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Cloning, Overexpression and in vitro Antifungal Activity of Zea Mays PR10 Protein.

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.

SUBMITTER: Zandvakili N

PROVIDER: S-EPMC5582252 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Cloning, Overexpression and <i>in vitro</i> Antifungal Activity of <i>Zea Mays</i> PR10 Protein.

Zandvakili Niloofar N Zamani Mohammadreza M Motallebi Mostafa M Moghaddassi Jahromi Zahra Z

Iranian journal of biotechnology 20170301 1

<h4>Background</h4>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.<h4>Objective</h4>The goal of this study is to assess the antifungal activity of maize PR10 against some of fungal phytopathogens.<h4>Materials and methods</h4><i>Zea mays PR10</i> gene (TN-05-147) was cloned from genomic DNA and cD ...[more]

PMID: 28959351

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Project description:BackgroundStresses (such as drought, salt, viruses, and others) seriously affect plant productivity. To cope with these threats, plants express a large number of genes, including several members of ERD (early responsive to dehydration) genes to synthesize and assemble adaptive molecules. But, the function of ERD3 gene hasn't been known so far.ObjectivesThe purpose of the present study was to clone the stress-resistance gene: ZmERD3, and to analyze its expression pattern in the maize plant organs at different stages and under various stress treatments.Materials and methodsMaizeGDB database search together with the bioinformatics analysis led to the identification of ZmERD3 gene in Zea mays. The cDNA sequence and promoter of ZmERD3 gene were obtained through PCR. Bioinformatics analysis was performed through online tools. The tissue-specific expression profile of the ZmERD3 gene in maize plant was carried out using the quantitative real time PCR (qRT-PCR) technique and its expression pattern in response to stress treatments (such as PEG, NaCl, ABA, and low temperature) was also analyzed through qRT-PCR method.ResultsBased on the homology alignment with AtERD3 (XP_002867953) in MaizeGDB (http://www. maizegdb.org/), the cDNA sequence and promoter region of the ZmERD3 gene were obtained. The bioinformatic analysis showed that ZmERD3 protein has one specific hit of methyltransferase and a high probability of location in the cytoplasm, and there are many cis-regulatory elements responsive to light, heat, cold, dehydration, as well as other stresses in its promoter sequence. Expression analysis revealed that the amount of ZmERD3 mRNA is different in all indicated organs of the maize plant. In addition, the ZmERD3 expression could be induced by abiotic stress treatments. Compared to the control, treatment with NaCl or PEG-6000 could significantly enhance the expression ability of ZmERD3 gene. As well, its expression level was increased about 20 times above the control after exposure to NaCl and PEG-6000 treatments for 3-6 h.ConclusionsOne putative methyltransferase gene, ZmERD3 was cloned. ZmERD3 expression exhibited an obvious tissue-specificity, and its expression could make a significant response to NaCl and PEG-6000 treatments.

Dataset Information

Cloning, Overexpression and in vitro Antifungal Activity of Zea Mays PR10 Protein.

Publications

Cloning, Overexpression and <i>in vitro</i> Antifungal Activity of <i>Zea Mays</i> PR10 Protein.

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