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

 

 

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