Cloning, Expression And SNP Application Analysis Of Calcium Regulating Genes From Hyriopsis Cumingii

Triangle pearl mussel (Hyriopsis cumingii) is an important freshwaterpearl mussel in China. In the present study, full-length cDNA ofMSP130-related-2and HcCRT gene from H. cumingii was obtained.Functions of these two genes were investigated by examiningtissue-specific expression, shell damage induced expression andprokaryotic expression. Then, partial DNA sequences of MSP130-related-2gene and the5′-flanking regions were amplified from genome DNAisolated from H. cumingii with PCR and genome walking technique.Several SNPs were obtained through direct sequencing these genes of H.cumingii from two populations (white stock and purple stock). The majorresults were as follows:1) cDNA cloning and analysis of MSP130-related-2and HcCRT geneFull-length sequence of MSP130-related-2cDNA was2343bp,comprising a5′-UTR of572bp, and a3′-UTR of475bp. The open readingframe (ORF) of1293bp encoded a polypeptide of430amino acids.Homology analysis indicated that the protein of MSP130-related-2sharedhighest identity with MSP130of Crassostrea talienwhanensis Crosse45%.A phylogenetic tree was constructed by neighbor joining, and indicated thatthe HcMSP130-rel-2in this study clustered with the MSP130family inCrassostreagigas.Full-length sequence of HcCRT cDNA was1437bp, comprising a5′-UTR of231bp, and a3′-UTR of616bp. The open reading frame (ORF)of591bp encoded a polypeptide of196amino acids. Homology analysis indicated that HcCRT amino acid had a conservative sequence ofcalreticulin family and was highly conserved with Danio rerio（77%）.TheSequence analysis showed that HcCRT shared two potential calreticulinfamily signature motifs with CRT from other species,KHEQNIDCGGGYLKVF and IMFGPDICG.2) Expression of MSP130-rel-2and HcCRT mRNA in different tissuesThe MSP130-rel-2transcripts were broadly detected in all tested tissues,including mantle, blood, gill, foot, liver, kidney, intestine and muscle, withthe highest expression in mantle followed by liver. The expressions ofMSP130-rel-2transcripts were low in blood and intestine. The effect ofshell damage of H. cumingii on HcMSP130-related-2gene expression inmantle and muscle was examined at2h、6h、12h、24h、2d、4d、7d、15dand30d after shell damage, using real-time PCR. In the mantle, theexpression of MSP130-related-2was up-regulated and then decrease.HcMSP130-related-2transcript levels in the mantle were up-regulatedsignificantly7days after shell damage (P<0.05), thus expression hadchanged in response to this injury; however, no significant difference inexpression was detected in adductor muscles after shell damage (P>0.05).The HcCRT transcripts were broadly detected in all tested tissues, withthe highest expression in mantle followed by blood and muscle. Theexpressions of HcCRT transcripts were very low in liver and kidney.Under37℃,1mmol/L IPTG inducted the expression ofHcMSP130-related-2recombinant plasmid in the expression strain, showed,protein existence in the form of inclusion body, expression increased withthe induction time extension, the largest expression at7h.3) Gene sequences,5′-flanking regions and SNP analysis ofHcMSP130-rel-2from H. cumingii The partial DNA sequence of HcMSP130-rel-2and the5′-flankingregion were amplified from genome DNA isolated from H. cumingii withPCR and genome walking technique. Sequence analysis of the5′-flankingregion revealed that it contained core promoter element (TATA-box) andother transcription regulation elements such as CF-1, Cap and CdxA so on.Single nucleotide polymorphisms were obtained through direct sequencinggenes of H. cumingii from white stock and purple stock. Forty-one SNPswere obtained of HcMSP130-rel-2gene and we investigated the associationof these polymorphic locis with two group. Among them, P-278A-TSNP、P-337G-A SNP in HcMSP130-rel-2intron were signifcant difference ofthe genotypes frequency and allelic frequency between two groups(P<0.05).Among them, P-140T-G SNP、P-158T-C SNPs were signifcantdifference of the allelic frequency between two groups (P<0.05); P-258A-GSNP were signifcant difference of the genotypes frequency between twogroups (P<0.05); T/T genotype present specifically in while stock atP-496C-T site, and T/T、 A/T、T/T genotype present specifically in purplestock at P-170G-C site P-4/91A/G/T site、and I-3092C-T site.