Efficient expression of laccase gene from white-rot fungus Schizophyllum commune in a transgenic tobacco plant.
Ligninolytic enzymes produced by white-rot fungi are effective degraders of recalcitrant aromatic environmental pollutants. However, gene sequences of these enzymes are rich in CpG dinucleotides, which are particularly unfavorable to efficient expression in plants. In order to develop a phytoremediation technique with a ligninolytic enzyme-producing transgenic plant, laccase cDNA (scL) from white-rot fungus Schizophyllum commune was used as a model ligninolytic enzyme, and we attempted to obtain the efficient expression of scL in a transgenic tobacco plant by decreasing the CpG-dinucleotide motif content. We constructed a mutagenized scL sequence, scL12, decreasing the CpG-dinucleotide motif content by 12%, and scL12 was introduced into the tobacco plant. Much higher laccase activity was detected in transgenic scL12 plants than in transgenic scL plants and wild-type plants. Using reverse transcriptase-PCR analysis, scL12 was translated in transgenic scL12 plants whereas mRNA of scL was not detected in the transgenic scL plants, and scL, which is the product of the scL12 gene, was produced in the transgenic scL12 plants using native-polyacrylamide gel electrophoresis analysis. Moreover, transgenic scL12 plants were able to remove trichlorophenol more effectively than transgenic scL plants and wild-type plants. These results suggest that decreasing CpG-dinucleotide motif content in fungal target genes is a useful method for efficient expression of these genes in transgenic plants.
Hirai
Hirofumi
H
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan. afhhirai@agr.shizuoka.ac.jp
Kashima
Yoshiyuki
Y
Hayashi
Katsuma
K
Sugiura
Tatsuki
T
Yamagishi
Kenji
K
Kawagishi
Hirokazu
H
Nishida
Tomoaki
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D012567:Schizophyllum / Q000201:enzymology*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D042845:Laccase