論文題目 1：Proteomic and targeted metabolomic analysis of virulence variations Xanthomonas campestris pv. campestris by multi-quantitative methods with mass spectrometry論文題目 2：Comparative proteomic analysis of physiological and molecular mechanism in Rice

Autor: Tao-Shan Chang, 章道姍
Rok vydání: 2016
Druh dokumentu: 學位論文 ; thesis
Popis: 105
論文 1： Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative plant pathogenic bacterium causing black rot in crucifers, and resulting in tremendous loss in agriculture. Depends on certain factors including extracellular enzymes and exopolysaccharides (EPS), Xcc would infect plants successfully. In addition, regulation of pathogenic genes expression through Xcc responds with population-wide by quorum sensing (QS) and two-component signal transduction systems (TCSTSs). A newly isolated pathogenic XC17 from an infected cabbage leaf has some phenotypic characteristics, which have previously been implicated as factors contributing to pathogenicity. A wild-type strain of X. campestris, XC11, was found to have lost its pathogenicity spontaneously after frequent subculturing for years. This non-pathogenic derivative, designated XC11A. X. campestris P20H was a non-mucoid mutant previously isolated from XC11A by mutagenesis with nitrous acid. Recently, proteomics techniques can offer insights into the quantity and quality of the final gene products, and have been developed more widespread. In this study, we aim to elucidate the physiological and molecular mechanism response to virulence variations in Xcc by using proteomics method. We first used two-dimensional gel electrophoresis (2-DE) to resolve the expressed proteome in total proteins of Xcc, and then used nanospray liquid chromatography/linear ion trap mass spectrometer (nano-LC/LTQ-MS) to identify the differentially expressed proteins in total proteins of XC17, XC11A and XCP20H. Meanwhile, proteins of extracellular, membrane and intracellular were extracted separately for proteomics identification via LTQ-MS and triple TOF 6600 mass spectrometer analysis. In addition, the interested proteins were quantified by multiple reaction monitoring (MRM) and sequential window acquisition of all theoretical fragment ion (SWATH). Moreover, metabolite strategies for low-molecular-weight signaling molecules DSF and cyclic-di-GMP that involved in TCSTSs were quantified by MRM. The result shows many proteins and signaling molecules involved in TCSTSs of XC17 had higher expression than XC11A and XCP20H. Therefore, TCSTSs could promote pathogenic genes expression. There were also higher expression of transcription and translation factor proteins, DNA polymerases, ribosome, and tRNA ligase of XC17. And extracellular enzymes and enzymes involved in the synthesis of exopolysaccharide of XC17 had upregulated expression as well. Simultaneously, NADH dehydrogenase and ATPase of membrane proteins had higher level expression, resulted in supplying more engery for secretion of extracellular enzymes by type II secretion system. Since XC17 had higher expression of Tol/Pal system which plays an important role in the maintenance and integrity of the bacterial cell envelope, such that XC17 had better survival ability. Through the above results, pathogenicity of Xcc originated in complex regulation of physiological processes. Although both XC11A and XC17 could secrete EPS, XC11A secreted far less. Besides, the protein expression of TCSTSs that regulates downstream pathogenic genes expression of XC11A is less than of XC17 as well. Thus, XC11A lacked of pathogenicity. Moreover, XCP20H had the least protein expression whether synthesis of EPS, involved in TCSTSs, or other pathogenesis related proteins, such that XCP20H also lacked of pathogenicity. 論文 2： In human diet, rice (Oryza sativa L.) is an important source of starch which is majorly accumulated in developing endosperm. Moreover, growth and productivity of rice are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt-stress tolerant strains. In this study, comparative proteomic analysis was used to study the different expression of proteins that affect the efficiency of starch biosynthesis between wild-type rice (TNG67) and its NaN3-induced pure line mutant (SA0419). In addition, rice seedlings of the same genetic species with various salt tolerances were also studied. We applied two-dimensional gel electrophoresis (2D-PAGE) proteomic approaches to profile the expressed proteome in developing endosperm, leaves and roots of rice and analyzed by nano-LC/MS/MS. We found several up-regulated enzymes, such as AGPase, PGM, PFP, PFK, PPDK, SBE and SDBE in SA0419, which were mainly involved in the pathways of starch metabolism and PPDK-mediated gluconeogenesis. Those up-regulated pathways explained that SA0419 had more starch accumulation and more rapid grain filling capacity than TNG67. Furthermore, The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, ATP synthase, quercetin 3-O-methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt-stress were glycolysis, purine metabolism, and photosynthesis. This study provided a detailed biochemical description of starch biosynthesis, the yield and quality of rice can be improved upon during the breeding of important agricultural cereal crop.
Databáze: Networked Digital Library of Theses & Dissertations
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