• BMB Reports
• /
• v.38 no.1
• /
• pp.82-88
• /
• 2005

Bacillus cereus 28-9 is a chitinolytic bacterium isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. At least two chitinases (ChiCW and ChiCH) were excreted by B. cereus 28-9. The ChiCW-encoding gene was cloned and moderately expressed in Escherichia coli DH5$\alpha$. Near homogenous ChiCW was obtained from the periplasmic fraction of E. coli cells harboring chiCW by a purification procedure. An in vitro assay showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis elliptica, a major fungal pathogen of lily leaf blight.

• Korean Journal of Plant Tissue Culture
• /
• v.26 no.3
• /
• pp.177-182
• /
• 1999

A fungal infection assay between normal and transgenic potato harboring chitinase gene in cultivar Belchip was investigated. In the first stage of experiment, seven transgenic lines having 12cm tall were tested for their resistance against potato late blight pathogen Phytophthora infestans by infection with the zoospores, artificially, Susceptibility to potato late blight infection could be classified into three types based on the rate. In terms of resistance to the disease, two lines were higher, two lines were more suppressive, and three lines were similar as compared with the control. In the following experiment, only 2 risistant lines and 1 suppressed line were used to confirm the resistance again. The results of both experiments were similar. Furthermore, two highly resistant transgenic lines grown in field exhibited a higher resistance than control under the conditions of natural ocurrence of the fungal disease.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 1999.04a
• /
• pp.36.1-36
• /
• 1999

• The Plant Pathology Journal
• /
• v.17 no.6
• /
• pp.363.5-364
• /
• 2001

• Plant Biotechnology Reports
• /
• v.2 no.1
• /
• pp.13-20
• /
• 2008

Potato (Solanum tuberosum L.), one of the most important food crops, is susceptible to a number of devastating fungal pathogens in addition to bacterial and other pathogens. Producing disease-resistant cultivars has been an effective and useful strategy to combat the attack of pathogens. Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus strain HUT 6037 and bialaphos resistance (bar) genes in a binary plasmid vector, pEKH1. Polymerase chain reaction (PCR) analysis revealed that the ChiC and bar genes are integrated into the genome of transgenic plants. Different insertion sites of the transgenes (one to six sites for ChiC and three to seven for bar) were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of the ChiC gene at the messenger RNA (mRNA) level was confirmed by Northern blot analysis and that of the bar gene by herbicide resistance assay. The results obviously confirmed that the ChiC and bar genes are successfully integrated and expressed into the genome, resulting in the production of bialaphos-resistant transgenic plants. Disease-resistance assay of the in vitro and greenhouse-grown transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight).

• Journal of Microbiology and Biotechnology
• /
• v.16 no.6
• /
• pp.952-960
• /
• 2006

As a unicellular green alga that possesses many of the metabolic pathways present in higher plants, Chlorelia offers many advantages for expression of heterologous proteins. Since strong and constitutive promoters are necessary for efficient expression in heterologous expression systems, the development of such promoters for use in the Chlorella system was the aim of this study. Proteins encoded by the early genes of algal viruses are expressed before viral replication, probably by the host transcriptional machinery, and the promoters of these genes might be useful for heterologous expression in Chlorella. In this study, putative promoter regions of DNA polymerase, ATP-dependent DNA ligase, and chitinase genes were amplified from eight Korean Chlorella virus isolates by using primer sets designed based on the sequence of the genome of PBCV-1, the prototype of the Phycodnaviridae. These putative promoter regions were found to contain several cis-acting elements for transcription factors, including the TATA, CAAT, NTBBF1, GATA, and CCAAT boxes. The amplified promoter regions were placed into Chlorella transformation vectors containing a green fluorescence protein (GFP) reporter gene and the Sh ble gene for phleomycin resistance. C. vulgaris protoplasts were transformed and then selected with phleomycin. The GFP fluorescence intensities of cells transformed with chitinase, DNA polymerase, and DNA ligase gene promoter-GFP fusion constructs were 101.5, 100.8, and 95.8%, respectively, of that of CaMV 35S-GFP-transformed Chlorella cells. These results demonstrate that these viral promoters are active in transformed Chlorella.

• Proceedings of the Korean Society of Life Science Conference
• /
• 2005.04a
• /
• pp.175-175
• /
• 2005

• Proceedings of the PSK Conference
• /
• 2000.10a
• /
• pp.205.2-206
• /
• 2000

• BMB Reports
• /
• v.44 no.3
• /
• pp.193-198
• /
• 2011

The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino-terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. $H_6SCChi$-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were $50^{\circ}C$ and pH 8.0, respectively.

• Journal of Microbiology
• /
• v.45 no.5
• /
• pp.422-430
• /
• 2007

Clonostachys rosea (syn. Gliocladium roseum) is a well-known biocontrol agent and widely distributed around the world. In this study, an endochitinase gene Crchi1 was isolated from the mycoparasitic fungus C. rosea using the DNA walking strategy. The Crchi1 ORF is 1,746 bp long and interrupted by three introns. The cloned gene Crchi1 encodes 426 amino acid residues and shares a high degree of similarity with other chitinases from entomopathogenic and mycoparasitic fungi. Several putative binding sites for transcriptional regulation of Crchi1 in response to carbon (5'-SYGGRG-3') and nitrogen (5'-GATA-3') were identified in the upstream of Crchi1. Expression of Crchi1 gene in different carbon sources was analyzed using real-time PCR (RT-PCR). We found that the Crchi1 expression was suppressed by glucose but strongly stimulated by chitin or solubilized components of the cell wall from Rhizoctonia solani. Phylogenetic analysis of chitinases from entomopathogenic and mycoparasitic fungi suggests that these chitinases have probably evolved from a common ancestor.