Comprehensive analysis of infection-specific proteins in Betula platyphylla var. japonica plantlets infected with a birch canker pathogen, Inonotus obliquus

本研究以白桦溃疡病菌Inonotus acuminus IO-U1和IO-B2菌株感染8号白桦（Betula platyphylla var.japonica No.8）幼苗为试材，检测其粗蛋白中的感染特异性蛋白，并利用数据库检索对检测到的蛋白进行鉴定。接种后一周每天收集感染I. aerobusIO-U1或IO-B2菌株的植株、对照1植株（未感染且未受伤）和对照2植株（受伤）。将这些试管苗用液氮深冻，粉碎，然后用缓冲液提取蛋白质。将所得蛋白溶液浓缩、脱盐，然后用于双向电泳（2DE）。电泳后，凝胶用银染色，并将图像输入带有扫描仪的个人计算机，然后对凝胶进行图像分析。免疫组织化学检测感染特异性蛋白的对应蛋白 ...更多信息 基于它们的分子量和等电点，在数据库中检索年龄分析。作为结果，碳酸酐酶、NADH-泛醌氧化还原酶、细胞色素C氧化酶、谷胱甘肽S-转移酶（GST）、L-抗坏血酸过氧化物酶和谷胱甘肽过氧化物酶被估计作为参与氧化爆发的蛋白质产生。此外，还推测形成了以下蛋白质：PR 10 -1、咖啡酰-CoA O-甲基转移酶、查耳酮-黄酮异构酶、热休克70 kDa蛋白、甘油醛3-磷酸脱氢酶、20 S蛋白酶体和钙网蛋白。在IO-U1感染的情况下，估计参与氧化爆发的蛋白质在大多数培养天数中被检测到。IO-B2感染还引起估计参与氧化爆发的蛋白质的产生。因此，它被认为是在与IO-U1和IO-B2的感染发生的氧化爆发作为一个主要的保护反应，在桦树苗感染后一周内。而在IO-U1感染的情况下，在感染后2天检测到许多感染特异性蛋白质，从感染的小植株获得蛋白质制剂。对样品进行2DE，并通过改进的方法用银染色凝胶用于MS分析。从凝胶上切下12个感染特异性蛋白点，将它们在凝胶内消化，然后用MALDI-TOF-MS分析。使用所获得的肽图，进行Mascot数据库搜索。结果表明，GST和60 kDa分子伴侣蛋白为感染特异性蛋白。从这些结果，它是澄清，氧化爆发发生作为一种保护机制和保护基因的表达活跃的白桦8号苗感染IO-U1。少

In the present study, infection-specific proteins were detected in the crude protein preparations from Betula platyphylla var.japonica No.8 plantlets infected with a birch canker pathogen, Inonotus obliquus IO-U1 or IO-B2 strain, followed by estimation and identification of the detected proteins with using data base search. The infected plantlets with I.obliquus IO-U1 or IO-B2 strain, control 1 plantlets (no infection and no injury), and control 2 plantlets (injured) were collected everyday after the inoculation for one week. These plantlets were deep-frozen with liquid N_2, powdered, and then subjected to extraction of proteins with buffer. The obtained protein solutions were concentrated, desalted, and then used for two-dimensional electrophoresis (2DE). After the electrophoresis, the gels were stained with silver, and the images were inco**rated into personal computer with scanner, followed by image analysis of the gels. Corresponding proteins to infection-specific ones dete** by im … More age analysis were searched on the data base, based on their molecular weight and isoelectric point. As the results, carbonic anhydrase, NADH-ubiquinone oxidoreductase, cytochrome C oxidase, glutathione S-transferase (GST), L-ascorbate peroxidase, and glutathione peroxidase were estimated to be produced as the proteins involved in oxidative burst. In addition, the following proteins were also presumed to be formed : PR10-1, caffeoyl-CoA O-methyltransferase, chalcone-flavonone isomerase, heat shock 70kDa protein, glyceraldehyde 3-phosphate dehydrogenase, 20S proteasome, and calreticulin. In the case of IO-U1 infection, the proteins estimated to be involved in oxidative burst were detected in the most of culture days. IO-B2 infection also caused production of the proteins estimated to be involved in oxidative burst. Hence, it is assumed that in both the infection with IO-U1 and IO-B2 oxidative burst occurred as a main protective reaction in birch plantlets within one week after infection. While in the case of IO-U1 infection many infection-specific proteins were detected 2 days after infection, protein preparation was obtained from the infected plantlets. The sample was subjected to 2DE, and the gel was stained with silver by the modified method for MS analysis. Twelve infection-specific protein spots were cut off from the gel, they were in-gel-digested, and then analyzed with MALDI-TOF-MS. Using the obtained peptide maps, Mascot data base search was performed. As the results, GST and 60kDa chaperonin were identified as infection-specific proteins. From these results, it is clarified that oxidative burst occurred as a protective mechanism and protective genes were actively expressed in the birch No.8 plantlets infected with IO-U1. Less