Tracing and sequencing of pathogenesis-related (PR) protein 10 gene in chickpea (Cicer arietinum L.)

پژوهش‌های حبوبات ایران

Article 2, Volume 11, Issue 1 - Serial Number 21, June 2020, Pages 26-37   

Document Type: Original Articles

DOI: 10.22067/ijpr.v11i1.73594

Authors

Maryam Noori Shekartoo¹; Fatemeh Zaker Tavallaie^{* 1}; Farhad Shokoohifar²

¹Complex Higher education of Shirvan, Shirvan, Iran

²Research Center for Plant Sciences. Ferdowsi University of Mashhad. Mashhad. Iran

Abstract

Introduction
Chickpea (Cicer arietinum L.) is an especial important crop among Pulse crops. One of the most important factors limiting the yield of chickpea is fungal diseases. Ascochyta blight is the most problematic disease of chickpea. The fungus Ascochyta rabiei (also called Didymella rabiei) causes ascochyta blight of chickpea. Pathogenesis related (PR) proteins are one of the ways to control plant pathogens. These proteins are encoded by the host plant, but their express done only in the presence of disease agent or the related conditions (Ghannadha et al., 2006). PR-proteins are a variety group of proteins including: glucanase, chitinases, endoproteinase, peroxidases, protease inhibitors, as well as small proteins such as osmotins, thionins, defensins, and lipid transfer proteins (Jain et al., 2012). So far seventeen PR proteins have been identified (Liu et al., 2006). PR-2 is the only PR-protein that identified in chickpea. The PR-10 has many functions. Direct evidence of antimicrobial activity induced by PR-10 is known in microbiological labs. The ability to connect to ligands is also observed. It also has an enzymatic activity in the secondary metabolism of plants and plays a role in abiotic stresses (Liu et al., 2006). This research was done to detect pr-10 gene in chickpea genome.
 
Materials & Methods
In this experiment, six genotypes of chickpea, including two sensitive genotypes (MCC506 and MCC507), two resistant genotypes (MCC142 and MCC528) and two tolerant genotypes (MCC20 and MCC150), against ascochyta blight were used. The seeds were surface sterilized using 2% sodium hypochlorite and germinated in sterile petri dishes. The germinated seeds were planted in pots (with 20 cm in diameter) containing combination of perlite, coco peat and vermicompost with a ratio of 1: 2: 1 at a depth of 2.5 cm. After one month, DNA extraction was down from leaves using CTAB method. RCR reaction was down using primers of PSH-91 and MN in 2 steps. These primers were designed using Primer software Ver.5. The first step was performed using PSH-91 primer on genomic DNA and second step using MN primer on first PCR product. The detected gene was extracted using a Gel extraction kit manufactured by Denazist Company. Then PR-10 gene constructs were sequenced. Sequencing results were analyzed using SeqMan software.
 
Results & Discussion
Germination of plants was almost simultaneously. The shoot production of resistant and tolerant plants was more than sensitive cultivars. Resistant and tolerant varieties reached to flowering stage earlier and produced more abundant seed rather than sensitive cultivars. The presence of a single-band 1350 bp in the first PCR and a single-band 1289 bp in the second PCR indicated to presence of the PR-10 gene in the chickpea genome. The results of this research indicated that there is no difference in sequence of coding gene of PR-10 protein in chickpea genome with different resistance levels.
 
 
 
Conclusion
Due to the lack of diversity in the nucleotide sequence of pr-10 gene among resistant and sensitive cultivars, it is likely that when the protein is expressed in the presence of the disease agent, the expression difference between the different varieties is shown. This seems reasonable considering research on the pr-10 rice genes (Babaiezadeh & Sayyari, 2012). The PR-10 gene in rice is naturally not expressed in leaves and does not respond to ulcers, but it has high expression potential in the presence of ethylene and jasmine acids (Heidarinejad et al., 2014). Also, in evaluating the resistant and susceptible cultivar of rice blight pods disease, PR-10 gene expression in resistant cultivars was significantly increased compared with susceptible cultivars (Babaiezadeh and Sayyari, 2012). It seems that additional experiments are necessary in disease condition on expression of pr-10 gene in chickpea.

Keywords

Cicer arietinum L; Pathogenesis related (PR) proteins; Ascochyta blight; pr-10 gene

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