- 學位論文
與菸草嵌紋病毒複製相關之阿拉伯芥基因PAP85啟動子功能性分析系統之建立
Establishment of the promoter assay system of arabidopsis gene PAP85 involved in replication of Tobacco mosaic virus
摘要
病毒本身基因體極小，包含之遺傳訊息不多，因此必須倚靠寄主系統完成其複製及增殖的過程；然而對於病毒透過何種方式調控寄主基因，目前所知仍然有限。先前實驗室研究，為探究這些寄主基因，利用菸草嵌紋病毒(Tobacco mosaic virus, TMV)感染阿拉伯芥原生質體後，收取0.5-24小時抽取原生質體RNA進行microarray試驗，篩選影響病毒複製早期的基因(Chen, 2010)。其中寄主基因PAP85，在感染後0.5-24小時表現量皆上升。而PAP85無論在轉基因植物或在原生質體進行PAP85 knock-down實驗，病毒累積量皆降低，可知PAP85影響病毒累積。本研究的目的，在於探討TMV調控寄主基因表現的可能方式。首先以快速增殖DNA尾端法(Rapid amplified of cDNA ends, RACE)找出PAP85轉錄起始點約在轉譯起始點上游24個核苷酸處，同時也利用電腦比較分析PAP85找出CIACADIANLELHC, NODCON1GM, OSE1ROOTNODULE, TATABOX4, TATAPVTRNALEU, TBOXATGAPB. CIACADIANLELHC可能為參與病毒誘導之cis-acting elements。接著嘗試建立原生質體過渡性短暫試驗(transient assay)系統，但因核酸轉染效率低，得到不一致之結果。為克服此一困難，以長度不一(107, 1227 及2000 bps) 之PAP85啟動子區域聯結GUS報導基因進行基因轉殖，以便進行病毒感染後PAP85啟動子功能性分析。目前已有轉基因植物可進行試驗，且在pCambia1301-PAP85-P2000與-P1227之轉基因植物中發現其GUS報導基因表現形式與內生性PAP85一致。另外，並分析四個在TMV感染前期類轉錄因子基因(transcription factor-like genes)的knock-out突變株，發現在At5g41140突變株中，病毒累積量減少，顯示At5g41140可能參與病毒累積的過程。
並列摘要
The genomes of plant viruses are small and encode limited genetic products. To complete their life cycles, plant viruses highly require assistance from host factors. In previous research, to study these host factors, Arabidopsis protoplasts infected by Tobacco mosaic virus (TMV) were used to screen the host genes responsive to TMV replication at initial stage of infection through microarray analysis (Chen, 2010). It is found that host gene PAP85 is continuously up-regulated during 0.5-24 hours post inoculation, and virus accumulation decreased both in PAP85 transient knock-down protoplasts and pap85-RNAi transgenic plants. In this thesis, we started to study how TMV induces the PAP85 in the initial stage during virus infection. Rapid amplification of cDNA ends (RACE) was conducted to determine the transcription start site of PAP85 and found that it is located at 24 bps upstream to translational start site. Computer assisted comparison analysis was conducted to identify the possible cis-acting elements, and CIACADIANLELHC, NODCON1GM, OSE1ROOTNODULE, TATABOX4, TATAPVTRNALEU, TBOXATGAPB. CIACADIANLELHC were identified that may be involved in regulation of PAP85. PAP85 promoter assay systems were initially established on transiently-expressed protoplasts; however, inconsistent results were obtained due to low transfection rates. The transgenic plants with different lengths (107, 1227 and 2000 bps) of PAP85 promoter fused GUS reporter gene were obtained. In plants transformed with pCambia1301-PAP85-P2000 and pCambia1301- PAP85-P1227, the GUS expression pattern is similar to the reported endogenous PAP85 expression. Besides, the knock-out mutants of 4 transcription factor-like genes (At1g09530, At2g38970, At4g29530, At5g41140) upregulated during TMV initial infection were obtained from the Arabidopsis Biological Resource Center (ABRC). After TMV inoculation, the virus accumulation decreased only in At5g41140 knockout plants, suggesting that At5g41140 was involved in virus accumulation.
參考文獻
延伸閱讀
- Chia, K. S. (2015). 探討阿拉伯芥PAP85與菸草嵌紋病毒P126之交互作用及其在抗病毒之應用 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2015.10630
- Kuo, W. H. (2014). 大岩桐SsCYC在圓葉菸草的表現及性狀觀察與其組織培養再生系統之優化 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2014.00535
- Wu, Y. (2015). 烯醇化酶與磷酸甘油酸變位酶在圓葉菸草中對竹嵌紋病毒累積量所扮演之角色 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2015.01842
- Huang, P. Y. (2012). 阿拉伯芥凝集素受體激酶VI.2在菸草轉殖植物的功能性分析及篩選參與阿拉伯芥防禦反應之富含半胱胺酸類受體激酶 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2012.02001
- Lin, S. F. (2012). Functional Study of Yeast MTM-like Genes in MnSOD Activation of Arabidopsis [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2012.01158