Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract

Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 μg and 200 μg.

A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins

“…The inhibition observed was lower than that reported for other plant chitinases ; however, it is known that plants overexpress a pull of chitinases, glucanases, and others PR proteins that act synergistically to fight pathogen infections . These inhibitory concentration values are similar to those reported for barley class I chitinase of 80 and 200 μg . Recently, the variation in the temporal and spatial induction of chitinases in response to infection was demonstrated in chickpea .…”

Chitinases—Potential Candidates for Enhanced Plant Resistance towards Fungal Pathogens

“…Recently, a 32 kDa recombinant chitinase was isolated from barley, which showed better antifungal activity upon expression in E. coli [17]. The produced recombinant chitinase was able to inhibit the growth of Alternaria solani, Fusarium sp., Rhizoctonia solani, and Verticillium dahlia [18]. Plant chitinases have also shown remarkable potential for tolerating abiotic stresses i.e., chitinase from Hippophae rhamnoides for cold stress [19] and chitinase from soybean for arsenic and cadmium stress [20].…”

Estabelecimento de sistema bacteriano de expressão de peptídeos derivados da enzima vegetal RuBisCO

“…Estas células são indicadas pelo fabricante para expressão de proteínas eucarióticas e/ou que possuem potencial de toxicidade à célula hospedeira (Novagen, 2004), ambas características do multímero que será expresso -sequência original proveniente da RuBisCO de soja e com atividade antimicrobiana prevista in silico. A cepa Rosetta vem sendo utilizada nos últimos anos no estabelecimento de sistemas de expressão de genes codificantes de proteínas de organismos eucarióticos, apresentando rendimento elevado (Yedahalli et al, 2016;Toufiq et al, 2018).…”