2008-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/685261摘要：在台灣許多重要病害諸如花生簇葉病、梨樹衰弱病等均為植物菌質體(phytoplasmas)所引起。因植物菌質體均無法人工培養，且其細胞的純化仍有許多困難，因此相關之生理、生化特性研究仍屬有限，而有關植物菌質體與寄主植物交互作用分子生物學之探討更是付之闕如。在本計畫中將利用differential display等技術，以期能找出日日春感染花生簇葉病菌質體後基因表現差異的mRNA，並加以選殖cDNA，製作探針對基因庫進行篩選、進行北方雜配分析、核酸解序與比對等工作，深入探討該等基因之功能，並綜合評析日日春與花生簇葉病菌質體之間交互作用之關係。本研究室已於93年度 (NSC93-2313-B-002-110-）及94年度（NSC 94-2313-B-002-106-）計畫項下進行先期研究，並已獲得10個相關基因之初步結果。本計畫將繼續執行後續相關研究，工作項目則包括：利用differential display等方式篩選日日春受到花生簇葉病菌質體感染後，表現有差異的mRNA，並完成cDNA之選殖、核酸解序與比對、進行北方雜配分析等工作，進而完成模式植物日日春cDNA基因庫之構築，並以篩選得到之cDNA製作探針對基因庫進行篩選，取得該基因之全長片段。並利用real-time PCR技術定量基因受到影響的程度，深入探討基因表現量之改變，以綜合評析日日春與花生簇葉病菌質體之間交互作用之關係，順利完成此一計畫。<br> Abstract: In Taiwan, many important plant diseases such as peanut witches` broom (PnWB), sweet potato witches` broom, pear decline, rice yellow dwarf were caused by phytoplasmas, former mycoplasmalike organisms (MLOs). Up to now, the phytopathogenic phytoplasmas still resist to be cultured in any available media. It`s also very difficult to purify phytoplasmas from affected plants without the contamination of plant antigens. The biological and biochemical data of phytoplasmas are also next to nothing. In this study, differential display strategies will be applied to isolate periwinkle cDNAs differentially expressed following infection with PnWB-phytoplasma. Differentially expressed cDNAs will be cloned and characterized by Northern blot and sequence analysis. Gene functions and interactions between periwinkle and PnWB- phytoplasma will be further revealed. In our Lab., previous studies supported by NSC in 2004 (NSC93-2313-B-002-110) and 2005 (NSC 94-2313-B-002-106-) were launched and many results have been obtained. Totally, 10 genes were primarily selected and are now under carefully evaluation. The proposed study will carry out further studies and be accomplished in one years. Differential display strategies and other approaches such as subtractive hybridization and cDNA-AFLP will be applied to isolate periwinkle cDNAs differentially expressed following infection with PnWB-phytoplasma. Differentially expressed cDNAs will be cloned and characterized by sequence analysis, and gene expression pattern will be analysised using Northern blot and real-time PCR. The cDNA library of periwinkle will be constructed in lambda vector then screened using the differentially expressed cDNA as probes to reveal the full length cDNA sequence. The roles of these genes involved in the interaction between periwinkle and PnWB- phytoplama will be studied.植物菌質體日日春交互作用phytoplasmaperiwinkleinteraction