Sains Malaysiana 34(1): 27-32 (2005)

Pembinaan dan Pencirian Perpustakaan Genom Siakap (Lates calcarifer)

(Construction and Characterisation of Genomic Library

for Sea bass Lates calcarifer)

Pan-Pan Chong, Adura Mohd-Adnan

& Kiew-Lian Wan

Pusat Pengajiain BioSains dan Bioteknologi

Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia

43600 UKM Bangi, Selangor, Malaysia

ABSTRAK

Siakap (Lates calcarifer) merupakan spesies ikan yang penting dari segi ekonomi di Malaysia. Dalam kajian ini, satu perpustakaan genom L. calcarifer telah dibina. DNA genom telah dipencil daripada tisu otot L. calcarifer, diserpih secara nebulisasi, dibaiki hujung tumpul dan diligasi ke dalam vektor plasmid pCR®4Blunt-TOP®. Campuran ligasi seterusnya telah ditransformasikan ke dalam Escherichia coli. Bagi mencirikan perpustakaan genom yang terhasil, DNA plasmid telah diekstrak daripada klon-klon rekombinan yang dipilih secara rawak. Elektroforesis gel agaros menunjukkan klon-klon rekombinan ini membawa selitan DNA yang bersaiz di antara 0.5 kb - 3.5 kb, dengan purata saiz 1.7 kb. Seterusnya, penjujukan DNA telah dijalankan dengan menggunakan DNA plasmid yang diperoleh sebagai templat. Sebanyak 121 jujukan rawak telah dihasilkan daripada perpustakaan genom L. calcarifer ini. Analisis jujukan rawak memberikan implikasi yang perpustakaan ini adalah representatif bagi genom L. calcarifer. Berdasarkan persamaan dengan jujukan dalam pangkalan data, 17% jujukan rawak ini didapati mempunyai keputusan BLAST yang bermakna, yang menunjukkan mereka mewakili gen L. calcarifer. Analisis dengan menggunakan perisian RepeatMasker menunjukkan sebanyak 25% jujukan rawak ini dikenal pasti sebagai unsur berulang. Pencirian awal terhadap perpustakaan genom ini menunjukkan ia berpotensi sebagai sumber yang berguna dalam kajian genom L. calcarifer.

Kata kunci: Perpustakaan Genom, Lates calcarifer

ABSTRACT

Sea bass (Lates calcarifer) is an economically important fish in Malaysia. In this study, a genomic library of L. calcarifer was constructed. Genomic DNA was isolated from the muscle tissue of sea bass, sheared by nebulisation, blunt-end repaired and ligated into the pCR®4Blunt- TOPO® plasmid vector. The ligation mixture was then transformed into Escherichia coli. In order to characterize the constructed genomic library, plasmid DNA was extracted from randomly selected recombinant clones. Gel electrophoresis showed that these recombinant clones contain inserts with a size range of 0.5 kb - 3.5 kb, with an average size of 1.7 kb. DNA sequencing was then carried out using the extracted DNA plasmid as template, and a total of 121 random sequences were generated from the L. calcarifer genomic library. Analysis of these sequences implies that the library is representative of L. calcarifer genome. Based on sequence similarity database searches, 17% of the random sequences were found to have significant BLAST hits, indicating that they represent L. calcarifer genes. Analysis using the Repeat Masker program reveals that 35% of the random sequences were identified as repeat elements. Initial characterization of this genomic library indicates that it is a potentially useful resource in the study of the L. calcarifer genome.

Keywords: Genomic library, Lates calcarifer

RUJUKAN/REFERENCES

Altschul, S. F, Madden, T. L. Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. 1997. Gapped BLAST and PSI­BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

Anderson, S. 1981. Shotgun DNA sequencing using cloned DNaseI-generated fragments. Nucleic Acids Res. 9: 3015-­3027.

Bodenteich, A. Chissoe, S., Wang, Y. F & Roe, B. A. 1994. Shotgun clonin as the strategy of choice to generate templates for high-throughput dideoxynucletide sequencing. Dlm. Adams, M.D. (pnyt.). Automated DNA sequencing and analysis. New York: Cold Spring Harbor Laboratory Press. 42-50.

Deininger, P. L. 1983. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal. Biochem. 129: 216-223.

Ewing, B. & Green, P. 1998. Base-calling of automated sequence traces using phred. II. Error probabilities. Genome Res. 8: 186-194.

Ewing, B., Hillier, L., Wendl, M.C. & Green, P. 1998. Base-calling of automated sequencer traces using phred. 1. Accuracy assessment. Genome Res. 8: 176-185.

Fitzgerald, M. C., Skowron, P., Van Etten, J. L., Smith, L. M. & Mead, D. A. 1992. Rapid shotgun cloning utilizing the two base recognition endonuclease CviJI. Nucleic Acids Res. 20: 3753-3762.

Garcia, L. M. B. 1990. Spawning response latency and egg production capacity of Lhrha-injected mature female sea bass, Lates calcarifer Bloch. 1. Appl. lchthyol. 6: 167-172.

Gingrich, J. C., Boeher, D. M. & Basu, S. B. 1996. Partial CviJI digestion as an alternative approach to generate cosmid sub­libraries for large-scale sequencing projects. BioTechiques 21: 99-104.

Grey, D. L. 1987. An overview of Lates calcarifer in Australia and Asia. Dlm. Copland, J.W. & Grey, D.L. (pnyt.). Management of Wild and Cultured Sea Bass/Barramundi (Lates calcarifer). Canberra: Australian Centre for International Agricultural Research. 15-21.

Hengen, P. N. 1997. Shearing On A for genomic library construction. Trend Biochem. Sci. 22: 273-274.

Kaufman, P. B., Wu, W., Kim, D. & Cseke, L. J. 1995. Handbook of molecular and cellular methods in biology and medicine. USA: CRC Press, Inc.

Kato, M. 1999. Structural bistability of repetitive DNA elements featuring CAITG dinucleotide steps and mode of evolution of satellite DNA. Eur. J. Biochem. 265: 204-209.

Oefner, P. J., Hunicke-Smith, S.P., Chiang, L., Dietrich, F Mulligan, J. & Davis, R.W. 1996. Efficient random subcloning of DNA sheared in a recirculating point-sink flow system. Nucleic Acids Res. 24: 3879-3886.

Sambrook, J. & Russel, D. W. 2001. Molecular cloning: a laboratory manual, ed. ke-3. New York: Cold Spring Harbor Laboratory Press.

Schriefer, L. A., Gebauer, B. K., Qui, L. Q., Waterson, R. H. & Wilson, R. K. 1990. Low pressure DNA shearing: a method for random DNA sequence analysis. Nucleic Acids Res. 18: 7455-7456.

Singer, M. F. 1982. SlNEs and LlNEs: highly repeated short and long interspersed sequences in mammalian genomes. Cell 28: 433-434.

Toledo, J.D., Marte, Castillo, A.R. 1991. Spontaneous maturation and spawning of sea bass (Lates calcifer) in floating net cages. J. Appl. Ichthyol. 7: 217-222.

Toh, G., Gaspari, Z. & Jurka, J. 2000. Microsatellites in different eukaryotic genomes: survey and analysis. Genome Res. 10: 967-981.

Weiner, A.M. 2002.  SINEs and LINEs: the art of biting the hand that feeds you. Curr. Opin. Cell Biol. 14: 343-350.