Hirai Hirofumi, Kashima Yoshiyuki, Hayashi Katsuma, Sugiura Tatsuki, Yamagishi Kenji, Kawagishi Hirokazu, Nishida Tomoaki
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan.
FEMS Microbiol Lett. 2008 Sep;286(1):130-5. doi: 10.1111/j.1574-6968.2008.01267.x.
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.
白腐真菌产生的木质素分解酶是顽固性芳香族环境污染物的有效降解剂。然而，这些酶的基因序列富含CpG二核苷酸，这对在植物中的高效表达尤为不利。为了开发一种利用产生木质素分解酶的转基因植物进行植物修复的技术，来自白腐真菌裂褶菌的漆酶cDNA（scL）被用作模型木质素分解酶，并且我们试图通过降低CpG二核苷酸基序含量来在转基因烟草植物中实现scL的高效表达。我们构建了一个诱变的scL序列scL12，其CpG二核苷酸基序含量降低了12%，并将scL12导入烟草植物。在转基因scL12植物中检测到的漆酶活性比转基因scL植物和野生型植物中高得多。使用逆转录酶 - PCR分析，scL12在转基因scL12植物中得以翻译，而在转基因scL植物中未检测到scL的mRNA，并且使用天然聚丙烯酰胺凝胶电泳分析在转基因scL12植物中产生了作为scL12基因产物的scL。此外，转基因scL12植物比转基因scL植物和野生型植物更有效地去除三氯苯酚。这些结果表明，降低真菌靶基因中的CpG二核苷酸基序含量是在转基因植物中高效表达这些基因的一种有用方法。
