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| Identifier |
uch.biology.phd//2001anastasopoulos |
| Title |
Συμβολή στη μελέτη της ευκαρυωτικής παγοπυρήνωσης. Ανάπτυξη μεθόδου επιλογής φυτών καπνού (Nicotiana tabacum L.) Ανθεκτικών στο πάγωμα και απομόνωση παγοπυρηνωτικού γονιδίου από τον μύκητα Fusarium acumitatum. |
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Author
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Αναστασόπουλος, Ηλίας
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Thesis advisor
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Πανόπουλος, Νικόλαος
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| Abstract |
The susceptibility of plants to frost is often due to ice nucleating bacteria and fungi. By producing certain proteins, these microorganisms cause plants to freeze at relatively warm subzero temperatures. This phenomenon is known as biological ice nucleation and has been a subject of research for more than 25 years. This study is part of a research effort aiming at the identification of the factors that relate to the biological ice nucleation and freezing injury in eukaryotic systems. In the first part of this study the use of transgenic tobacco plants expressing the ice nucleation gene inaZ from Pseudomonas syringae is described. The first part of this study refers to the development of the required methodology for the large screening of heterozygous (inaZ +/-) plants for the identification of mutants with reduced freezing susceptibility after γ ray irradiation. The methodology that has been developed is based on two freezing assays. The first is applied on small seedlings whereas the second is applied on leaf discs from selected plants. Both freezing assays are carried out in microtitter plates floating the surface of a cooling bath. The second part of this study deals with the cloning of an ice nucleation cDNA from the fungus Fusarium accuminatum. Two cDNA libraries were constructed with different average insert sizes. The identification of the ice nucleation cDNA was accomplished using a combination of heterologous hybridization and a new functional assay. This assay is simple and rapid and can be used for the cloning of other ice nucleation genes. It is based on the induced expression of cDNA clones from the library and the functional screening a) of phage plaques, after blotting of phage expressed proteins on filters and b) bacterial colonies after in vivo phagemids excision. The cloning of the ice nucleation cDNA clone was confirmed by transforming bacteria and checking for ice nucleation activity. The ice nucleation activity of the isolated cDNA was compared to that of the inaZ gene and of the fungus from which it was isolated. The cDNA was sequenced and the structure of the predicted protein product was analysed in silico. The protein lacks conserved amino acid repeats similar to these fount in the bacterial counterparts, though there are some imperfect repeats of different nature. The protein also shows segment homology to glycosylhydrolases and contains antifreeze motifs in different parts of the molecule.
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| Language |
Greek |
| Issue date |
2001-01-25 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Doctoral theses
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Type of Work--Doctoral theses
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| Views |
250 |
Συμβολή στη μελέτη της ευκαρυωτικής παγοπυρήνωσης. Ανάπτυξη μεθόδου επιλογής φυτών καπνού (Nicotiana tabacum L.) Ανθεκτικών στο πάγωμα και απομόνωση παγοπυρηνωτικού γονιδίου από τον μύκητα Fusarium acumitatum.
