Cloning and analysis of the molecularly characterized chitinase genes of Daphnia carinata and Simocephalus vetulus

Abstract

Daphniidae plays an important role in maintaining the integrity and sustainability of food chains of freshwater ecosystems. However, studies on the intact genes of chitinase in Daphniidae have not been carried out till date. In this study, we obtained the full-length complementary DNA (cDNA) from Daphnia carinata and Simocephalus vetulus, members of the Family Daphniidae, by rapid amplification of cDNA ends and polymerase chain reaction. The two cDNAs were named DcChi and SvChi, respectively. The result showed that DcChi and SvChi were 1404 and 1319 bp in length and they encoded 383 and 382 amino acids, respectively. Based on their cDNA sequences, the genomic structures of the two chitinases were characterized. Sequence analysis revealed that DcChi was composed of three exons and two introns, while SvChi had four exons and three introns. Based on the presence of conserved catalytic domain sequences, the two chitinases could be clustered within the same phylogenetic group. Homology analysis showed that the two deduced proteins had a high similarity (65–88%) to those from published species in Family Daphniidae and a low similarity to those from the species in Class Malacostraca Subclass Copepoda (38–42%) and those from the species in Class Insecta Order Diptera (33–38%). Based on the results of multiple alignments of chitinases, we designed peptides that were specific to chitinases from the Family Daphniidae. The quantitative real time PCR analysis indicated that the transcript level of the chitinase gene increased with the growth of the two species.

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