- 學位論文
疫病菌木聚醣酶基因的選殖與功能性分析
Cloning and functional characterization of genes encoding xylanase in Phytophthora parasitica
摘要
病原菌侵染植物寄主時，會分泌各種細胞壁分解酵素以促進侵染進程，其中包含木聚醣酶 (endo-beta-1,4-xylanase)，能夠水解半纖維素的主要成分－長鏈木聚醣，達到破壞植物細胞壁的目的。本研究探討木聚醣酶在疫病菌(Phytophthora parasitica)致病過程中所扮演的角色，將疫病菌木聚醣酶基因家族中共四個基因進行選殖，並將之個別命名為ppxyn1、ppxyn2、ppxyn3與ppxyn4。胺基酸序列分析顯示這四個疫病菌木聚醣酶皆具有木聚醣酶的保守性酵素活性區，親緣關係分析顯示，有別於真菌木聚醣酶，疫病菌木聚醣酶全都隸屬於醣苷水解酶家族10 (glycoside hydrolases family 10, GH10)的成員。以嗜甲醇酵母菌(Pichia pastoris)表現重組蛋白進行測試，證實ppxyn1、ppxyn2及ppxyn4重組蛋白皆具有水解樺木木聚醣之酵素活性，並且在耐熱特性及所需最適反應條件(包括酸鹼性及作用溫度等)皆有不同。以定量反轉錄-聚合酶連鎖反應測定基因表現情形，發現ppxyn1及ppxyn2在疫病菌感染番茄葉片初期即大量表現，ppxyn3在靜止子(cyst)表現量較高，而ppxyn4則在萌發靜止子被誘導表現。為進一步了解疫病菌木聚醣酶在疫病菌致病過程的的重要性，本研究應用Gateway選殖系統改造靜默載體，並建立疫病菌轉型系統，可以雙股 RNA 驅動疫病菌之基因靜默。分析結果顯示ppxyn1及ppxyn2靜默轉型株在圓葉煙草(Nicotiana benthamiana)或番茄(Solanum lycopersicum)上所造成的病徵皆有減弱現象，顯示ppxyn1及ppxyn2是疫病菌侵染植物時重要的細胞壁分解酵素。此外，利用農桿菌注入法所進行的分析發現ppxyn1及ppxyn3在圓葉煙草造成壞死斑，ppxyn2則在煙草(Nicotiana tabacum cv. Xanthi)上造成壞死斑，顯示這些基因可能具有導致細胞壞死的活性。
並列摘要
To infect their host plants, pathogens need to secret a variety of cell wall degrading enzymes, including endo-beta-1,4-xylanase that is crucial for the initial breakdown of xylan, the major hemicellulose component of the plant cell wall. To investigate the role of xylanase in Phytophthora parasitica, an oomyceteous plant pathogen with a wide host range, four genes encoding endo-b-1,4-xylanase, which named ppxyn1, ppxyn2, ppxyn3, and ppxyn4, were cloned and characterized respectively. Analysis of the deduced amino acid sequences indicated that all these genes contain active site signature for xylanase and belong to glycoside hydrolases family 10 (GH10). Phylogenic analysis demonstrated that all these genes form a cluster distinct from xylanases of the fungal pathogens. Analysis of the recombinant proteins obtained from the yeast Pichia pastoris indicated that ppxyn1, ppxyn2, and ppxyn4 encode a functional protein with a hydrolase activity toward birchwood xylan. However, each protein behaved differently regarding to their thermostability and conditions for optimal enzymatic activity. Analysis by quantitative reverse transcriptase-PCR indicated that ppxyn1 and ppxyn2 were up-regulated upon infection of tomato leaves by P. parasitica, suggestive of their involvement in the process of host infection. In contrast, ppxyn3 was highly expressed in cysts and ppxyn4 in germinating cysts. To further investigate the role of ppxyn genes in P. parasitica, silencing vectors were modified to facilitate Gateway cloning and CaCl2¬¬-PEG-mediated protoplast transformation method was established, which allow dsRNA mediated gene silencing in this pathogen. Both ppxyn1- and ppxyn2-silenced transformants show reduced virulence on tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum) plants. These results demonstrate the crucial role of xylanase-encoding ppxyn1 and ppxyn2 in the infection process of P. parasitica. In addition, transient expression of ppxyn1 and ppxyn3 by agroinfiltration caused necrosis on the leaves of N. benthamiana. As well, ppxyn2 caused necrosis on the leaves of Nicotiana tabacum cv. Xanthi. These results suggest that these genes might have necrotizing activities.
參考文獻
延伸閱讀
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