ANNOUNCEMENTS
Pathogens are a major challenge for the growth of agronomically and economically useful crops. Pathogens responsible for causing Alternaria blight disease (ringspot disease) in Brassica species include Alternaria brassicae and Alternaria brassicicola and causing decreased crop productivity and yield quality. A wild species Diplotaxis erucoides showed significant resistance against A. brassicae. However, role of certain important genes like IGMT (INDOLE GLUCOSINOLATE O-METHYLTRANSFERASE) mediating resistance against pathogens in D. erucoides remains unclear. The focus of this study is on the functional validation of the gene IGMT of D. erucoides in stress response using reverse genetics approach. Post transcriptional gene silencing can be achieved via siRNA (short interfering RNA) to develop molecular insights about the candidate gene. Transcriptional attenuation of the target gene and thereby loss of function mediates functional characterisation of the gene. Herein, we report cloning of IGMT in plant transformation vector pHANNIBAL mainly used for generation of RNAi based silencing constructs. Cloning was done by restriction enzyme method in pHANNIBAL to form an intron hairpin RNA loop. Results were confirmed via PCR and restriction-based screening. Successful cloning of IGMT in pHANNIBAL be validated using Agrobacterium infiltration and transient expression studies in D. erucoides. The vector construct and its validation would reveal useful functional insights about the involvement of the gene in pathogen resistance. If validated, the gene can then be used for expression in crops like B. juncea which is not resistant to Alternaria.
Keywords: Diplotaxis erucoides, Gene silencing, Hairpin RNA, pHANNIBAL, RNAi.
