Abstract:
Identification and characterization of genes and proteins involved in signal transduction pathway during ovarian development in the giant tiger shrimp (Penaeus monodon) were identified and characterized. Screening of expression patterns of genes functionally related to ovarian development of P. monodon by RT-PCR, 4 genes including Mitogen-activated protein (MAP) kinases (MEK), Large Tumor Suppressor1 (LATS1), Kinase Suppressor of RAS (KSR) and RAS with differentially expresses pattern as reveal by RT-PCR were characterized. The full length cDNAs of PmMEK was 1559 bp in length containing the ORFs of 1227 bp to polypeptide of 408 amino acid. Molecular mass and pI of the deduced PmMEK protein was 45.27 kDa and 6.13, respectively. PmMEK contained Serine/Threonine protein kinases (S_TKc). In situ hybridization indicated that PmMEK was localized only in the cytoplasm of previtellogenic oocytes while PmLATS1 was localized in ooplasm of late cortical rod oocytes as well as previtellogenic oocytes. Quantitative real-time PCR indicated that In normal broodstock, expression levels of PmMEK at stages IV ovaries was greater than those other stages (P >0.05) and this transcript was not significantly different during development of eyestalk-ablated brooder P. monodon. The expression level of PmLATS1 was significantly increased at the final stage (IV) of ovarian development in normal broodstock. And the expression level of PmKSR transcript was higher in stage I, II and III ovaries of eyestalk-ablated broodstock than that of intact broodstocks P. monodon. Results suggested that PmKSR gene may be used as bioindicators for monitoring progression of oocyte maturation. Recombinant PmMEK protein was successfully expressed in vitro. Polyclonal antibodies of these recombinant proteins were successfully produced. Western blot analysis indicated that PmMEK expressed at the vitellogenic ovaries and early cortical rod. In the present study, the expression profiles of interesting genes/proteins were examined in different stages of ovarian development of normal and eyestalk-ablated P. monodon broodstock implied that several key genes from the signal transduction pathway may contribute ovarian development and maturation in P. monodon. Functionally analysis of genes/proteins involving ovarian development can be further carried out for better understanding of the reproductive maturation of female P. monodon in captivity.
