Molecular Cloning, Tissue Distribution and Prokaryotic Expression of Crustacyanin A2 Gene from Procambarus clarkii

Abstract:

Crustacyanin plays an important role in the formation and regulation of the color in crustacean aquatic products. To study the gene structural feature and prokaryotic expression of Procambarus clarkii (P. clarkii) crustacyanin A2 (PcCRA2), the coding sequence of PcCRA2 gene (cra2) was obtained by gene cloning. The expression patterns of cra2 in 9 different tissues of P. clarkii were detected by real-time fluorescence quantitative PCR (RT-qPCR). Escherichia coli BL21 (DE3) was used as the host to conduct the heterologous recombinant expression of PcCRA2. The results showed that the full length of cra2 cDNA was 573 bp, encoding 190 amino acids. Bioinformatics analysis indicated that the relative molecular weight of PcCRA2 was 21 158.9, with a theoretical isoelectric point of 5.59. Amino acid alignment revealed that the protein coding sequence of cra2 had the highest similarity to that of C. quadricarinatus (91.58%). RT-qPCR demonstrated that cra2 was expressed in all tested tissues, with the highest expression in epidermis (P<0.05). The prokaryotic expression vector pET28a-cra2 was constructed and induced in DE3. The optimal expression condition was adding 0.5 mmol/L IPTG at 30 ℃ for 6 h after 4 h of inoculation. Results indicated that the recombinant protein could specifically bind to astaxanthin, with a maximum absorption peak at 505 nm. These results provide a foundation for further studies on the biological function of crustacyanin.