瘤背石磺FMRFamide基因的克隆、分析及其在炎症刺激下的表达变化

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

该文研究了瘤背石磺(Onchidium reevesii)在炎症刺激后体内FMRFamide基因的表达水平变化,以及FMRFamide多肽保持其自身稳态的分子机制。以瘤背石磺转录组中FMRFamide基因片段为基础,通过RACE(Rapid-amplification of cDNA ends)技术克隆得到该基因cDNA的全长为2618 bp,开放阅读框(Open reading frame,ORF)为882 bp,编码293个氨基酸,系统进化树显示该基因与静水椎实螺(Lymnaea stagnalis)FMRFamide基因的亲缘关系最近,这和传统形态学分类相吻合。实时荧光定量结果显示FMRFamide基因在瘤背石磺的不同组织中均有表达,但在神经节部位的相对表达量极显著高于皮肤、腹足、肝胰腺、血细胞、性腺和肌肉组织(P<0.01)。免疫组织化学验证了FMRFamide多肽在组织中与mRNA分布的一致性。炎症刺激实验结果表明,注射脂多糖(LPS)后,实验组神经节、肝胰腺、血细胞和皮肤中FMRFamide基因的mRNA表达水平显著高于对照组(P<0.05),且在刺激后第12小时相对表达量达到最高。综上,瘤背石磺FMRFamide主要存在于神经节中,通过神经内分泌系统参与肌体的免疫调节,对炎症刺激下的瘤背石磺保持体内稳态具有重大作用。

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 2618 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 12th 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.