Molecular cloning and characterization of a ste20-like gene in Phytophthora parasitica

以真菌的訊息傳導來說，酵母菌Saccharomyces cerevisiae是研究最普遍的生物，目前已經知道S. cerevisiae中有五條mitogen activated protein kinase (MAPK)訊息傳遞路徑，分別負責酵母菌的mating、osmotolerance及invasive/filamentous growth等。參與在此訊息傳遞路徑之蛋白由上游到下游分別為MAPK kinase kinase (MAP3K)、MAPK kinase (MAP2K)、MAPK，MAPK藉由活化下游之transcription factor，將訊息傳遞至細胞核，以誘導特定基因表現。在其他病原真菌的相關研究也發現此MAPK訊息傳遞在Magnaporthe grisea、Ustilago maydis、Cryptococcus neoformans、Candida albicans等的致病過程扮演重要角色。Phytophthora parasitica是台灣重要病原菌，會在蕃茄及枇杷等多種經濟重要作物造成根腐、莖腐、果腐等病害，嚴重時甚至會造成整株植物萎凋。為了探討MAPK訊息傳遞途徑在疫病菌致病機制的重要性，首先自P. infestans及P. sojae 的EST database (Phytophthora Functional Genomics Database) 搜尋MAPK訊息傳遞途徑相關基因，包括活化MAP3K的蛋白Rac及Ste20，還有MAPK下游的transcription factor Ste12，其中ste20-like基因在其他病原真菌如Magnaporthe grisea、Ustilago maydis、Neurospora crassa、Fusarium graminearum、Cryptococcus neoformans、Candida albicans也有發現到，並參與在mating、appressorium formation、infectious growth、virulence等過程。接著根據這些MAPK pathway上相關蛋白的序列設計degenerate primer，進行genomic DNA PCR及TA cloning。序列分析結果顯示所增幅的核酸片段中包含一ste20-like gene，命名為ppste20。後續以RACE進行分子選殖的結果顯示，ppste20全長度cDNA包含一1134 bp的ORF，可轉譯出一378 amino acid的蛋白質，其含有ser/thr protein kinase domain，且在P. parasitica基因體中只存在單一copy。北方雜合分析結果顯示，ppste20在P. parasitica菌絲期表現量很低， real-time RT-PCR之分析結果顯示ppste20在疫病菌zoosporogenesis的過程中似乎會被誘導表現。為了確定此基因確實具有ser/thr protein kinase activity，將此基因送到E. coli進行表現，並進行蛋白質純化，然後進行in-gel kinase activity assay。但截至目前為止，仍然沒有偵測到Ppste20的活性。Phytophthora parasitica, being able to attack a wide variety of plants, is a very important plant pathogen in Taiwan. It is an oomyceteous fungus. It causes root rot, foot rot, leaf blight, and fruit rot in a variety of economically important crops. It has been shown that isolates of P. parasitica from tobacco and loquat differed significantly from other isolates in morphology and pathogenicity and were recognized as "atypical" types of the fungus. Ste20 is a key regulator in the mating pheromone response pathway in Saccharomyces cerevisiae. It responses via the activation of a MAPK cascade. It also can influence osmotic response, invasive growth, and polarized growth. Recently, it was also toward that Ste20-like protein could influence the virulence in Cryptococcus neoformans and Candida albicans. We designed the degenerate primers according to the Ste20 consensus amino acid sequence, and obtained a 888bp DNA fragment from Phytophthora parasitica after PCR. Then, we performed the RACE to obtain the full length ste20-like gene, which was named ppste20 gene. The amino acid sequence of the Ppste20 encodes a ser/thr protein kinase. Southern blot analysis has demonstrated that Phytophthora parasitica has only one copy of ppste20, and the RT-PCR showed that ste20-like gene could express in the mycelium stage of P. parasitica. The result of quantitative RT-PCR showed that the gene was induced at the zoosporogenesis stage in P. parasitica. The Ppste20 protein was expressed in and purified from E. coli for in-gel kinase assay. So far no kinase activity was detected.中文摘要………………………………………………………………..……..…….1 英文摘要……………………………………………………………..…….…….….3 序 言…………………………………………………………………...…….….4 壹、前人研究………………………………………………………………...….….5 一、疾病菌Phytophthora parasitica 的研究:……………………….…..…...5 1.分類地位…………………………………………………………....…..5 2.型態特徵……………………………………………………………….6 3.發生生態…………………………………..…………………....……....6 4.台灣發生現況與危害作物疾病徵………………..…………….…..…7 5.疾病菌之訊息傳導研究:……………………………….……….....…..8 二、真核生物的MAPK pathway:…………………………….…………......10 三、酵母菌的訊息傳導研究:………………………………..…………. ….11 1.酵母菌的生活史………………………………………………..….…11 2.Pheromone訊息傳導路徑……………………..………...…….…..….12 3.酵母菌中的ste20…………………………………..…………………14 四、Ste20在其他植物病原真菌中所扮演的角色…...………......…..…..….15 貳、材料與方法……………………………………………….………..….……...17 一、菌株來源與保存:……………………………………………….….…....17 二、Phytophthora parasitica 之核酸製備:……………………….......……..17 1. DNA的製備………………………………………….….……...……17 2. RNA及poly(A)+RNA之製備………………………...….……..……18 三、ppste20基因選殖:…………………………………………. .….…....….19 1. ppste20基因序列擴增………………………………….……....….…19 2. 純化DNA片段…………………………………………..….………19 3. TA選殖方法…………………………………………….……...…….20 4. 定序反應…………………….…………………...…….…………….20 5. RACE (Rapid amplification of cDNA ends)…………..........…….……21 6. ppste20 gene 序列比對分析………………………….…..…….……23 四、南方雜合分析…………………….…………………………..…...….…23 1、Genomic DNA 之酵解與瓊脂膠體電泳…………………..…. …..23 2、DNA轉漬法…………………………………….……………….…24 3、核酸探針之製備….………………………………….……….….…24 4、雜合前置反應…….……………………………………..……….…25 5、雜合反應…………….………………….…………....………..….…25 6、雜合訊息之偵測…………..….…………………….……….………25 五、北方雜合分析:……………………………………………..….. ….……26 1. RNA電泳分析…………………………………………….…….……26 2. RNA轉漬………………………………………………..…....………27 3. 核酸探針製被、前置雜核反應、雜核反應 及標示核酸探針之冷光偵測………………………………..…….....27 六、ppste20 基因表現:……………………………………………….……..27 1. RNA 材料製備…………………………….…..…………......………27 2. 以 real-time PCR 偵測ppste20 表現情形……….……...………….29 3. 分析real-time PCR數據………………………………………..……29 七、測試Ppste20蛋白質活性…………………………………...……....…..30 1. 建構ste20 ORF表現載體………………………………....……...….30 2. 基因轉殖入Escherichia coli M15 strain……………...…………...…31 3. 誘導基因表現……………………………………...……………..…..31 4. 抽取誘導Ppste20蛋白……………………………....………….....…31 5. 蛋白質SDS聚丙烯胺膠體電泳分析………………….……….........32 6. in-gel-kinase assay……………………………….......………….……....33 參、結果………………………………………………………...………….……...34 一、 ppste20全長度基因序列選殖……………………………….………..34 1. 利用degenerate primer增幅ppste20部分片段…...………………..34 2. 利用RACE得到ppste20基因5’以及3’端序列…...…………....….34 二、Ppste20序列比對分析…………………………………..…………..….35 三、南方雜合反應分析ppste20在Phytophthora parasitica 基因體中的分佈………………………………………….…………….36 四、北方雜合反應分析ppste20基因訊息RNA表現情形…..……......…..36 五、Phytophthora parasitica接種試驗………………………......…………36 六、Phytophthora parasitica游走子被誘導釋放的情形……….....……….37 七、定量PR-PCR分析ppste20基因表現情形……………………...….….37 八、Ppste20蛋白質活性測試…………………………………………...…..37 1. 建構ppste20 ORF表現載體………………………………………..38 2. 誘導Ppste20蛋白質表現並純化…………………………………...38 3. in-gel kinase assay………………………………………..………….....39 肆、討論……………………………………………………………….…...….….40 伍、圖表……………………………………………………………..…..…..……47 陸、參考文獻………………………………………………………..…..….…….70 柒、附錄………………………………………………………………..…..……..7