite RNA, satBaMV),satBaMV RNA為單股,正極性,含有一個轉譯區,可表現一個非結構性的20 KD蛋白質 (P20),此轉譯區非複製所必需,可為外源蛋白所取代,已發展成載體系統。本實驗的轉殖構築包括T-DNA的兩端序列、抗生素基因外,主要加上可以被BaMV感染誘變複製的BaMV 衛星核酸卡匣,此表現的卡匣帶有GFP基因,轉殖株經BaMV接種後,若未破壞BaMV或satBaMV複製所需的寄主因子,GFP的訊號會因衛星核酸的複製而被放大;若剛好破壞BaMV或satBaMV複製所需的寄主因子,則會使BaMV或satBaMV的複製受阻,而GFP也不會被大量表現,因此以GFP當作一個指示訊號,可以較快速的找到與BaMV或satBaMV複製有關的N. benthamiana轉殖突變株。目前已篩選出405株GFP轉殖植物,而BaMV在其中三株轉殖株 (GFP-16, GFP-78及GFP-104 )中有較少的累積量,且在其子代F1中皆可獲得相同的結果,因次推測此三株轉殖株之T-DNA所破壞的N. benthamiana染色體基因,應該與BaMV的複製有關。
The infection of plants by viruses usually occurs through complex interactions between virus-encoded and host-encoded factors. Some evidences reveal that host factors are involved in the process of virus RNA replication, such as those interacted with RdRP and other cellular translational factors. But the information about the host factors that directly participate in the virus RNA replication is limited. The aim of this study is to generate a large number of Nicotiana benthamiana mutants by T-DNA insertion mutagenesis, for screening of mutant lines in which BaMV replication are affected. These mutants can provide the further study of the host genes, which have been inserted by T-DNA and participated in the BaMV replication. Bamboo mosaic potexvirus (BaMV) is a member of the potexvirus group. A satellite RNA (satBaMV) associated with BaMV is a liner RNA molecule of 836 nts (excluding poly(A) tail) encoding a protein of 183 amino acids (P20). In this study, the plasmid construct for transformation includes both borders of T-DNA、Kanamycin-resistant gene and BaMV satellite cassette with GFP gene. This cassette can be amplified upon BaMV challenge in plant cells. If the transgenic plant with out any disrupted genes related BaMV replication, the GFP will be overexpression after challenged by BaMV, because of the BaMV satellite replication. At present, 405 transgenic lines have been screened; there are three lines (GFP-16, GFP-78 and GFP-104) in which the BaMV accumulation is lower than the control plants. The F1 plants of these three lines also reveal the same result. Presumally, the chromosome genes of N. benthamiana destroyed by T-DNA insertion in these three lines are concerted with BaMV replication.
URI: http://hdl.handle.net/11455/36067
Appears in Collections:生物科技學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.