Cloning And Characterization Of Flps Gene From Pine Wood Nematode Bursaphelenchus Xylophilus And A Rs-eng-1 Gene From Burrowing Nematode Radopholus Similes

FMFRamide-like peptides(FLPs) occur widely across the phylum Nematoda and FLPs involved neurotransmission plays a central role in plant nematode biology.FLPs appear integral to how these parasites move,feed,sense their environment and perhaps establish parasitism within the plant.Therefore,extensive study of FLPs genes and their functions will greatly improve our understanding of the nematode parasitism and might lead to novel and efficient ways in plant-parasitic nematodes control,β-1, 4-endoglucanase is a large class of cellulases secreted by parasitic nematodes that can degrade cellulose to alter the structure and function of the plant cells.The encoding genes of these secretions,so called parasitism genes,have been thought to greatly enhance the parasitism of the nematodes in the plant.Our study isolated and analyzed the full-length cDNA of flp and ENG genes from two alien invasive plant-parasitic nematodes Bursaphelenchus xylophilus and Radopholus similis,respectively.The study provided possible targets for anti- plant-parasitic nematodes drugs.The main results were as follows:1.Based on the EST sequences of the target genes from the gene databases.9 full-length cDNA and 1 cDNA fragment of flp genes(Bx-flp-3b) from B.xylophilus were RACE amplified and analyzed.The 9 obtained flp genes are designated Bx-flp-1, Bx-flp-2,Bx-flp-3a,Bx-flp-6a,Bx-flp-6b,Bx-flp-6c,Bx-flp-6d,Bx-flp-12,Bx-flp-14, respectively,with Genebank access number EU026161,EU930826,EF422867, FJ151415,FJ151416,FJ151417,FJ151418,EF622046,respectively.According to the gene structure analysis,Bx-flp-1 was composed of four 4 exons and 3 introns;Bx-flp-3a was composed of 2 exons and 1 intron;Bx-flp-6 had two different editing mechanisms, one in middle and the other in the C terminus,resulting 4 different selectively edited isomeric products.The 5 flanking sequences of three genes were also obtained by Tail-PCR,with length of 857 bp,377 bp,and 1630 bp,respectively.2.Six flp genes from B.xylophilus were localized by in-stiu hybridization and the result revealed that Bx-flp-1 and Bx-flp-3a were expressed in the pharyngeal ring and the head;Bx-flp-2,Bx-flp-3b,Bx-flp-6a～d and Bx-flp-14 were expressed mainly in the tail;the expression patter of Bx-flp-12 was very complicated. 3.RNA interference(RNAi) experiments were performed to study the functions of the flp genes,dsRNA fragments were synthesized in vitro and the B.xylophilus nematodes were treated by soaking.Q-PCR was used to compare the differences between the treated and control groups.The results showed that the transcription level of the target genes with treatment was significantly reduced(p＜0.05%) to 22%,41% and 29%,respectively.The microscopic observations showed that the mobility of the interfered nematodes was remarkably decreased,appearing to be in rigor and apparent death.However,after incubation at 40℃for 1 h,the mobility could be recovered.The reproductive capability assay demonstrated that the interference of Bx-flp-1,Bx-flp-3a and Bx-flp-6 could not inhibit the reproduction rate.4.The full length cDNA sequence ofβ-1,4-endoglucanase gene was obtained by RT-PCR and RACE from R.similes,named Rs-eng-1(GenBank:EU414839).It consisted of 1630 bp,with a 1404 bp ORF encoding a 467 amino acid protein.The putative protein,with theoretical molecular weight 48.22 kDa and pI 6.04,was classified as a member of cellulase(glycosyl hydrolase family 5),and had a 22 amino acids long singal sequence at N terminus and a high similarity to a bacterial type cellulose-binding domainâ…¡at C terminus.Southern blot analysis showed that there were mμLtiple copies of Rs-eng-1 within R.simile genome.In situ hybridization showed RS-ENG-1 was secreted from R.simile's esophageal gland cells.Genomic analysis suggested Rs-eng-1 contained six introns demarcated by 5'-GT...AG-3' in the splicing sites and phylogenic analysis suggested that Rs-eng-1 had a strong homology to Bacillus.subtilis and Erwina carotovora,which might be indicative of an ancient horizontal gene transfer.5.Prokaryotic expression system was tried first to express Rs-eng-1.Rs-eng-1 was sub-cloned into vector pET28a,pET42a and pGEX-6p-1,respectively,using EcoRâ… and Notâ… restriction sites.After transformed into E.coli BL21 component cells and induced by IPTG,no specific bands were observed on SDS-PAGE gels.A different expression vector pMAL-c2x/Rs-eng-1 was thus constructed using EcoRâ… and Pstâ… restriction sites and transformed into E.coli BL21 component cells.After induced by IPTG,it started to produce inclusion bodies based on SDS-PAGE analysis.The failure of the above trials suggested that Rs-eng-1 coμLd not be expressed in prokaryotic expression systems.Considering the eukaryotic origin of Rs-eng-1,a eukaryotic expression vector pPIC9K/Rs-eng-1 was constructed and transformed into yeast cells by electroporation.After induced by methanol,Rs-eng-1 had a large amount of extracellular expression and showed an expected apparent molecular size of 60 kD on SDS-PAGE gel.The expressed Rs-eng-1 also showed cellulase activity.6.The function of Rs-eng-1 was also analyzed by RNAi experiments.Q-PCR resμLts demonstrated that the mRNA abundance of Rs-eng-1 was significantly decreased,only 15%compared to the control.CellμLase activity assays showed that the average size of the decomposition circles of the treated nematodes was 17 cm, significantly different from that of 22 cm of the control nematodes.The reproductive capability assay suggested RNAi of Rs-eng-1 could defer the reproduction of R.similis and increase the proportion of the male nematodes.7.Plant expressing vector of Rs-eng-1 fragment was successfully constructed and applied to tomatoes transformation.We obtained many hygromycin-resistant transgenic plants expressing dsRNA by Agrobacterium tumefaciens EHA105 mediated transformation with plasmid pFGC5941-RSENG-RNAi.PCR analysis showed that transgenic plants could amplify the target gene fragment,and southern blot indicated that the Rs-eng-1 fragment was integrated into the genomes of the tomato plants. RT-PCR showed that the target gene was effectively expressed in tomato plants. Compared with the control group,the number of root knots in transgenic tomatoes was significantly less 25 dpi when J2 were inoculated to T₁ plants,suggesting that the transgenic plants exhibited strong resistance to J2.The method described here provided a new way for the control and prevention of plant-parasitic nematodes.