Identification of testis-relevant genes from testes ESTs and cDNA microarray in the black tiger shrimp

Identification of testis-relevant genes using in silico analysis from testes ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon) A total of 4,803 ESTs from the testis library were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. A total of 2,702 unique sequences from 424 contigs and 2,278 singletons were identified. Approximately, 1,134 sequences from the total of 2,702 unique sequences (contigs and singletons) are homologous to genes with known functions. Some of which (mago nashi, insulin-degrading enzyme, actin-depolymerizing factor) are related to testicular development in studies of other organisms. Moreover, the sequences were further characterized according to the gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). By comparing with the EST libraries of other tissues, a total of 1,579 possible testis-specific ESTs were identified. Some of these testis-specific ESTs, such as Phospholipase A2 and Matrix Metalloproteinases, have functions relevant to testicular development. In addition, novel ESTs (621 ESTs) that have never been identified in any ESTs from the Penaeidae family were found in this study. Through cDNA microarray analysis, some of testis-specific ESTs were preferentially expressed in testes to ovary; one of which was a saposin homolog. Therefore, saposin was further characterized by real-time PCR and its full-length sequence was obtained by RACE-PCR. A cDNA microarray was constructed from the EST libraries of P. monodon, consisting of 5,376 features (1,125 features were amplified from testis EST libraries). RNA samples were extracted from testes and ovaries of juveniles (4 month-old, pooled from n=112 and n=98 for testes and ovaries, respectively) and broodstocks from Andaman Sea. The RNA from ovary was labeled with Cy3 dye as a reference and that from testes was labeled with Cy5 dye.