Identification And Regulating Role Study Of Long Non-coding RNA Related To The Fruit Development Of Cucumis Melo

Cucumis melo L.is a horticultural crop widely planted all over the world.Melon fruit is an important food resource for human and animal nutrition owing to its high vitamin,mineral,fibers,and antioxidants.It has been considered as an attractive Cucurbitaceae model plant for investigation of important biological processes,including fruit ripening,sex determination,and stress tolerance.Thus,the study on the regulation mechanism of melon fruit development has significant scientific value and application effect.Plant long non-coding RNAs(lncRNAs)are linked to biological processes such as gene silencing,flowering time regulation,abiotic stress response,and numerous other developmental pathways.However,the regulation mechanism of lncRNA on melon fruit development has not been reported yet.The transcriptome of Cucumis melo L.cv.Hetao fruit at four typical developmental stages was sequenced(stage G,R,C,and P).By doing this,an informative lncRNA library of melon fruit was constructed.Target genes of the lncRNAs and the regulatory relationship(cis-or trans-acting)were analyzed.The regulatory functions of lncRNAs to their target genes were predicted based on the GO and KEGG enrichment analysis.Focusing on the GO terms like response to auxin stimulus,hormone biosynthetic process,etc,and KEGG pathways like plant hormone signal transduction,carotenoid/ethylene biosynthesis,etc.,systematic cluster analysis of DE-mRNAs which were correlated with these functions and their targeted DE-lncRNAs was performed.Protein-PPI analysis on target-mRNAs was analyzed using the BlastX program.A network was constructed including target genes,trans-lncRNAs,and PPI interactions.Functions of two lncRNAs that were correlated with plant hormone signal transduction were validated by using transgenic methods(gene editing and overexpression analysis).(1)By mapping reads to the melon genome,a total of 3857 candidate lncRNAs were identified,of which 1601 were differentially expressed between any two periods,and 142 were highly expressed(Average FPKM > 100).The number of DE-lncRNAs between the ripening stage and the climacteric stage was 510.KEGG enrichment analysis of co-expressed mRNA showed that the most enriched pathway between the growing stage and the ripening stage was planted hormone signal transduction.It was suggested that plant hormones play crucial roles during C.melo fruit ripening.The glycosaminoglycan degradation pathway was enriched between the ripening stage and the climacteric stage,which explained the soluble solids content increased at stage C.(2)Of the up-and down-stream of genes correlated with plant hormone pathways,109 DElncRNAs were located,and 422 DE-lncRNAs were co-expressed with these genes,indicating lncRNAs getting involved in plant hormones transduction regulation.(3)The co-expression network showed that the highly expressed lncRNA LNC-002345 and LNC-000154 were co-expressed with multiple genes associated with auxin signal transduction,and may act as regulatory factors in the pathway.The highly expressed lncRNAs(LNC-000987,LNC-000693,LNC-001323,LNC-003610,LNC-001263,and LNC-003380)may participate in the regulation of ethylene biosynthesis and ABA signaling pathway in the fruit ripening and climacteric of C.melo.(4)The differentially expressed transcription factors might play a role in melon fruit development and ripening.LNC-003066 interacted with bHLH130,LNC-000126 and LNC-001263 were co-expressed with bHLH93 and WRKY70 respectively,suggesting they might regulate the fruit development of C.melo.(5)By comparing with CKs at 43 DAP,Oe-3380 s and Oe-3610 s had harder fruits and less soluble solids.The average mature day of Oe-3610 s was delayed for 7 days,indicating that LNC-003610 might be the negative regulator of melon fruit ripening.LNC-003380 was no obvious regulating function on melon fruit development,due to there were no differences between Oe-003380 s and CKs in the mature days,fruit pressure,and soluble solids.(6)qRT-PCR analysis indicated that LNC-003380 was co-expressed with PDC1,SAPK4,and AG2 probably,and LNC-003610 was co-expressed with TF3 A and IAA13.