Preliminary Study On Cholinergic Immune Regulatory System In Pinctada Fucata Martensii

The cholinergic immune regulatory system mediated by neurotransmitter acetylcholine play an important role in inhibiting the release of inflammatory cytokines which invovled in regulating the immune response of organisms. With our previous research based on the transcriptom database of Pinctada fucata martensii, expression of nicotinic acetylcholine receptors from haemocytes were significant increase after nucleus inserted surgery, therefore, indicating that cholinergic immune regulatory system was involve in the regulation of the immune response of pearl oyster after nucleus insertion. In this study, we selected the key genes of the cholinergic immune regulatory system through analyzing the genome and transcriptome database, and then these genes were cloned, the expression was detected in different tissues, their localization in the haemocytes was confirmed, and immune regulatory mechanism was analysed. The main results are as follows:(1) Cloning and sequence analysis of nicotinic acetylcholine receptor genes: The six nicotinic acetylcholine receptor genes Pmn AChRα1, PmnAChRα2, PmnAChRα3, PmnAChRα4, PmnAChRα5 and PmnAChRα6 were cloned from Pinctada fucata martensii, which encoded for protein of 465, 446, 445, 446, 445 and 438 amino acids respectively. All of them had four typical transmembrane structure domains(TM), a cysteine loop(Cys~loop) and a conservative extracellular ligand binding domain(LBD). The expression level detected by Real-time PCR showed that these six genes are distributed in different tissues with high expressed level in gill; In situ hybridization have demonstrated that all presented a specific expression in the different hemocytes, according to the classification criteria of hemolymph. We hypothesized that PmnAChRα6 presented a specific expression not only in small granulocytes but also in large granulocytes. However others are present with a strong positive signal in large granulocytes. The results indicated that a typical cholinergic immune regulatory system may exist in pearl oyster.(2) The analysis of immune regulatory mechanism of cholinergic system: The six nicotinic acetylcholine receptor genes had varying degrees of regulation during early(6 h, 12 h or 24 h) or late(18 d) phase after nucleus insertion in Pinctada fucata martensii; After lipopolysaccharide(LPS) stimulation, the expression levels of PmnAChRα1 increased sharply at 12 h, the remaining five genes showed highly expression level at 6 h; The downstream regulators of cholinergic signaling pathway: NF-KB, Ikk, and IL-17 showed a significant increase of expression at 12 h after nucleus insertion; after LPS stimulation, these three genes also expressed highly at 12 h, however, when the acetylcholine receptor activator Nicotine was injected in pearl oyster followed with LPS, the expression of NF-kB and Ikk were obviously inhibited at 12 h. Which illustrated that cholinergic system may be involved in immune response by regulating the NF-kB signaling pathway.(3) The analysis of tissue repair mechanism of cholinergic system: The exoression of BMP7, Wnt, Collagen I-α2, Collagen IV-1 and Collagen IV-2 all appeared a significant increase 18 d after nucleus insertion. After nicotine stimulation, BMP7 and Wnt genes showed highly expression level at 12 h and 24 h respectively; while Collagen I-α2, Collagen IV-1 and Collagen IV-2 genes level increased significantly at 6 h, the result indicated that cholinergic immune regulatory system may also be adjusted by BMP7, Wnt and collagen involved in the regulation of the tissue repair in organism.(4) The analysis of expression and regulation of acetylcholine receptor genes: Based on the regulatory mechanism between microRNAs and target genes, and the miRNA transcriptome data obtained in our previous research, the analysis of the target softwares RNAhybrid and microTar showed that mi R-873, miR-669 m, miR-4577, miR-103 a, miR-6753 and miR-516 b were respectively predicted as the candidate miRNAs of PmnAChRα1, PmnAChRα2, PmnAChRα3, PmnAChRα4, PmnAChR5 and PmnAChRα6; Dual luciferase analysis illustrated that the luciferase activity of the reporter containing 3’ UTR of PmnAChRα1 gene was significantly downregulated about 36.2% by miR-873; miR-669 m had no effect on PmnAChRα2; the luciferase activity of the reporter containing 3’ UTR of PmnAChRα3 gene was significantly downregulated about 29.6% by miR-4577; PmnAChRα4 was about 10.7% by miR-103a; PmnAChRα5 was about 27.3% by miR-6753; and PmnAChRα6 was about 57.3% by miR-6753. In summary, the result of this study can provide backgroud information for the further study of cholinergic immune regulatory mechanism in immune response in pearl oyster.