title: Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon).
abstract: BACKGROUND
  Poor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation.
  
  
  RESULTS
  In this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon.
  
  
  CONCLUSIONS
  Without a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
authors.forename: Thidathip
authors.initials: T
authors.lastname: Wongsurawat
authors.forename: Rungnapa
authors.initials: R
authors.lastname: Leelatanawit
authors.forename: Natechanok
authors.initials: N
authors.lastname: Thamniemdee
authors.forename: Umaporn
authors.initials: U
authors.lastname: Uawisetwathana
authors.forename: Nitsara
authors.initials: N
authors.lastname: Karoonuthaisiri
authors.forename: Piamsak
authors.initials: P
authors.lastname: Menasveta
authors.forename: Sirawut
authors.initials: S
authors.lastname: Klinbunga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D033561:Penaeidae / Q000235:genetics*; D010802:Phylogeny; D049231:Saposins / Q000235:genetics; D013737:Testis / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D049231:Saposins; C074556:DMC1 protein, human