In-Silico Analysis and Genomic Tracking of CaDRRG Gene Promoter Region in Chickpea Genotypes with Different Levels of Resistance to Ascochyta Blight

Abstract

Identification of early responsive genes is very useful in determining the plant's defense system against pathogens. It also has a great interest from the aspect of identifying early responsive inducible promoters and their application in the breeding of resistant cultivars. In the present study, based on the expression data analysis of chickpea genes against the infection with the fungus Ascochyta rabiei that causes chickpea Ascochyta blight, a transcript was identified with high inducibility 3 hours after inoculation with this pathogen. The position of this transcript on the chickpea genome was identified by comparing the transcriptomic and genomic data and the corresponding predicted gene sequence was selected. However, the comparison of these proteins with the predicted protein sequence in the chickpea genome led to the mentioned transcript being investigated under the name of CaDRRG. In order to analyze the sequence of this gene inducible promoter, its upstream sequence was retrieved from genomic data and the position of consensus regulatory elements such as TATA-box and CAAT-box was determined on it. Moreover, the position and number of Cis-regulatory elements known to be inducible against pathogens such as, AS-1, W-box, and G-box, were identified in the promoter sequence. In addition, by comparing the upstream sequence of CaDRRG gene with other genes with the highest induction level in response to A. rabiei, three new potential regulatory elements were identified in the promoter sequence of this gene. The fragment containing these regulatory elements was tracked and sequenced in resistant and sensitive chickpea genotypes. Multiple sequence alignment of the CaDRRG gene promoter sequence in different chickpea genotypes led to the identification of several point mutations outside the positions of the identified regulatory elements. In a preliminary experiment to analyze the induction capacity of the CaDRRG gene promoter sequence, about 700 bp of this sequence was cloned upstream of the beta-glucuronidase reporter gene and its basal expression and inducibility in response to the treatment of A. rabiei fungal extract was investigated on Nicotiana benthamiana leaves by agroinjection method. The assessment of beta-glucuronidase enzyme activity in tobacco leaf discs after treatment with fungal extract and its comparison with the basal expression level confirmed the inducibility of this fragment which was observationally compared with the expression level of CaMV 35S constitutive promoter. Although the results of the preliminary analysis showed that the selected fragment has the potential to be used as an inducible promoter for the expression of resistance genes against A. rabiei, more additional studies are necessary to identify the regulatory elements responsible for responding to this fungal pathogen.