بررسی الگوی بیان نسبی ژن‌های کاندیدای تحمل به شوری در مرحله گیاهچه‌ای برنج با استفاده از پی‌سی‌آر در زمان واقعی

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه بیوتکنولوژی، دانشکده کشاورزی و منابع طبیعی، دانشگاه بین المللی امام خمینی (ره)، قزوین، ایران

2 دانشگاه شهید بهشتی- دانشکده علوم و فناوری زیستی- گروه علوم و زیست فناوری گیاهی

چکیده

هدف: با توجه به این که برنج منبع تغذیه بیش از نیمی از مردم جهان است. همچنین رشد و عملکرد این گیاه به شدت تحت تاثیر تنش شوری قرار می‌گیرد، تحقیق برای توسعه واریته‌های متحمل به شوری امری ضروری است. لذا در این پژوهش، الگوی بیانی هفت ژن دخیل در تحمل به شوری با روش پی‌سی‌آر در زمان واقعی در ارقام برنج حساس (#6) ARC6578 و (#178) Shoemed و متحمل Bombilla (#48) مورد بررسی قرار گرفت.
مواد و روش‌ها: نمونه RNA از گیاهچه‌های ۲۰ روزه برنج تحت تیمار NaCl (۱۰۰ میلی‌مولار) در سه زمان ۲۴، ۴۸ و ۷۲ ساعت پس از اعمال تنش شوری استخراج شد. بررسی بیان ژن بر روی هفت ژن کاندیدا (شامل ژن­های  SOD, Cat1, 14-3-3 like protein GF14، Proxidase BP1 precursor، Zinc ion binding protein Plasma membrane H+-ATPase, و OsSIPK) با روش پی‌سی‌آر در زمان واقعی انجام شد. از ژن اکتین به عنوان ژن مرجع استفاده شد.
نتایج: بیان ژن OsSIPK در رقم متحمل، ۲۴ و ۴۸ ساعت بعد از تنش افزایش و ۷۲ ساعت بعد از تنش شوری کاهش یافت در حالی که بیان ژن Zinc ion binding protein در تمام ارقام و در همه ساعات پس از تنش کاهشی بود. بیان ژن PM H+-ATPase در ساعات اولیه پس از تنش در هر دو رقم حساس و متحمل افزایش یافت ولی با گذشت ۷۲ ساعت از تنش بیان این ژن در رقم حساس کاهش و در رقم متحمل افزایش یافت. بیان ژن 14-3-3 like protein GF14-6 ۴۸ ساعت پس از تنش در هر دو رقم حساس و متحمل به شدت افزایش یافت ولی با گذشت ۷۲ ساعت از تنش بیان این ژن در رقم حساس کاهش و در رقم متحمل افزایش یافت. بیان ژن Similar to Peroxidase BP1 precursor  ۷۲ ساعت پس از تنش در رقم متحمل حدود ۴۰ برابر افزایش یافت. بیان ژن کاتالاز ۷۲ ساعت پس از تنش فقط در رقم متحمل افزایش معنی‌دار نشان داد. بیان ژن SOD از الگوی زمانی خاصی تبعیت نکرد هرچند بیان آن ۷۲ ساعت پس از تنش در رقم متحمل بیشتر از ارقام حساس بود.
نتیجه‌گیری: برخی ژن‌های مورد مطالعه در این تحقیق با حذف گونه‌های فعال اکسیژن (ROS) در پاسخ عمومی گیاه به تنش شوری نقش دارند (مانند ژن‌های SOD و CatA). از طرفی بین تحمل به شوری و میزان بیان برخی دیگر از ژن‌های مورد مطالعه می‌توان نوعی ارتباط وابسته به رقم-زمان را تشخیص داد (مانند ژن‌های OsSIPK، PM H+-ATPase، 14-3-3 like protein GF14-6 و Peroxidase BP1).

کلیدواژه‌ها


عنوان مقاله [English]

Investigation of relative expression of candidate genes related to salinity tolerance at seedling stage of rice using real-time PCR

نویسندگان [English]

  • Leila Nayyeripasand 1
  • Ghasemali Garoosi 1
  • Asadollah Ahmadikhah 2
1 Dept. of Biotechnology, faculty of Agriculture & Natural Resources, Imam Khomeini International University, Qazvin, I.R. Iran.
2 Chamran Expw., Velenjak, Shahid Beheshti University, Faculty of Life Sciences and Biotechnology
چکیده [English]

Objective
Rice is a food source for more than half of the world's population and it is important as a model plant for monocots, and also its growth and yield is strongly affected by salinity stress, hence comprehensive research is essential for the development of salt tolerant varieties. In the present study, the expression pattern of several new genes involved in salt tolerance of rice was investigated by Real-Time PCR in sensitive (ARC6578 and Shoemed) and tolerant (Bombilla) rice cultivars.
Materials and methods
RNA samples were extracted from 20-day-old seedlings treated with NaCl (100 mM) at three times of 24, 48 and 72 h after salt stress. Gene expression analysis was performed on 7 candidate genes (including SOD, CatA, 14-3-3 like protein GF14, Proxidase BP1 precursor, Zinc ion binding protein and Plasma Membrane H+-ATPase, OsSIPK) using real-time PCR. The Actin gene was used as the reference gene.
 
         Results
The expression of OsSIPK in tolerant cultivar was increased 24 and 48 h after salt stress and was decreased 72 h after stress, while the expression of Zinc ion binding protein was decreased in all cultivars at all times after stress. The expression of PM H+-ATPase at early times after stress in both tolerant and sensitive cultivars was increased but it decreased after 72 h in sensitive cultivar and increased in tolerant cultivar. The expression of 14-3-3 like protein GF14-6 was highly increased at 48 h after stress but it was decreased after 72 h in sensitive cultivars and increased in tolerant cultivar. The expression of Peroxidase BP1 precursor was increased up to 40 times at 72 h after stress in tolerant cultivar. The expression of CatA was significantly increased at 72 h after stress only in tolerant cultivar. The expression of SOD didn't show a special time pattern, although its expression in tolerant cultivar was higher than that in sensitive cultivar at 72 h after stress.
Conclusions
Some genes in this research did function in general response of the rice plant to salt stress via scavenging the reactive oxygen species (ROS) (such as SOD and CatA). In another hand, a time-cultivar dependent relationship between salt tolerance and expression level of other studied genes (such as OsSIPK, PM H+-ATPase, 14-3-3 like protein GF14-6 and Peroxidase BP1) could be recognized.

کلیدواژه‌ها [English]

  • Candidate Gene
  • Gene Expression
  • Sensitive
  • Stress Response
  • Tolerant
توحیدی نژاد فاطمه، محمدآبادی محمدرضا، اسمعیلی زاده کشکوئیه علی، نجمی نوری عذرا (۱۳۹۳) مقایسه سطوح مختلف بیان ژنRheb  در بافت های مختلف بز کرکی راینی. مجله بیوتکنولوژی کشاورزی ۶(۴)، ۵۰-۳۵.
جعفری دره در امیر حسین، محمدآبادی محمدرضا، اسمعیلی زاده کشکوئیه علی، ریاحی مدوار علی (۱۳۹۵) بررسی بیان ژن CIB4  در بافت‌های مختلف گوسفند کرمانی با استفاده از Real Time qPCR. مجله پژوهش در نشخوارکنندگان ۴(۴)، ۱۳۲-۱۱۹.
محمدآبادی محمدرضا، کرد محبوبه، نظری محمود (۱۳۹۷) مطالعه بیان ژن لپتین در بافت‌های مختلف گوسفند کرمانی با استفاده از Real Time PCR. مجله بیوتکنولوژی کشاورزی ۱۰(۳)، ۱۲۲-۱۱۱.
References
Ahsani MR, Mohammadabadi MR, Asadi Fozi M et al. (2019a) Effect of roasted soybean and canola seeds on peroxisome proliferator‐activated receptors gamma (PPARG) gene expression and cattle milk characteristics. Iran J Appl Anim Sci 9, 635-642 (In Persian).
Ahsani MR, Mohammadabadi MR, Asadi Fozi M et al. (2019b) Leptin gene expression in subcutaneous adipose tissue of Holstein dairy cattle using Real Time PCR. Agric Biotechnol J 11, 135-150 (In Persian).
Ayala F, Ashraf M, O'Leary JW )1997( Plasma membrane H+‐ATPase activity in salt‐tolerant and salt‐sensitive lines of spring wheat (Triticum aestivum L.). Acta Botanica Neerlandica 46, 315-324.
Benitez LC, da Maia LC, Ribeiro MV et al. )2013) Salt induced change of gene expression in salt sensitive and tolerant rice species. J Agric Sci 5, 251.
Bennetzen J )2002) Opening the door to comparative plant biology. Science 296, 60-63.

Chen G, Hu Q, Luo LE et al. )2015) Rice potassium transporter OsHAK1 is essential for maintaining potassium‐mediated growth and functions in salt tolerance over low and high potassium concentration ranges. Plant Cell Environ 38, 2747-2765.

Cho K, Agrawal GK, Jwa NS et al. (2009) Rice OsSIPK and its orthologs: a “central master switch” for stress responses. Biochem Biophys Res Commun 379, 649-653.
Dai Yin CH, Luo YH, Min SH et al. (2005) Salt-responsive genes in rice revealed by cDNA microarray analysis. Cell Res 15, 796-810.

Dudziak K, Zapalska M, Börner A et al. (2019) Analysis of wheat gene expression related to the oxidative stress response and signal transduction under short-term osmotic stress. Sci Rep 9, 2743.

Du H, Zhou X, Yang Q et al. (2015) Changes in H+-ATPase activity and conjugated polyamine contents in plasma membrane purified from developing wheat embryos under short-time drought stress. Plant Growth Regul 75, 1-10.
Falhof J, Pedersen JT, Fuglsang AT, Palmgren M (2016) Plasma membrane H+-ATPase regulation in the center of plant physiology. Mol Plant 9, 323-337.
Fan HF, Du CX, Guo SR (2013) Nitric oxide enhances salt tolerance in cucumber seedlings by regulating free polyamine content. Environ Exp Bot 86, 52-59.
Finnie C, Borch J, Collinge DB (1999) 14-3-3 proteins: eukaryotic regulatory proteins with many functions. Plant Mol Biol 40, 545-554.
Ganguly M, Datta K, Roychoudhury A et al. (2012) Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance. Plant Signal Behav 7, 502-509.
Gévaudant F, Duby G, Von Stedingk E et al. (2007) Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance. Plant Physiol 144, 1763-1776.
Gong DS, Xiong YC, Ma BL et al. (2010) Early activation of plasma membrane H+-ATPase and its relation to drought adaptation in two contrasting oat (Avena sativa L.) genotypes. Environ Exp Bot 69, 1-8.
Gossett DR, Millhollon EP, Lucas M (1994) Antioxidant response to NaCl stress in salt-tolerant and salt-sensitive cultivars of cotton. Crop Sci 34, 706-714.
Grover A, Pental D (2003) Breeding objectives and requirements for producing transgenics for major field crops of India. Current Sci 84, 310-320.
Hernandez JA, Olmos E, Corpas FJ et al. (1995) Salt-induced oxidative stress in chloroplasts of pea plants. Plant Sci 105, 151-167.
Hu L, Li H, Chen L et al. (2015) RNA-seq for gene identification and transcript profiling in relation to root growth of bermudagrass (Cynodon dactylon) under salinity stress. BMC Genomics 16, 575.
Islam F, Wang J, Farooq MA et al. (2019) Rice responses and tolerance to salt stress: deciphering the physiological and molecular mechanisms of salinity adaptation. In: Advances in Rice Research for Abiotic Stress Tolerance. Woodhead Publ, UK, pp. 791-819.
Jafari Darehdor AH, Mohammadabadi MR, Esmailizadeh AK, Riahi Madvar A (2016) Investigating expression of CIB4 gene in different tissues of Kermani sheep using real time qPCR. J Rumin Res 4, 119-132 (In Persian).
Kennedy B, De Filippis LF (1999) Physiological and oxidative response to NaCl of the salt tolerant Grevillea ilicifolia and the salt sensitive Grevillea arenaria. J Plant Physiol 155, 746-754.
Khare T, Kumar V, Kishor PK (2015) Na+ and Cl ions show additive effects under NaCl stress on induction of oxidative stress and the responsive antioxidative defense in rice. Protoplasma 252, 1149-65.
Kim CY, Liu Y, Thorne ET et al. (2003) Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. Plant Cell 15, 2707-2718.
Kim YH, Khan AL, Waqas M et al. (2014) Silicon application to rice root zone influenced the phytohormonal and antioxidant responses under salinity stress. J Plant Growth Regul 33, 137-49.
Kim D, Shibato J, Agrawal GK (2007) Gene transcription in the leaves of rice undergoing salt-induced morphological changes (Oryza sativa L.). Mol Cells 24(1), 45.
Lakra N, Nutan KK, Das P et al. (2015) A nuclear-localized histone-gene binding protein from rice (OsHBP1b) functions in salinity and drought stress tolerance by maintaining chlorophyll content and improving the antioxidant machinery. J Plant Physiol 176, 36-46.
Lee MO, Cho K, Kim SH et al. (2008) Novel rice OsSIPK is a multiple stress responsive MAPK family member showing rhythmic expression at mRNA level. Planta 227, 981-990.
Lee DH, Kim YS, Lee CB (2001) The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.). J Plant Physiol 158, 737-745.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402-408.
Martz F, Sutinen ML, Kiviniemi S, Palta JP (2006) Changes in freezing tolerance, plasma membrane H+-ATPase activity and fatty acid composition in Pinus resinosa needles during cold acclimation and de-acclimation. Tree Physiol 26, 783-790.
Mishra P, Bhoomika K, Dubey RS (2013) Differential responses of antioxidative defense system to prolonged salinity stress in salt-tolerant and salt-sensitive Indica rice (Oryza sativa L.) seedlings. Protoplasma 250, 3-19.
Mittova V, Tal M, Volokita M, Guy M (2002) Salt stress induces up‐regulation of an efficient chloroplast antioxidant system in the salt‐tolerant wild tomato species Lycopersicon pennellii but not in the cultivated species. Physiol Plant 115, 393-400.
Mittova V, Tal M, Volokita M, Guy M (2003) Up‐regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt‐induced oxidative stress in the wild salt‐tolerant tomato species. Acta Bot Neerl 26, 845-856.
Mishra S, Singh B, Panda K et al. (2016) Association of SNP haplotypes of HKT family genes with salt tolerance in Indian wild rice germplasm. Rice 9, 15.
Mohammadabadi MR, Kord M, Nazari M (2019) Studying expression of leptin gene in different tissues of Kermani sheep using Real Time PCR. Agric Biotechnol J 10, 111-122 (In Persian).
Morgan SH, Maity PJ, Geilfus CM et al. (2014) Leaf ion homeostasis and plasma membrane H+-ATPase activity in Vicia faba change after extra calcium and potassium supply under salinity. Plant Physiol Biochem 82, 244-253.
Nayyeripasand L, Garoosi GA, Ahmadikhah A (2019) Selection for salinity tolerance in an international rice collection at vegetative stage. Aust J Crop Sci 13, 837-846.
Niu X, Narasimhan ML, Salzman RA et al. (1993) NaCl regulation of plasma membrane H+-ATPase gene expression in a glycophyte and a halophyte. Plant Physiol 103, 713-718.
Niu X, Damsz B, Kononowicz AK et al. (1996) NaCl-induced alterations in both cell structure and tissue-specific plasma membrane H+-ATPase gene expression. Plant Physiol 11, 679-686.
Paul AL, Liu L, McClung S et al. (2009) Comparative interactomics: analysis of arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants. J Proteome Res 8, 1913-1924.
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45-e45.
Rahman A, Nahar K, Hasanuzzaman M, Fujita M (2016) Calcium supplementation improves Na+/K+ ratio, antioxidant defense and glyoxalase systems in salt-stressed rice seedlings. Front Plant Sci 7, 609.
Rhodes D, Hanson AD (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Biol 44, 357-384.
Roy P, Niyogi K, Sengupta DN, Ghosh B (2005) Spermidine treatment to rice seedlings recovers salinity stress-induced damage of plasma membrane and PM-bound H+-ATPase in salt-tolerant and salt-sensitive rice cultivars. Plant Sci 168, 583-591.
Sasaki T, Sederoff RR (2003) Genome studies and molecular genetics: the rice genome and comparative genomics of higher plants. Curr Opin Plant Biol 6, 97-100.
Sehmer L, Alaoui-Sosse B, Dizengremel P (1995) Effect of salt stress on growth and on the detoxifying pathway of pedunculate oak seedlings (Quercus robur L.). J Plant Physiol 147, 144-151.
Shankar R, Bhattacharjee A, Jain M (2016) Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses. Sci Rep 6, 23719.
Mishra S, Singh B, Panda K et al. (2016) Association of SNP haplotypes of HKT family genes with salt tolerance in Indian wild rice germplasm. Rice 9,15.
Svennelid F, Olsson A, Piotrowski M et al. (1999) Phosphorylation of Thr-948 at the C terminus of the plasma membrane H+-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11, 2379-2391.
Swain DM, Sahoo RK, Srivastava VK et al. (2017) Function of heterotrimeric G-protein γ subunit RGG1 in providing salinity stress tolerance in rice by elevating detoxification of ROS. Planta 245, 367-83.
Tajti J, Németh E, Glatz G et al. (2019) Pattern of changes in salicylic acid‐induced protein kinase (SIPK) gene expression and salicylic acid accumulation in wheat under cadmium exposure. Plant Biol 21, 1176-1180.
Tohidi Nezhad F, Mohammadabadi MR, Esmailizadeh AK et al. (2015) Comparison of different levels of Rheb gene expression in different tissues of Raini Cashmir goat. Agric Biotechnol J 6, 35-50 (In Persian).
Vallee BL, Falchuk KH (1993) The biochemical basis of zinc physiology. Physiol Rev 73, 79-118.
Vitart V, Baxter I, Doerner P, Harper JF (2001) Evidence for a role in growth and salt resistance of a plasma membrane H+‐ATPase in the root endodermis. Plant J 27, 191-201.
Visconti S, Camoni L, Fullone MR et al. (2003) Mutational analysis of the interaction between 14-3-3 proteins and plant plasma membrane H+-ATPase. J Biol Chem 278, 8172-8178.
Yamane K, Mitsuya S, Taniguchi M, Miyake H (2010) Transcription profiles of genes encoding catalase and ascorbate peroxidase in the rice leaf tissues under salinity. Plant Prod Sci 13, 164-8.
Yoshida S, Forno D, Cock JH, Gomez K (1976) Routine procedure for growing rice plants in culture solution. Laboratory manual for physiological studies of rice (3rd edn), IRRI, Los Baños, Phllippines, pp. 61-66.
Zeng L, Shannon MC, Lesch SM (2001) Timing of salinity stress affects rice growth and yield components. Agric Water Manag 48, 191-206.
Zhao K, Tung CW, Eizenga GC et al. (2011) Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat Commun 2, 467.
Zhang S, Klessig DF (1997) Salicylic acid activates a 48-kD MAP kinase in tobacco. Plant Cell 9, 809-824.