Cloing And Sequence Analysis Of Flp-14, Flp-21 From Radopholus Similis (the Burrowing Nematode)

The neuromuscular system control infection,feeding and reproduction of plant parasitic nematode, FMRFamide-like peptides(FLPs) play an important role in regulation and transmission of parasite neuromuscular system.FLPs signaling systems also are well-established targets for parasite control,so people study the neuropeptide signaling of nematode neuromuscular system's important ingredient in order to seek and develop new drug targets.FMRFamide-like peptides(FLPs) represent the largest family of neuropeptides known in invertebrates.In this research,the full length cDNA sequence of flp-14,flp-21 gene was obtained through RT-PCR and RACE from Radopholus similis designated Rs-flp-14,Rs-flp-21.Their amino acids sequences,copy number in genome,intron and expression patterns were analyzed.1.Full-length cDNA of flp-14 and flp-21 gene from the migratory plant-parasitic nematodes R.similis were obtained using RACE technique according to the EST of flp-14 and flp-21 in the GenBank designated Rs-flp-14(GenBank:FJ534855) and Rs-flp-21(GenBank:AW871675) respectively. Rs-flp-14 consisted of 569 bp in length,with a 357 bp ORF,encoding a 119 amino acids protein, the putative protein with the molecular weight 12.85 KD and pI 9.41.Sequence analysis showed that Rs-flp-14 coded two copies of the FLPs(2x KHEYLRFG),and it had a 27 amino acids long signal sequence at N terminal.Rs-flp-21 consisted of 567 bp in length,with a 297 bp ORF, encoding a 99 amino acids protein,the putative protein with the molecular weight 10.5 KD and pI 9.80.Sequence analysis showed that Rs-flp-21 coded one copy of the FLP(AKGSRMRF),and it had a 26 amino acids long signal sequence at N terminal.According to cluster analysis,As the closest homologues of Rs-FLP-14 and Rs-FLP-21 had been assigned to As-FLP-14 and Ce-FLP-21 respectively,The amino acid sequence of Rs-flp-14 showed significant similarity to As-FLP-14(64%).2.To obtain the introns of Rs-flp-14 and Rs-flp-21 gene,we performed a PCR amplification of the R. similis genomic DNA using a pair of the full-length Rs-flp-14,Rs-flp-21 cDNA primer.The result showed that Rs-flp-14 and Rs-flp-21 gene had three and two introns respectively.The Daucus carota and R.similis genomic DNA samples were digested with EcoRI,Hindâ…¢and Sacâ… , Southern blot was performed with a partial cDNA fragment labeled with DIG-High prime as a probe,the result indicated that Rs-flp-14 likely presents as single copy gene in genome.3.Expressed localisation using in situ hybridisation of digoxigenin-labelled cDNA probes to the Rs-flp-14 gene,micrographs showed that the sites of expression of Rs-flp-14 gene was in the circumpharyngeal nerve ring,which is the central of nervous system in nematodes,this indicates Rs-flp-14 is closely related to nervers systems.Understanding the sites of expressions is very important to study the functions of the gene.The Rs-flp-14,Rs-flp-21 gene structure characteristic will provide valuable information for a comprehensive understanding of the functions of these genes,and will lay the foundation for the gene function and provide theoretical basis for developing novel plant parasite control targets.