ردیابی و توالی‌یابی ژن کُدکنندة پروتئین مرتبط با بیماریزایی Pr10 در نخود (Cicer Arietinum L.)

در بین حبوبات، نخود از جایگاه ویژه‌ای برخوردار است و از جمله عوامل محدودکننده عملکرد آن، می‌توان به برق‌زدگی نخود اشاره کرد. استفاده از پروتئین های مرتبط با بیماریزایی یکی از راهکارهای مقابله گیاهان با عوامل بیماریزا به‌شمار می رود. یکی از ژن‌های کُدکننده این نوع پروتئین ها، ژن کدکننده pr10 است. این پژوهش با هدف ردیابی ژنpr10 در ژنوم گیاه نخود انجام شد. بدین منظور dnaی ژنومی شِش ژنوتیپ نخود با مقاومت متفاوت نسبت به بیماری شامل دو رقم مقاوم mcc142 و mdd528، دو رقم متحمل mcc150 و mcc20 و دو رقم حساس mcc507 و mcc506 به روشctab استخراج گردید. با استفاده از آغازگرهای psh91 و mn واکنش pcr طی دو مرحله انجام شد. مرحله اول با استفاده از dnaی ژنومی نخود و با آغازگرpsh91 و مرحله دوم با استفاده از محصولات مرحله اول و آغازگرmn انجام شد. سپس تخلیص باندها به وسیله کیت صورت گرفت و جهت توالی‌یابی ارسال شد. حضور تک‌باند 1350جفت بازی در مرحله اول و حضور تک‌باند 1289بازی در مرحله دوم بیانگر حضور ژنpr10 در ژنوم نخود می باشد. نتایج این آزمون حاکی از عدم تفاوت در توالی های ژن کدکننده پروتئینpr10 در ارقام نخود با سطوح مقاومتی متفاوت بود. با توجه به نتایج به‌دست‌آمده، می توان نتیجه گرفت که تفاوت در واکنش ارقام نسبت به بیماری، به تفاوت در توالی این ژن مربوط نیست و دلایل دیگری مقاومت متفاوت را ایجاد می‌کنند.

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

IntroductionChickpea (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). PRproteins 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). PR2 is the only PRprotein that identified in chickpea. The PR10 has many functions. Direct evidence of antimicrobial activity induced by PR10 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 pr10 gene in chickpea genome. Materials MethodsIn 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 PSH91 and MN in 2 steps. These primers were designed using Primer software Ver.5. The first step was performed using PSH91 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 PR10 gene constructs were sequenced. Sequencing results were analyzed using SeqMan software. Results DiscussionGermination 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 singleband 1350 bp in the first PCR and a singleband 1289 bp in the second PCR indicated to presence of the PR10 gene in the chickpea genome. The results of this research indicated that there is no difference in sequence of coding gene of PR10 protein in chickpea genome with different resistance levels. ConclusionDue to the lack of diversity in the nucleotide sequence of pr10 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 pr10 rice genes (Babaiezadeh Sayyari, 2012). The PR10 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, PR10 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 pr10 gene in chickpea.