Isolation and characterization of gene involving ovarian development of the black tiger shrimp Penaeus monodon

Abstract

RT-PCR were carried out to examine the expression patterns of functionally important genes in ovaries (N = 6) and testes (N = 6) of the giant tiger shrimp (Penaeus monodon) A total of 158 pairs of primers were designed from EST of heamocyte (85 pairs), ovaries (71 pairs) and testes (2 pairs). Of these, 110 primers provided the positive amplification product. Nine gene homologues including female sterile (PMFS), ATP/GTP binding protein, adipose differentiation related protein (PMADRP), broad complex Z4 isoform, ovarian lipoprotein receptor (PMOVLR), carbonic anhydrate, aminopeptidase, Wolf hirschorn syndrome candidate 1 protein, and proactivator polypeptide precursor) were only expressed in ovaries but not testes of P.monodon broodstock where homologues of female sterile and ovarian lipoprotein receptor were ovarian-specific. Sixty-four gene homologues for example, 3-oxoacid CoA transferase, NADP-dependent leukotriene B4 12 hydroxy dehydrogenase (PMLTB4DH), dolichyl diphosphooligocharide protein glycotransferase (PMDDPG), asparaginyl tRNA synthetase (PMATRS), aspartase amino transferase(PMAST), endotherial cell growth factor I (PMECGFI) and nuclear autoantigenic sperm protein (PMNASP) were preferentially expressed in ovaries than testes of P.monodon broodstock. SSCP analysis was applied for examination of polymorphism of the amplified RT-PCR product of 22 gene homologues across different individuals of P. monodon. PMOVLP, PMNASP and tetrasparinD 107 were highly polymorphic suggesting the possibility to study correlations between SNP of these gene homologues and expression levels (or phenotypes) of the corresponding genes in P. monodon. Asparaginyl tRNA synthetase was also polymorphic and additional forms of this transcript were found in the female shrimp possessing the GSI of 1.90 - 2.13 compared with those having the GSI between 0.65 - 1.43%. This indicated that asparaginyl tRNA synthetase (PMATRS) is required for rapid protein synthesis during vitellogenesis. A fixed SSCP pattern of TATA binding protein associated factor 9 was found in ovaries and testes of P.monodon suggesting its sex-specific isotypes in P.monodon. The full length cDNA of 9 gene homologues including 3- oxoacid CoA trasferase, ATP/GTP binding protein, PMFS, PMADRP, PM, PMDDPG, PMATRS, PMAST, PMECGFI and PMNASP were successfully characterized and reported for the first time .The partial ORF (3318 bp), the complete 3’ UTR(116 bp) and the polyA tail of PMOVLR were also isolated. Functional analysis of these gene homologues will be further studied. 5-HT modulates the release of neuropeptide hormones from the sinus gland of shrimp and its effect on ovarian maturation of shrimp has been proposed. A time course effect of 5-HT(single or double injection of 50 microgram.g[superscript -1] of the body weight of 5-HT) on expression of PMFS, PMADRP, PMNASP PMAST, PMDDPG and 3-oxoacid CoA tranferase in ovaries of juvenile P.monodon were examined using semiquantitative RT-PCR. Results indicate that 5-HT potentially stimulated the expression level of all investigated genes (p is less than 0.05). PMFS and PMOVLP did not require the repeat injection of 5-HT as the second injection adverse the positive effect of the first injection. Nevertheless, repeat injection of 5-HT extended its effects on the expression level of PMADRT, PMNASP, 3-oxoacid CoA trasferase, PMDDPG and PMAST.