Sains Malaysiana 47(7)(2018): 1465–1471

http://dx.doi.org/10.17576/jsm-2018-4707-14

 

Transformasi Gen Proteolisis 6 (PRT6) Berperantarakan Agrobacterium tumefaciens ke dalam Kotiledon Tomato kultivar Micro Tom

(Agrobacterium tumefaciens Mediated Transformation of the Proteolysis 6 (PRT6) Gene
into Cotyledons of Tomato cv. Micro Tom
)

 

INTAN ELYA SUKA1, NUR FARHANA ROSLAN1, BEE LYNN CHEW2, HOE HAN GOH3,

ZAMRI ZAINAL1,3 & NURULHIKMA MD ISA1*

 

1Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

2Pusat Pengajian Sains Kajihayat, Universiti Sains Malaysia, Minden, 11800 Georgetown, Penang, Malaysia

 

3Institut Biologi Sistem, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

 

Diserahkan: 15 September 2017/Diterima: 7 Mac 2018

 

ABSTRAK

Gen Proteolisis 6 (PRT6) merupakan gen yang memainkan peranan penting dalam tapak jalan N-end rule dan berfungsi sebagai enzim E3 ligase. PRT6 berperanan dalam pengenalan protein sasaran bagi proses degradasi. Objektif utama kajian ini adalah untuk mentransformasi konstruk RNAi PRT6 ke dalam tomato berperantarakan Agrobacterium tumefaciens. Ini bertujuan untuk memahami peranan tapak jalan N-end rule semasa proses pemasakan buah. Beberapa faktor yang memberi kesan kepada transformasi seperti masa ko-penanaman dan juga kepekatan antibiotik yang digunakan telah dioptimumkan. Keputusan kajian menunjukkan pengeraman kotiledon selama 48 jam pada medium ko-penanaman dapat meningkatkan penghasilan kalus sebanyak 61% manakala penggunaan 500 mg/L antibiotik karbenisilin dalam medium regenerasi pucuk dapat mengurangkan kontaminasi A. tumefaciens sehingga 5.2%. Selain itu, strain A. tumefaciens C58 merupakan strain A. tumefaciens yang paling sesuai digunakan sebagai perantara dalam kajian ini. Tindak balas berantai polimerase (PCR) telah dijalankan pada pucuk yang terhasil untuk mengesahkan integrasi fragmen PRT6 ke dalam genom tomato. Berdasarkan analisis PCR, kesemua tujuh pucuk putatif transgenik adalah merupakan transforman positif.

 

Kata kunci: Antibiotik; A. tumefaciens; kalus; ko-penanaman; Proteolisis 6

 

ABSTRACT

Proteolysis 6 (PRT6) gene plays an important role in the N-end rule pathway which functions as an E3 ligase enzyme. PRT6 functions to recognise target proteins for degradation. The main objective of this study is to transform the PRT6 RNAi construct through Agrobacterium tumefaciens into tomato. The purpose of this study was to understand the role of the N-end rule pathway during fruit ripening. Several factors affecting transformation efficiency such as co-cultivation time and concentration of antibiotics were optimised. The results from this study showed that pre-cultured cotyledons incubated for 48 h in co-cultivation medium increased the callus formation to 61% while using 500 mg/L carbenicillin antibiotic in the shoot regeneration medium reduced the contamination of A. tumefaciens to 5.2%. Besides, A. tumefaciens strain C58 was shown to be the most suitable A. tumefaciens strain to be used in this study. Polymerase Chain Reaction (PCR) was performed on the regenerated shoots to confirm integration of the PRT6 fragment into the tomato genome. Based on the PCR analysis, all putative transgenic shoots were positive transformants.

 

Keywords: Antibiotic; A. tumefaciens; callus; co-cultivation; Proteolysis 6

RUJUKAN

Carvalho, R.F., Marcelo, L.C., Lilian, E.P., Simone, L.C., Agustin, Z., Joni, E.L., Vagner, A.B., & Lázaro, E.P.P. 2011. Convergence of developmental mutants into a single tomato model system: Micro-Tom as an effective toolkit for plant development research. Plant Methods 7(1): 1-14.

Chetty, V.J., Ceballos, N., Garcia, D., Narváez-Vásquez, J., Lopez, W. & Orozco-Cárdenas, M.L. 2013. Evaluation of four A. tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom. Plant Cell Reports 32(2): 239-247.

Costa, M.G.C., Nogueira, F.T.S., Figueira, M.L., Otoni, W.C., Brommonschenkel, S.H. & Cecon, P.R. 2000. Influence of the antibiotic timentin on plant regeneration of tomato (Lycopersicon esculentum Mill.) Cultivars. Plant Cell Reports 19(3): 327-332.

Gibbs, D.J., Mark, B., Hannah, M.T. & Michael, J.H. 2016. From start to finish: Amino-terminal protein modifications as degradation signals in plants. New Phytologist 211(4): 1188-1194.

1188-1194.

Gibbs, D.J., Jorge, V.C., Sophie, B., Geeta, P., Guillermina, M.M. & Michael, J.H. 2015. Group VII ethylene response factors coordinate oxygen and nitric oxide signal transduction and stress responses in plants. Plant Physiology 169(1): 23-31.

Gibbs, D.J., Md Isa, N., Movahedi, M., Lozano-Juste, J., Mendiondo, G.M., Berckhan, S., Marín-de, L.R.N., Vicente, C.J., Sousa, C.C., Pearce, S.P., Bassel, G.W., Hamali, B., Talloji, P., Tomé, D.F., Coego, A., Beynon, J., Alabadí, D., Bachmair, A., León, J., Gray, J.E., Theodoulou, F.L. & Holdsworth, M.J. 2014. Nitric oxide sensing in plants is mediated by proteolytic control of group vii erf transcription factors. Molecular Cell 53(3): 369-379.

Gibbs, D.J., Seung, C.L., Nurulhikma, M.I., Silvia, G., Takeshi, F., George, W.B., Cristina, S.C., Corbineau, F., Theodoulou, F.L., Bailey-Serres, J. & Holdsworth, M.J. 2011. Homeostatic response to hypoxia is regulated by the n-end rule pathway in plants. Nature 479(7373): 415-418.

Godwin, I., Gordon, T., Brian, F. & Newbury, H.J. 1991. The effects of acetosyringone and pH on agrobacterium-mediated transformation vary according to plant species. Plant Cell Reports 9(12): 671-675.

Graciet, E. & Frank, W. 2010. The plant n-end rule pathway: Structure and functions. Trends in Plant Science 15(8): 447-453.

Grewal, D., Raman, G. & Satbir, S.G. 2006. Influence of antibiotic cefotaxime on somatic embryogenesis and plant regeneration in Indica rice. Biotechnology Journal 1(10): 1158-1162.

Haddadi, F., Maheran, A.A., Siti, N.A.A., Soon, G.T. & Hossein, K. 2015. An efficient agrobacterium-mediated transformation of strawberry cv. camarosa by a dual plasmid system. Molecules 20(3): 3647-3666.

Holford, P. & Newbury, H.J. 1992. The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus. Plant Cell Reports 11(2): 93-96.

Martí, E., Carmina, G., Gerard, J.B., Mark, S.D. & José, L.G. 2006. Genetic and physiological characterization of tomato cv. Micro-Tom. Journal of Experimental Botany 57(9): 2037-47.

Mendiondo, G.M., Gibbs, D.J., Szurman-Zubrzycka, M., Korn, A., Marquez, J., Szarejko, I., Maluszynski, M., King, J., Axcell, B., Smart, K., Corbineau, F. & Holdsworth, M.J. 2016. Enhanced waterlogging tolerance in barley by manipulation of expression of the n-end rule pathway E3 ligase PROTEOLYSIS6. Plant Biotechnology Journal 14(1): 40-50.

Nakano, T., Suzuki, K., Fujimura, T. & Shinshi, H. 2006. Genome-wide analysis of the ERF gene family. Plant Physiology 140(February): 411-432.

Qin, Y.H., Jaime, A., Teixeirada, S.J.H.B, Zhang, S.L. & Hu, G.B. 2011. Response of in vitro strawberry to antibiotics. Plant Growth Regulation 65(1): 183-193.

Rao, A.M., Padma, S.K. & Kavi, K.P.B. 1995. Enhanced plant regeneration in grain and sweet sorghum by asparagine, proline and cefotaxime. Plant Cell Reports 15(1-2): 72-75.

Saito, T., Ariizumi, T., Okabe, Y., Asamizu, E., Hiwasa-Tanase, K., Fukuda, N., Mizoguchi, T., Yamazaki, Y., Aoki, K. & Ezura, H. 2011. TOMATOMA: A Novel tomato mutant database distributing Micro-Tom mutant collections. Plant and Cell Physiology 52(2): 283-296.

Sun, H.J., Sayaka, U., Shin, W. & Hiroshi, E. 2006. A highly efficient transformation protocol for Micro-Tom, a model cultivar for tomato functional genomics. Plant and Cell Physiology 47(3): 426-431.

Tan, L.W., Zuraida, A.R., Hoe, H.G., Duk, J.H., Ismanizan, I. & Zamri, Z. 2017. Production of transgenic rice (Indica Cv. MR219) overexpressing ABP57 gene through agrobacterium-mediated transformation. Sains Malaysiana 46(5): 703-711.

Varland, S., Camilla, O. & Thomas, A. 2015. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects. Proteomics 15(14): 2385-2401.

Varshavsky, A. 2011. The N-- End rule pathway and regulation by proteolysis. Protein Science 20(8): 1298-1345.

Wood, D.W., Setubal, J.C., Kaul, R., Monks, D.E., Kitajima, J.P., Okura, V.K., Zhou, Y., Chen, L., Wood, G.E., Almeida, N.F. Jr., Woo, L., Chen, Y., Paulsen, I.T., Eisen, J.A., Karp, P.D., Bovee, D. Sr., Chapman. P., Clendenning, J., Deatherage, G., Gillet, W., Grant, C., Kutyavin, T., Levy, R., Li, M.J., McClelland, E., Palmieri, A., Raymond, C., Rouse, G., Saenphimmachak, C., Wu, Z., Romero, P., Gordon, D., Zhang, S., Yoo, H., Tao, Y., Biddle, P., Jung, M., Krespan, W., Perry, M., Gordon-Kamm, B., Liao, L., Kim, S., Hendrick, C., Zhao, Z.Y., Dolan, M., Chumley, F., Tingey, S.V., Tomb, J.F., Gordon, M.P., Olson, M.V. & Nester, E.W. 2001. The genome of the natural genetic engineer A. tumefaciens C58. Science 294(5550): 2317-2323.

 

 

*Pengarang untuk surat-menyurat; email: hikma@ukm.edu.my

 

 

 

 

sebelumnya