The Cloning And Functional Study Of Collagen â…¥ (COLâ…¥) Genes Of Pinctadafucata Martensii

Type Ⅵ collagen(COLⅥ) is a vital member of the collagen family and it is ubiquitously expressed throughout all ECMs in animals. It plays an important rolein bone formation of vertebrates. In this study, to investigate the function of COLⅥ in shell formation of Pinctada fucata martensii, six selected COLⅥs belong to collagen Ⅵ super famly, which have high content in shell, based onprevious analysis of the shell matrix protein datebase of Pinctada fucata martensii. The polymerase chain reaction and rapid amplification of cDNA ends technology was used to clone the full-length sequences of COLⅥ genes, Real-time PCR was used to detect the tissue-specific expression of these genes, then detected the express location of these genes by situ hybridization. RNA interference was used to investigate the function of COLⅥ in shell formation, and expess it in Escherichia coli to obtained the recombinant protein.1. In this study, the full-length of PmCOLⅥA4-1, PmCOLⅥA6-1 and PmCOLⅥA6-2 were obtained. The total length of PmCOLⅥA4-1gene was 3136 bp, including 2841 bp of the open reading frame(ORF) which encoding 946 amino acids, a 5’UTR of 93 bp and a 3’UTR of 202 bp. The total length of PmCOLⅥA6-1 gene was 2100 bp, including 1875 bp of the open reading frame(ORF) which encoding 624 amino acids, a 5’UTR of 56 bp and a 3’UTR of 169 bp. And the total length of PmCOLⅥA6-2 gene was 2192 bp, including 1941 bp of the open reading frame(ORF) which encoding 646 amino acids, a 5’UTR of 41 bp and a 3’UTR of 210 bp. And verify the open reading frames of PmCOLⅥA3, PmCOLⅥA4-2 and PmCOLⅥA5 from transcriptomeo of Pinctada fucata martensii. It is collagen domain that is lost in the six COLⅥ genes, but the vWA domain show stronger conservation.2. The pearl sac,the centeral and pallial zone are the organ forming nacreous layer. PmCOLⅥA3, PmCOLⅥA6-1 and PmCOLⅥA6-2 have the highest expression in peal sac(P<0.05), PmCOLⅥA4-1 and PmCOLⅥA-2 have highest expression in mantal center(P<0.05), and PmCOLⅥA5 have higher expression in peal sac and mantle edge(P<0.05), according the result of Real-Time PCR. In situ hybridization experiment, we found that mRNA of PmCOLⅥA4-1, PmCOLⅥA6-1 and PmCOLⅥA6-2 intensively expressed in the outer of mantle center epithelium. It suggest that COLVLs may be related to the nacreous layers formation.3. We respectively injected the six synthesised ds RNA of COLⅥ into the adductor muscle. After silencing of the PmCOLⅥA3、PmCOLⅥA4-1、PmCOLⅥA4-2、PmCOLⅥA5、PmCOLⅥA6-1 and PmCOLⅥA6-2 by injecting the dsRNA, the surfacestructure of nacreous layers appear distortedbut the prismatic layers are nomal. It is evidenced that COLⅥs are the matrix protein of shell which regulate the formation of nacre.4. The PmCOLⅥA4-1 was cloned into the expression vector pET30 a and successfully express the vWA domain of PmCOLⅥA4-1. To obtain large guantity recombinant PmCOLⅥA4-1protein, it was induced at 15℃ overnight with 0.5mM IPTG. About 3.5mg fusion PmCOLⅥA4-1protein was obtained from inclusion body extract after washing the Ni-IDA column. The recombinant protein was detected with the molecular weight of 20 kDa by Western blotting, which is corresponded to the theoretic weight of 20.9 kDa.In conclusion, PmCOLⅥA3、PmCOLⅥA4-1、PmCOLⅥA4-2、PmCOLⅥA5、PmCOLⅥA6-1 and PmCOLⅥA6-2 are the matrix protein of shell, which regulate the formation of nacre in Pinctada fucata martensii. The study is the first to confirm that COLⅥ protein related to the formation of shell in mollusks, which enrich the datebase of the nacreous layer-special matrix protein.