Molecular cloning, characterization and recombinant expression of crustacean hyperglycemic hormone in white shrimp Litopenaeus vannamei

Abstract

Crustacean hyperglycemic hormone (CHH) plays an important role in crustacean. In the present study, a full-length cDNA of CHH was cloned from the eyestalk of Litopenaeus vannamei by RACE approach for the first time. The full-length cDNA of LvCHH was 846bp, containing a 5′ untranslated region (UTR) of 65bp, a 3′ UTR of 436bp with a canonical polyadenylation signal-sequence AATAA and a poly (A) tail, and an open reading frame (ORF) of 345bp. The ORF encoded a polypeptide of 114 amino acids including a 24 amino acid signal peptide. The calculated molecular mass of the mature protein (74 amino acids) was 8.76kDa with an estimated pI of 6.78. The sequence of LvCHH was submitted in NCBI GenBank under the accession number HM748790.2. Phylogenetic analysis revealed that LvCHH was clustered with CHH of other crustaceans. Tissue distribution analysis revealed that the expression of LvCHH mRNA was observed in all tissues but gill, and was highest in heart. Specific primers containing Xho I and BamH I restriction sites respectively, were designed based on the obtained ORF sequence of LvCHH gene and the cloning sites of expression vector pET-32a (+). The recombinant plasmid LvCHH-pET32a, was used to transform Escherichia coli BL21 (DE3). LvCHH was successfully expressed by means of SDS–PAGE and western blot analysis. We detected gill Na+/K+-ATPase activity after rLvCHH protein injection and found that All the experimental group Na+/K+-ATPase activity presented peak change among 0–6h, and the peaks of all treated groups occurred in 1h. 20 and 30μg/shrimp−1 groups showed significant increase (P<0.05) in 1h post-injection. L. vannamei were exposed for 96h to hypo- and hyper-salinity challenge. Hypo-salinity caused a significant rise (P<0.05) in the mRNA expression of CHH and gill Na+/K+-ATPase activity at 12h and 24h respectively, then the CHH mRNA expression declining by 24h, and returned to control group level by 48h, and the Na+/K+-ATPase activity tended to be stable after 72h, and higher than that of control. The hyper-salinity challenge had the same trend at mRNA expression with the hypo-salinity group. The Na+/K+-ATPase activity had no significant change under the low salinity challenge. All these results indicate that LvCHH is an important hormone involved in the osmosis responses of swimming shrimps, and can provide further information of crustacean osmoregulation physiological mechanism.