Genome-wide Identfication And Characterization Of Novel LncRNAs And Extracellular Vesicle Preliminary Study In Strongyloides Stercoralis

Strongyloides stercoralis is a type of zoonotic parasite that prevalent in the subtropics.S.stercoralis has a complex gene expression regulation system with a complex life cycle,including free living stages and parasitic stages.The infective third-stage larvae(iL3)is the only larvae with the ability to invade the host in the free-living stage of S.stercoralis.The process of developing iL3 into a parasitic female(pF)is not only a simple development process,but also a very important transformation process from the free-living stage to the parasitic stage.Studying the developmental regulation mechanisms between these two periods and the invasion mechanism of iL3 plays an important role in the search for drug targets and vaccine candidate molecules.In recent years,long non-coding RNA(lncRNA)has attracted much attention from researchers because of its important role in transcriptional regulation.Studies have confirmed that lncRNAs are involved in parasitic infection and the expression of virulence factors in the process of invading the host,which plays an important role in the successful parasitization of the parasite.And many studies have shown that parasites communicate with host by secreting extracellular vesicles to regulate host immunity.In this thesis,i mainly studied the expression of lncRNA and EVs of S.stercoralis to study the developmental regulation mechanism between iL3 and pF and obtained the following results: 1.Sequencing quality of S.strongyloidesTwo different developmental stages of S.stercoralis RNA samples were tested.After the RNA samples were tested,the samples were removed by rRNA,reverse transcription,first strand synthesis,second strand synthesis,end repair,3 plus A,and joints.The fragment selection,the U-chain degradation and the like are performed to construct a library.After the quality control of the library was qualified,more than 89% of the high-quality sequencing fragments were obtained in the worms using the transcriptome sequencing technology.The 90% for the infective third-stage larve(iL3)and 83% for the parasitic females(pF)of the sequencing fragments can be compared to the reference genome,respectively.On the genome,the quality of sequencing is high,which can reflect the transcriptome of S.stercoralis.2.lncRNA screeningLncRNA is a long-chain non-coding RNA of >200 bp in length,which is screened according to the characteristics of lncRNA.First,x,j,o,u,i and transcripts with a length of 200 bp or more were retained,and 7126 transcripts were screened.Secondly,transcripts with the expression levels FPKM>1,readscount>10,and the coding ability prediction software CPC(score<0)and CNCI(score<0)were all annotated as non-encoded transcripts,and finally the intersections were obtained with a higher degree of confidence.3.lncRNA feature analysisThe basic characteristics of the classified lncRNA were including,length distribution and number of exons were classified.According to its position on the genome,lncRNA can be divided into four categories,including 497 intergenic long-chain non-coding RNA,17 intron LncRNA,586 antisense lncRNA and 106 sense lncRNA.Most of these(41%)lncRNAs are antisense lncRNAs,which is consistent with most reported studies.Gene structure analysis revealed that most lncRNAs(79%)contained two exons,and only about 1% of the lncRNA exons had more than six exons.Compared to mRNA,lncRNA contains fewer exons.The length distribution of lncRNA ranged from 200 nt to over 3000 nt,of which 61% lncRNA was less than 1500 nt,indicating that the majority of lncRNA has a sequence length shorter than the length of the protein-coding gene.4.lncRNA function analysis635 differential lncRNAs at two different developmental stages,of which 312 LncRNAs were down-regulated in infective third stage larve and 322 lncRNAs were upregulated in parasitic females.The function of lncRNA was analyzed by cis and trans.The function of KEGG database indicated that the target gene of lncRNA of S.stercoralis might be involved in many biological processes such as signal transduction,transcription,transport and metabolism.5.Validation of differentially regulated genesAccording to the results of lncRNA differential analysis,8 lncRNA genes in two periods were selected for fluorescence quantitative PCR experiments to analyze the expression differences of lncRNA in two different developmental stages.The results showed that the expression of 8 lncRNA were consistent with the expression of RNA-seq and bioinformatics,which proved the accuracy of the data analysis.This work laid the foundation for the future study of lncRNA molecular function of S.stercoralis.6.Identification results of exosome in stage iL3 of S.stercoralisThis study was to isolate and molecularly identify the infective third-stage larve extracellular vesicles of S.stercoralis.First,the larve of iL3 was collcted and the vitro culture conditions were established,and then collected the supernatant for 20-24 h culture were collcted,and the extracellular vesicles were isolated by ultracentrifugation.Transmission electron microscopy(TEM)was used to observe the extracellular vesicles of phosphotungstic acid negative staining.The typical cup-shaped vesicle structure with a diameter of about 100-200 nm was observed.Subsequently,the total protein of extracellular vesicles of S.stercoralis were analyzed by shotgun-tandem mass spectrometry(Shotgun LC-MS/MS)and the Uniprot protein database were searched.A total of 17 proteins were identified,including the extracellular vesicles marker protein elongation factor.In summary,this thesis not only expands our understanding of lncRNA and extracellular vesicls of S.stercoralis,but also builds the theoretical basis for screening the molecular markers of disease in future.