Antiviral and Antifungal Activity of Biologically Active Recombinant Bouganin Protein from Bougainvillea spectabilis Willd

Abstract

Bouganin antiviral protein (BAP) gene, one of the ribosome inactivating proteins, isolated from Bougainvillea spectabilis Willd. was cloned, expressed and the antiviral and antifungal activities were investigated. The full-length bouganin antiviral protein gene was amplified by reverse transcription-PCR using mRNA as template extracted from mature leaves. The coding region of bouganin gene was cloned into prokaryotic expression vector pETDuet-1 after amplification with end to end gene specific primers. The recombinant plasmid was transformed into Escherichia coli cells BL21(DE3)pLysS and the expression of BAP gene was induced by isopropyl β-D thiogalactopyranoside (IPTG). Bouganin antiviral protein having a molecular mass of 28 kDa has been isolated from transformed bacterial colonies. Antiviral activity of bouganin was assayed against Zucchini yellow mosaic virus (ZYMV) by a mechanical inoculation test. The antifungal activity of purified recombinant protein was tested against pathogenic and non-pathogenic Rhizoctonia solani, Trichoderma harzianum, and Fusarium oxysporum fungi using disc diffusion method. The increased amount of antiviral protein reduced the disease severity caused by ZYMV. The bouganin antiviral protein was inhibited the growth of R. solani by 30.7% and of T. harzianum by 20% after 72 h compared to control. No growth inhibition was observed for F. oxyporum. All plants including controls treated with in vitro expressed BAP protein exhibited severe growth reduction compared with negative control (not treated) plants. 

Keywords

• bougainvillea spectabilis; antimicrobial protein; bap gene; expression; antiviral and antifungal activities