Six Chitinases CDNA Cloning And Expression Analysis During Post-Embryonic Development And Moulting Cycle In Macrobrachium Nipponense

Oriental river prawn, Macrobrachium nipponense is an economically important freshwater prawn in China. The aquaculture production of the prawn reached 237,431 tons in China, and annual value of production was over 15 billion. The growth, development and breeding of the prawn need to be accomplished by molting. Chitinase is one of the most important enzymes in crustacean molting, which hydrolyze chitin polymer into chitin monomer jointly action with β-N-acetylglucosaminidase. In addition, Chitinase was used to digest chitin-containing food and defense of shrimp against viruses. Given important function of the chitinase gene, I decide to clone the family gene of chitinase. The expression profiles of each MnCht in different tissues, moulting cycle and larval development were determined using quantitative polymerase chain reaction (qPCR).28 ESTs of chitinase gene were found in testis cDNA library, and these sequences were assembled to five contigs. In addition, the other on was acquired by homology-based cloning. The full-length cDNA sequences of the six chitinase genes (MnChtlA、MnCht1B、 MnCht3A、MnCht3B、MnCht3C and MnCht4) were cloned by rapid-amplification of cDNA ends technology. The full-length of six cDNA sequences was 1554,1529,2459, 1359,1547 and 1941bp, and encoded 408,410,380,389,480 and 618 amino acids respectively. Chitinase and chitinase-like proteins from crustacean could be classified into six groups on the basis of the phylogenetic analysis. Six distinct cDNA sequences encoding chitinases were cloned from M. nipponense were divided into three categories (Group1, Group2 and Group3) and designated as MnChtlA, 1B,3A,3B,3C and 4 respectively. In the multiple sequence alignment, MnCht3C and MnCht4 shared almost the same architecture characteristics, a signal peptide, a catalytic domain with a S/T-rich linker region and one or two chitin-binding domains (CBDs), whereas the latter two features were absent in the remaining four MnCht genes.Expression patterns and tissue distribution of the six MnCht genes was analyzed in each of ten selected tissues using real-time quantitative RT-PCR. MnCht1A and MnCht1B had a similar tissue distribution and expression profile, and were distributed in five (cuticle, testis, hepatopancreas, gut, eyestalk) and four (cuticle, testis, hepatopancreas, gut) tissues, respectively. The expression levels were much higher in hepatopancreas and gut than in other tissues, and expression in hepatopancreas was several times higher than that in the gut Each MnCht in Group 3 had a different tissue distribution pattern; the only common feature of these three genes was the expression level in the gut and hepatopancreas which was higher than in other tissues, and gut much higher than hepatopancreas, which was the opposite of MnChtlA and MnCht1B in Group 1. MnCht3A was expressed in testis, hepatopancreas and gut; MnCht3B was expressed in cuticle, testis, hepatopancreas, gut and eyestalk; MnCht3C was only expressed in hepatopancreas and gut, with no distribution in testis. This may be the reason why the non-EST sequence of MnCht3C can be found in the testis cDNA library of M. nipponense. MnCht4 was broadly expressed in the various tissues, with no expression in brain and gut, which was different from the other five MnCht genes.Expression of six MnCht genes during post-embryonic development (larval and post-larval stages) revealed that the six MnCht genes were not expressed during the larval stage, before metamorphosis. After metamorphosis, six MnCht began to express, and the expression of MnChtlA, MnCht1B, MnCht3B and MnCht4 during the post-larval stage was relatively low at PL1 (first day of the post-larval stage), spiked at PL5, decreased significantly at PL 10, increased again at PL15, and then decreased significantly at PL20. However, MnCht3A and MnCht3C did not display such a varied pattern. In the molting cycle, the expression of MnChtlA, MnCht1B, MnCht3B and MnCht4 had a remarkable fluctuation in the cuticle. The uppermost expression level was determined in the ecdysis. MnCht1B was up-regulated when stimulated by temperatures which were lower or higher than the normal growth temperature; however, MnCht1A did not show such diversification.Temperature is an important factor affecting crustacean molting. When the living environment of crustacean changes in sudden (temperature swells and dips), often cause molting. The magnitude of the temperature change too large can also cause death. The expression levels of the six MnCht transcripts at different temperature were monitored by real-time quantitative RT-PCR. Expession of MnChtlA did not change both in high temperature (30℃) and low temperature (20℃). MnCht1B was opposite to MnChtlA, the expression level of MnCht1B changed significantly in the two experimental groups more than the control. Expression of MnCht3B was up-regulated in higher temperature, and up-regulated slightly in lower temperature. MnCht4 was only slightly up-regulated both in high and low temperature.MnCht3A shared 82% identity with MnCht3C, and no expression level was detected in the cuticle. So, MnCht3A and MnCht3C may only function in the digestion of chitinous foods and modification of the gut peritrophic membrane. However, MnChtlA, MnCht1B, MnCht3B and MnCht4 had a pivotal role in the moulting cycle of adult prawn. This study advanced our understanding of the multiple biological functions of the chitinase genes, and lays the foundation for further research on chitinase genes in this important aquaculture species and other crustaceans.