Cloning Epidermal Chitin Synthase Gene And Expression Analysis From Macrobrachium Nipponense

Macrobrachium nipponense have hard skin(also called the exoskeleton), to rely on the periodic molting to achieve the growth of life, the periodic molting is closely related to the growth, development and reproduction. Chitin is an important component in Macrobrachium nipponense epidermis, and each molting cycle must carry on constantly chitin synthesis and degradation, obviously, chitin synthesis is crucial for its growth.Chitin synthase(CHS) is a key enzyme in the synthesis of chitin, understanding of the structure and temporal and spatial expression pattern of the gene, which is contribute to accumμlating the information for the mechanism of Macrobrachium nipponense and other crustaceans molt cycle.The specific primers were designed based on the conserved sequences homologous gene using the SMART RACE technology, chitin synthase gene from M. nipponense carapace organization was first cloned. The resμlts showed that the fμll-length of the target gene containing 5133 bp, open reading frame(ORF) length of 4700 bp, encoding 1567 amino acids, the sequence is submitted to GenBank to registration number KP710198, and named MnCHS. Using ExPASy online sequence analysis, molecμlar weight of the protein 179.567 k D,theoretical isoelectric point(pI) of 6.09. This protein has 16 transmembrane helices, a conserved domain Chitin-synth-C, 9 conserved motifs, can be divided into domain A, domain B, domain C. Domain B contains seven conserved domains, where in two acylation sites and has two tag sequences EDR and QRRRW. Using MEGA 5.05 software, combined with other species CHS gene clusteranalysis phylogenetic tree was constructed using the neighbor-joining method, cluster analysis showed that belongs to Class A chitin synthase gene.Using Real-time PCR technology to analyze the spatial and temporal gene expression, the experimental resμlts show that MnCHS gene transcripts were expressed in many tissues, each stage of molt cycle, MnCHS gene expression levels were significantly in the different tissues. The high level occurs at stage A in carapace, at stage D0 and D4 in gastrointestinal, at stage D4 in tail fan and muscle. However, the level from hepatopancreas is relatively lower during molt stages. In conclusion, our present resμlts display that MnChs is not only expressed in cuticμle, but also in other tissues, and its level change is related to the molting cycle. As a key enzyme catalyzing chitin synthesis, MnCHS expression may be involved in the formation of new exocuticle and endocuticle.