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

白桦感染特异性蛋白的综合分析

• 批准号: 15580119
• 负责人: YOKOTA Shinso
• 金额: $ 1.79万
• 依托单位: Utsunomiya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580119/
• 关键词: Inonotus obliquus; Betula platyphylla var.japonica; infection-specific proteins; proteome; forest pathology; two-dimensional electrophoresis; oxidative burst; chaperonin; 樹病; 宿主特異的抵抗性

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

在演讲研究中，在贝氏铂型脂肪蛋白制剂中检测到感染特异性蛋白质，这些蛋白质的蛋白质制剂是8号japonica No.8 No. 8植物园，这些植物会感染了桦木病原体，Inonotus io-U1或IO-B2菌株，随后是使用数据基础搜索的检测蛋白进行估计和鉴定。每天在接种后每天收集一周后，每天收集带有I.obliquus io-U1或IO-B2菌株，对照1个植物性植物（无感染和无损伤）的感染植物园，并收集对照2个植被（受伤）（受伤）。这些植物会用液体N_2深冻，粉末，然后用缓冲液提取蛋白质。将所获得的蛋白质溶液浓缩，脱盐，然后用于二维电泳（2DE）。电泳后，将凝胶用银染色，并用扫描仪将图像插入个人计算机中，然后对凝胶进行图像分析。根据IM的相应蛋白质与特定于感染的蛋白质有关……根据其分子量和等电点，在数据库上搜索了更多的年龄分析。结果，碳酸酐酶，NADH-偶普酮氧化还原酶，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估计被估计作为蛋白质与氧化脉络产生。此外，还提出了以下蛋白质形成：PR10-1，咖啡酰-COA O-甲基转移酶，chalcone-氟烯酮异构酶，热休克70KDA蛋白，3-磷酸盐3-磷酸甘油醛，脱氢酶，20S蛋白质和钙含蛋白。在IO-U1感染的情况下，在大多数培养日中都检测到估计参与氧化爆发的蛋白质。 IO-B2感染还导致估计参与氧化爆发的蛋白质的产生。因此，假定在感染IO-U1和IO-B2氧化爆发的感染中都是在感染后一周内作为桦木植物的主要保护反应。在感染后2天，在IO-U1感染的情况下检测到许多感染特异性蛋白，但从感染的植物园中获得了蛋白质制备。将样品进行2DE进行，并通过修饰方法将凝胶用银染色。从凝胶中切除了十二个特异性蛋白质斑点，它们被凝胶消化，然后用MALDI-TOF-MS进行分析。使用获得的肽图，进行了吉祥物数据库搜索。结果，GST和60KDA伴侣蛋白被确定为感染特异性蛋白。从这些结果中可以澄清，氧化爆发是作为一种受保护的机制发生的，受保护的基因在感染了IO-U1的桦木8号植物中被积极表达。较少的