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类FMRF酰胺多肽(FMR Famide-like peptides,FLPs)在控制寄生性线虫侵染、取食和繁殖过程中起着重要作用。在防治线虫时,FLPs信号系统经常成为药物的作用靶标。本研究以香蕉相似穿孔线虫(Radopholus similis)为材料,用RT-PCR和RACE方法,获得了类FMRF酰胺多肽flp-14基因的cDNA全长序列,命名为Rs-flp-14。此基因cDNA序列全长为569bp,包括一个357bp的开放阅读框(0RF),编码含119个氨基酸的蛋白,分子量与等电点分别为12.85KD和9.41。序列分析表明Rs-flp-14编码2个FLP肽(2xKHEYLRFG),N端具有27个氨基酸残基组成的信号肽。Southern杂交显示其为单拷贝基因。cDNA与基因组DNA重叠分析表明基因包含三个内含子,四个外显子。聚类分析表明该基因与猪蛔虫的As-flp-14同源,相似度最高(64%)。原位杂交结果显示Rs-flp-14基因表达位置在线虫的神经环部位。本研究结果将为研究该基因功能奠定基础,并为以FLPs为作用靶标的防治线虫药物提供理论依据。
FMR Famide-like peptides (FLPs) play an important role in the control of parasitic nematode infection, feeding and reproduction. In the control of nematodes, FLPs signaling systems often become the target of drugs. In this study, the full-length cDNA sequence of flp-14 gene of FMRF amide was obtained by RT-PCR and RACE using Radopholus similis as Rs-flp-14. The full-length cDNA sequence of this gene was 569bp, including a 357bp open reading frame (ORF) encoding a protein of 119 amino acids with molecular weights and isoelectric points of 12.85KD and 9.41, respectively. Sequence analysis showed that Rs-flp-14 encodes two FLP peptides (2xKHEYLRFG) and has a signal peptide consisting of 27 amino acid residues at the N-terminus. Southern blot shows that it is a single copy gene. Overlapping analysis of cDNA with genomic DNA showed that the gene contains three introns and four exons. Cluster analysis showed that the gene was homologous to As-flp-14 of Ascaris suum as the highest similarity (64%). In situ hybridization results showed that the Rs-flp-14 gene was expressed in the nematode neuro-annulus. The results of this study will lay the foundation for the study of gene function and provide a theoretical basis for the prevention and treatment of nematodes with FLPs as the target.