Cloning And Expressed Localisation Using In Situ Hybridization Of FMRFamide-like Peptides Encoding Genes Mi-flp-16/27/31 Of Meloidogyne Incognita

FMFRamide-like peptides (FLPs) occur widely across the phylum Nematoda. FLP neurotransmission is central of plant nematode biology. FLPs appear integral to how these parasites move, feed, sense their environment and perhaps establish parasitism within the plant. Better understanding of the functions of FLPs is very important to prevent plant-parasitic nematodes. The study cloned the flp genes Mi-flp-16,Mi-flp-27,Mi-flp-31, from southern root-knot nematodes (Meloidogyne incognita) juvenile 2 stage using the RACE technique and employed an in situ hybridization technique to detect the expression of the Mi-flp genes ,which shed light on understanding the function and application of FLPs .The main results were as follows:Target genes were selected by bioinformatics methods .Three transcripts of FLP encoding genes,designated Mi-flp-16,Mi-flp-27,Mi-flp-31,were characerised using RACE.In terms of the full-length cDNA , Mi-flp-16 was 690 bp long and coded two copies of the FLP,(×2 AQTFVRF,GQTFVRF) , containing an open reading frame of 522 bp, which predicted to encode a protein of 174 amino acids with calculated molecular weight of 20.7 kDa and pI of 9.45, and the GenBank number is EU549831. The same datum of Mi-flp-27 and Mi-flp-31 were respecitively 429bp, 279bp, 93, 10.5kDa ,9,80 , AW871075 and 358bp, 267bp, 89, 10.5kDa , 9.51, BM882182. Each of the two genes coded one copy of the FLP, respecitively AKESRMRFamide and LYRPRGPPRF.All of the three genes which were cloned in this research were novel to the parasitic plant nematodes,moreover,the full length cDNA of Mi-flp-31 was the first time to report in the phylum Nematoda .Expressed Localisation using in situ hybridisation of digoxigenin-labelled DNA probes to the three genes, micrographs showed that the sites of expression of three genes were all in the circumpharyngeal nerve ring,which was the central of nervous system in nematodes. The study suggests that FLPs play an important role in the nervous system of nematodes, Understanding the sites of expressions is very important to study the functions of the genes,and making clear the functions of the genes will support the idea to find out the novel approach to control of plant parasitic nematodes.