香蕉相似穿孔线虫Rs-flp-14基因克隆与特征分析

Cloning and sequence analysis of Rs-flp-14 gene from Radopholus similis

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。

FMRFamide-like peptides （FLPs） play an important role in infection, feeding and propagation of parasitic nematodes. FLPs signaling systems are also well-established targets for nematode control. In this research, the full length cDNA sequence of Rs-flp-14 was obtained through RT-PCR and RACE from Radopholus similis designated Rs-flp-14 （ Gene Bank： FJ534855 ）. It consisted of 569 bp in length, with a 357 bp ORF encoding a 119 amino acids protein. The putative protein had the molecular weight of 12.85 KD and a pI of 9.41. Sequence analysis showed that Rs-flp-14 coded two copies of FLPs （2x KHEYLRFG）, with a 27 amino acids signal sequence at the N terminus. Southern blot analysis indicated that there was single copy of Rs-flp- 14 within R.similis genome. Genomic analysis suggested that Rs-flp-14 contained three introns and four extrons. According to the cluster analysis, the gene exhibited homology to As-tip-14 with the highest extent （64%）. In situ hybridization, it was showed Rs-flp-14 was expressed in neurons associated with the nerve ring. This study will provide the foundation and theoretical basis for research on the gene function and developing novel plant parasite control targets.