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

本研究从白桦溃烂病病原菌IO-U1或IO-B2菌株侵染的日本白桦8号试管苗的粗蛋白中检测到侵染特异性蛋白，并利用数据库检索对检测到的蛋白进行了估计和鉴定。接种1周后，每天收集感染斜纹夜蛾IO-U1或IO-B2菌株的植株、对照1株(无感染和损伤)和对照2株(受伤)。这些试管苗用液氮冷冻，粉末状，然后用缓冲液提取蛋白质。得到的蛋白质溶液经浓缩、脱盐后进行双向电泳(2DE)。电泳后，用银染色凝胶，用扫描仪将图像输入个人计算机，然后对凝胶进行图像分析。IM…检测感染特异性蛋白对应蛋白的研究根据它们的相对分子质量和等电点，在数据库上搜索到更多的年龄分析。结果表明，与氧化猝发有关的蛋白质有碳酸酐酶、NADH-泛醌氧化还原酶、细胞色素C氧化酶、谷胱甘肽S转移酶、L抗坏血酸过氧化物酶和谷胱甘肽过氧化物酶。此外，还推测形成了以下蛋白质：PR10-1、咖啡酰辅酶A O-甲基转移酶、查尔酮-黄酮异构酶、热休克70 kDa蛋白、甘油醛3-磷酸脱氢酶、20S蛋白酶体和钙网蛋白。在IO-U1感染的情况下，大多数培养天数都检测到与氧化猝发有关的蛋白质。IO-B2感染还导致了估计与氧化爆发有关的蛋白质的产生。因此，推测IO-U1和IO-B2在白桦试管苗感染后一周内都发生了氧化猝发作为主要的保护反应。而在IO-U1感染的情况下，在感染2天后检测到许多感染特异的蛋白，并从感染的植株中获得蛋白制剂。样品经双向凝胶电泳法提取后，用改良的银染色法进行MS分析。从凝胶中切下12个感染特异性蛋白点，经凝胶内消化后，进行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分子伴侣蛋白是感染特异性蛋白。这些结果表明，在感染IO-U1的8号桦树试管苗中，氧化猝发是一种保护机制，保护基因的表达活跃。较少

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