Identification of genes and proteins related to ovarian development of the giant Tiger Shrimp Penaeus monodon

Abstract

Two dimensional gel electrophoresis (2-DE) of proteins in different ovarian stages (I, II, III and IV ovaries, respectively) of normal and eyestalk-ablated giant tiger shrimp (Penaeus monodon) broodstock was carried out. Proteomic patterns between different stages of ovaries were clearly different. Protein spots after 2-DE were further analyzed by mass spectrometry. Initially, 35 protein spots from various ovarian stages were selected and analyzed by peptide mass fingerprinting (PMF) using MALDI-TOF. All protein spots did not match any protein in the database. Additionally, 90 protein spots were analyzed by peptide fragmentation using MALDI-TOF/TOF and only 4 protein spots significantly matched calreticulin, protein disulfide isomerase, transketolase and allergen pen m2. Afterwards, nanoLC-MS/MS was used for identification of ovarian proteins separated by 2-DE. A total of 375 protein spots (215 spots from stage II and 160 spots from stage IV) from ovaries of normal P. monodon broodstock were examined. Of which, 90 (41.86%) and 102 (63.75%) spots of respective ovarian stages were significantly homologous to known proteins. The remaining 183 (125 and 58 from stages II and IV, 58.14 and 36.25%, respectively) protein spots did not match any protein and were regarded as novel uncharacterized proteins of P. monodon. In addition, 300 protein spots (180 and 120 spots from stages II and IV) from ovaries of eyestalk-ablated P. monodon broodstock were also characterized. A total of 85 (47.22%) and 41 (34.16%) proteins matched proteins in the databases, respectively. A large number of unknown protein; 95 and 79 protein spots accounting for 52.77 and 65.83%, were observed. Results clearly indicated that additional unknown proteins expressed at stage IV ovaries were induced by eyestalk ablation. Comparative analysis using MW and pI of protein spots can classify proteins in ovaries of P. monodon to 4 categories; those expressed in stages II, IV, both stages II and IV ovaries of normal and eyestalk-ablated broodstock and those expressed only in ovaries of eyestalk-ablated broodstock. The full length cDNA of DEAD box 52 (ORF of 1824 bp deduced to a polypeptide of 607 amino acids), DEAD box ATP-dependent RNA helicase (1209 bp, 402 aa), ATP-dependent RNA helicase (1206 bp, 401 aa) and L-3-hydroxyacyl coenzyme A dehydrogenase (933 bp, 301 aa) and protein disulfide isomerase (PDI, 1293 bp, 430 aa) and the partial sequence of valosin containing protein (VCP) were successfully characterized. Tissues distribution analysis of various genes was examined. Generally, these genes were constitutively expressed in all examined tissues of P. monodon broodstock. Nevertheless, ATP-dependent RNA helicase was not expressed in eyestalk whereas L-3-hydroxyacyl coenzyme A dehydrogenase was expressed in intestine. PDI and helicase lymphoid-specific isoform 2 were not expressed in both eyestalk and thoracic ganglion. Semiquantitative RT-PCR of DEAD box 52, DEAD box ATP-dependent RNA helicase, ATP-dependent RNA helicase, helicase lymphoid specific isoform 2 and PDI in ovaries of normal and eyestalk-ablated were carried out. DEAD box ATP-dependent RNA helicase, ATP-dependent RNA helicase and PDI were differentially expressed during ovarian development of normal broodstock (P < 0.05). However, expression of helicase lymphoid-specific isoform 2 was comparable during ovarian development of both normal and eyestalk-ablated P. monodon broodstock (P > 0.05). In addition, expression of DEAD box ATP-dependent RNA helicase, VCP and PDI were examined by quantitative real-time PCR. DEAD box ATP-dependent RNA helicase were up-regulated at stage III and IV ovaries of normal shrimp and at stage IV ovaries of eyestalk-ablated shrimp (P < 0.05). VCP was comparably expressed during ovarian development of normal P. monodon but was up-regulated at the final stage of ovarian development in eyestalk-ablated P. monodon broodstock. Considering expression levels of PDI in both normal and eyestalk-ablated P. monodon simultaneously, PDI was up-regulated at stage III ovaries of normal shrimp (P < 0.05) but its levels were not significant different during ovarian development of eyestalk-ablated broodstock (P > 0.05).