Protein antigen-i parasit ikan C. irritans berpotensi tinggi digunakan sebagai calon dalam pembangunan vaksin komersial terhadap C. irritans. Walau bagaimanapun, kewujudan variasi pada antigen-i serotip C. irritans yang berbeza mempengaruhi tahap perlindungan yang bakal diberikan terhadap varians C. irritans yang berbeza apabila antigen-i digunakan sebagai vaksin. Kajian ini dijalankan untuk membandingkan jujukan pelbagai antigen-i pencilan C. irritans di Malaysia berbanding antigen-i pencilan C. irritans yang pernah dilaporkan. Perbandingan filogenetik dijalankan untuk meramalkan potensi protein tersebut dalam usaha membangunkan calon serodiagnostik dan pemvaksinan terhadap pencilan C. irritans yang berlainan. Penjajaran jujukan berbilang bagi jujukan asid amino antigen-i dilakukan dengan perisian CLUSTALX dan analisis filogenetik antigen-i dilakukan menggunakan kaedah parsimoni maksimum (MP) dan kaedah Bayes. Sembilan transkrip unik (TU) C. irritans yang mempunyai padanan signifikan dengan antigen-i di pangkalan data protein NCBI didapati mempunyai peratus kesamaan antara 41% hingga 71%. Kedua-dua pohon MP dan Bayesian yang dijana menunjukkan varians antigen-i cn56 and cn57 terkelompok bersama dalam satu kumpulan manakala varians antigen-i yang lain terbahagi kepada dua kumpulan berasingan dan pengkelompokan ini disokong oleh kehadiran asid amino yang terpulihara dalam kumpulan masing-masing. Kajian lanjutan boleh dilakukan untuk mengenal pasti varians antigen-i yang sesuai sebagai calon serodiagnosis dan juga dapat memberi perlindungan silang terhadap pelbagai pencilan C. irritans di serata dunia.
The lysosomal aspartic proteinase cathepsin D is an acute phase protein involved in various physiological processes, including vitellogenesis, yolk processing and immune responses. In this study, we characterised the cathepsin D from the Asian seabass Lates calcarifer and examined its expression profile during infection. The complete coding sequence of L. calcarifer cathepsin D consists of 1191 nucleotides, encoding a 396 amino acid protein molecule that is made up of a putative signal peptide, a leader peptide and a mature peptide. Phylogenetic analyses showed that two types of cathepsin D are present in the teleost lineage i.e. cathepsin D1 and D2, whereas higher vertebrates possess only one type of cathepsin D. L. calcarifer cathepsin D was clustered together with cathepsin D1 from other teleosts. Compared to mammalian sequences, L. calcarifer cathepsin D lacks the β-hairpin loop that forms the double chain and is present as a single chain peptide with conserved aspartic active sites like other fish. Both multiple sequence alignment and phylogenetic analysis indicated that the L. calcarifer cathepsin D sequence codes for cathepsin D1 and suggested that it shares the same functions with cathepsin D from other fish. Expression profiling analysis of cathepsin D in L. calcarifer infected with Aeromonas hydrophila showed that it is up-regulated in immune-related tissues such as gills, spleen and liver, suggesting that cathepsin D plays an important role in the innate immune response of L. calcarifer against pathogens.
In recent years, there has been considerable interest in simple sequence repeats (SSRs) particularly as molecular markers with applications in many different fields. We have carried out an effort to identify and analyse SSRs in the genome of the Asian seabass, Lates calcarifer by random sequencing. Genomic DNA was isolated from the muscle tissue of L. calcarifer, sheared by nebulisation and ligated into plasmid vector. Recombinant clones were selected randomly from the genomic libraries constructed. Subsequently, plasmid DNA was extracted and subjected to one-pass sequencing. A total of 4175 random sequences, also known as genome survey sequences (GSSs), with a total length of 1.7 Mb was generated. Screening of the whole L. calcarifer GSS data set allowed for the identification of a total of 151 perfect (100% similarity) SSRs. These SSR consensus patterns spread over a wide range of size (1 to 226 bp). The most frequent consensus pattern is dinucleotide, which represents 60% of all SSRs identified. The dinucleotides (AC)n, (AT)n and (AG)n were also found to occur frequently in the L. calcarifer genome. Sequence comparison between L. calcarifer and other fish species showed variation in repeat content, indicating the different ways in which repeats may evolve in the genome of these species. Data generated from this random sequencing of the L. calcarifer genome should serve as a valuable resource for further studies of this organism.
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.
[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].
The Asian seabass (Lates calcarifer) is one of the most economically important aquaculture fish species in South East Asia. While the biology of the Asian seabass is widely studied, relatively little information is available at the molecular level. This lack of molecular information represents one obstacle to rapid progress in the study of immune responses particularly under aquaculture conditions. In light of this situation, we have undertaken an expressed sequence tag (EST) project on the Asian seabass spleen, the secondary lymphoid organ, for the identification of immune-related genes. A
total of 2932 ESTs were generated and grouped into 1063 unique transcripts (UTs), which consisted of 104 consensi and 959 singletons. Of these, 51.3% (545/1063) matched to previously identified genes, while 48.7% (518/1063) showed no match. Of the 545 homologous UTs, 102 (9.6%) can be putatively identified as immune-related genes. The identification of the putative immune-related genes provides a meaningful framework in the effort to comprehend the Asian seabass immune system that may lead to an increase in the understanding of the defense mechanisms of and our abilities to
manage this fish species.
Teknologi DNA mikroatur merupakan salah satu teknologi penting dalam penyelidikan biologi kini. Teknologi tersebut membolehkan analisis pengekspresan gen pelbagai sistem model pada skala genom dijalankan. Prestasi DNA mikroatur ditentukan oleh parameter seperti kepadatan titik, ciri-ciri titik (morfologi, kepadatan prob dan keamatan isyarat penghibridan), latar belakang, kespesifikan dan kesensitifan. Dengan menggunakan mesin pemegun GeneTac™G3, slaid mikroatur cDNA ikan siakap (Lates calcarifer) yang mengandungi 1920 klon-klon cDNA terpilih daripada perpustakaan cDNA hepar dan limpa yang dipegunkan secara duplikasi telah difabrikasikan. Klon-klon yang dipilih mempunyai fungsi putatif berkaitan dengan keimunan, aruhan tekanan, metabolisme, pengangkutan, transkripsi dan translasi. Kawalan negatif seperti oligonukleotida (A)50 dan beberapa gen daripada Saccharomyces cerevisiae dan Escherichia coli serta kawalan positif seperti beberapa siri pencairan DNA genom dan cDNA L. calcarifer juga dipegunkan ke atas slaid kaca. Diperhatikan bahawa kepekatan prob dalam julat antara 150-250 μg/mL, kesesuaian jenis kawalan positif dan negatif yang digunakan berjaya menghasilkan slaid yang berkualiti. Selain itu, perlakuan pengeringan slaid secara semalaman dan rehidrasi semula slaid menghasilkan morfologi titik DNA yang sekata dan membulat. Kawalan kualiti ke atas slaid yang terhasil membuktikan keberkesanan slaid yang difabrikasi untuk kajian respons transkriptom L. calcarifer terhadap jangkitan Cryptocaryon irritans.