J. Sci. Dev. 2009, 7 (Eng.Iss.1): 9 - 16 HA NOI UNIVERSITY OF AGRICULTURE 9 Application of PCR to identify the transgene (Arabidopsis thaliana allene oxide synthase 2) in transgenic rice plants Ứng dụng PCR để phát hiện sự có mặt của gen AtAOS 2 trong genome của lúa đã được chuyển gen Nguyen Thi Thuy Hanh 1 , Han Oksoo 2 1 Faculty of Agronomy, Hanoi University for Agriculture 2 College of Agriculture and Life sciences, Chonnam National University, South Korea TÓM TẮT Allene oxide synthase (AOS) là một enzyme quan trọng trong số các enzyme tham gia vào quá trình tổng hợp axit jasmonic thông qua con đường sinh hoá octadecanoid. Ở thực vật, axit jasmonic đóng vai trò then chốt trong việc hình thành các phản ứng đáp lại các tác động của môi trường trong và ngoài tế bào. Để làm tăng hàm lượng enzyme allene oxide synthase nội sinh ở lúa, gen allene oxide synthase 2 của Arabidopsis thaliana (AtAOS 2) đã được chuyển vào genome của lúa (Oryza sativa L.Japonica cv. Nakdong). Phản ứng PCR đã được sử dụng để phát hiện sự có mặt của gen AtAOS2 trong genome của lúa đã được chuyển gen. Cụ thể là phản ứng PCR đã được sử dụng để phát hiện gen tổng hợp chất kháng sinh hygromycin, promoter CaMV35S (promoter này nằm ngay phía trước gen AtAOS2 trong cấu trúc của vector pCAMBIA1201/AtAOS) và gen AtAOS 2 trong genome của lúa. Từ khoá: Allene oxide synthase, axit jasmonic, con đường sinh hoá otadecanoid, thực vật chuyển gen. SUMMARY Allene oxide synthase (AOS) is an important regulator that steers the octadecanoid pathway toward jasmonic acid (JA) synthesis. Plant jasmonic acid (JA) plays pivotal roles regulating cellular responses against environmental cues, including the innate immune response(s). The Arabidopsis thaliana allene oxide synthase 2 (AtAOS 2) gene was introduced into rice (Oryza sativa L.Japonica cv.Nakdong) genome. The polymeraza chain reactions (PCRs) were applied to indentify the transgene - AtAOS 2 in transgenic rice plants. A set of PCRs was carried out to detect the hygromycin resistance gene, CaMV35S promoter (the pomoter located infront of the AtAOS 2 gene in pCAMBIA1201/AtAOS binary vector) and the AtAOS 2 gene in the transgenic rice genome. Key words: Allene oxide synthase, jasmonic acid, octadecanoid pathway, transgenic plants. 1. INTRODUCTION Plants have evolved defense strategies to protect themselves from various abiotic and biotic stresses. One of the most prominent defense mechanisms is the octadecanoid pathway, which leads to jasmonic acid (JA) biosynthesis. JA is a genuine plant hormone that has various physiological functions, including growth control, senescence promotion, responses to wounding and drought, and defenses against insects and pathogens (Schaller et al., 2005). Furthermore, JA signaling interacts with signaling of other plant hormones such as ethylene and salicylic acid to form a complex signaling network (Devoto et al., 2005). JA is synthesized through the octadecanoid pathway (Figure 1). In the octadecanoid pathway, 13 (S) - HPOT is the pivotal intermediate, from which several metabolic pathways branch out. For the JA synthesis, 13 (S) - HPOT is converted to an unstable allene oxide, 12,13 - epoxy - 9Z, 11E, 15Z - octadecatrienoic acid (12,13 - EOT), by an allene oxide synthase (AOS). The allene oxide is readily converted to cis-(+)-OPDA by allene oxide cyclase Application of PCR to identify the transgene (Arabidopsis thaliana allene oxide synthase2) 10 (AOC); cis-(+)-OPDA is further metabolized through reduction and β-oxidation to JA. On the other hand, 13(S)-HPOT can also be metabolized by other enzymes, including hydroperoxide lyase, peroxygenase, hydroperoxide reductase, and divinyl ether synthase. A typical function of these branch pathways is defence against insect and pathogens (BlÐe, 2002). Therefore, AOS is an important regulator that steers the octadecanoid pathway to JA synthesis, thus affecting the synthesis of all JA- related compounds (Kongrit et al., 2007). Rice AOS (OsAOS) and Arabidopsis thaliana AOS belong to the CYP74A subfamily. A database search indicates that, besides the characterized OsAOS1 (Agrawal et al., 2004), there are at least four additional OsAOS (OsAOS2-5) genes in the rice genome (Figure 2). Most of the rice AOS genes are located on chromosome 3, except for the OsAOS3 gene, that is located on chromosome 2. The OsAOS proteins show putative localization sites such as mitochondria (MC), endoplasmic reticulum (ER), plasma membrane (PM), microbody (MB), and cytosol (CS) (Agrawal et al., 2004). Three Arabidopsis thaliana AOS genes (AtAOS1-3) have been identified. All AtAOS genes are located on chromosome 5. Arabidopsis thaliana AOS proteins localize on chloroplast (CP), microbody (MB), mitochondria (MC). In order to increase the level of endogenous JA in rice, we try to over express AOS genes by transforming AtAOS2 gene to the rice genome Fig. 1. The octadecanoid pathway leading to jasmonic acid (JA) biosynthesis in plants AOC: allene oxide cyclase; OPR: 12-oxo-phytodienoic acid reductase; 13 (S)-HPOT: (9Z, 11E, 15Z, 13S)-13 - hydroperoxy - 9, 11, 15-octadecatrienoic acid; 12, 13 - EOT: (9Z, 11E, 15Z, 13S, 12R) - 13 - Nguyen Thi Thuy Hanh, Han Oksoo 11 epoxy - 9, 11, 15 - octadecatrienoic acid; OPDA: 12 - oxo- 10, 15 (Z) - octadecatrienoic acid; OPC - 8:0: 3-oxo-2 (2' (Z) -pentenyl)-cyclopentane-1-octanoic acid (Schaller, 2001). Fig. 2. Phylogenetic tree of AOS homologues and related proteins from other species CP: chloroplast; CPS: chloroplast stroma; CS: cytosol; ER: endoplasmic reticulum; MB: microbody; MC: mitochondria; PM: plasma membrane (Agrawal et al., 2004). Application of PCR to identify the transgene (Arabidopsis thaliana allene oxide synthase2) 12 Fig. 3. Structure of the pCAMBIA1201/AtAOS2 binary vector (pCAMBIA1201 from CAMBIA,USA; pCAMBIA1201/AtAOS2 was contructed by Dr.Eunsun Kim) In this study, a set of PCRs was carried out to detect the hygromycin resistance gene, CaMV35S promoter (the promoter located in front of the AtAOS2 gene), and the AtAOS2 gene in the transgenic rice genome. The results of PCRs would confirm the appearance of the AtAOS2 gene in the transgenic rice genome. 2. MATERIALS AND METHODS 2.1. Extraction of rice DNA The leaves of one-month-old wild-type (Oryza sativa L. Japonica cv. Nakdong) and 5 lines of T 1 transgenic rice plants (T 1 transgenic seeds were obtained from Dr.Eunsun Kim) were harvested, frozen in liquid nitrogen, and ground to fine powder with a mortar and pestle under liquid nitrogen. Total genomic DNA from non-transgenic and transgenic rice plants were extracted by using the DNeasy Plant Mini Kit (QIAGEN) according to the instructions of the manufacturer. 2.2. PCR analysis 2.2.1. Design of Primers The Primer 3 program was used to design all primers for PCRs analysis. 2.2.2. PCR analysis to detect hygromycin resistance gene The primer pair employed to detect the selectable marker (hygromycin phosphotransferase) was: foward primer 5'- ACAGCGTCTCCGACCTGATGCA-3' and reverse primer 5'- AGTCAATGACCGCTGTTATGCG-3'. PCR was carried out according to the PCR Amplification Kit by using HiPi PCR Premix (ELPis Biotech, Korea) in a 20 µl reaction volume, with thermal cycling parameters as follows: initial denaturation at 94 0 C for 5 min; following by 35 cycles of 94 0 C for 1 min, 61 0 C for 1 min, 72 0 C for 2 min; and 72 0 C for 5 min for final extension. 2.2.3. PCR analysis to detect the CaMV 35S promoter The primer pair, forward primer 5'- GACCTAACAGAACTCGCCGTA-3' and reverse primer 5'- CACTTGCTTTGAAGACGTGGT-3', was designed from the sequence of CaMV35S promoter which located infront of AtAOS2 gene in pCAMBIA1201/AtAOS binary vector (Fig. 3). PCR was carried out according to the PCR Amplification Kit by using HiPi PCR Premix (ELPis Biotech, Korea) in a 20 µl reaction volume. Template DNA were initially denatured at 94 0 C for 5 min, followed by 35 cycles with thermal cycling parameters of 94 0 C for 50 second, 58 0 C for 50 seconds, 72 0 C for 1 min.30 seconds. A final 5 min. incubation at 72 0 C was allowed for completion of primer extension. 2.2.4. PCR analysis to detect the introduced gene- AtAOS2 The primer pair employed to detect the introduced genes was: forward primer 5'- ACGACCAAGGAGCTGAAGAG-3' and reverse primer 5'-CGCCGGTGGTAGACTAAATG-3'. PCR was carried out according to the PCR Amplification Kit by using HiPi PCR Premix (ELPis Biotech, Korea) in a 20 µl reaction volume with thermal cycling parameters as follows: 94 0 C for 5 min; following by 35 cycles of 94 0 C for 40 seconds, 53 0 C for 40 seconds, 72 0 C for 1 min.40 seconds; and 72 0 C for 5 min. 3. RESULTS AND DISCUSSION The agarose gel banding patterns of the PCR products were shown in figure 4. For the hygromycin resistance gene, a 590 base pair amplified segment was detected in pCAMBIA1201/AtAOS plasmid and transgenic rice genome with the specific designed primers, but was not detected in non-transgenic (Fig. 4a). For the CaMV35S promoter, which was located infront of the AtAOS2 gene, the amplified band size was 399 base pairs, and it was detected in the plasmid, transgenic rice genome (Fig. 4b) but was not detected in the non-transgenic genome. Nguyen Thi Thuy Hanh, Han Oksoo 13 A 599 base pair segment that was located in the Arabidopsis thaliana cDNA region was detected in the plasmid genome and transgenic rice genome, but was not detected in the non- transgenic rice genome (Fig. 4c). fsghfjgykh Fig. 4. Representative PCR analysis for the presence of the hygromycin resistance gene (a); CaMV35S promoter (b) and AtAOS2 gene (c) in transgenic rice plants Lane 1: molecular size marker; Lane 2: pCAMBIA1201/AtAOS plasmid (positive control); Lane 3: untransformed plant (negative control); Lanes 4 - 8: transformed plants. Application of PCR to identify the transgene (Arabidopsis thaliana allene oxide synthase2) 14 The result in fig.4c shows that beside expected band (599 bp) in transgenic rice plants, a strong band (about 200 bp) was also detected in both non - transgenic and transgenic rice plants. This is because there are AOS genes in the rice genome. As shown in fig 2, five different rice AOS genes have been identified. The alignment of the rice AOS genes with a primer pair, which was used to detect the Arabidopsis thaliana AOS2 gene in the rice genome, shows that rice AOS5 (AK066137) may have a 208 base pair segment with this primer pair (Fig. 5). Nguyen Thi Thuy Hanh, Han Oksoo 15 Application of PCR to identify the transgene (Arabidopsis thaliana allene oxide synthase2) 16 Fig. 5. Alignment of rice AOS5 (AK066137) with the primer pair that was used to detect the Arabidopsis thaliana AOS2 gene in the rice genome 4. CONCLUSION From the above results, the hygromycin resistance gene, CaMV35S promoter (the promoter located in front of the AtAOS2 gene), and the AtAOS2 gene were clearly detected in the transgenic rice genomes. These results strongly confirmed the appearance of the transgene - Arabidopsis thaliana AOS2- in transgenic rice plant genomes. REFERENCES Agrawal, G.K., Tamogami, S., Han, O., Iwahashi, H., Rakwal, R (2004). Rice octadecanoid pathway, Biochem. Biophys. Res. Commun. 317, 1-15. Blée, E (2002). Impact of phyto-oxilipins in plant defense, Trends Plant Sci. 7, 315-321. Devoto, A., Turner, J.G (2005). Jasmonate- regulated Arabidopsis stress signaling network, Physiol. Plant. 123,161-172. Kongrit, D., Jisaka, M., Iwanaga, C., Yokomichi, H., Katsube, T., Nishimura, K., Nagaya, T., Yokota, K (2007). Molecular cloning and functional expression of soybean allene oxide synthases, Biosci. Biotechnol. Biochem. 71(2), 491-498. Schaller, F (2001). Enzymes of the biosynthesis of octadecanoid-derived signaling molecules, Journal of experimental botany., 52, 11-23. Schaller, F., Schaller, A., and Stintzi, A (2005). Biosynthesis and metabolism of jasmonates. J. Plant Growth Regul., 23, 179-199. . transgenic rice genome. The results of PCRs would confirm the appearance of the AtAOS2 gene in the transgenic rice genome. 2. MATERIALS AND METHODS 2.1. Extraction of rice DNA The leaves of one-month-old. CaMV35S promoter (the pomoter located infront of the AtAOS 2 gene in pCAMBIA1201 /AtAOS binary vector) and the AtAOS 2 gene in the transgenic rice genome. Key words: Allene oxide synthase, jasmonic. Kim) In this study, a set of PCRs was carried out to detect the hygromycin resistance gene, CaMV35S promoter (the promoter located in front of the AtAOS2 gene), and the AtAOS2 gene in the transgenic