ABSTRACT
BACKGROUND: Plants have various defense mechanisms such as production of antimicrobial peptides, particularly pathogenesis related proteins (PR proteins). PR10 family is an essential member of this group, with antifungal, antibacterial and antiviral activities. OBJECTIVE: The goal of this study is to assess the antifungal activity of maize PR10 against some of fungal phytopathogens. MATERIALS AND METHODS: Zea mays PR10 gene (TN-05-147) was cloned from genomic DNA and cDNA and overexpressed in Escherichia coli. The existence of a 77- bp intron and two exons in PR10 was confi rmed by comparing the genomic and cDNA sequences. The PR10 cDNA was cloned in pET26b (+) expression vector and transformed into E. coli strain Rosetta DE3 in order to express PR10 recombinant protein. Expression of the recombinant protein was checked by western analysis. Recombinant PR10 appeared as insoluble inclusion bodies and thus solubilized and refolded. PR10 was isolated using Ni- NTA column. The activity of the refolded protein was confi rmed by DNA degradation test. The antifungal activity of PR10 was assessed using radial diff usion, disc diff usion and spore germination. The hemolytic assay was performed to investigate the biosafety of recombinant PR10. RESULTS: Recombinant maize PR10 exerted broad spectrum antifungal activity against Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Verticillium dahlia and Alternaria solani. Hemolysis biosafety test indicated that the protein is not poisonous to mammalian cells. CONCLUSIONS: Maize PR10 has the potential to be used as the antifungal agent against diff erent fungal phytopathogens. Therefore, this protein can be used in order to produce antifungal agents and fungi resistance transgenic plants.
ABSTRACT
BACKGROUND: Selectable marker gene (SMG) systems are critical for generation of transgenic crops. Transgenic crop production without using SMG is not economically feasible. However, SMGs are non-essential once an intact transgenic plant has been established. Elimination of SMGs from transgenic crops both increases public acceptance of GM crops and prepares gene stacking possibility for improvement of complex traits. Synthetic inducible promoters provide an efficient and flexible strategy to regulate transgene expression. OBJECTIVES: This study aimed to construct a transformation vector based on Cre/loxP recombination system to enhance efficiency of SMG-free transgenic plant production followed by post-excision expression of gene of interest in transgenic plants by a pathogen inducible promoter. MATERIALS AND METHODS: In pG-IPFFDD-creint-gusint construct, cre recombinase and selectable marker gene (nptII) cassettes were placed between the two loxP recognition sites in direct orientation. Seed-specific Napin promoter was used for regulation of Cre expression in transgenic seeds. In the construct, loxP flanked sequence containing nptII and recombinase cassettes, located between a pathogen inducible promoter containing FFDD cis-acting elements and ß-glucuronidase coding region. The cunstuct was transformed into Nicotiana tabaccum via Agrobacterium-mediated transformation. RESULTS: The results showed that both cre and nptII excision occurs in T1 progeny tobacco plants through seed-specific cre expression. The excisions were confirmed by methods activation of the gus gene, germination test on kanamycin-containing medium and molecular analysis. Inducibility of gus expression by FFDD-containing promoter in T1 leaf tissues was confirmed by histochemical Gus staining assay. CONCLUSIONS: The established system is not only an efficient tool for marker gene elimination but also provides possibility for inducible expression of the transgene.
