Cloning, overexpression and in vitro antifungal activity of zea mays PR10 protein

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 confirmed 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 confirmed by DNA degradation test. The antifungal activity of PR10 was assessed using radial diffusion, disc diffusion 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 different fungal phytopathogens. Therefore, this protein can be used in order to produce antifungal agents and fungi resistance transgenic plants

Sugarcane [nco310] transient transformation using uida reporter gene

Sugarcane is a monocotyledonous crop that is cultivated in the tropical and subtropical regions of the world. One of the most important criteria, influencing the efficiency of the sugarcane transformation is known to be related to physical and biological factors during the transformation procedure. The objective of this research was to study the response of callus induction and embryogenic callus production and to identify the major parameters controlling DNA delivery by particle bombardment into sugarcane [Saccharum officinarum L.] cv. NCo310. For callus induction and embryogenic callus production, leaf base segments were subjected to in vitro culture medium supplemented with two plant growth regulators [2,4-D and Dicamba]. Results showed that 1 mg.L -1 2,4-D was significantly influential in callus induction and embryogenic callus production. Considering both physical and biological factors, the efficiency of DNA [uidA gene] delivery was assessed by scoring transient GUS [gene [beta-glucuronidase]] expression in bombarded tissues. The highest transient GUS expression was obtained when callus was bombarded with the construct harboring rice Act1 promoter, and having 9 cm target distance, 25 inHg vacuum pressure, 1 microm gold particles, 12.5 micro g DNA per bombardment and one day pre-culture prior to the bombardment. A bombardment procedure suitable for elite sugarcane varieties was developed, which allowed high-efficiency DNA delivery combined with reduced damage to target tissues.