Transcriptome Analysis Of Two Foliar Nematodes And Identifiction Of FAR Protein In Aphelenchoides Ritzemabosi

The rice white tip nematode,Aphelenchoides besseyi and the chrysanthemum foliar nematode,Aphelenchoides ritzemabosi belonging to Nematoda,Secernentea,Aphelenchida,Aphelenchoididae,Aphelenchoidinae,are migratory plant parasitic nematodes that infect the aboveground parts of plants,they are also known as foliar nematode.De novo transcriptome sequencing is a method of creating a transcriptome profiling of organisms or species through the high-throughput technologies without a reference genome,including library construction,sequencing and bioinformatics analysis.Fatty acid-and retinoid-binding(FAR)protein is a nematode-specific protein and it is involved in many important biological processes of nematodes.In order to understand the developmental and reproductive characteristics of A.besseyi and A.ritzemabosi at the molecular level,and the mechanism of interaction between these two nematodes and host plants,a transcriptome of two mixed-stage populations with different pathogenicity of A.besseyi and a transcriptome of mixed-stage population of A.ritzemabosi were sequenced on the Illumina HiSeq 2000 platform.Based on the transcriptome analysis of A.ritzemabosi,a FAR gene(Ar-far-1)was cloned and the function of FAR protein was studied.The main results of this study were listed as following:1.This study obtained the gene expression profiles of two mixed-stage populations with different pathogenicity of A.besseyi.A total of 24,896 and 27,690 unigenes corresponding to N10 and S24 populations were obtained from the high-quality clean reads mapped back to the contigs and paired-end reads,a total of 25,163 unigenes with a mean length of 1,192 bp and N50 of 1,739 bp were obtained after assembled two populations of A.besseyi.A total of 17,087 unigenes were annotated in six databases.20,011 codingsequences(CDs)were annotated after the A.besseyi unigenes were aligned to protein databases by BLASTX,6,969 unigenes were mapped to the protein database,3,042 unigenes cannot be aligned to any database were predicted by ESTScan to produce amino sequence.The result of A.besseyi unigenes orthologues present in seven selected completely sequenced genomes of nematode showed A.besseyi and Bursaphelenchus xylophilus had a closer phylogenetic relationship.In addition,the number of SSRs was 278 after A.besseyi unigenes was used as a reference to detect the SSR and SNP,the number of SNPs was 26,150 in the population of N10 and 5,311 in the population of S24.We had used a rigorous algorithm to identify differentially expressed genes(DEGs)between two populations of A.besseyi from N10 and S24.There were a total number of 1,696 unigenes which were significant differences,681 unigenes were up-regulated and 1,015 unigenes were down-regulated in the populations of N10 comparing to the populations of S24,respectively.Thirty DEGs were confirmed with qPCR and the results were consistent with the results of transcriptome sequencing.In all A.besseyi unigenes,1,198 unigenes matched to six families of CAZymes,23 types of genes in the A.besseyi transcriptome were predicted as orthologues of 7 of 8 gene families compared with C.elegans RNAi pathway.2.This study obtained the gene expression profiles of mixed-stage population of A.ritzemabosi.A total of 26,817 unigenes with a mean length of 1,032 bp were obtained after further clustering and assembly and 16,467 unigenes were annotated against in six databases.20,311 CDs were annotated after the A.ritzemabosi unigenes were aligned to protein databases by BLASTX,16,293 unigenes were mapped to the protein database,4,018 unigenes cannot be aligned to any database were predicted by ESTScan to produce amino sequence.The result of A.ritzemabosi unigenes orthologues present in four selected completely sequenced genomes of nematode showed A.ritzemabosi and B.xylophilus had a closer phylogenetic relationship.Approximately 495 of the A.ritzemabosi unigenes were predicted as SSRs and a total of 8,353 SNPs were detected.In total,1,199 unigenes were assigned to six classes of CAZymes,fewer transcripts were predicted as homologues of plant cell wall-degrading enzymes than fungal cell wall-degrading enzymes.The phylogenetic analysis of GH5,GH16,GH43 and GH45 proteins between A.ritzemabosi and other organisms showed A.ritzemabosi and other nematodes have a closer phylogeneticrelationship.In the A.ritzemabosi transcriptome,sixteen types of genes orthologues with seven classes of protein families involved in the RNAi pathway in C.elegans were predicted.3.This study analyzed and predicted the EST sequence of a FAR protein from transcriptome sequencing data of A.ritzemabosi.The full-length cDNA sequence of Ar-far-1 was obtained,which included a 546 bp of open reading frame(ORF).A 766 bp full-length DNA sequence of Ar-far-1 were amplified and obtained five exons and four introns.The deduced amino acid(aa)sequence of Ar-FAR-1 contains 181 aa with a molecular weight of 20.52 KDa and the theoretical isoelectric point of Ar-FAR-1 is 6.22.An18 aa signal peptide with a cleavage site between Ala18 and Glu19 was predicted and the phylogenetic tree showed that Ar-FAR-1 of A.ritzemabosi and Ab-FAR-1 of A.besseyi were grouped in the same branch of the phylogenetic tree and had a closer phylogenetic relationship.The purified Ar-FAR-1 was tested for lipid binding using fluorescent analogs DAUDA and retinol and showed that this protein has the ability to bind fatty acids and vitamin A.qPCR results showed that Ar-far-1 mRNA was expressed highest in females than other development stages of A.ritzemabosi.The result of in situ hybridization with showed that Ar-far-1 mRNA was expressed in the oesophageal gland,intestine,hypodermis,medium bulb and ovaries.This research provides comprehensive gene expression information of A.besseyi and A.ritzemabosi at the transcriptional level,which will facilitate the elucidation of the molecular mechanisms of A.besseyi and A.ritzemabosi.It also provides a new target to control A.besseyi and A.ritzemabosi.