Cloning and characterization of a rhamnogalacturonan hydrolase gene from Botrytis cinerea.
Rhamnogalacturonan hydrolase (Rgase A) cleaves alpha 1--2 linkages between rhamnosyl and galacturonosyl residues in pectin. A 1.9 kb RGase A cDNA clone (BCRHGA) was isolated from a B. cinerea cDNA library using a PCR-amplified Aspergillus aculeatus RGase A probe. It's 1.7 kb open reading frame had 62% identity at the amino acid level with A. aculeatus RGase A. Northern blots of B. cinerea total RNA probed with BCRHGA revealed a 2 kb band, suggesting the cDNA clone is full or nearly-full length. To determine mRNA expression of the gene, B. cinerea was grown in media containing 0.5% apple pectin, 0.5% rhamnogalacturonan-I and 1% glucose carbon sources. Northern analysis revealed the BCRHGA gene was expressed on all carbon sources, but with different patterns of expression. B. cinerea RGase A appeared to be coded for by a single or low copy number gene based on Southern analysis.
Chen
H J
HJ
Horticultural Crops Quality Laboratory, USDA, Agricultural Research Service, Beltsville, MD 20705, USA.
Smith
D L
DL
Starrett
D A
DA
Zhou
D
D
Tucker
M L
ML
Solomos
T
T
Gross
K C
KC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004271:DNA, Fungal / Q000032:analysis; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003904:Mitosporic Fungi / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D006026:Glycoside Hydrolases; C067959:rhamnogalacturonase A