Zhang Rui, Qin Mengting, Shi Jie, Tan Lu, Xu Jiamin, Tian Zhenyan, Wu Yuhui, Li Yuxuan, Li Yitian, Wang Ning
1School of Medicine, Jiangsu University, Zhenjiang City, 212013 China.
2School of Food and Biological Engineering, Jiangsu University, Zhenjiang City, 212013 China.
3 Biotech. 2018 Apr;8(4):214. doi: 10.1007/s13205-018-1233-z. Epub 2018 Apr 9.
In the present study, the Pif gene of the freshwater pearl aquaculture mussel, (HcPif) was successfully cloned and functionally characterized. The full sequence of HcPif gene consists of 3415 base pairs, which putatively encode two proteins, HcPif90 and HcPif80. A sequence analysis revealed that HcPif contained a von Willebrand factor type A domain and a chitin-binding domain, and shared many functional residues with other Pif homologues. A highly conserved sequence, FKGLDEIELML, at the C-terminus of Pif80s was identified as the key functional site. The corresponding peptide fragment markedly modified the morphology of calcite crystallites in CaCO crystallization assay and might play an essential role in the interactive binding between HcPif80 and CaCO. Moreover, real-time PCR results showed that HcPif gene was dominantly expressed in the pearl secreting tissues and its expression changed in response to the different development status of the pearl sac during pearl aquaculture. The gene expression of HcPif was maximum 7 days after mantle grafting and declined to about the control level on day 30. Our in vitro and in vivo experimental data indicated that HcPif gene possessed the inherent characteristics of a nacre formation gene and its expression might faithfully reflect the pearl secretion status of the pearl mussels examined. Our findings may extend the understanding of the biomineralization mechanism of nacre formation and provide a potential biomarker for pearl farming.
在本研究中，成功克隆了淡水珍珠养殖贻贝的Pif基因（HcPif）并对其进行了功能表征。HcPif基因的全长序列由3415个碱基对组成，推测编码两种蛋白质，即HcPif90和HcPif80。序列分析表明，HcPif包含一个血管性血友病因子A结构域和一个几丁质结合结构域，并与其他Pif同源物共享许多功能残基。在Pif80s的C末端鉴定出一个高度保守的序列FKGLDEIELML，作为关键功能位点。在碳酸钙结晶试验中，相应的肽片段显著改变了方解石微晶的形态，可能在HcPif80与碳酸钙的相互结合中起重要作用。此外，实时定量PCR结果表明，HcPif基因在珍珠分泌组织中占主导表达，并且在珍珠养殖过程中，其表达随珍珠囊不同发育状态而变化。HcPif的基因表达在套膜移植后7天达到最大值，并在第30天降至对照水平左右。我们的体外和体内实验数据表明，HcPif基因具有珍珠质形成基因的固有特征，其表达可能忠实地反映所检测珍珠贻贝的珍珠分泌状态。我们的研究结果可能会扩展对珍珠质形成生物矿化机制的理解，并为珍珠养殖提供一个潜在的生物标志物。
