The Identification Of Early Pre-parasitic Genes And The Cloning And Functional Analysis Of FMRF-amide Like Peptides (FLPs) From Soybean Cyst Nematode Heterodera Glycines

Soybean(Glycine max)is most important food and oil crop.The soybean cyst nematode(Heterodera glycines,SCN)is an obligatory plant parasite that causes enormous losses to soybean production each year.It is difficult to control this pathogen due to various races in soil and the cyst that can survive longtime.Although pesticides can effectively prevent soybean cyst nematodes,most have been restricted t highly toxic and residue.Most resistant sources of soybean only effect for the constant races and the resistance is easily broken with consistant growing same resistant soybean varieties in the field.Therefore,the application of these methods were limited.Currently,hot research spots are focuses on the interaction between nematode and host to explore safe,broad-spectrum and highly efficient nematicides.One key stage for parasitism is the recognition process,which involves in the allelopathic identification,locomotion adjustment,metabolism and immune regulation of the second-stage juveniles.The interruption at this stage can effectively prevent the infection of nematodes.Previous studies suggest that the early interaction between nematodes and plants was affected not only by the chemical signals released from plant roots or rhizosphere microorganisms but also nematode itself(e.g.pheromones,neuron peptides).However,little is known about early SCN-plant interaction and involved nematode genes.In addition,the family of FMRF-amide like peptides(FLPs)is conserved in nematodes and flp is a hot research spot to be utilized to develop new human medicine.Yet the knowledge of SCN flp genes was not clear.Based on these issues,this study contained the two aspects of research,(?)including identifying pre-parasitic genes involved in early nematode recognition to plant signals by RNA-seq,and(??)screening SCN flp genes and functionally characterizing these flp genes.The results were listed below.In this study,second stage of juveniles(J2s)of soybean cyst nematode were exposed to soybean roots in Pluronic F127 gel.Nematodes were collected at 3 and 24 h after the assay.Nematodes treated without plant in gel were collected as control.The differentially expressed genes among samples were analyzed through RNA-Seqtechnique.The identified genes were cloned using PCR.RNAi technologies and nematological methods were used to further confirm their potential functions.This study will provide theoretical basis for establishing new control strategy against nematodes.The results in this research are listed as follows:1.Through transcriptomic data analysis,a total of 56,947 Unigenes were obtained.Out of them 29,031 were annotated into different databases.Among 373 differentially expressed genes,236 genes are up-regulated,137 down-regulated genes.14 genes(cathepsin S-like cysteine proteinase,thiazole synthase Thi G,effectors,heat shock protein,Acireductone dioxygenase domain containing protein,metalloprotease m41 ftsh,nucleotide diphosphate kinase ndk)might be involved in parasitism and host finding.Of them,three genes(Ndk,Ftsh,and Thi G)were verified by q RT-PCR.Further,these potential candidate genes will be cloned and verified with RNAi.The understanding of the nematode-plant interaction will provide new control strategy.2.Thirteen candidate genes of FLPs were screened from transcriptome data,including Hg-flp-1,Hg-flp-3,Hg-flp-5,Hg-flp-6,Hg-flp-7,Hg-flp-11,Hg-flp-12,Hg-flp-13,Hg-flp-14,Hg-flp-16,Hg-flp-18,Hg-flp-22 and Hg-flp-27.All of 13 candidate genes were cloned,and putative H.glycines FLP-encoding gene homolog are compared with those in phylum nematode.Sequence alignment and phylogenies reveal 13 putative FLPs that are highly conserved among nematodes.The sequences of Hg-flp-1,Hg-flp-6,Hg-flp-7,Hg-flp-11,Hg-flp-12,Hg-flp-14,Hg-flp-16,Hg-flp-18 and Hg-flp-27 are highly conserved within the plant parasitic nematode species.Structure chacteristic of FLP in SCN indicated that mature FLP peptide contains three cleavage sites(R\K/KR),implying that FLPs precursors require post-translational modification for active function.Candidate gene expression levels between different developmental stages were confirmed by Real-time PCR.We found that all genes are maintained at high level in juveniles,speculated that these neuropeptide are related to recognition and parasitism.3.FLP-1 and FLP-11 were selected for further functional verification.In situ hybridization showed that Hg-flp-1 and Hg-flp-11 were expressed in ventral ganglion of nematodes.In vitro RNAi assay indicated that Hg-flp-1 expression was inhibitedand female index decreased,but Hg-flp-11 ds RNA did not affect infection and reproduction,which may due to functional redundancy of FLPs.We constructed transgenic hair root to express Hg-flp-1 and Hg-flp-11 respectively,the results of Hg-flp-11 were consistent with in vitro.After in vivo Hg-flp-1 interference,the number of females was significantly increased when compared to the control.4.Functional confirmation of Hg-flp-1 and Hg-flp-11 were conducted by the addition of exogenous synthesized FLP-1 and FLP-11 peptides.The locomotion ability of juvenile was found to be enhanced with FLP-1(a\b\c\d\e)and FLP-11(a\b)subunit treatments with dosage effect,which might be caused by excessive excitability of nematodes.The reproductive ability of SCN was enhanced with treatments of FLP-1e(100 ?M,10 ?M,1 ?M),10 ?M FLP-11 a and 100 ?M FLP-11 b,suggesting that treatments with FLP-1e,FLP-11 a,FLP-11 b can stimulate J2 s and help formation of feeding sites.Application of FLP-1b\c\d\e and FLP-11a/b didn't affect J2 infection,but the number around root tip of FLP-1b/c/d/e treatment was reduced.The reason might be that J2 s become more active after synthesized FLP peptides.Meanwhile,FLP-11a/b could increase the mobility and reproductive capacity of nematodes but with dosage effect,which might depend on the efficiency of nematode absorption of exogenous peptides by body surface.Above all,differentially expressed genes identified from the above transcriptome data help us increase the understanding of nematode-host interaction,and provide more information for functional analysis of these candidate genes.The cloning of 13Hg-flp genes and the functional study of Hg-flp-1 and Hg-flp-11 provides scientific basis for uncovering the role of FLPs in SCN.Our results showed that Hg-flp-1regulate not only the nematode recognition,but also post-parasitism.Additionally,this study will provides a new direction for developing new control strategies based on FLPs.