Isolasi Gen Penyandi Peroksidase melalui Penapisan terhadap Pustaka Genom Kedelai Kultivar Slamet

Suharsono Suharsono, Faisal Rachman, Utut Widy Astuti

Abstract


Screening to genomic library of soybean cultivar Slamet in ë phage to isolate the gene encoding for peroxidase(per) was performed by two steps. cDNA of per gene from Arabidopsis thaliana labeled by alkalin phosphatasewas used as probe. The first screening was done by using 105 recombinant pfu (plaque forming unit). The secondscreening was carried out on 100 recombinant pfu isolated from the first screening. The result of second screeningshowed that all screened clones were supposed containing per gene. The ë phage resulted from second screeningwas infected into E. coli BM 25.8. Due to the Cre recombinase in E. coli BM 25.8, two lox P sites flanking the insertDNA of soybean created recombination. The recombination in loxP sites resulted the excision and formedrecombinant plasmid containing per gene of soybean. To multiply, the recombinant plasmid was introduced into E.coli DH5á. Analysis southern hibridization of recombinant plasmid cut by Bam HI showed that Bam HI 3.7 kbfragmen of S52121R clone, 5,3 kb of S2412R clone, 2,4 and 5,3 kb fragments of S2532R clone were supposed tocontain per genes.

Keywords


genomic library, peroxidase gene, screening, soybean

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References


Anggraini, V. 1999. Identifikasi gen-gen toleran aluminium pada kedelai menggunakan gen pelacak dari Arabidopsis dan gandum. Tesis Program Pascasarjana. Bogor: Institut Pertanian Bogor.

Hong, C-Y., Hsu, Y.T., Tsai, Y-C., & Kao, C.H. 2007. Expression of ascorbate peroxidase 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl. J. Exp. Bot. 58: 3273-83.

Intapruk, C., Takano, M., & Shinmyo, A. 1994. Nucleotide sequence of a new cDNA for peroxidase from Arabidopsis thaliana. Plant Physiol. 104: 285-286.

Islam, M.M., Hoque, M.A., Okuma, E., Banu, M.N., Shimoishi, Y., Nakamura, Y., & Murata, Y. 2009. Exogenous proline and glycinebetaine increase antioxidant enzyme activities and confer tolerance to cadmium stress in cultured tobacco cells. Plant Physiol. (In Press, Available online 7 May 2009).

Lee, S.H., Ahsan, N., Lee, K.W,, Kim, D.H., Lee, D.G., Kwak, S.S., Kwon, S.Y., Kim, T.H., & Lee, B.H. 2007a. Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. J. Plant Physiol. 164: 1626-38.

Lee, Y.P., Kim, S.H,, Bang, J.W., Lee, H.S., Kwak, S.S., & Kwon, S.Y. 2007b. Enhance tolerance to oxidative stress in transgenic tobacco plants expressing three antioxidant enzymes in chloroplast. Plant Cell Rep. 26: 591-598.

Richards, K.D., Schott, E.J., Sharma, Y.K., Davis, K.R., & Gardner, R.C. 1998. Aluminum induced oxidative stress genes in Arabidopsis thaliana. Plant Physiol. 116: 409-418.

Ritter, D., Allen, R.D., Trolinder, N., Hughes, D.W., & Galau, G.A. 1993. Cotton Cotyledon cDNA Encoding a Peroxidase. Plant Physiol. 102: 1351.

Sato, Y., Murakami, T., Funatsuki, H., Matsuba, S., Suruyama, H., & Tanida, M. 2001. Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings. J. Exp. Bot. 52: 145-151.

Suharsono. 2002. Kontruksi pustaka genom kedelai kultivar Slamet. Hayati 9: 67-70.

Suharsono, Anwar, S., Jusuf, M., & Widyastuti, U. 2003a. Isolasi dan karakterisasi gen-gen dari tanaman kedelai yang mendapat cekaman aluminium. Laporan riset RUT VIII bidang bioteknologi. Jakarta: Kementerian Riset dan Teknologi dan Lembaga Ilmu Pengetahuan Alam.

Suharsono, Julianto, T., & Jusuf, M. 2003b. Isolasi gen penyandi peroksidase dari kedelai kultivar lumut melalui penapisan terhadap pustaka genom. Simposium Bioteknologi dan Sumberdaya Genetik, Balitbiogen Pertanian. Bogor, 11-12 Desember 2003.

Syarifuddin, A. & Abdurachman, A. 1993. Optimasi pemanfaatan sumberdaya lahan berwawasan lingkungan. Prosiding Simposium Penelitian Tanaman Pangan III. Pusat Penelitian dan Pengembangan Tanaman Pangan dan Badan Litbang Deptan. Jakarta/Bogor, 23-25 Agustus 1993.

Tang, L., Kwon, S.Y., Kim, S.H., Kim, J.S., Choi, J.S., Cho, K.Y., Sung, C.K., Kwak, S.S., & Lee, H.S. 2006. Enhance tolerance of transgenic potato plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against oxidative stree and high temperature. Plant Cell Rep. 25: 1380-1386.

Tsuduki, M., Takano, T., Nakatsubo, F., Yoshida, K., Shinmyo, A., & Asao, H. 2006. Resistance to insects in transgenic Solanum plants expressing a peroxidase gene from horseradish. Plant Biotechnol. 23: 71-74.

Zhao, C.R., Ikka, T., Sawaki, Y., Kobayashi, Y., Suzuki, Y., Hibino, T., Sato, S., Sakurai, N., Shibata, D., & Koyama H. 2009. Comparative transcriptomic characterization of aluminum, sodium chloride, cadmium and copper rhizotoxicities in Arabidopsis thaliana. BMC Plant Biol 9: 32

Xu, W., Li, W., He, J., Balwant, S., & Xiong, Z. 2009. Effect of insoluble Zn, Cd, and EDTA on the growth, activities of antioxidant enzymes and uptake of Zn and Cd in Vetiveria zizanioides. J. Environ. Sci. (China) 21: 186-92.

Yoshimura, K., Yabuta, Y., Ishikawa, T., & Shigeoka, S. 2000. Expression of spinach ascorbate peroxidase isoenzymes in response to oxidative stresses. Plant Physiol. 123: 223- 233.




DOI: http://dx.doi.org/10.31258/jnat.12.1.38-43

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