Identification Of LncRNA During Eclosion Of Sogatella Furcifera And Functional Study Of MSTRG.16087.1

Sogatella furcifera is a typical long-distance migratory rice pest that causes severe damage to rice crops.Long noncoding RNA（lncRNA）is a class of endogenous non-coding RNA with a transcript length of over 200 bp that regulate gene expression at both the transcriptional and post-transcriptional levels,and thus play an important role in insect development.Little research has been reported on the developmental transition of lncRNA in S.furcifera.In this thesis,the White-backed planthopper as a research target,the full-length transcriptome of lncRNA during nymph-to-adult developmental transition of S.furcifera was constructed.Through in-depth analysis and screening of sequencing data,the key lncRNA involved in the eclosion process of S.furcifera were identified.The spatial and temporal expression dynamics of the differential lncRNA in S.furcifera were analyzed by RT-q PCR,and the biological functions of the target gene MSTRG.16987.1 in the developmental molting were investigated by RNAi technology,and the co-expressed genes of MSTRG.16987.1 were predicted.The main results are as follows:1.Analysis of lncRNA transcriptome data during eclosion of S.furciferaBased on the full-length transcriptome of the nymph-to-adult developmental transition of S.furcifera,4649 lncRNAs were identified,classified into intergenic lncRNA,bidirectional lncRNA,intronic lncRNA,antisense lncRNA and sense lncRNAs,of which the number of intergenic lncRNA was the highest and intronic lncRNAs the lowest.Compared to m RNA,lncRNA transcripts were shorter in length and open reading frames,and the relative expression of genes was lower.Differential expression analysis revealed 795 lncRNA and 3543 m RNA differentially expressed genes during eclosion of S.furcifera.Trend analysis showed that the differential lncRNA and m RNA were clustered into eight expression dynamics involved in the molting process of S.furcifera and most of the differentially expressed genes clustered in significant clusters profile 1 and profile 6 with up-and down-regulated expression trends.Target gene predictions showed that 574 lncRNA were predicted to 2719（target）m RNA（cis（cis）lncRNA located within 10 kb upstream and downstream of gene:156trans（trans）lncRNA compared with m RNA correlation coefficients:2875 both cis and trans:156）for prior to ecdysis（PE）vs.during ecdysis（PE）,627 lncRNA were predicted to 2816（target）m RNA（cis:164 trans:2978 both cis and trans:163）for prior to ecdysis（PE）vs.after ecdysis（AE）,and 35 lncRNA were predicted to 51（target）m RNA（cis:1trans:52 both cis and trans:1）for during ecdysis（PE）vs.after ecdysis（AE）.GO functional enrichment results showed that cis and trans target genes were mainly focused on inorganic anion transmembrane transporter protein activity,protein binding involved in cell adhesion,structural composition of the stratum corneum and fatty acid synthase activity pathways.KEGG functional enrichment results showed that cis target genes were significantly enriched in pantothenate and coenzyme A biosynthesis,fatty acid degradation and protein digestion and absorption pathways;trans target genes were significantly enriched in metabolic pathways,amino and nucleotide sugar metabolism,fatty acid metabolic pathways and c AMP signaling pathways.In-depth analysis of the target gene annotation results revealed that MSTRG.16086.1,MSTRG.16087.1,MSTRG.19835.2,MSTRRG.23212.1,MSTRG.2447.1,MSTRG.8295.1 and MSTRRG.11199.1 genes were most likely to target the chitin synthesis and degradation pathway,which in turn is involved in regulating the nymph-to-adult developmental transition of S.furcifera.The lncRNA-m RNA interaction analysis showed that MSTRG.16086.1,MSTRG.16087.1,MSTRG.2447.1,MSTRG.11199.1 and cuticle protein genes,chitin synthase and ecdysone pathway with Pearson coefficients exceeding 0.98,which will be the focus of subsequent studies.2.Analysis of spatiotemporal expression patterns of lncRNAThe spatiotemporal expression characteristics of 12 differential lncRNA selected from trend analysis profile 1（down-regulated expression trend）,profile 6 and profile 7（up-regulated expression trend）were studied in S.furcifera using RT-q PCR.The results of the expression patterns at different developmental stages showed that the lncRNA of profile 6 and profile 7 were differentially expressed at all developmental stages of S.furcifera,among which MSTRG.4465.1,MSTRG.22069.1,MSTRG.19835.2 and MSTRG.22544.1 were significantly highly expressed in AE,MSTRG.23212.1 was significantly highly expressed at 3^rd-3d.The expression of profile 1 lncRNA was mostly concentrated in the 3rd and 4th instars nymph and showed a decreasing and then increasing trend during eclosion of S.furcifera,with the lowest expression at DE.The expression patterns in different tissues showed that the 12 lncRNA in profile 1,profile6 and profile 7 showed different expression patterns in the tissues of S.furcifera.Among which MSTRG.22544.1,MSTRG.19835.2 and MSTRG.23212.1 in profile 6 and profile7 were significantly expressed in integument,MSTRG.17223.1 gene was significantly expressed in integument of S.furcifera in profile 1.3.Functional analysis of MSTRG.16087.1 gene in S.furciferaRNAi technology was used to investigate the biological function of MSTRG.16087.1 during the nymph-to-adult developmental transition of S.furcifera.The results showed that the expression levels of the target gene were significantly down-regulated at 48 h and 72 h after the injection of ds MSTRG.16087.1 into 4^th-1d instar nymph of S.furcifera.Compared with the control group,and the silencing efficiency of the experimental groups was above 80%,indicating that the MSTRG.16087.1 gene was successfully suppressed.After the silencing of MSTRG.16087.1 gene,compared with the control group,the treated insects showed a lethal phenotype with the old epidermis not completely shed and adhering to the edge of the body,and the successfully fledged adult showed curled wings,and the survival and fledging rates were significantly reduced.These results suggest that the MSTRG.16087.1 gene performs an important biological function during the nymph-to-adult developmental transition of S.furcifera.In addition,the chitin deacetylase 4 gene Sf CDA4 was predicted to be the trans target gene of MSTRG.16087.1,and the RF Classifier and SVM Classifier values of both were greater than 0.5,indicating that the association is of research significance.In different developmental stage,MSTRG.16087.1 and Sf CDA4 showed the same expression trend from 1^st-1d to 3^rd-2d instar,and 4^th-2d to DE instar,with an opposite expression trend from 3^rd-2d to 4^th-2d instar.MSTRG.16087.1 showed differential expression with Sf CDA4 in different tissues.After silencing MSTRG.16087.1,Sf CDA4 transcript levels were significantly down-regulated to 77%at 48 h and up-regulated at 72 h.In summary,in this thesis,based on the full-length transcriptome of lncRNA and m RNA during the nymph-to-adult developmental transition of S.furcifera.The sequencing data were analyzed,screened and identified key lncRNA that may be involved in the eclosion processes S.furcifera,revealed potential pathways through which lncRNA may exercise regulatory functions by targeting chitin metabolic pathways.The expression patterns of 12 differentially expressed lncRNA in different developmental stages and different tissues of S.furcifera were analyzed.The biological functions of the MSTRG.16087.1 gene during the nymph-to-adult developmental transition of S.furcifera were clarified.The potential trans target genes co-expressed with MSTRG.16087.1 were predicted.The results enriched the basic research on lncRNA related to growth and development of S.furcifera,and laid the foundation for studying the regulatory relationship between lncRNA and target genes.