劉瑞芬臺灣大學:植物病理與微生物學研究所丁善焉Ding, Shan-YanShan-YanDing2010-05-112018-06-292010-05-112018-06-292008U0001-0302200823503800http://ntur.lib.ntu.edu.tw//handle/246246/181910hytophthora parasitica Dastur (疫病菌，又名P. nitcotianae Breda de Haan) 廣泛分布於全球各地。疫病菌的孢囊與游走子能靠著環境中些微水分迅速傳播，且生活史極短，能在同一植株上進行重複感染，寄主範圍又廣，經常造成重大農業損失，是世界上重要的植物病原菌之一。近年來許多研究顯示絲裂原活化蛋白激酶 (mitogen-activated protein kinases，MAPK) 訊息傳導與植物病原菌的病原性有關，例如炭疽病菌 (Colletotrichum spp.) 與稻熱病菌 (Magnaporthe grisea)。 Protein phosphatase 2C 在MAPK訊息傳遞路徑中扮演負向調控的角色，藉由去除磷酸根來抑制MAPK的活性。我們根據 Phytophthora ramorum 與 P. sojae 的 PP2C 同源性序列設計引子對，從 P. parasitica 基因體中增幅得到一長為 350 bp 之部份序列；再由 RACE (rapid amplification of cDNA ends)得到全長基因，其ORF (open reading frame)可轉譯出 344 個胺基酸序列，並將此基因命名為 ppptc1 (Phytophthora parasitica phosphatase two C 1)。ppptc1與人類 PP2Cα的相似度高達 40%。在 P. parasitica 基因體中，ppptc 1的拷貝數為一，但可能有其他同源基因存在。利用 pMAL (pMALTM Protein Fusion and Purification System) 載體於大腸桿菌中表現之PPPTC1重組蛋白能夠對 pNPP進行去磷酸化反應。PPPTC1在in vitro 情況下能將已磷酸化的ppmk3去磷酸化，但在in vivo下是否能對ppmk3進行逆向調控，有待進一步驗證。Ppptc1基因表現受酸性環性、過氧化物及高濃度鈣離子等逆境因子所抑制，且在基礎環境下即有表現，極有可能參與疫病菌MAPK訊息傳導途徑。Phytophthora parasitica Daster (=P. nitcotianae Breda de Haan) is an oomycete plant pathogen. Spreading easily by water, short life cycle, wide host range contributes P. parasitica a worldwide disease. Recent studies show that MAPK pathway is related with the pathogenicity of plant pathogens, such as Colletotrichum spp and Magnaporthe grisea. PP2C negatively regulate MAPK pathway by inactive phospo-MAPKs. We designed specific primers to amplify partial sequence of PP2C from P. parasitica genomes. The entire open reading frame (ORF) was gained by Rapid Amplify cDNA ends (RACE) and named as ppptc1 (Phytophthora parasitica phosphatase two C 1), which encoded 344 amino acids. Ppptc1, which has 40% similarity to human PP2Cα gene, is an one-copy number gene but other homologuesin may exist in the genome of P. parasitica. To express the PPPTC1 protein, the pMAL vector was used to express heterogeneous fusion protein from E. coli. As an alkaline phosphatase, PPPTC1 can dephosphorylate pNPP (para-nitrophenyl phosphate), a non-protein substrate. PPPTC1 also seems to dephosphorylate in vitro with ppmk3, a MAP kinase in P. parasitica. The ppptc1 gene is down-regulated under oxidation, low pH, and high concentration of Ca2+, thus it may involved in MAPK pathway in P. parasitica.中文摘要………………………………………………………………………… i 文摘要………………………………………………………………………… ii言 ……………………………………………………………………………… iii、前人研究 …………………………………………………………………… 1、材料與方法 ………………………………………………………………… 9、結果 …………………………………………………………………………19、討論 …………………………………………………………………………24、參考文獻 ……………………………………………………………………27 、圖 …………………………………………………………………………37錄……………………………………………………………………………… 52application/pdf733052 bytesapplication/pdfen-USMAPKPhytophthora parasiticapNPPppmk3protein phosphatase 2Calkaline phosphatase[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/181910/1/ntu-97-R94633016-1.pdf