Identification and expression pattern analysis of Piwi genes during the spermiogenesis of Portunus trituberculatus

The Piwi genes have an important role in stem cell development, gametogenesis and RNA interference in diverse organisms. So far, most of the studies have focused on the function of Piwis in vertebrates, but their function during spermiogenesis in invertebrates still remains largely unclear. In order to investigate the function of Piwis during spermiogenesis in the crab Portunus trituberculatus, we use RT-PCR and RACE to identify three Piwi complete cDNA sequences from the total RNA of the testis in P. trituberculatus. The deduced amino acid sequences of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 showed that each contains a well-conserved PAZ domain and PIWI domain. RT-PCR analyzed the tissue expression pattern of P. trituberculatus Piwi-1, Piwi-2 and Piwi-3 in the testis, heart, muscle, hepatopancreas and gill. All of the Piwis are found in germ cells of adult testis in P. trituberculatus by in situ hybridization, suggesting that these genes may play function during spermiogenesis in this species.     

Cloning and expression of arginine kinase from a swimming crab, <Emphasis Type="Italic">Portunus trituberculatus</Emphasis>

Arginine kinase (AK) is an important phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the cDNA of AK (designated as PtAK) was identified from the eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA was 1,479 bp, containing an open reading frame of 1,074 bp that coded for 357 amino acids. The estimated molecular mass of mature PtAK was 40.30 kDa and theoretical isoelectric point was 6.18. Amino acid sequence alignment showed that PtAK had very high similarity with other shrimp and crab AKs ranging from 0.876 to 0.983. The genomic DNA fragments of about 1,434 bp consisted of two exons interrupted by an intron. Totally 24 SNPs, including 17 in the coding region and seven in the non-coding region, were detected by direct sequencing of 19 genomic samples. In exon 1, the coding SNPs (cSNPs) were only found in the disease-resistant specimens. The fluorescent real-time PCR analysis revealed that the expression of PtAK was detected in all the examined tissues with the highest expression in the muscle and the lowest in the eyestalk. The expression of PtAK after Vibrio alginolyticus injection was tested in haemocytes, showing that two peak values were 5.01-fold (at 3 h) and 3.60-fold (at 24 h) compared with the control values, respectively. The results suggested that AK might play an important role in the immune response in crabs.     

Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89 %). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.