Studies on the detection of blue pigment and mechanism of color development on MacConkey medium for Erwinia chrysanthemi.

Autor: CHEN , CHIA-HSIN, 陳家欣
Rok vydání: 2008
Druh dokumentu: 學位論文 ; thesis
Popis: 96
chapter-1-Abstract The blue pigment, indigoidine, was discovered in Pseudomonas indigofera, Erwinia chrysanthemi can also produce this blue pigment. E. chrysanthemi is a plant-pathogenic enterobacterium responsible for soft rot diseases in many crops. The blue pigment has the potential to prevent the oxygen radicals of plant defence mechanisms. Until now, we don’t confirm indigoidine existence in plant soft rot tissues, because the blue pigment is difficult to purify and without standard. At first, E. chrysanthemi produce abundant indigoidine in NGM medium, then we purified blue pigment by a method early devised in laboratory. The blue pigment structure agree with indigoidine by Nuclear Magnetic Resonance Spectroscopy (NMR) and Infrared Spectroscopy (IR) structure determination. The blue pigment standard could be dissolved in DMSO solution. HPLC with methanol as mobile phase and absorption at 615 nm. HPLC separation showed a clear peak at 3 minute after sample injection. The plant soft rot tissues by E. chrysanthemi-infected had been purified, and the HPLC separation showed a minim peak at 3 minute after sample injection. In contrast, E. chrysanthemi suspension in NGM medium added into plant soft rot tissues, HPLC separation showed a clear peak at 3 minute as well as standard. We add standard to purified product of plant soft rot tissues for HPLC detection, HPLC separation showed the minim peak increase significantly at 3 minute. The result demonstrate the existence of indigoidine in the E. chrysanthemi-infected plant tissues. When we added hydrogen peroxide and Ferrous Sulphate into blue pigment, Fenton reaction, oxidated and changed the color. The blue pigment within hydrogen peroxide via 65℃ sonication could enhance oxidation and the color turned into colorless at 90 min. chapter-2-Abstract Erwinia carotovora subsp. carotovora (Erc) and E.chrysanthemi (Ech) were first grown in MacConkey medium. Erwinia carotovora subsp. carotovora produces brick red colonies but E. chrysanthemi produces colorless colonies. MacConkey medium was used for differentiating lactose-fermenting from lactose-nonfermenting bacterium. Erwinia carotovora subsp. carotovora and E. chrysanthemi have different color on the MacConkey medium. Accordingly, we have focused on lactose fermentation related gene in Erwinia carotovora subsp. carotovora and E. chrysanthemi. Specific oligonucleotide primers lacZ-F+R (671 bp) and lacY-F2+R2 (500 bp) could be used to detecte by PCR amplification and Southern hybridization for Erwinia carotovora subsp. carotovora and E. chrysanthemi. These data indicated Erwinia carotovora subsp. carotovora possess lacZ and lacY gene, but E. chrysanthemi only possess lacZ gene. We suggest that lacZ and lacY gene are concerned in fermentation of lactose. The other Erwinia spp. grown in MacConkey medium, we discovered only E. rhapontici ER1 and E. rhapontici ER120(T) could develop red colonies. Other Erwinia spp. are also detected lacZ and lacY gene by PCR amplification and Southern hybridization. These data indicated E. cypripedii EC160 、 E. cypripedii EC155(T) 、 E. rhapontici ER1 、 E. rhapontici ER120(T) and E. nigrfluensi EN105 contain lacZ gene and E. rhapontici ER1 is the only one can be detected with lacY gene. We construct lacZ gene by Dickeya dianthicola 1200 (E.chrysanthemi pv. dianthicola) genomic library. LA medium contain X-gal and chloramphenicol was used to screen blue colonies. We found 2 clonies with the specific primer lacZ-F+R3 318 bp fragment. The plasmid of blue clone digest with EcoRI and EcoRI+HindIII. We used Dig- labeled lacZ fragment as probe in Southern hybridization. The result show only 2 clones which have lacZ hybridize fragment. We choosed EPI300-1200-A-10 clone for gene subcloning.
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