Chitin Metabolic Enzyme Genes From Hyriopsis Cumingii: Cloning, Characterization And Potential Functions

Hyriopsis cumingii is one of the most important freshwater pearl mussel in China.In this study, two chitin metabolic enzyme genes were cloned from H. cumingii usingtwo defined ESTs: chitinase-3（Chi-3） and chitin deacetylase （Cda）. These enzymeswere characterized and the potential physiological functions of these two genes wereinvestigated by examining tissue-specific expression, shell damage induced expressionand prokaryotic expression. SSR markers from Lamprotula leai were developed bymagnetic beads hybridization and5’anchored PCR and part of them randomly selectedwere applied in allied species Hyriopsis cumingii for cross-amplification. The mainfindings were as follows:1. Full-length cDNA cloning and molecular characterization of two chitin metabolicenzyme genes from Hyriopsis cumingiiThe Chi-3cDNA was2,523bp in length, and it consisted of a196bp5’-untranslated region （UTR）, a1962bp ORF encoding653amino acid residues, and a365bp3’-UTR. BLAST analysis revealed that the deduced amino acid sequence of H.cumingii Chi-3（HcChi-3） shared21.3–34.4%identity with known genes from otherorganisms and relatively higher identity27.2–34.7%with five other Chi-3s. Similar toother known chitinases, the HcChi-3peptide was predicted to consist of a signal peptideand three conserved domains: a catalytic domain （Glycoside hydrolase family18domain-GH18domain） followed by two chitin-binding domains （Carbohydrate-bindingmodule family14domain-CBM14domain）. The phylogenetic tree showed thatHcChi-3clustered with Chi-3from C. gigas, which suggests that HcChi-3in thispresent study belongs to the bivalvia Chi-3group.The Cda cDNA was2654bp in length, and this contained a347bp5’-UTR, a1938bp ORF that encodes645amino acid residues, and a369bp3’-UTR and wasfound to contain a signal peptide, three predicted N-glycosylation sites （NTS, NIT andNET） and a catalytic domain （Polysacc_deac₁domain）. In our study, the phylogenetic tree of Cda in invertebrates was constructed based on the Cda domain patterns. Asshown, HcCda clustered in group4, which contains peptides that consist only of asignal peptide and a Polysacc_deac₁domain.2. Expression analysis of two chitin metabolic enzyme genes from Hyriopsis cumingiiIn tissue specific expression experiment, taking β-actin gene as inner control. Asshown, Chi-3was expressed in a wide range of tissues with a relatively high levelexpression in mantle, liver, stomach, kidney, relatively low level expression in intestine,gill and foot and no expression in blood while Cda was expressed in each of thesetissues with relatively high level expression in mantle, stomach, foot.In shell damage induced experiment, taking β-actin gene as inner control. Chi-3transcript levels were up-regulated significantly （p<0.05）12h after shell damage andexpression reduced gradually thereafter, however, transcript levels of Cda did notchange significantly （p>0.05） in response to shell damage.In prokaryotic expression experiment, the optimum condition for Chi-3gene was1mmol/L of IPTG at24℃. As shown in SDS-PAGE, the expression level ofrecombinant protein was in a clear rising trend in the first four hours and grew at aslower pace in the last four hours. With regard to Cda, the optimum condition for Chi-3gene was1mmol/L of IPTG at37℃. The expression level of Cda recombinant proteinrose steadily in eight hours. In this study, pGEX-5X-1vector which expressed a GSTtag （26KD） was selected for generating recombinant protein including GST tag and a10KD Chi-3domain. Also, a7KD Cda domain for Cda gene. Our finding could lay afoundation for enzyme assay.3. Development and characterization of30microsatellite loci in Lamprotula leai, withcross-amplification in Hyriopsis cumingiiColonies from library constructed by magnetic beads hybridization method wereselected and screened, of which69.2%were positive; Number of replications over10were taking up70.2%, and28pairs of primers were designed according to sequencesobtained, of which11primers could be applied in DNA polymorphism analysis, rating39.3%. Results of genetic diversity in cultured population of L.leai using11primersfrom magnetic beads hybridization method indicated that the number of alleles per locusranged from4to13. The value of the observed heterozygosity （Ho）, expectedheterozygosity （He） ranged from0.2051-0.7381and0.5656-0.8393, respectively. Colonies from library constructed by5’anchored PCR method were selected andscreened, of which97.8%were positive; Number of replications over10were taking up24.7%, and56pairs of primers were designed according to sequences obtained, ofwhich19primers can be applied in DNA polymorphism analysis, rating30.4%. Resultsof genetic diversity in cultured population of L.leai using19primers from5’anchoredPCR indicated that the number of alleles per locus ranged from3to10. The value of theobserved heterozygosity （Ho）, expected heterozygosity （He） ranged from0.2083-0.8944and0.4305-0.8961, respectively. Process and result of the research indicated thatmicrosatellite sequences from it could be higher quality, and the magnetic beadsenriched method could be a higher efficiency method, whereas5’anchored PCR methodwas more easily performed and genetic diversity index of primers obtained was higher.In addition,18SSR markers developed by two methods were selected applying ingenetic diversity of Hyriopsis cumingii, of which10were relatively high polymorphism.This indicated that all the microsatellite markers obtained could be useful in geneticdiversity analysis of L.leai and related species.