Differential Screening of Randomly Amplified cDNAs Using RAPD Primers in Salt Tolerance and Sensitive Wheat Majeed A. Sabbah Biotechnology Research Center/ Alnahrain University/ Baghdad/ Iraq Received in : 6 May 2013, Accepted in : 26 August 2013 Abstract The identification of salinity-tolerance genes is a critical aspect of the new molecular technology. In this work cDNA-RAPD is used for the identification of genes expressed in salt tolerant but not in salt sensitive wheat. Two cultivars wheat, salt tolerance (Dijla) and sensitive (Tamooz2) were used for the preparation of RNA and cDNA synthesis. Eight primers were used for random amplification of cDNA constructed from RNA and three primers were differentially expressed in salt tolerant cultivars. Genes related to salt tolerant were predicted using NCBI blast for the three primers. The predicted genes were involved in salt tolerance of wheat and other plants as well. This indicates the suitability of the primers and the method for salt tolerance genes identification in wheat under study. Key words: wheat, salt, RAPD-PCR 43 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 Introduction Salinity is a major factor limiting plant growth and leads to lower agricultural production in arid and semi-arid regions [1]. Several technologies were used for the identification of differential expressed salt tolerance genes in wheat. Typically, closed systems such as microarrays and real-time polymerase chain reaction (PCR) have been extensively followed in gene expression analysis in plants [2]. In open systems, there is no need for previous knowledge of the genome or transcriptome of the organism. cDNA-AFLP (cDNA-Amplified fragment length polymorphism) have been successfully used to quantify transcript abundance and generate expression data across different types of tissue or developmental stages in wheat [3,4]. cDNA-RAPD PCR is a simple method established by other investigators [5] for the identification of differentially expressed genes in rice. This method was used to identify novel drought tolerance gene in Gossypium hirsutum [6], and evaluation of cellulolytic filamentous fungi phenotypes [7] and the identification of a novel Getah virus [8]. This study aimed to use this method to identify differentially expressed genes in salt tolerance and sensitive wheat cultivated in Iraq. Materials and methods Wheat genotypes cultivation Two cultivars of wheat were used in this study, salt tolerance (Dijla) and sensitive (Tamooz2). Seed of both cultivars were washed with tap water for 30 min, immersed in 50% of sodium hypochlorite then treated with 2-3 drop of Tween 20 for 10 min, seeds were washed once with 70% ethanol and rinsed many times with sterile water. Five sterilized seeds from each plant were placed in culture bottle containing 15 ml of agar solidified, hormones- free MS medium [9] with each of 0 ds/m, 15 ds/m and 25 ds/m of (NaCl) salt concentrations. Each treatment for wheat was replicated three times. All cultures were kept in the light for 16h and dark for 8h at 25oC. Data were recorded 15 days after treatment steps. RNA isolation and cDNA synthesis Total RNA was isolated by using Geneaid total RNA purification mini kit (Taiwan) according to the manufacturer's instructions. Isolated RNA was treated with RNase-free DNase I (Biobasic, Canada) for 20 min at 37°C, DNase I was inactivated at 65°C for 10 min. The integrity of the RNA was verified after separation by electrophoresis on a 1.5% agarose gel containing 0.5% (v/v) ethidium bromide. First-strand cDNA was synthesized from 500 ng of total RNA by using Reverse Transcription system (Bioneer, Korea) with an oligo-dT15 primer. The reaction solution was used as templates for reverse transcriptase polymerase chain reaction (RT-PCR) [3]. cDNA-RAPD Eight primers (table1) were used for amplification of cDNA using optimized PCR protocols and master mixes. Polymerase chain reaction was initiated with hot start method by using the single strand cDNA template on Labnet Thermocycler (USA). The PCR reaction was carried out according to the program of 35 amplification cycles (94°C for 30 s, 61°C for 45 s and 72°C for 90 s). Ethidium bromide agarose gel electrophoresis (1%) is used for the analysis of PCR products. The generated bands were compared, the differential amplified bands were recorded and the sequences of these bands aligned to related sequences in NCBI blast database [3]. Results Total RNA isolated from both cultivars and DNase treated in order to eliminate genomic DNA (figure1). Eight RAPD primers were used for the amplification of cDNAs generated 44 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 from both genotypes. Three primers produced differential bands between salt resistance and sensitive cultivars were 7, 13, and 20 (figure 2, 3). To predict the differential expressed genes in salt resistant but not sensitive genotype, NCBI blast was performed with the three primers. The predicted genes were listed in table (2). Discussion Investigating the function of the predicted genes in published; revealed the role of these genes in salt tolerance in different ways in several plants. Of the predicted proteins are kinases and phosphatases which are major posttranslational regulators of numerous cellular processes. These enzymes regulate metabolic pathways and are intimately involved in cellular signaling networks as shown in many studies which have involvement in salt tolerance [10]. Cytokinin oxidase dehydrogenase involved in cytokinins synthesis which showed they are involved in stress responses [11,12]. Thioredoxin is involved in the stress response through the regulation of the apoplastic reactive oxygen species in rice [13]. These genes need to be verifies its contribution in salt tolerance by other techniques such as real time PCR. References 1-Bai, R. ; Zhang, Z.; Hu, Y.; Fan. M. and Schmidhalter, U. (2011). Improving the salt tolerance of Chinese spring wheat through an evaluation of genotype genetic variation. Aust J Crop Sci. 5: 1173-1178. 2-Monroy, A. ; Dryanova, A.; Malette, B.; Oren, D.; Ridha Farajalla, M. and Liu, W. (2007) Regulatory gene candidates and gene expression analysis of cold acclimation in winter and spring wheat. Plant Mol. Biol. 64:409-423. 3-Chen, G.; Ma, W.; Huang, Z. ; Xu, T.; Xue, Y.; and Shen, Y. (2003) Isolation and characterization of TaGSK1 involved in wheat salt tolerance. Plant Sci. 165: 1369–1375. 4-McIntosh, S. L.; Watson, P. ; Bundock, A.; Crawford, J.; White, G.; Cordeiro, D.; Barbary, L.; Rooke and Henry, R. (2007) SAGE of the developing wheat caryopsis. Plant Biotechnol. J. 5:69-83. 5-Yoshida, K.; Naito, S. and Takeda, G. (1994) cDNA cloning of regeneration-specific genes in rice by differential screening of randomly amplified cDNAs using RAPD primers. Plant Cell Physiol. 35(7):1003-9. 6-Selvam, J.; Kumaravadivel, N.; Gopikrishnan, A.; Kumar, B.; Ravikesavan, R. and Boopathi, M. (2009) Identification of a novel drought tolerance gene in Gossypium hirsutum L. cv KC3. Commun. Biometry Crop Sci. 4: 9–13. 7 – Oliveira, E.; Barros, N.; Ferreira, T.; Oliveira, T.; Terzi, S. and Damaso, M. (2010). Evaluation of cellulolytic filamentous fungi phenotypes using randomly amplified cDNA with RAPD primers. 32nd symposium on biotechnology for fuel and chemicals, USA. 8-Hu, T.; Zheng, Y.; Zhang, Y.; Li, G.; Qiu, W.; Yu, J.; Cui, Q.; Wang, Y.; Zhang, C.; Zhou, X.; Feng, Z.; Zhou, W. and Fan, Q. (2012) Identification of a novel Getah virus by Virus- Discovery-cDNA random amplified polymorphic DNA (RAPD). BMC Microbiol. 12:305. 9- Murashige, T. and Skoog, F.A. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473-497. 10-Wang, H.; Chevalier, D.; Larue, C.; Cho, S. and Walkera, J. (2007). The Protein Phosphatases and Protein Kinases of Arabidopsis thaliana. The Arabidopsis Book. American Society of Plant Biologists First. 11-Tran, L.S.; Urao, T.; Qin, F.; Maruyama, K.; Kakimoto, T.; Shinozaki, K. and Yamaguchi- Shinozaki, K. (2007). Functional analysis of AHK1/ATHK1 and cytokinin receptor histidine kinases in response to abscisic acid, drought, and salt stress in Arabidopsis. Proc. Natl. Acad. Sci. USA 104: 20623–20628. 45 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 http://pubget.com/search?q=author:%22K%20T%20KT%20Yoshida%22&from=7820372 http://pubget.com/search?q=author:%22S%20S%20Naito%22&from=7820372 http://pubget.com/search?q=author:%22G%20G%20Takeda%22&from=7820372 http://pubget.com/search?q=latest%3APlant+and+Cell+Physiology&from=7820372 http://pubget.com/search?q=latest%3APlant+and+Cell+Physiology&from=7820372 http://pubget.com/search?q=issn%3A0032-0781+vol%3A35+issue%3A7&from=7820372 12-Argueso, C.T.; Ferreira, F.J. and Kieber, J.J. (2009) Environmental perception avenues: the interaction of cytokinin and environmental response pathways. Plant Cell Environ. 32: 1147–1160. 13-Zhang, C.; Zhao, B.; Ge, W.; Zhang, Y.; Song, Y.; Sun, D. and Guo, Y. (2011) An apoplastic H-type thioredoxin is involved in the stress response through regulation of the apoplastic reactive oxygen species in rice. Plant Physiol. 157: 1884–1899. Table (1): RAPD primers used. Primers Sequence Maj-OPA-07 5'-AAGTCCGCTC-3' Maj-OPA-09 5'-GGGTAACGCC-3' Maj-OPC-08 5'-TGGCGGTG-3' Maj-OPN-16 5'-CAAGGTGGGT-3' Maj-OPC-12 5'-TGTCATCCCC-3' Maj-OPA-11 5'-CAATCGCCGT-3' Maj-OPA-13 5'CAGCACCCAC-3' Maj-OPD-20 5'-TGTCATCCCC-3' Table (2): NCBI Blast- predicted expressed genes and their Genbank accession numbers Primer Predicted gene Genbank accession number Primer No. 7 purple acid phosphatase JX501672.1 vacuolar proton inorganic pyrophosphatsae mRNA AY296911.1 thioredoxin AJ005840.1 Primer No. 13 Galactosyltransferase B3 JN165358.1 Galactosyltransferase B2 JN165357 Galactosyltransferase B1 jn165356 Galactosyltransferase A JN165355 vaculor proton inorganic pyrophosphatase JQ180506.1 Cytokinin oxidase dehydrogenase (Ckx2.4) JN381555.1 Galactosyltransferase GQ 231955.1 ABA binding protien1 HQ166718.1 Primer No. 20 Low affinity nitrate transporter HF544988.1 Cytokinin oxidase/dehydrogenase salt tolerant protein EF415486.1 Storage protein activator FM242575.1 Plastid acetulye coA carboxylase EU660902.1 Lon1 protease AY494984.1 Serine therionine kinase AY036609.1 46 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 28S 18S rRNA M 1 2 3 4 Figure (1): ethidium bromide stained agarose gel electrophoresis (1%), M: 100bp DNA ladder, Lanes 1 to 4: DNase- treated total RNA preparations. Figure (2): Ethidium bromide stained agarose gel electrophoresis (1%) of PCR products. M: 100bp DNA ladder. Lanes (1-2): PCR products of primer number (07), lanes (3-4): PCR products of primer number (08), lanes (5-6): PCR products of primer number (09), lanes (7,8): PCR products of primer number (13), lanes (9-10): PCR products of primer number(11), N: negative control. Figure (3): Ethidium bromide stained agarose gel electrophoresis (1%) of PCR products. M: 100bp DNA ladder. Lanes (1-2): PCR products of primer number (20), lanes: PCR products of primer number (12), lanes (5-6): PCR products of primer number (16), E: Empty lane. 47 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 26 (3) 2013 المضخمة باستخدام بادئات عشوائیة في cDNAالمسح التفریقي لقطع ال الحنطة المتحملة والحساسة للملوحة مجید ارشید سباح جامعة النھرین / مركز بحوث التقنیات االحیائیة 2013آب 26، قبل البحث في : 2013آیار 6في : استلم البحث الخالصة لتحدید الجینات التي یعبر عنھا اصناف الحنطة المتحملة للملوحة والیعبر cDNA-RAPD PCRاستخدمت تقنیة ) إلعداد الحمض النووي 2الحنطة المتحملة للملوحة (دجلة) والحساسة (تموز اصنف استعمالالحساسة. االصناف عنھا في ض النووي الریبي. مصنعة من الحم cDNAثماني بادئات للتضخیم العشوائي من استعملت. cDNAالریبي وتصنیع ال عمالحمل الملوحة باستتوقع الجینات المتعلقة بت بادئات تعبیر تفاضلي في الصنف المتحمل للملوحة. تم وقد أظھرت ثالث blast NCBI دورھا بتحمل الملوحة في الحنطة أو غیرھا من النباتات وئات الثالثة. تبین أن الجینات التي تم توقعھا دللبا شیر إلى اھمیة ھذه البادئات والطریقة المستخدمة في تحدید الجینات التي لھا عالقة بتحمل الملوحة في الحنطة تحت ت الدراسة. cDNA-RAPD PCRالحنطة، الملوحة، الكلمات المفتاحیة: 48 | Biology @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@ÚÓ‘Ój�n€a@Î@Úœäñ€a@‚Ï‹»‹€@·rÓ:a@Âig@Ú‹©@Ü‹1a26@@ÖÜ»€a@I3@‚b«@H2013 Ibn Al-Haitham Jour. for Pure & Appl. Sci. 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