日日春受花生簇葉病植物菌質體感染後之基因表現差異性研究

Abstract

本研究利用差異性顯示法 (differential display, DD) 技術獲得日日春 (Catharanthus roseus) 為花生簇葉病 (peanut witches'' broom, PnWB) 植物菌質體感染後表現具有差異性的 mRNA，並完成 64 個 cDNA 片段之選殖。以反向北方雜配反應 (reverse northern hybridization) 分析，篩選出 10 個選殖株，經核酸解序與比對分析，推測其基因功能。經同步聚合酵素連鎖反應 (real-time polymerase chain reaction, PCR) 分析上述 10 個基因於健康日日春及感染花生簇葉病植物菌質體之日日春體內的表現情形，顯示 7 個基因之表現具差異性。其中一個與 phi-1 相似基因之表現量上升 (up-regulated) 外，其餘基因之表現量則均下降 (down-regulated)。表現量下降的基因中，有 2 個為預測蛋白基因 (predicted protein gene)，其餘則分別與 psaDa gene, ML (MD-2-related lipid recognition) domain protein gene, eukaryotic translation initiation factor SUI1 gene 及 plastidic aldolase NPALDP1 gene 具有高相似度。進而完成模式植物日日春 cDNA 基因庫之構築，並以菌落聚合酵素連鎖反應 (colony PCR) 篩選 cDNA 基因庫，取得 3 個基因之 cDNA 全長序列，並加以比對以確認基因名稱，分別為ML domain protein gene, eukaryotic translation initiation factor SUI1 gene 及 plastidic aldolase NPALDP1 gene，其功能分別與植物先天性免疫反應 (innate immune response)、逆境反應 (stress response) 與光合作用 (photosynthesis) 相關。在基因表現時序 (time course) 的研究中，發現在接種後第六週時，該三個基因的表現量呈現表現量下降的情形，與差異性顯示法所得之結果一致。此三個基因的功能及可能的交互作用機制經由文獻探討進行討論，以綜合評析日日春與花生簇葉病植物菌質體間交互作用之關係。

The differential display (DD) strategy was applied to isolate periwinkle (Catharanthus roseus) cDNAs differentially expressed following infection with peanut witches'' broom (PnWB) phytoplasma. Sixty four clones have been selected from differentially expressed cDNA fragments. After screening by reverse northern hybridization, 10 transcripts were selected and sequenced. The expression levels of them was quantified by real-time polymerase chain reaction (PCR), and 7 DD transcripts were identified as ones truly differentially expressed after PnWB phytoplasma infection. One showing homology with phi-1 gene was up-regulated, while the others were down-regulated. Except two of which were predicted protein genes, the other down-regulated genes share homology with psaDa gene, ML domain protein gene, eukaryotic translation initiation factor SUI1 gene, and plastidic aldolase NPALDP1 gene. Homologous genes were further confirmed for 3 DD transcripts by isolating full-length cDNA fragments from the cDNA library constructed in this study. Confirmed genes included ML domain protein gene, translation initiation factor SUI1 gene, and plastidic aldolase NPALDP1 gene, and were mainly involved in innate immune responses, stress responses, and photosynthesis. In the time course experiments, expression levels of these three genes were down-regulated at 6th week after inoculation, and the results agreed with those of differential display. The possible role of these genes in the periwinkle that was infected by PnWB phytoplasma is discussed.