Cloning and expression analysis of NatF gene from Pinctada fucata martensii

【Objective】 By exploring the expression pattern of different tissues, different development stages, and pathogen-related molecular model(PAMPs) stimulation of Pinctada fucata martensii would provide a theoretical basis for the follow-up study of the N acetyltransferase 60(NatF) gene function and the molecular mechanism of response to stimulation.【Method】 The full length of NatF from P. fucata martensii(PmNatF) cDNA was obtained using rapid amplification of cDNA ends(RACE) technology. Bioinformatics analysis was carried out by online softwares such as ProtScale, ProtParam, PSITE-Search, SignalP 4.1, SMART, and Cell-PLoc 2.0 Package. And real-time fluorescence quantitative PCR (RT-PCR) was used to detect the expression of different tissues, different developmental stages, and performance after being stimulated by PAMPs of PmNatF.【Result】 The results showed that the full length of PmNatF cDNA was 1142 bp, containing a 5' end non coding region(5'-UTR) of 181 bp, a 3' end noncoding region(3'-UTR) of 268 bp and an open reading frame(ORF) of 693 bp which encoded 230 amino acids. It was predicted that its molecular weight of PmNatF protein was about 26.64 kD and its theoretical isoelectric point(pI) was 8.54.The overall average hydrophilicity coefficient was -0.068, which was an unstable hydrophilic protein. Analysis of deduced amino acids showed that it had no signal peptide and transmembrane domain, and contained an acetyltransf_1 domain, and subcellular localization in the cytoplasm. In the secondary structure of PmNatF protein, α-helix accounted for 36.52%, β-turn accounted for 6.96%, extended chain accounted for 22.91%, and random coil accounted for 33.91%. Its three-dimensional structure was similar to that of Pacific oyster(Crassostrea gigas) NatF protein. The amino acid sequence of PmNatF was highly conservative among species, among which the amino acid sequence similarity with the NatF of American oyster(C. virginica) and European scallop (Pecten maximus) were high, 76.52% and 75.22%, respectively. RT-PCR showed that PmNatF was expressed in all the tested tissues, with the highest expression in gonads, and the expression was also found in different developmental stages, with highest expression in the egg stage and lowest in trochophore stage. After stimulated by lipopolysaccharide(LPS), the relative expression reached the highest level at 24 h;under peptidoglycan(PGN) stimulation, PmNatF was greatly up-regulated, peaked at 6 h;under PolyI:C stimulation, the relative expression reached the highest at 72 h.【Conclusion】 The PmNatF gene is highly conserved and expresses in various tissues and in different developmental stages, especially the highly expresses in gonads and egg stage. It is differentially expressed in the gill tissue after stimulation with PAMPs, indicating that the gene may be involved in the process of in the division and maturation of germ cells and its immune response of P. fucata martensii.