Prediction, isolation, overexpression and antifungal activity analysis of Medicago truncatula var. truncatula putative thaumatin like proteins (TLP-1,-2,-3,-4 and-5)

Authors : Maryam SAEİDİ, Reza ZAREİE

Pages : 176-187

View : 16 | Download : 9

Publication Date : 2020-12-01

Article Type : Research Paper

Abstract :Pathogenesis-related proteins insert ignore into journalissuearticles values(PR-proteins); are induced in response to environmental stresses such as osmotic and drought stress, wounding, microbial infections and treatment with specific plant hormones and elicitors. These proteins are classified into several groups insert ignore into journalissuearticles values(PR-1 through PR-17); based on their amino acid sequence and biochemical functions. The present study focuses on prediction, isolation, over-expression and analysis of the antifungal activities of the thaumatin-like proteins insert ignore into journalissuearticles values(i.e. PR-5); in the model legume M. truncatula var. truncatula. Analysis of M. truncatula genome sequence, available freely on the NCBI website, indicated the presence of at least 15 PR-5 Open Reading Frames insert ignore into journalissuearticles values(ORFs);, 5 of them insert ignore into journalissuearticles values(dubbed TLP-1, -2, -3, -4 and -5); were selected for this study. Using gene-specific primers, the genomic coding sequences were isolated, sequenced and all confirmed to match with those reported in the database. All the fragments were, then, cloned in Escherichia coli isolate BL21 insert ignore into journalissuearticles values(DE3);, using pET-21cinsert ignore into journalissuearticles values(+); plasmids for subsequent overexpression insert ignore into journalissuearticles values(overexpression);. All 5 genes were expressed as inclusion bodies insert ignore into journalissuearticles values(IBs); with masses, estimated by SDS PAGE, corresponding to the theoretical values. As expected, none of the protein IBs had no detectable effect on the phytopathogenic fungi Rhizoctonia solani, Alternaria alternata, Fusarium graminearum, Fusarium solani, Verticillium sp. and Phytophtora spp. However, when the in vitro refolded IB preparations were applied, all displayed comparable strong antifungal activities against the tested fungi. The current study is the first report of overexpression and evaluation of antifungal activities of PR-5 family of proteins from M. truncatula Var. truncatula, and provides experimental evidence that all investigated proteins have the potential for enhancing resistance against some important fungal pathogens.
Keywords : Antifungal activity, Medicago truncatula, pathogenesis related proteins, protein expression, thaumatin like protein