• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1178-1187
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• 2024

Cordyceps militaris is a significant edible fungus that produces a variety of bioactive compounds. We have previously established a uridine/uracil auxotrophic mutant and a corresponding Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic characterization in C. militaris using pyrG as a screening marker. In this study, we constructed an ATMT system based on a dual pyrG and hisB auxotrophic mutant of C. militaris. Using the uridine/uracil auxotrophic mutant as the background and pyrG as a selection marker, the hisB gene encoding imidazole glycerophosphate dehydratase, required for histidine biosynthesis, was knocked out by homologous recombination to construct a histidine auxotrophic C. militaris mutant. Then, pyrG in the histidine auxotrophic mutant was deleted to construct a ΔpyrG ΔhisB dual auxotrophic mutant. Further, we established an ATMT transformation system based on the dual auxotrophic C. militaris by using GFP and DsRed as reporter genes. Finally, to demonstrate the application of this dual transformation system for studies of gene function, knock out and complementation of the photoreceptor gene CmWC-1 in the dual auxotrophic C. militaris were performed. The newly constructed ATMT system with histidine and uridine/uracil auxotrophic markers provides a promising tool for genetic modifications in the medicinal fungus C. militaris.

• Korean Journal of Plant Tissue Culture
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• v.27 no.5
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• pp.387-393
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• 2000

The process by which Agrobacterium tumefaciens genetically transforms plants involves a complex series of reactions communicated between the pathogen and the plants. To identify plant factors involved in agrobacterium-mediated plant transformation, a large number of T-DNA inserted Arabidopsis thaliana mutant lines were investigated for susceptibility to Agrobacterium infection by using an in vitro root inoculation assay. Based on the phenotype of tumorigenesis, twelve T-DNA inserted Arabidopsis mutants(rat) that were resistant to Agrobacterium transformation were found. Three mutants, rat1, rat3, and rat4 were characterized in detail. They showed low transient GUS activity and very low stable transformation efficiency compared to the wild-type plant. The resistance phenotype of rat1 and rats resulted from decreased attachment of Agrobacterium tumefaciens to inoculated root explants. They may be deficient in plant actors that are necessary for bacterial attachment to plant cells. The disrupted genes in rat1, rat3, and rat4 mutants were coding a arabinogalactan protein, a likely cell wall protein and a cellulose synthase-like protein, respectively.

• Applied Biological Chemistry
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• v.28 no.2
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• pp.92-97
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• 1985

UV irradiation method was applied to Agrobacterium tumefaciens A 136 to obtain arginine auxotrophic mutant which is applicable as a host of Ti-plasmid. When the bacterial growth was measured at 600 nm, it showed the exponential phase between 7 and 16 hours after 2% inoculation (v/v) in TY medium and the generation time of 4.8 hours. Survival rate of $1{\sim}0.1%$ was reserved when irradiated at the intensity of $800\;{\mu}w/cm^2$ for $30{\sim}50sec$. Fifteen mutants were selected among 5,000 colonies after UV irradiation. Two of them were identified as arginine auxotrophs, three of them as asparagine auxotrophs, ana the other not as arginine, asparagine, glycine nor cysteine auxotrophs.

• KSBB Journal
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• v.24 no.1
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• pp.75-79
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• 2009

The effect of inorganic, organic nitrogen sources and amino acids on the coenzyme $Q_{10}$ production and coenzyme $Q_{10}$ component ratio was investigated. Among the nine organic nitrogen sources, CSP showed a remarkable enhancing effect on the production of coenzyme $Q_{10}$. But this enhancement was not observed in medium containing Bacto peptone, tryptone, casamino acid and soybean meal. These differences on the production of coenzyme $Q_{10}$ may be due to differences in kinds and amounts of component amino acids and peptides in the various organic nitrogen sources tested. In the addition of inorganic nitrogens, only $(NH_4)_2SO_4$ increase the coenzyme $Q_{10}$ production by 2.0 times compare to the control group. The addition of L-tyrosine to the medium containing Bacto tryptone, was also determined to be crucial for the coenzyme $Q_{10}$ production. But phenylalanin and tryptophan, other aromatic amino acids, had no stimulatory effect on the coenzyme $Q_{10}$ production. These results show that the production and components ratio of coenzyme $Q_{10}$ was greatly affected by the kinds and the concentration of inorganic, organic nitrogen sources as well as amino acids.

• Mycobiology
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• v.43 no.1
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• pp.37-42
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• 2015

A mutant library of Cordyceps militaris was constructed by improved Agrobacterium tumefaciens-mediated transformation and screened for degradation features. Six mutants with altered characters in in vitro and in vivo fruiting body production, and cordycepin formation were found to contain a single copy T-DNA. T-DNA flanking sequences of these mutants were identified by thermal asymmetric interlaced-PCR approach. ATP-dependent helicase, cytochrome oxidase subunit I and ubiquitin-like activating enzyme were involved in in vitro fruiting body production, serine/threonine phosphatase involved in in vivo fruiting body production, while glucose-methanol-choline oxidoreductase and telomerase reverse transcriptase involved in cordycepin formation. These genes were analyzed by bioinformatics methods, and their molecular function and biology process were speculated by Gene Ontology (GO) analysis. The results provided useful information for the control of culture degeneration in commercial production of C. militaris.

• KSBB Journal
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• v.14 no.4
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• pp.429-433
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• 1999

$\beta$-Glucosidaes from Cellvibrio gilvus(CG) was successfully overproduced in soluble form in E. coli with the coexpression of GroEL/ES/. Without the GroEL/ES protein, the $\beta$-glucosidase overexpressed in E. coli constituted a huge amount(80%) of total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the $\beta$-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. Similar effects of GroEL/ES were also observed on the overexpressed $\beta$-glucosidase from Agrobacterium tumefaciens(AT). And pET28(a)-RGRAR, partially deleted mutant lacking 5-amino acid residues at carboxy teminus also could be folded into an active form when expressed with the molecular chaperonin GroEL/ES, and its activity was higher than that of the without GroEL/ES system, In addition, the synergistic effect of GroEL/ES and the low induction temperature were important factors for solubilization of the inclusion body from overproduced $\beta$-glucosidases.

• Journal of Life Science
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• v.11 no.5
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• pp.423-431
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• 2001

Genomic and cDNA clones containing the RAT3 gene involving in Agobacterium-mediated plant transformation were identified using plant DNA flanking the righ border of a T-DNA rescued from the rat3 mutant as hy-bridization probe. Two highly homologous cDNA clones were identified; one (RAT3-1) weakly hybridized with the probe whereas another (RAT3-2) strongly hybridized with the probe. Both Rat3-1 and Rat3-2 proteins contain a putative signal peptide for secretion. The deduced molecular weights of encoded proteins are 15 kDa. The results of genomic DNA blot analysis and DNA sequencing indicated that RAT3-1 and RAT3-2 exist as single copy genes and they were arranged side by side with just 600 bp distance between them. RAT3-1 was disrupted by the integration of T-DNA into the 3 untranslated region in rat3 mutant. A BLAST search showed that both RAT3-1 and RAT3-2 proteins have homology with only the C-terminal region of $\beta$-1,3-glucanase homologues from Triticum aestivum and Arabidopsis thaliana. Thses $\beta$-1,3-glucanase homologues contain an unusually long C-terminal region with no sig-nificant homology to other $\beta$-1,3-glucanase.

• Horticultural Science & Technology
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• v.28 no.3
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• pp.442-448
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• 2010

The objectives of this study were to analyze mutant lines of Chinese cabbage ($Brassica$ $rapa$ ssp. $pekinensis$) using gene tagging system (plasmid rescue and inverse polymerase chain reaction) and to observe the phenotypic characteristics. Insertional mutants were derived by transferring DNA (T-DNA) of $Agrobacterium$ for functional genomics study in Chinese cabbage. The hypocotyls of Chinese cabbage 'Seoul' were used to obtain transgenic plants with $Agrobacterium$ $tumefaciens$ harboring pRCV2 vector. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. These techniques were successfully conducted to Chinese cabbage plant with high efficiency, and as a result, T-DNA of pRCV2 vector showed distinct various integration patterns in the transgenic plant genome. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. Compared with wild type, the $T_1$ progenies showed varied phenotypes, such as decreased stamen numbers, larger or smaller flowers, upright growth habit, hairless leaves, chlorosis symptoms, narrow leaves, and deeply serrated leaves. The polyploidy level analysis showed the change in phenotypic characteristics of 13 mutant lines was not due to variation in somatic chromosome number. To tag T-DNA from the Chinese cabbage genomic DNA, plasmid rescue and inverse PCR were applied for multiple copies and single copy insertional mutants. Mutants that showed distinct phenotypic difference compared to wild type with 1 copy of T-DNA by Southern blot analysis, and with 2n = 20 of chromosome number were selected. These selected mutant lines were sequenced flanking DNA, mapped genomic loci, and the genome information of the lines is being recorded in specially developed database.

• Journal of the Korean Society of Tobacco Science
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• v.20 no.1
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• pp.57-65
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• 1998

This study was conducted to develop a resistant tobarro against Potato virus Y (PVY) by transformation of the plants with genetically engineered viral genes. The complimentary DNAs (cDNAS) of potato virus Y-necrosis strain (PVY-Vn) replicase mutant genes (3'-deleted, 5'-deleted and ADD-mutant Nlbs) were synthesized through RT-PCR by using purified PVY-VN RNA and synthesized primers, and cloned in the sense orientation into a plant expression vector (pMBPI), The cDNAS of the genes were transferred into Agrobacterium tumefaciens LBA 4404, and then transformed into tobacco (Nicotiana tabacum cv. Burley 21) plants. Regenerated plants were tested for PVY resistance by inoculation test; 13 transgenic plants including 7 for 3'-deleted Nlb, 3 for 5'-deleted Nlb, and 3 for ADD-mutant Nlb appeared to be resistant at 4 weeks after inoculation with PVY-VN. Among the 13 transgenic tobacco plants, 8 plants had no symptom up to 14 weeks after inoculation. The progenies ($T_1$) from self-fertilization of the transgenic lines varied 0.0% to 81.2% in their resistance (% of resistant plants). The analysis of Nlb-31deleted, -5'deleted and -ADD mutant in the $T_1$ plants by polymerase chain reaction (PCR) showed that Nlb-3'deleted, -5'deleted and -ADD mutants were detected in all of the resistant plants. These results suggest that the PVY resistance was inherited in the $T_1$ generation.

• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.240-244
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• 2022

Precisely reliable and quantitative reporters can provide phenotypes that are consistent with research goals in protein expression. Here, we developed an improved reporter mATglu III 5 by directed evolution using a versatile β-glucosidase ATglu derived from Agrobacterium tumefaciens. When expressed in hosts, a vector containing this mutant distinctly showed a colored or fluorescent phenotype, according to the supplemented substrate, without any inducer. Analysis of mATglu III 5 showed it to be fully functional in fusion state with oligomeric proteins, especially under non-induction conditions, thereby offering an alternative to conventional reporters.