疫病菌之ppste20基因分子選殖與特性分析

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。

關鍵字

疫病菌；訊息傳遞路徑；孢囊；游走子

並列摘要

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.

並列關鍵字

signal transduction ； zoosporogenesis ； ste20-like protein、Phytophthora parasitica

參考文獻

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疫病菌之ppste20基因分子選殖與特性分析

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