Proteomic analysis of ovarian proteins and characterization of thymosin-β and RAC-GTPase activating protein 1 of the giant tiger shrimp Penaeus monodon

Jun 20, 2014Comparative biochemistry and physiology. Part D, Genomics & proteomics

Protein study of giant tiger shrimp ovaries focusing on thymosin-beta and RAC-GTPase activating protein 1

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Abstract

A total of 1638 proteins were matched in the ovaries of giant tiger shrimp.

Of the identified proteins, 1253 (76.50%) significantly matched known proteins.
Several reproduction-related proteins, including Cdc2 and Cyclin B, were found.
The full-length cDNA of thymosin-β and Rac-GTPase activating protein 1 was characterized, revealing distinct lengths and deduced amino acids.
Thymosin-β was expressed in all tissues, while Rac-GTPase activating protein 1 had higher expression in gonads compared to non-reproductive tissues.
Expression levels of thymosin-β and Rac-GTPase activating protein 1 were significantly greater in ovaries of wild adult broodstock than in juveniles.
Eyestalk ablation led to a significant reduction in thymosin-β expression during certain ovarian stages, but did not significantly affect Rac-GTPase activating protein 1 levels.

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Cellular proteomics of total proteins in ovaries of domesticated and wild giant tiger shrimp (Penaeus monodon) were examined using GeLC-MS/MS. In total, 1638 proteins matched those previously deposited in databases and 1253 (76.50%) of these significantly matched known proteins. Several reproduction-related proteins (e.g. Cdc2, Cyclin B, Cdc25, 14-3-3, thymosin-β and Rac-GTPase activating protein 1) were identified. In addition, the full-length cDNA of P. monodon thymosin-β (PmTmsb; 1084 bp with an ORF of 387 bp and 128 deduced aa) and Rac-GTPase activating protein 1 (PmRacgap1; an ORF of 1881 bp and 626 deduced aa) were further characterized. PmTmsb was constitutively expressed in all tissues. In contrast, PmRacgap1 was more abundantly expressed in gonads than in several non-reproductive tissues (e.g. subcuticular epithelium, hepatopancreas, intestine, pleopods, stomach and thoracic ganglion). The expression levels of PmTmsb and PmRacgap1 in ovaries of wild adult broodstock were significantly greater than those in ovaries of juveniles (P<0.05). However, their expression levels did not vary significantly during ovarian development stages in intact broodstock. However, eyestalk ablation resulted in a significant reduction in PmTmsb expression at stages I and III ovaries (P<0.05), although it did not affect PmRacgap1 transcription significantly at these stages. On the other hand, use of polyclonal antibodies derived from recombinant PmTmsb and PmRacgap1 revealed that levels of both proteins decreased at the late stage (IV) of ovarian development. Our results suggested that PmTmsb and PmRacgap1 may act as negative effectors during ovarian development in P. monodon.

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Proteomic analysis of ovarian proteins and characterization of thymosin-β and RAC-GTPase activating protein 1 of the giant tiger shrimp Penaeus monodon

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