大珠母贝珍珠质相关基因N36和N45的克隆与序列特征分析

Cloning and characterization of nacre-related genes in silver-lip pearl oyster Pinctada maxima

采用同源克隆从大珠母贝外套膜中扩增到2种编码N66类似蛋白的基因,分别命名为N36和N45。N36的开放阅读框为987 bp,编码329个氨基酸,推测分子量为35.7ku。N45的开放阅读框为1 164 bp,编码387个氨基酸,推测分子量为44.6 ku。二者都富含甘氨酸、天冬酰胺和天冬氨酸。N36蛋白不含信号肽,N45蛋白在N-端有一段22个氨基酸的信号肽。N36蛋白和N45蛋白分别有2个和1个糖基化位点,分别有13个和16个潜在的磷酸化位点,它们的二级结构都由α螺旋、β折叠和无规卷曲组成。系统进化分析显示N36和N45与N66进化关系最近,而与其他nacrein蛋白则距离较远。与合浦珠母贝的nacrein和大珠母贝的N66类似,N36和N45蛋白都包含2种功能结构域:1个α-碳酸酐酶结构域和1个Gly-Xaa-Asn(Xaa=Asp,Asn or Glu)重复结构域。推测N36和N45与珍珠层碳酸钙晶体的形成有关。

We have cloned two cDNAs that encode N66-like proteins in Pinctada maxima and are named N36 and N45,respectively.The open reading frame（ORF） of N36 was 987 bp long,encoding 329 amino acids with molecular mass estimated to be 35.7 kD while the ORF of N45 was 1 164 bp,encoding 387 amino acids with estimated molecular mass of 44.6 kD.Both proteins were rich in Gly,Asn and Asp residues.There was no signal peptide in N36 protein while the amino acid residues 1-22 in N45 showed a typical signal peptide.N36 and N45 contained 2 and 1 putative glycosylation sequences,9 and 16 putative phosphorylation sites,respectively.Their secondary structure consisted of α-helix,β-sheet and coil.Phylogenetic analysis indicated that N36 and N45 were more closely related to N66 than other nacrein-like proteins.Similar to nacrein in P.fucata and N66 in P.maxima,N36 and N45 proteins contained two functional domains： α-carbonic anhydrase domain and Gly-Xaa-Asn（Xaa = Asp,Asn or Glu） repeat domain,suggesting that both N36 and N45 are likely to participate in calcium carbonate crystal formation in nacreous layer.