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Transient gene expression and promoter analysis in basidiomycete
Yoichi Honda,Eiji Tanigawa,Takahito Watanabe,Takashi Watanabe 한국버섯학회 2010 한국버섯학회지 Vol.8 No.4
Ceriporiopsis subvermispora is a unique white rot fungus degrading plant cell wall lignin without severe loss of cellulose. Recombinant plasmids containing homologous gene expression signals fused to the coding sequence of Escherichia coli hph which encodes for hygromycin phosphotransferase were introduced in protoplasts of wild type C. subvermispora using PEG/CaCl2 protocol. A number of hygromycin B resistant strains were isolated on a screening plate containing 100㎍/ml of hygromycin B: whereas, no colonies were observed when protoplasts were treated with no DNA as a negative control. It was demonstrated that most of the isolates lost their drug resistance during successive cultivations in the presence of the same concentration of hygromycin B, but some of the isolates showed stable drug resistance after five times repeated screening. They did not lose the drug resistance even after the cultivation in the absence of hygromycin B and incorporation of the hph sequence was confirmed by specific amplification of the target sequence in PCR experiments and Southern hybridization analysis. The stable transformation system will make it possible to do molecular genetic analysis, as well as breeding of genetically modified strains, in C. subvermispora. Moreover, it was demonstrated that recombinant constructs with truncated promoter showed reduced number of the drug resistant isolates on the first screening plate, in response with the length of the remaining promoter sequence. These findings indicated that unstable drug resistance observed in these isolates should originate from transient expression of the introducing marker genes, and that a promoter assay system has been developed for the first time in basidiomycete. This system is practically not affected by the positional effect of the integrated recombinant gene in the host chromosome.
Recent Progress in Molecular Genetics of Mushrooms
Yoichi Honda 한국버섯학회 2017 버섯 Vol.21 No.2
Molecular genetic approaches have advantages in investigating various physiological aspects of mushrooms. We have been engaged in developing genetic transformation system mainly in wood rot mushrooms. In this presentation, our recent trials to accelerate mushroom science beyond the post-genomic era will be presented. They include development of research tools both in forward and reverse genetics; a transfection system and its utilization in gene expression signal analysis in the selective white rot fungus Ceriporiopsis subvermispora, multiple gene targeting system in Pleurotus ostreatus and Coprinopsis cinerea, which was used to do knock-out or knock-in of reporter constructs on the chromosome of these fungi, and a “powered” forward genetics combined with genome information was used to identify new gene responsible for lignin-degradation in P. ostreatus. They may contribute to elucidate uncovered molecular mechanisms in unique processes in the mushrooms and to make them as a new industrial tool in the next era. Furthermore, our recent results in on-going genome editing experiments will be also reported.
Nguyen Dong Xuan,Nishisaka Emi,Kawauchi Moriyuki,Nakazawa Takehito,Sakamoto Masahiro,Honda Yoichi 한국미생물학회 2020 The journal of microbiology Vol.58 No.12
Terminators and introns are vital regulators of gene expression in many eukaryotes; however, the functional importance of these elements for controlling gene expression in Agaricomycetes remains unclear. In this study, the effects of Ceriporiopsis subvermispora terminators and introns on the expression of a recombinant hygromycin B phosphotransferase gene (hph) were characterized. Using a transient transformation system, we proved that a highly active terminator (e.g., the gpd terminator) is required for the efficient expression of the hph gene. Mutational analyses of the C. subvermispora gpd terminator revealed that hph expression was dictated by an A-rich region, which included a putative positioning element, and polyadenylation sites. In contrast, our results indicated that introns are not required for the expression of hph directed by the Csβ1-tub and Csgpd promoters in C. subvermispora. This study provides insights into the functions and cis-element requirements of transcriptional terminators in Agaricomycetes, which may be relevant for designing recombinant genes for this important fungal class.
Development of a Molecular Marker for Fruiting Body Pattern in Auricularia auricula-judae
( Fang-jie Yao ),( Li-xin Lu ),( Peng Wang ),( Ming Fang ),( You-min Zhang ),( Ying Chen ),( Wei-tong Zhang ),( Xiang-hui Kong ),( Jia Lu ),( Yoichi Honda ) 한국균학회 2018 Mycobiology Vol.46 No.1
The fruiting body pattern is an important agronomic trait of the edible fungus Auricularia auricula-judae, and an important breeding target. There are two types of fruiting body pattern: the cluster type and the chrysanthemum type. We identified the fruiting body pattern of 26 test strains, and then constructed two different near-isogenic pools. Then, we developed sequence characterized amplified region (SCAR) molecular markers associated with the fruiting body pattern based on sequence-related amplified polymorphism (SRAP) markers. Ten different bands (189-522 bp) were amplified using 153 pairs of SRAP primers. The SCAR marker “SCL-18” consisted of a single 522-bp band amplified from the cluster-type strains, but not the chrysanthemum strains. This SCAR marker was closely associated with the cluster- type fruiting body trait of A. auricula-judae. These results lay the foundation for further research to locate and clone genes controlling the fruiting body pattern of A. auricula-judae.