Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36097
標題: 竹嵌紋病毒於類甲基轉移酶(NbMTs1) 大量表現或靜默之菸草轉殖株內的複製能力
BaMV Replication in NbMTs1-overexpressed or NbMTs1-silenced Transgenic Nicotiana benthamiana
作者: 黃士韋
Huang, Shih-Wei
關鍵字: BaMV;甲基轉移酶;NbMTs1;甲基轉移酶
出版社: 生物科技學研究所
摘要: 
竹嵌紋病毒(Bamboo mosaic virus)為正股RNA病毒,基因體含有五個轉譯區(ORFs)。第一個轉譯區可轉譯出大小約155kDa的複製酵素,從N端到C端分別為戴帽酵素活性區 (CAP domain) ,類解旋酵素活性區 (HLD domain),以及核糖核酸聚合酵素活性區 (RdRp domain)。本實驗室先前已利用酵母菌雙雜交系統,以RdRp為釣餌蛋白於菸草葉片所建構的cDNA基因庫中,篩選得到一類甲基轉移酶 (NbMTs1),經先前實驗證實可抑制竹嵌紋病毒之複製,但是對於其他植物病毒能否產生影響目前尚不清楚。因此本實驗擬建構大量表現及基因靜默之NbMTs1轉殖菸草,以pEpyon質體為載體,pEpyon質體上有35S 啟動子能大量轉錄接入序列,建構pEpyon-MTs,帶有正股NbMTs1基因片段以大量表達NbMTs1蛋白質,pEpyon-MTs-rev則接入負股NbMTs1基因片段以達到基因靜默作用,以農桿菌系統 (Agrobacterium tumefaciens LBA4404) 轉殖方式建立基因轉殖菸草,已分別獲得約60株pEpyon-MTs植株及20株pEpyon-MTs-rev植株,以PCR方式確定這些轉殖植株含有轉殖基因片段,再以RT-PCR實驗,已獲得8株pEpyon-MTs植株能大量表現NbMTs mRNA及6株pEpyon-MTs-rev植株能靜默NbMTs mRNA 表現量。大量栽種轉殖植株,接種不同的植物病毒,分析及探討菸草抵抗植物病毒感染能力和NbMTs1表達量是否具有相關關係。

Bamboo mosaic virus has a single-strand RNA genome which consists of five open reading frames (ORFs). ORF1 encodes a ~155 kDa replicase that contains a capping enzyme domain, a helicase-like domain, and a RNA-dependent RNA polymerase domain (RdRp). In previous study, a putative Nicotiana benthamiana methyltransferase (NbMTs1) was found to interact with RdRp in a yeast two-hybrid system screening against a leaf cDNA library of N. benthamiana. The NbMTs1 can inhibit the replication of virus, and it may be the part of defense mechanism in plants. However, the effects of the NbMTs1 overexpression or NbMTs1 gene silence on other plant virus replication are not clear yet. In this study, we generated N. benthamiana lines overexpressing NbMTs1 and gene silencing transgenic plants, respectively. We constructed pEpyon-MTs by inserting NbMTs1 fragment into pEpyon plasmid to overexpress NbMTs1, and constructed pEpyon-MTs-rev by inserting antisense NbMTs1 fragment into pEpyon plasmid to silence the NbMTs1 expression in the transgenic plants. The recombinant plasmids were transformed into N. benthamiana via Agrobacterium LBA4404-mediated transformation. The transgenic plant lines were first selected by Kanamycin resistance phenotype, and further confirmed by genomic PCR analysis. The quantity of NbMTs1 mRNA in transgenic plants was detected by RT-PCR. We had obtained 8 pEpyon-MTs and 6 pEpyon-MTs-rev transgenic plant lines. We will inoculate transgenic N. benthamiana with several different plant virus and analyze the effect of NbMTs1 expression on plant virus replication in transgenic plants.
URI: http://hdl.handle.net/11455/36097
Appears in Collections:生物科技學研究所

Show full item record
 

Google ScholarTM

Check


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