| Long non-coding RNA(lncRNA)participates in many crucial biological processes,such as epigenetics modification,transcription regulation,post-transcription regulation,cell differentiation,immune response,and protein degradation.As a class of non-coding RNA,the length of lncRNA is longer than 200 nucleotides(nt).Recently,the identification of lncRNA is widely conducted in many species with the rapid development of sequencing technology and bioinformatics analysis.Studies focusing on lncRNA have become the hot topic in life science.lncRNA is widely studied in mammals,while there is less information in insects.A lot of studies show that lncRNA exhibits significant tissue-specific expression,and testis has the most proportion of specifically expressed lncRNAs,of which the functional analyses are relatively abundant.The melon fly,Zeugodacus cucurbitae(Coquillett),belonging to Tephritidae Diptera,is regarded as one of the most destructive agricultural pests.Z.cucurbitae is widely distributed in subtropical,tropical,and temperate regions of the world,and mainly damages cucurbitaceous plants,including cucumber,bitter gourd,pumpkin,watermelon,and papaya.By far,there is little information about lncRNAs in Z.cucurbitae.Thus,the identification of lncRNAs and further functional studies on the tissue-specific lncRNAs of Z.cucurbitae appear to be very important.Collectively,multiple transcriptome datasets were obtained from different tissues of female and male Z.cucurbitae using high-throughput sequencing.The lncRNAs were systematically identified in Z.cucurbitae using bioinformatics methods.Then a series of techniques were comprehensively utilized,including quantitative realtime polymerase chain reaction(RT-qPCR),fluorescent in situ hybridization(FISH),RNA interference(RNAi),target gene prediction,dual-luciferase reporter assay,to discover the physiological functions of vital testis-specific lncRNAs.The main results were as follows:1 Construction of RNA-seq libraries from different tissues of Z.cucurbitae and identification of lncRNADifferent tissues were separately collected from female and male Z.cucurbitae adults,including midgut,Malpighian tubules,fat body,ovary and testis.RNA-seq data were obtained using high-throughput sequencing technology.As a result,a total of 487.83 Gb clean reads were generated,GC content ranged from 40.10% to 46.69%,and Q30 values were all above 93.00%.Lnc RNAs were identified from the whole transcriptome data using a computational pipeline.A total of 22,159 candidate lncRNAs were firstly filtered,and then 3,124 lncRNAs were obtained after deleting the transcripts with extremely low FPKM values(FPKM < 1)in all tissues.Each tissue of female and male Z.cucurbitae had a certain number of lncRNAs.In female and male Z.cucurbitae,the most abundant lncRNAs existed in Malpighian tubules and fat body,respectively.Features and characterizations of Z.cucurbitae lncRNAs were analyzed,and the long intergenic non-coding RNA(linc RNA)had the most abundant amount in four types of lncRNA.Most of Z.cucurbitae lncRNAs had two exons and one isoform.2 Identification and analysis of tissue-specific lncRNAs in Z.cucurbitaeA total of 1,554 differentially expressed lncRNAs(DELs)were obtained from differential expression analyses of transcriptome data.Among female tissues,Malpighian tubules vs.ovary had the largest number of DELs,among which 151 DELs showed higher expression in ovary and 103 DELs showed higher expression in Malpighian tubules.Among male tissues,the largest number of DELs were found in Malpighian tubules vs.testis,of which 604 DELs showed higher expression in testis and 202 DELs showed higher expression in Malpighian tubules.In the differential expression analysis between similar tissues of female and male Z.cucurbitae,ovary vs.testis harbored the largest number of DELs,of which 472 DELs showed higher expression in testis and 151 DELs showed higher expression in ovary.The hierarchical clustering of DELs was generated in a heatmap using FPKM value,the results showed that testis owned more highly expressed DELs,compared to other tissues.Expression profiles of the randomly selected DELs were validated by RT-qPCR,which showed a consistency.Several highly enriched pathways were found in functional annotation of cis-regulated targets of DELs,suggesting essential functions of these lncRNAs.Tissue-specific lncRNAs were screened under the stricter criteria of ratio ≥ 10 based on the differential expression analyses.As a result,a total of 8,5,9,and 42 specifically highly expressed lncRNAs were screened in midgut,Malpighian tubules,fat body,and ovary of female Z.cucurbitae;in male Z.cucurbitae,a total of 8,7,21,and 364 specifically highly expressed lncRNAs were screened in midgut,Malpighian tubules,fat body,and testis.3 Testis-specific lncRNA—lnc94638 mediates the fertility of Z.cucurbitaeExpression profiles of the top 10 highly expressed testis-specific lncRNAs in different developmental stages of Z.cucurbitae were investigated by RT-qPCR,the results showed that they were highly expressed in the pupa and male adult,while hardly expressed in other stages.Localization of lnc94638,the most highly expressed testisspecific lncRNA,was captured at the region close to the vas deferens in Z.cucurbitae testis using FISH assay,the transformation of spermatids to mature spermatozoa took place in this region.Thus,we hypothesized lnc94638 might participate in the spermatogenesis of Z.cucurbitae.RNAi-mediated knockdown of lnc94638 resulted in reduction of spermatozoa number of Z.cucurbitae and hatchability of the eggs,demonstrated that lnc94638 regulated spermatozoa number and affected the fertility of male Z.cucurbitae.4 lnc94641 acts as “molecular sponge” adsorbing miR-957-3p to regulate the fertility of Z.cucurbitaeFunctional analysis was undertaken on lnc94641,the testis-specific lncRNA with the second abundant expression.RNAi-mediated knockdown of lnc94641 caused the reduction of spermatozoa number and hatchability of the eggs.The subcellular distribution analysis indicated that lnc94641 was mainly distributed in the cytoplasm of Z.cucurbitae cell,suggesting the mediation in “competing endogenous RNA,(ceRNA)”regulation.In this molecular mechanism,lnc94641 may serve as a “molecular sponge” to adsorb miR-957-3p,then play its physiological function.The small RNA libraries of Z.cucurbitae tissues were further analyzed.A total of2,380 miRNAs were identified in Z.cucurbitae.Four softwares were utilized to predict the targeted miRNA of lnc94641,and two common miRNAs were predicted by four softwares,namely miR-957-3p and miR-5735-3p.Only the over-expression of miR-957-3p in vivo reduced expression of lnc94641,indicating lnc94641 may bind tomiR-957-3p.The result of dual-luciferase reporter assay was performed,compared with mimic-NC,the relative luciferase activity in co-transfection of mimic-miR-957-3p and the recombinant plasmid with lnc94641 binding sites to HEK293 T cells significantly decreased,which demonstrated the combination of lnc94641 and miR-957-3p.The result of dual-luciferase reporter assay using mutation of miR-957-3p binding site in recombinant plasmid confirmed the targeted region of miR-957-3p and lnc94641.Colocalization of lnc94641 and miR-957-3p showed that there were many co-signals in the region close to the vas deferens in Z.cucurbitae testis via FISH assay.This co-location provided a visual clue of their interaction in testis.Over-expression of miR-957-3p reduced the spermatozoa number and hatchability of eggs,indicated that miR-957-3p bind to lnc94641,and then regulated the spermatozoa number and fertility of Z.cucurbitae.In summary,this dissertation was conducted based on RNA-seq data of Z.cucurbitae tissues,lncRNAs were systematically identified from the RNA-seq data;tissue-specific lncRNAs were screened based on differential expression analyses.Two testis-specific lncRNAs,lnc94638 and lnc94641,were proved to affect spermatogenesis of Z.cucurbitae.Furthermore,lnc94641 acted as a “molecular sponge” to bind to miR-957-3p then play its physiological function.The results enriched lncRNA catalogs in insect,and promoted the study of ceRNA mechanism in insects,and also provided new insight into genetic-based sterile insect technique. |
