Molecular cloning and characterization of β-glucan biosynthesis related genes, glp5-1 and glp5-2, in Ganoderma lucidum
Date Issued
2005
Date
2005
Author(s)
Wang, Chih-Wei
DOI
zh-TW
Abstract
Polysaccharides from Ganoderma lucidum have been reported to exhibit immuno-modulation and antitumor activties. The molecular basis of polysaccharides biosynthetic pathway was not yet clear. Sequences of potentially related genes were retrieved from the G. lucidum genome database and primers were desigened to isolate the corresponding clones. Full-length cDNA sequences of target genes were initially obtained by RACE (rapid amplification of cDNA ends). Among them, two genes named glp5-1 and glp5-2 were further characterized in this sudy. glp5-1 cDNA contained an open reading frame of 1,077 nucleotides, which encodes a protein with 359 amino acid residues and an estimated molecular mass of 39.7 kDa. Glp5 has 70% amino acid sequences similarity with SHS4, reported in Agaricus bisporus, and a putative exo-β-1,3 glucanase domain was also identified. SHS4 has been reported to have 42% amino acid similarity with 1,6-β-glucan synthase gene in Pseudomonas putida KT2400. Expression level of shs4 gene is correlated with the increase of 1,6-β-linked glucan side branches and considered to participate in the biosynthesis of 1,6-β-glucan. Genes with such domain include scw4, scw10, scw11, and bgl2 reported in yeast. These four genes were classified as glycoside hydrolase family 17 by CAZy database. Bgl2 has been reported to have endo-1,3-β-glucanase and glucanosyltransferase activity. Others were reported to have putative 1,3-β-glucanase or 1,3-β-glucanosyltransferase activity by genetic analysis. In searching available genome database of other fungi, we have found that glp5-1 homologous genes were highly conserved in many basidiomycetes. Further study also found that glp5-2, a homologous gene of glp5-1, was located about 400 nucleotides downstream of glp5-1 gene. glp5-2 cDNA had an open reading frame of 1,242 nucleotides encoding a 414-amino acid protein with a predicted size of 44.6 kDa. Northern blot analysis showed that glp5-1 mRNA both expressed in mycelium and fruit body stage of G. lucidum, and was decreased in mycelium stage with increasing culture period. In fruit body stage, glp5-2 mRNA expression was greatly induced in pileus of growth area than in stipe. Glp5-1 recombinat protein expressed by E. coli was purified and used to raise antibody for further experiments. Western blot analysis showed a size of 63 kDa protein was detected in cell wall fraction of both mycelium and fruit body. Indicated Glp5-1 is a cell wall protein. The estimated size of Glp5-1 protein based on western blot signals was larger than that predicted from amino acid sequences. Glycosylation of Glp5-1 protein may occurr in G. lucidum. The functions of Glp5-1 and Glp5-2 proteins need to be further investgated.
Subjects
靈芝
葡聚醣
Ganoderma lucidum
beta-glucan
Type
other