菸草基因NbSCL6對竹嵌紋病毒感染周期的影響

竹嵌紋病毒(Bamboo mosaic virus; BaMV)為一外型絲狀的單一正意股RNA病毒，屬於Flexiviridae科，Potexvirus屬。本實驗主要是針對在病毒的感染週期中，菸草(Nicotiana benthamiana)基因差異性表現的基因對於BaMV的影響，並進而探討其影響病毒的機制。在初步的研究中，利用cDNA amplified fragment length polymorphism (cDNA-AFLP)的技術，將這些具差異性表現的基因片段篩選出來，目前已有90個基因片段被篩選出來，其中一個命名為ACTC7-1的基因片段被選擇出來進行後續的研究。菸草基因ACTC7-1在竹嵌紋病毒感染後，會有表現量增加的現象，表示它可能是為了防禦病毒或是被病毒利用而造成表現量增加。首先，我利用VIGS (virus-induced gene silencing)的技術將ACTC7-1的表現量進行靜默，然後在ACTC7-1基因靜默的菸草進行病毒的接種，發現竹嵌紋病毒的外鞘蛋白累積量有增加的現象。因此猜測ACTC7-1應該是屬於防禦病毒的相關基因。為了釐清這個基因的影響層次是作用在病毒的複製階段還是在病毒的移動階段，我在將病毒RNA接種在ACTC7-1基因靜默的原生質體中，發現病毒的外鞘蛋白累積量也有增加的現象。由此結果可以猜測ACTC7-1是作用在病毒的複製階段。最後，利用RACE的技術，我得到了ACTC7-1的全長基因，透過NCBI的資料庫比對，發現ACTC7-1跟SCL-6 (SCARECROW-LIKE-6) 蛋白的相似度很高。SCL屬於GRAS家族，是一種轉錄因子(transcription factor)，對根部發育的調控有關。未來會大量表現ACTC7-1蛋白，判斷是否會對病毒的累積量有影響。並觀察ACTC7-1蛋白在菸草細胞中的位置，將可以更進一步的探討ACTC7-1對病毒影響的真正機制為何。Bamboo mosaic virus ( BaMV) is a single-stranded positive-sense RNA virus with a 5￠cap and a 3￠ poly(A) tail. BaMV belongs to the genus Potexvirus and the family Flexiviridae. The gene expression profile in Nicotiana benthamiana may be altered after BaMV infection. To identify the possible host genes involving the infection cycle of BaMV, our lab used cDNA-AFLP technique to screen the differentially expressed genes in BaMV-inoculated N. benthamiana plants. ACTC7-1 is an upregulated gene when BaMV infects N. benthamiana. To characterize the function of ACTC7-1 involving in BaMV infection cycle, I used the virus-induced gene silencing (VIGS) technique to known down the expression of ACTC7-1 leveling N. benthamiana plant and then inoculated BaMV onto the knockdown leaves. The accumulation levels of BaMV coat protein was determined by Western blotting analysis. The accumulation of BaMV was enhanced when after the expression of ACTC7-1 was knocked down. To further analyze the effect of ACTC7-1 on BaMV infection is on viral RNA replication or virus movement, I infected BaMV RNA into the ACTC7-1-knockdown protoplasts which were derived from the knockdown plants. The result of the accumulation of BaMV coat protein in N. benthamiana protoplasts was also increased. Overall of these preliminary results suggest that ACTC7-1 is probably involved in the replication of BaMV. Furthermore, I used RACE technique to clone the full-length of ACTC7-1 and blasted the sequence to WORKBENCH. The identity of ACTC7-1 could be the SCARECROW-LIKE transcriotion factor in GRAS family. The future work of this research will focus on the transient expression the full-length of ACTC7-1 fused with Orange fluorescent protein in N. benthamiana to localize this protein and examine the effect on BaMV accumulation中文摘要 i Abstract ii Introduction 1 Materials and Method 5 Virus-induced gene silencing (VIGS) and mechanical inoculation of viruses 6 Protein detection 6 Total RNA extraction 7 cDNA synthesis 8 Real-time RT-PCR 8 Viral RNA extraction 9 Protoplasts preparation and viral RNA inoculation 9 mRNA purification 10 The 3? Rapid amplification of complementary DNA ends (RACE) 11 The cloning of the ACTC7-1 full-length cDNA 11 Results 12 The accumulation of BaMV coat protein in ACTC7-1-knockdown plants is reduced. 12 ACTC7-1 could be involved in the viral RNA replication step of BaMV infection cycle 13 The full-length of ACTC7-1 fragment might encode a scarecrow-like protein in N. benthamiana. 13 Discussion 15 Figures 17 Reference 2