Preliminary Study On Carbonic Anhydrase Gene Related With Pearl Color And Formation In Hyriopsis Cumingii

Hyriopsis cumingii is most important freshwater mussel cultured in China.it is necessary to study the function of pearl formation related gene,which would be important significance on the understanding of the pear formation mechanism.This study obtained the full length of HcCA3(carbonic anhydrase 3 in Hyriopsis cumingii)cDNA by RT-PCR and RACE according to the partial cDNA sequence of a carbonic anhydrase from the transcriptome library of H.cumingii.The expression and function of HcCA3 gene were studied by qRT-PCR,RNAi,fluorescence in situ hybridization and western blot.1.Cloning and analysis of HcCA3 gene from H.cumingiiThe full length of HcCA3 cDNA was obtained by RT-PCR and RACE and the genbank number is KX181539.The full length of HcCA3 cDNA was 1628 bp,including 3’ untranslated regions(3’ UTR)of 511 bp,5’ UTR of 65 bp and open reading frame(ORF)of 1053 bp encoding 350 amino acids.The protein included a carbonic anhydrase domain,a small low-complexity regions,N-Terminal signal peptide with a 19 amino acid,4 glycosylation sites and 6 sites of phosphorylation,and without transmembrane structure.Multiple sequence alignment showed that the similarity of amion acid sequences among HcCA3 in H.cumingii and carbonic anhydrases of other species was from 24.15% to 46.61%.Meanwhile,these carbonic anhydrase amino acids sequences had three same Zn2+ site on the same position.It was deduced that the Zn2+ sites were the key to the function of HcCA3 protein.2.Real time fluorescence quantitative(qRT-PCR)analysis of HcCA3 genefrom H.CumingiiqRT-PCR showed that the HcCA3 cDNA was highly expressed in the mantle,whereas its expression was low in other tissues(foot,hepatopancreas,gill and intestines).The expression level of HcCA3 in the posterior mantle pallial(pMP)was the highest and that of the anterior mantle pallial(aMP)was the lowest.In purple mussel,the expression level of pMP was significantly higher compared with aMP,whereas no significant difference between the aMP and pMP in white mussel.The expression level of pMP,aMP and mantle center(MC)in purple mussel were significantly higher than that of white mussel.It was deducted that HcCA3 gene was involved in the formation of shell and nacre layer color.qRT-PCR detected the expression of HcCA3 cDNA in various tissues during the formation process of pear.The result showed that there are similar expression in the aMP,pMP and pear sac(PS),which decreased firstly and then increased,and the lowest was 24 h,96 h and 7 d respectively.The expression level of MC increased firstly and then decreased,and the highest was 3 h.We deduced that HcCA3 participated in the formation process of pear.The expression level of the foot,gill and intestines reached the highest at 28 d,12 h and 24 h respectively,whereas it was low at other times.Organisms was a complex system and needed the inter coordination of various tissues to accomplish some physiological process.3.The analysis of fluorescence in situ hybridization and western blot of HcCA3 from H.CumingiiFluorescence in situ hybridization showed that the hybridization signals were seen outer epidermal cell of the pMP,both outer and inner epidermal cell of outer fold and middle fold.It was deduced that HcCA3 gene participated in the formation process of periostracum layer,prismatic layers and nacre layer at the same time.Prokaryotic expression showed that one part of recombinant protein existed in the form of inclusion bodies,another part in the form of soluble protein.By constructing the recombinant vector,expression and purification of protein,animal immunization and antibody purification,HcCA3 polyclonal antibodies was obtained ultimately.Western blot detected the expression of HcCA3 protein in various tissues from H.cumingii.The results showed that HcCA3 protein expressed in the aMP,pMP and MC.It was similar with the result of qRT-PCR.It was speculate that it participated inthe bio-mineralization process of H.cumingii.4.The analysis of RNAi from H.cumingiiThree objective gene interfere chains were screened and the interference chain: siRNA-HcCA3-1 was used for formal experiment.At the same time,four negative control chain were screened,but no proper negative control chain was obtained because the expression level of various tissues showed different degrees of decreased significantly.After injecting interfere chain siRNA-HcCA3-1 cumulatively,the expression levels of aMP and pMP were knocked down at 12 d(28% and 30% of control group respectively);that of MC decreased at 12 d(84% of control group)and 18 d(76% of control group).It provided the basic materials for the study of HcCA3 function further.