Cloning and expression analysis of FMRFamide gene in Onchidium reevesii and inflammatory stimulaton on its gene expression

In this study, we investigated the expression of FMRFamide in vivo of Onchidium reevesii after inflammatory stimulus, and the molecular mechanism of FMRFamide polypeptide maintaining homeostasis. Based on a fragment of FMRFamide gene from the transcriptome of O. reevesii, we obtained the full-length cDNA of FMRFamide gene as 2 618 bp by RACE (Rapid-amplification of cDNA ends), including an open reading frame (ORF, 882 bp) which encoded a total of 293 amino acids. The results of the phylogenetic tree suggest that the FMRFamide gene of O. reevesii was most closely related to that of Lymnaea stagnalis, which is consistent with traditional morphological classification. The qRT-PCR results indicate that FMRFamide mRNA was distributed in different tissues of O. reevesii, but the expression in ganglion was very significantly higher than that in skin, hemocyte, hepatopancreas, muscle, gonad and pleopod (P<0.01). Immunohistochemistry verified the consistency of the FMRFamide peptide with mRNA distribution in tissues. The inflammatory stimulation experiment shows that the mRNA expression levels of FMRFamide gene in ganglia, hepatopancreas, hemocyte and skin were significantly higher in the experimental group than in the control group after lipopolysaccharide (LPS) injection (P<0.05), reaching the maximum value at 12^th hour after stimulation. In conclusion, O. reevesii FMRFamide is mainly found in ganglia and is involved in collective immune regulation through neuroendocrine system, playing a significant role in maintaining the stability of tumefaciens in vivo under inflammatory stimuli.