| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 89 === Xanthomonas campestris pv. campestris (Xcc) is the causing agent of black rot of crucifies. The purpose of this study is to find out the role which orf F gene plays in pathogenicity. Insertion of a Kmr DNA fragment in the orf F gene was first made with an orf F-containing Xcc genomic DNA fragment, and the fragment was introduced into two strains of Xcc via a suicide vector. The orf F::Kmr recombinants were selected and confirmed by Southern hybridization. The orf F::Kmr mutants of one Xcc strain showed reduced pathogenicity toward turnip plants, whereas those of the other Xcc strain completely lost the pathogenicity.
By using the T7 RNA polymerase/T7 promoter system and 35S Methionine, it was found that the orf F-containing Xcc DNA fragment was capable of expressing a protein with expected size of Orf F. With the protein expression plasmid pET21b, an Orf F-His protein was generated in vivo and purified through affinity chromatography. However, further HPLC analysis indicated that the protein solution contained two forms of Orf F-His with same molecular weight.
The Orf F-His protein solution was used to prepare polyclonal antibody which was then to probe the total proteins of two Xcc strains and their cultural filtrates. Both strains demostrated hybridization signals with their cellular proteins and the cultrual filtrates, indicating that Orf F is a secretion protein.
The orf F gene was cloned into a plant expression vector with GUS reporter gene and delivered into onion epidermal cells via particle biolistic system. It was shown that the Orf F-GUS protein was localized in the nucleus of the onion dermal cells. The 28th to 30th residues of the predictive Orf F protein sequence are lysine residues and likely constitue of a nuclear localization signal. Deletion or mutation of the three residues abolished the nuclear localization capability of Orf F. Further yeast one-hybrid study indicated that Orf F did not have transcriptional activation activity in yeast.
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