Chitinases are enzymes that catalyze the degradation of chitin, a major component of the cell walls of pathogenic fungi and cuticles of insects, gaining increasing attention for the control of fungal pathogens and insect pests. Production of recombinant chitinase in a suitable host can result in a more pure product with less processing time and a significantly larger yield than that produced by native microorganisms. The present study aimed to express the synthetic chi42 gene (syncodChi42), which was optimized from the chi42 gene of Trichoderma asperellum SH16, in Escherichia coli to produce 42 kDa chitinase (Ta-CHI42); then determined the activity of this enzyme, characterizations and in vitro antifungal activity as well as its immunogenicity in mice. The results showed that Ta-CHI42 was overexpressed in E. coli. Analysis of the colloidal chitin hydrolytic activity of purified Ta-CHI42 on an agar plate revealed that this enzyme was in a highly active form. This is a neutral chitinase with pH stability in a range of 6–8 and has an optimum temperature of 45°C with thermal stability in a range of 25–35°C. The chitinolytic activity of Ta-CHI42 was almost completely abolished by 5 mM Zn2+ or 1% SDS, whereas it remained about haft under the effect of 1 M urea, 1% Triton X-100 or 5 mM Cu2+. Except for ions such as Mn2+ and Ca2+ at 5 mM that have enhanced chitinolytic activity; 5 mM of Na+, Fe2+ or Mg2+ ions or 1 mM EDTA negatively impacted the enzyme. Ta-CHI42 at 60 U/mL concentration strongly inhibited the growth of the pathogenic fungus Aspergillus niger. Analysis of western blot indicated that the polyclonal antibody against Ta-CHI42 was greatly produced in mice. It can be used to analyze the expression of the syncodChi42 gene in transgenic plants, through immunoblotting assays, for resistance to pathogenic fungi.

The present work aims to establish an efficient protocol for in vitro regeneration of peanut (Arachis hypogaea) cultivar L14. The study showed that de-embryonated cotyledon was a suitable explant for shoot multiplication on MS medium containing 4 mg/L BAP. The highest number of shoots per explant obtained after 4 weeks of culture was up to 6.8 shoots. Shoots in vitro were able to produce a large number of approximately 11 roots on MS medium supplemented with 0.5 mg/L NAA. These results will be very useful in establishing an in vitro regeneration protocol for peanut cultivar L14 during gene transfer in the next studies to improve their disease resistance.

Enhanced Green Fluorescent Protein (eGFP) shows much stronger fluorescence than its ancestor, Green Fluorescent Protein (GFP), thus has been widely applied as a reporter for biomedical research. In this study, we reported the expression of a synthetic codon optimized gene encoding eGFP in Escherichia coli (E. coli). The gene was cloned into two expression vectors, pQE30 and pColdII and the resulting recombinant vectors were transformed into E. coli M15 and BL21 De3 RIL codon plus strains, respectively. The expression levels of functional eGFP showed a temperature dependent pattern, in which lowering the induction temperature increased the amount of functional eGFP. Surprisingly, eGFP showed a phenomenon called auto-induction when E. coli TOP10 cells carrying recombinant pQE30 and pColdII were grown on Luria Broth plates. The recombinant eGFP showed robust stability even at room temperature, thus greatly facilitated its purification and handling. Mouse polyclonal antibodies were conveniently generated against the protein. Besides its potential application as a reporter gene in E. coli, the gene and its expression systems reported here are extremely useful as models for teaching recombinant DNA technology at undergraduate level.

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This study reports the expression of 42 kDa chitinase genes from Trichoderma asperellum SH16 in peanut (Arachis hypogaea) roots under the regulation of tissue-specific Asy promoter through Agrobacterium tumefaciens-mediated transformation. The 42 kDa chitinase genes, including one wild-type sequence (Chi42) and two synthetic sequences (syncodChi42-1 and syncodChi42-2) which were optimized for codon usage for plant expression, were incorporated into the peanut genome and successfully expressed in their roots. The investigation revealed that the enzyme chitinase from two synthetic genes had higher activity than that from the wild-type gene, about 901 U/mg (140 U/mL) and 1124 U/mg (197 U/mL) vs about 508 U/mg (87 U/mL). Transgenic peanut roots also exhibited extracellular chitinase activity which was driven by signal peptide of rice amylase 3D gene against the pathogenic fungus Sclerotium rolfsii under in vitro conditions. The higher chitinase activity of two synthetic genes in peanut roots promises potential applications in the field of transgenic crops against phytopathogenic fungi.