Gene cloning and expression analysis of cuticular chitin synthase from Macrobrachium nipponense

In order to study the role of chitin synthase(Chs) gene from Macrobrachium nipponense in the molting cycle,the M.nipponense chitin synthase(MnChs) of the cuticular tissue was first cloned using rapid amplification of cDNA ends(RACE) method,and its sequence was analyzed with a biological software.Spatio-temporal expression of the MnChs was determined by RT-PCR.The full-length contains 5 133 bp with a 283 bp of 5'-untranslated region(UTR),a 159 bp of 3'UTR and a 4 701 bp of open reading frame encoding a putative MnChs protein of 1566 amino acids with a predicted molecular mass of 179.57 ku and pI of 6.09.It includes two specific tag sequence of Chs(EDR,QRRRW) and a Chitin-synth-C domain.Sequence comparison shows that the MnChs deduced amino acid sequence shares an overall similarity of 89% to Pandalopsis japonica and of 63% to Daphnia pulex.The significant difference of MnChs mRNA levels among tissues is observed during molt stages.The high level occurs at stage A in carapace,at stage D₀ and D₄ in gastrointestine,at stage D₄ in tail fan and muscle.However,the level from hepatopancreas is relatively lower during molt stages.In conclusion,our present results show that MnChs is not only expressed in cuticle,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.