To investigate the function of nuclear-localized plant HSP70, we used NtHSP70-1 isolated from Nicotiana tabacum. The subcellular localization of NtHSP70-1 was identified by fluorescence microscopy for NtHSP70-1/GFP or smGFP fusion proteins in onion epidermal cells, obtained using particle gun bombardment. To analyze the drought-stress tolerance and thermoprotective role of NtHSP70-1, we obtained transgenic tobacco plants that constitutively expressed elevated levels of NtHSP70-1 as well as transgenic plants containing either the vector alone or else having NtHSP70-1 in the antisense orientation. From analysis for genomic DNA in transgenic seedlings after heat stress, NtHSP70-1 helps to prevent the fragmentation and degradation of nuclear DNA during heat stress. In addition, seedlings constitutively overexpressing NtHSP70-1 grew to be healthy plants, whereas transgenic vector or antisense seedlings resulted in death after heat-/drought-stress.
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. H6SCChi-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 o C and pH 8.0, respectively. [BMB reports 2011; 44(3) : 193-198]
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