Identification Of LncRNA And Their Function In The Oogenesis Of Migratory Locust,Locusta Migratoria

Locusta migratoria,a worldwide agricultural pest,is characterized by high fecunity and strong migratory ability.The outbreak of locust plagues cause huge losses to agricultural production.In adult females of L.migratoria,JH promotes Vg synthesis in the fat body,secretion into hemolymph and deposition into developing oocytes.Our previous studies have shown that JH can stimulate Vg synthesis and egg production by non-coding RNA?microRNA?in addition to inducing a series of cascaded genes.lncRNAs are a class of non-coding RNAs transcribed by RNA polymerase II or III with a length greater than 200 nt,which play a role in regulating physiological activities and development of animals by influencing chromatin status,transcription factor activity,alternative splicing of mRNA,competitive binding sites of miRNA.Although it has been shown that non-coding RNA as miRNA plays a role in the JH signaling pathway,whether lncRNA is related to the JH pathway and whether lncRNA is involved in JH-induced insect egg production remains unclear.Through high-throughput sequencing,we obtained the lncRNA transcriptome of female fat body at 0 PAE?12h within adult eclosion?,6 PAE?6 days post adult eclosion?,and treated with a JH analog?JHA?for 24 h.Through bioinformatic analysis,we found the following characteristics of locust lncRNAs:?1?lncRNAs were mainly derived from intergenic at 0 PAE,but distributed in exons at 6 PAE;?2?Compared to protein-coding genes,the expression level of lncRNAs was relatively lower,and most lncRNAs were single exon transcripts with a length around 1 kb;?3?The AU content of lncRNAs was significantly higher than that of protein-coding genes,suggesting that it may play a role in gene regulation.Compared to that at 0 PAE,208 lncRNAs were significantly up-regulated at 6 PAE,while 10304 lncRNAs were significantly down-regulated.After JHA treatment for 24 h,555 lncRNAs were up-regulated and 353 were down-regulated.Subseqeut qRT-PCR validation indicates that the expression of these lncRNAs were in response to JH.To explore the function of lncRNAs in JH signaling pathway,we focused on lncRNAs in the range of 100 kb near the JH receptor gene Met,its heterodimer Taiman,and an early JH response gene Kr-h1.Ten lncRNAs were identified,including 4 lncRNAs in the promoter and 5' UTR of Met,2 lncRNAs in the intron of Taiman,and 4 lncRNAs derived from the promoter of Kr-h1.Interestingly,these lncRNAs have a poly?A?and are localized in the nucleus,suggesting that their regulatory role in JH pathway as well as JH-dependent vitellogenesis and egg production.dsRNAs-mediated RNAi demonstrated that knockdown of Met-related TCONS₁26004 downregualted the transcription of Met mRNA precursor,leading to decreased levels of Met mRNA.Consequently,ovarian development and oocyte maturation were inhibited after TCONS₁26004 was depleted.When Met-related TCONS₁26084 was knocked down,the transcription of Met mRNA precursor was not affected,but Met mRNA levels significantly decreased.As a result,Vg expression significantly declined,accompanied by arrested ovarian development and oocyte maturation.Knockdown of Met-related intMet16 and Kr-h1-related TCONS₁22063 also resulted in blocked ovarian development.ChIRP and mass spectrometry showed that heat shock protein 70,ATP synthase-? subunit,endoplasmic reticulum protein bound to TCONS₁26004.The data suggest that TCONS₁26004 is likely to regulate vitellogenesis and egg production via energy metabolism,protein synthesis and folding.In summary,based on lncRNA transcriptome of locust fat body at 0 PAE vs 6 PAE,we found that the expression of some lncRNAs in the fat body of adult female locust was regulated by JH.By using RNAi,ChIRP and mass spectrometry analysis,we demosntrated that lncRNA TCONS₁26004 at the 5' end of Met plays an important role in vitellogenesis and egg maturation.This dissertation study has revealed that:?1?some lncRNAs are regulated by JH and involved in JH-dependent vitellogenesis and egg production;and?2?lncRNAs such as lncRNA TCONS₁26004 are produced along with Met transcription,which acts on Met expression and regulates expression,vitellogenesis and egg production.