In silico analysis and overexpression of chitinase class IV gene in Brassica juncea improves resistance against Alternaria brassicae

Abstract

Indian mustard (Brassica juncea), an elite oilseed crop in the Brassicae family is challenged by various biotic stresses. Alternaria leaf blight is the most devastating disease in B. juncea caused by Alternaria brassicae. Hence engineering B. juncea plants for improved Alternaria disease resistance becomes imperative. Plant chitinases are key players in the plant immune system that offers successful protection to fungal infections over a broad spectrum of diseases. Chitinase class IV gene was isolated and functionally characterized in Indian mustard (Brassica juncea). Based on phylogenetic analysis, Bj chitinase class IV gene was closely related to Brassica napus followed by other Brassica species. To understand the defensive role of this gene, it was cloned in (Pet28a+) vector and expressed in the prokaryotic expression host Escherichia coli BL21. The purified protein showed antifungal activity against A. brassicae. To further discern its role in disease resistance, B. juncea transgenic lines were raised by overexpressing Bj chitinase IV gene and further confirmed by PCR and southern blotting. In addition, independent B. juncea transgenic lines with high transcript levels of chitinase class IV gene were selected for disease assays against A. brassicae infection. Pathological studies showed that the rate of A. brassicae lesions development and size was significantly reduced in the Bj chitinase transgenic lines compared with non transgenic plants. Interestingly, Bj chitinase transgenic lines showed increased activity of ROS scavenging enzymes when compared to control plants. Collectively our results suggest that overexpression of Bj chitinase IV gene in B. juncea improves resistance against Alternaria leaf blight.