Kloning Gen Coat Protein SMV dengan Pendekatan PCR

https://doi.org/10.22146/jpti.9365

Sismindari Sismindari(1*), Sudjadi Sudjadi(2)

(1) Pusat Antar Universitas Bioteknologi Universitas Gadjah Mada
(2) Pusat Antar Universitas Bioteknologi Universitas Gadjah Mada
(*) Corresponding Author

Abstract


SMV is RNA virus had to be converted to the first strand DNA using oligo (dT) and murine reverse transcriptase. Amplification of coat protein gene region was carried out by Polimerase Chain Reaction (PCR) with two primers, 5’-TACATCTTGGAACCAATGGCAGGCAAGGAGAGAAG-3’ and 5’-AGGACAACAAACATTGCCG-3’. The PCR product was blund ended by S1 nuclease, and ligated into SmaI digested pUC18 and phosphatase treatment by calf intestine phosphatase. Ligation mixture was used to transform E. coli DH5α. Recombinant plasmid was digested with EcoRI and HindIII showed 0,8 kb fragment. Southern blot analysis at high strigency using PCR using PCR product as a probe shows that the 0,8 kb fragment produced intense signal.


Keywords


SMV; coat protein

Full Text:

PDF


References

Beachy, RN., Loesch-Fries, and N.E. Turner. 1990. Coat Protein Mediated resistence Against Virus Infection. Annu. Rev. Phytopathol 28: 451-474.

Eggenberger, AL., D.M. Stark, and RN. Beachy, 1989. The Nucleotide Sequence of Soybean Mosaic Virus Coat Protein Coding Region and Its Expression in Escherichia coil, Agrobacterium tumefaciens and Tobacco Callus, J. Gen. Virol. 70, 1853-1860.

Hollings, M. and A.A.. Brunt, 1981, Potyvirus in: E. Kurtsak (ed.) Plant Virus Infections. Elsevier/ North-Holand Biomedical Press, Amsterdam, 733-807.

Kushner, S.R. 1978. An Improved Method for Transformation of Escherechia coli with Co1E1-Derived Plasmid, In: H.B. Boyer and S. Nocosia (Eds.), Genetic Engineering, Elsevier/ North-Holand Publishing Co, Amsterdam, 17-23.

Ling, K, Namba, S., Gonsalves. C, Slinghtom, L., Gonsalves, D. 1991. Protection Against Detrimental Effects of Potyvirus Infection in Transgenic Tobacco Plants Expressing the Papaya Ringspot Virus Coat Protein Gene, Biotechnology 9:752-758

Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989. Molecular Cloning, A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, New York.

Stark, D.M. and R.N. Beachy. 1989. Protection Against Potyvirus Infection in Transgenic Plants Evidence for Broad Spectrum Resistance. Biotechnology 7: 1257 - 1262.

Sumardiyono, Y.B., Sudjadi. Sismindari dan S. Sulandari, 1994. Laporan RUT Tahun I. (Tidak dipublikasikan).

Yanisch-Perron, C., J, Viera and J. Messing. 1983. Improved M13 phage cloning vector and host strains: nucleotide sequence of the M l3mp19 and pUCI9 vectors, Gene, 33: 103-1 19.



DOI: https://doi.org/10.22146/jpti.9365

Article Metrics

Abstract views : 3564 | views : 2531

Refbacks

  • There are currently no refbacks.


Bookmark and Share


Copyright (c) 1996 Jurnal Perlindungan Tanaman Indonesia

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Jurnal Perlindungan Tanaman Indonesia ISSN 1410-1637 (print), ISSN 2548-4788 (online) is published by the Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, in collaboration with Indonesian Entomological Society (Perhimpunan Entomologi Indonesia, PEI) and Indonesian Phytopathological Society (Perhimpunan Fitopatologi Indonesia, PFI). The content of this website is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.  

View website statistics