Cloning and characterization of a rhamnogalacturonan hydrolase (RGase A) gene from Botrytis cinerea
Chen, Huey-Jen
Ph.D., University of Maryland College Park, 1996, 78 pages
1996
บทคัดย่อ
Rhamnogalacturonan hydrolase, also known as RG-hydrolase (RGase A), cleaves α-1®2 linkages between non-esterified galacturonosyl and rhamnosyl residues of the pectic backbone. RGase A is suspected to be involved in pathogenesis of Botrytis cinerea, an important postharvest pathogen, because of its ability to degrade the highly branched pectic backbone.
The aims of this study were to isolate and characterize the B. cinerea RGase A gene. The initial approach was to purify RGase A from B. cinerea. A new assay method, using α -rhamnosidase, was developed to assay for RGase A activity in column fractions during purification. Using the α -rhamnosidase assay method, a 60 kDa RGase A was purified from crude B. cinerea extract by anion exchange, cation exchange and gel filtration. Because of a low yield of pure RGase A protein, an alternate approach was taken.
Isolation of a cDNA clone for the B. cinerea RGase A gene was achieved using a published nucleotide sequence for the Aspergillus aculeatus RGase A gene transcript. Three gene-specific primers were designed and used to synthesize the RGase A cDNA from A. aculeatus. A 1100 bp PCR fragment was obtained using these three primers. Using the PCR-amplified A. aculeatus RGase A clone as a probe, a 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a B. cinerea cDNA library.
The coding region of the BCRHGA clone shares 62% identity at the amino acid level with A. aculeatus RGase A. Northern blot analysis using B. cinerea total RNA revealed a 2 kb band when the BCRHGA cDNA was used as a probe, showing that the BCRHGA clone is a nearly full length cDNA clone. To determine the expression profile of the RGase A gene, B. cinerea spores were grown in Richmond media containing either 0.5% apple pectin, 0.5% rhamnogalacturonan-I (RG-I) or 1% glucose as a carbon source. Northern blot analyses revealed that the B. cinerea RGase A gene was expressed in cells grown on all three carbon sources. With 0.5% apple pectin and RG-I, maximum expression was reached in three-day-old cells and then dropped after day three. A different gene expression pattern was obtained when cells were grown on 1% glucose, in which RGase A expression increased progressively from day one to six. B. cinerea RGase A appeared to be coded for by a single or low copy number gene based on Southern blot analysis. Finally, the B. cinerea RGase A clone has been expressed in Escherichia coli, for use to obtain sufficient protein for antibody production.