ردیابی و توالی‌یابی ژن کُدکنندة پروتئین مرتبط با بیماریزایی PR10 در نخود (Cicer arietinum L.)

نوع مقاله : مقالات پژوهشی

نویسندگان

1 مجتمع اموزش عالی شیروان

2 مجتمع آموزش عالی شیروان

3 استادیار بیوتکنولوژی کشاورزی و عضو هیأت علمی پژوهشکده علوم گیاهی، دانشگاه فردوسی مشهد

چکیده

در بین حبوبات، نخود از جایگاه ویژه‌ای برخوردار است و از جمله عوامل محدودکننده عملکرد آن، می‌توان به برق‌زدگی نخود اشاره کرد. استفاده از پروتئین­های مرتبط با بیماریزایی یکی از راهکارهای مقابله گیاهان با عوامل بیماریزا به‌شمار می­رود. یکی از ژن‌های کُدکننده این نوع پروتئین­ها، ژن کدکننده PR-10 است. این پژوهش با هدف ردیابی ژنPR-10 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 PSH-91 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 به­وسیله کیت صورت گرفت و جهت توالی‌یابی ارسال شد. حضور تک‌باند 1350جفت بازی در مرحله اول و حضور تک‌باند 1289بازی در مرحله دوم بیانگر حضور ژنPR-10 در ژنوم نخود می باشد. نتایج این آزمون حاکی از عدم تفاوت در توالی­های ژن کدکننده پروتئینPR-10 در ارقام نخود با سطوح مقاومتی متفاوت بود. با توجه به نتایج به‌دست‌آمده، می­توان 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.

کلیدواژه‌ها


عنوان مقاله [English]

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

نویسندگان [English]

  • Fatemeh zaker tavallaie 1
  • Maryam Noori Shekartoo 2
  • Farhad Shokoohifar 3
1 Complex Higher education of Shirvan
2 Complex Higher education of Shirvan
3 Research Center for Plant Sciences. Ferdowsi University of Mashhad.
چکیده [English]

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.

کلیدواژه‌ها [English]

  • Cicer arietinum L
  • Pathogenesis related (PR) proteins
  • Ascochyta blight
  • pr-10 gene
1. Ahmadi, K., Gholizadeh, H., Ebadzadeh., H.R., Hosseinpour, R., Hatami, F., Fazli, B.,kazemian, A. and Rafiee, M. 2016. Agricultural Statistics. First Volume. Ministry of Jihad-e-Agriculture, Deputy Director of Planning and Economics, ICT Center. Iran.(In Persian).
2. Babaeizadeh, V., and Sayyai, M. 2013. Intervention of Lipoxigenase gene and multiple pathogenesis-related (PR) genes in the resistance of tarom and anonymous cultivars of rice to Rhizoctonia solani Disease. 12th Iranian Congress of Genetics. May 21-23, 2012. Tehran. (In Persian).
3. FAO, 2014. FAOSTAT Database Results from FAO Website. Rome: Food and Agriculture Organization of the United Nations.
4. Fernandes, H., Michalska, K., Sikorski, M., and Jaskolski, M. 2013. Structural and functional aspects of PR‐10 proteins. FEBS Journal 280(5): 1169-1199.
5. Ghiai, S., Razavi, M., and Shahriary, D. 2012. Study on pathogenic and molecular variability in some isolates of Ascochyta rabiei causal agent of Ascochyta blight of chickpea in Iran. Journal of Applied Entomology and Phytopathology 79: 199-218.
6. Hanselle, T., and Barz, W. 2001. Purification and characterisation of the extracellular PR-2b β-1, 3-glucanase accumulating in different Ascochyta rabiei-infected chickpea (Cicer arietinum L.) cultivars. Plant Science 161(4): 773-781.
7. Heidarinezhad, M., Babayei Zadeh, A., and Rahimian, H.A. 2015. The role of genes related to pathogenicity PR10 and PR2 in rice in resistance to Acidovorax avenae subsp. Avenae. First National Congress of Biology and Natural Sciences of Iran. https://www.civilica.com/Paper-BSCONF01-BSCONF01_101.html ( in Persian).
8. Jain, S., Kumar,D., Jain, M., Chaudhary, P., Deswal, R., and Sarin, N.B. 2012. Ectopic overexpression of a salt stress-induced pathogenesis-related class 10 protein (PR10) gene from peanut (Arachis hypogaea L.) affords broad spectrum abiotic stress tolerance in transgenic tobacco. Plant Cell, Tissue and Organ Culture (PCTOC) 109(1): 19-31.
9. Ghannadha, K., and Zahrawi, M. 2006. Areas of construction, operation and engineering of resistance genes in plants. The 9th Iranian Congress of Agronomy and Plant Breeding. Tehran University. Pardis Abooreihan. (in Persian). https://www.civilica.com/Paper-NABATAT09-NABATAT09_007.html
10. Ghazanfar, M.U., Wakil, W., and Sahi, Sh.T. 2011. Induction of resistance in chickpea (Cicer arietinum L.) against Ascochyta rabiei by applying chemicals and plant extracts. Chilean Journal of Agricultural Research 71(1): 52-62.
11. Hassan, S.I., Hameed, A., Tariq, M., and Arshad, M. 2012. Pearl: A high yielding and full season white maiz variety. Journal of Agricultural Research 50(3): 329-334.
12. Hashemi, S., Babaeizad, V., Tajik, M.A., and Rahimian, H. 2013. Studying of several Pathogenesis-related genes role in rice resistance Bipolaris oryzae. Iranian Journal of Plant Pathology 49(2): 57.
13. Kaseb, A. 2017. Tracking and sequence analysis of the PR10 gene promoter from chickpea plant (MSc. Thesis). Retrieved from IRANDOC Database. (Accession No. 2443873).
14. Katile, S.O., Perumal, R., Rooney, W.L., Prom, L.K., and Magill, C.W. 2010. Expression of pathogenesis‐related protein PR‐10 in sorghum floral tissues in response to inoculation with Fusarium thapsinum and Curvularia lunata. Molecular Plant Pathology 11(1): 93-103.
15. Leo, A.E., Linde, C.C., and Ford, R. 2016. Defence gene expression profiling to Ascochyta rabiei aggressiveness in chickpea. Theoretical and Applied Genetics 129: 1333-1345.
16. Li, H., Rodda, M., Gnanasambandam, A., Aftab, M., Redden, R., Hobson, K., Rosewarne, G., Materne, M., Kaur, S., and Slater, A.T. 2015. Breeding for biotic stress resistance in chickpea: progress and prospects. Euphytica 204: 257-288.
17. Liu, J.J., and Ekramoddoullah, A.K. 2006. The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiological and Molecular Plant Pathology 68(1): 3-13.
18. McGee, J.D., Hamer, J.E., and Hodges, T.K. 2001. Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Molecular Plant-Microbe Interactions 14(7): 877-886.
19. Moosa, A., Farzand, A, Sahi, S.T., and Aleem Khan, A. 2017. Transgenic expression of antifungal pathogenesisrelated proteins against phytopathogenic fungi-15 years of success. Israel Journal of Plant Sciences, ISSN: 0792-9978 (Print) 2223-8980 (Online) Journal homepage: http://www.tandfonline.com/loi/tips20. DOI: 10.1080/07929978.2017.1288407.
20. Nourollahi, K., Javannikhah, M., Naghavi, M.R., and Okhvt, S.M. 2009. Pathogenic diversity in Didymella rabiei from the western Iranian Ilam and Kermanshah provinces. Journal of Plant Protection 23: 56-65.
21. Saikia, R., Singh, B.P., Kumar, R., and Arora, D.K. 2005. Detection of pathogenesis-related proteins- chitinase and β -1,3-glucanase in induced chickpea. Current Science 89: 659-663.
22. Sharma, M., and Ghosh, R. 2016. An update on genetic resistance of chickpea to ascochyta blight. Agronomy 6: 18. doi:10.3390/agronomy6010018.
23. Shokoohifar, F., Bagheri, A., and Falahati Rastgar, M. 2003. Identification of genetic diversity in the Ascochyta blight pathogen of chickpea [Ascochyta rabiei (Pass.) Lab.] Using RAPD markers. JWSS-Isfahan University of Technology 7(2): 193-204.
24. Shokoohifar, F., Bagheri, A.R., and Falahati Rastgar, M. 2006. Identification of resistant chickpea lines against pathotypes causing Ascochyta blight disease in Iran. Iranian Journal of Biology 19(1): 29-42.
25. Soh, H.C., Park, A.R., Park, S., Back, K., Yoon, J.B., Park, H.G., and Kim, Y.S. 2012. Comparative analysis of pathogenesis-related protein 10 (PR10) genes between fungal resistant and susceptible peppers. European Journal of Plant Pathology 132(1): 37-48.
26. Taran, B., Warkentin, T., and Vandenberg, A. 2013. Fast track genetic improvement of Ascochyta blight resistance and double podding in chickpea by marker-assisted backcrossing. Theoretical and Applied Genetics 126(6): 1639-1647.
27. Udupa, S., and Baum, M. 2003. Genetic dissection of pathotype-specific resistance to Ascochyta blight disease in chickpea (Cicer arietinum L.) using microsatellite markers. Theoretical and Applied Genetics 106(7): 1196-1202.
28. Vidhyasekaran, P. 2002. Bacterial Disease Resistance in Plants: Molecular Biology and Biotechnological Applications. Routledge. 322pp
29. Wu, W.Q., Fan, H.Y., Jiang, N., Wang, Y., Zhang, Z.Y., Zhang, Y.L., Wang, X.B., Li, D.W., Yu, J.L., and Han, Ch.G. 2014. Infection of Beet necrotic yellow vein virus with RNA4-encoded P31 specifically up-regulates pathogenesis-related protein 10 in Nicotiana benthamiana. Virology Journal 11(1): 118.
30. Yadav, P., Tripathi, D.K., Kafeel Khan, K., and Yadav, A.K. 2015. Determination of genetic variation and heritability estimates for morphological and yield traits in chickpea (Cicer arietinum) under late sown conditions. The Indian Journal of Agricultural Sciences 85(7): 116-129.
CAPTCHA Image