Isolation And Function Definition Of Flp-32&flp-27 Genes Of Bursaplelenchus Xylophilus

In this paper, two ESTs (Expression Sequence Tag) of the pine wood nematode Bursaphelenchus xylophilus were loaded from GenBank of NCBI, CJ988312 and CJ991966, belonged to FMRFamide-related peptides (flp). The CJ988312 and CJ991966 own the feature of theoretical FMRFamide-related peptides C-termini. The B. xylophilus was cultured on potato dextrose agar (PDA) plates that had been inoculated with Botrytis cinerea at 25℃. B. xylophilus worms were washed off PDA plates. Total RNA preparation was made by the way of TRizol reagent method, and reverse-transcribed using a cDNA synthesis kit after oligo (dT) priming. CJ988312 and CJ991966 were isolated from cDNA, and total cDNA sequence of CJ988312 with 648bp and 3’termini sequce of CJ991966, were cloned using RACE kits. Peptide deduced by cDNA sequence of CJ988312 had specific C-termini of FLP-32 of Caenorhabditis elegans, CJ991966 versus flp-27, using DNAMAN and Primerpremier5.0 softwares. CJ991966 had owned 3’termini sequence.In situ hybridisation technique was employed to detect the expression of Bx-flp-32 and Bx-flp-27, previously isolated from B.xylophilus, in whole-mount preparations of the all stages of this worm. Bx-flp-32, deduced it could encode the FMRFamide-related peptide (FaRP) AMRNSLVRFGamide, was expressed in only male worms’neurones associated with the circumpharyngeal nerve ring, the lumbar ganglia of the perianal nerve ring, the retrovesicular ganglion, ventral cord and maybe have something with gonad. Bx-flp-27 (deduced it could encode GGRMRFGamide) expression was localised to cell bodies of the perianal nerve ring and the cells of tails of both female and male worms.In order to detect the two flp genes’function, double-stranded RNA (dsRNA) was delivered to L4 larvae and adults worms by soaking, with 1% resorcinol as a stimulus. After suspensibly soaked 24h by rolled, the activity of worms was much lower than control worms, but not than gfp control worms. No gene-specific phenotypes were observed for Bx-flp-32gene. Worms’tails had spasm, which were treated by dsRNA of Bx-flp-27 gene. DsRNA is poisonous, which can cause stiffness, the rise in death rates, the depression of genes’normal expression, but no effection on the worms’offspring.