Phytophthora parasitica多聚半乳糖醛酸酶基因族之分子選殖與功能性分析

Abstract

植物病原菌侵染植物過程所涉及的機制相當複雜，果膠質分解酵素(pectic enzymes, pectinases, or pectinolytic enzymes)或其他細胞壁分解酵素可能在病原菌致病過程佔有關鍵性地位。多聚半乳糖醛酸酶(polygalacturonase, PG)是其中一類的果膠質分解酵素，可以水解果膠質主要成分galacturonan 分子間之鍵結，並且在許多的植物病原真菌當中已被證實和致病力有關。Phytophthora parasitica是一分佈廣泛且寄主範圍相當廣的卵菌，可以在許多重要的經濟作物上造成病害，是相當重要的植物病原菌。在本研究室先前的研究當中，由P. parasitica中選殖到一在侵染植物的過程當中會大量表現的多聚半乳糖醛酸酶基因，命名為pppg1。南方雜合分析顯示在P. parasitica基因體當中應有其它pppg1的同源性序列存在(Yan and Liou, 2005)。為了進一步的了解這些基因的功能特性，我們針對P. parasitica基因庫進行篩選，並應用反轉錄聚合酶連鎖反應的方式，一共得到了十三個多聚半乳糖醛酸酶基因。親緣分析的結果顯示Phytophthora spp.的多聚半乳糖醛酸酶和真菌的多聚半乳糖醛酸酶有較高的同源性，且在不同種的Phytophthora spp.當中可以找到相對應的同源性基因，推測Phytophthora spp.在種化前即具有多個多聚半乳糖醛酸酶基因。為了解這些不同的多聚半乳糖醛酸酶的特性，我們針其中八個基因，利用甲醇酵母菌(Pichia pastoris)進行重組蛋白質表現及活性測定，證實其具有分解果膠質的酵素活性。進一步利用PVX agroinfection技術將這些蛋白質於菸草(Nicotiana benthamiana)上進行系統性的表現，並配合石蠟切片觀察其對於菸草的影響，結果發現不同的多聚半乳糖醛酸酶會在菸草上造成不同的影響，這些影響和其酵素的活性有關。除此之外，將其中一些蛋白質在蕃茄(Lycopersicon esculentum)及菸草(Nicotiana tabacum)上進行系統性的表現，亦會造成不同的影響。顯示這些蛋白質在in planta的情況下具有不同的生物特性，可能在P. parasitica侵染植物的過程中扮演不同的角色。

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of plant species. It has been shown in our previous study that pppg1, which encodes endopolygalacturonase (endoPG), is highly induced during interaction of P. parasitica with the host plant, suggesting its involvement in the process of plant infection. Southern blot analysis using pppg1 as the probe indicated the presence of other sequences homologous to pppg1 in the genome of P. parasitica (Yan and Liou, 2005). To gain insight into to the functional characteristics of these genes, we screened the genomic library of P. parasitica using the fragments amplified by degenerate primers which were designed from the conserve region of Phytophthora endoPGs. The results indicated that there exists an endoPG multigene family with at least 13 members in P. parasitica. Besides, analysis of eight recombinant proteins obtained from the yeast Pichia pastoris by heterologous expression demonstrated that these proteins process polygalacturonase activity. By using PVX agroinfection to express these endoPGs systemically in Nicotiana benthamiana, we showed that these endoPGs caused distinct effects on plants and these effects were abolished when hydrolysis activity and signal peptide were lost by site direct mutagenesis or deletion. Moreover, the effects caused by these endoPGs were different when expressed in tomato (Lycopersicon esculentum) and Nicotiana tabacum. The results here showed that these endoPGs have distinct biological characteristics in planta.