Molecular cloning and pattern of expression of an alpha-L-fucosidase gene from pea seedlings.
alpha-L-Fucosidase is a cell wall protein purified from pea (Pisum sativum) epicotyls. The alpha-L-fucosidase hydrolyzes terminal fucosyl residues from oligosaccharides of plant cell wall xyloglucan. alpha-L-Fucosidase may be an important factor in plant growth regulation, as it inactivates fucose-containing xyloglucan oligosaccharides that inhibit growth of pea stem segments. The amino acid sequences of the NH2-terminal region and one internal peptide were used to design redundant oligonucleotides that were utilized as primers in a polymerase chain reaction (PCR) with cDNA, generated from pea mRNA, as the template. A specific PCR amplification product containing 357 base pairs was isolated, cloned, and sequenced. The deduced amino acid sequence included the two peptides used to design the primers for PCR plus two other peptides obtained by proteinase digestion of alpha-L-fucosidase. No sequence homology to other alpha-L-fucosidases was apparent, although the NH2-terminal region is strongly homologous to Kunitz-type trypsin inhibitors. cDNA and genomic copies were isolated and sequenced. In pea, the gene is present in two or three copies. Its mRNA is present in roots, leaves, and elongating shoots. The spatial pattern of expression of the alpha-L-fucosidase was determined by in situ hybridization.
Augur
C
C
ORSTOM, Institute of Scientific Research for Development in Cooperation, Los Morales, Mexico D.F., Mexico.
Stiefel
V
V
Darvill
A
A
Albersheim
P
P
Puigdomenech
P
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D008969:Molecular Sequence Data; D018532:Pisum sativum / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D005644:alpha-L-Fucosidase / Q000737:chemistry / Q000235:genetics*
D000596:Amino Acids; D018076:DNA, Complementary; D012333:RNA, Messenger; D005644:alpha-L-Fucosidase