Genome-wide Identification And Expression Regulation Of LncRNAs During Early Somatic Embryogenesis In Dimocarpus Longan Lour.

Longan(Dimocarpus longan Lour.)belongs to the genus Longan of the Sapindaceae family and is a famous tropical and subtropical fruit tree.The longan somatic embryogenesis(SE)system is a good system for research on longan embryo development.Long non-coding RNAs(lncRNAs)are involved in variable cleavage,transcriptional interference,regulation of DNA methylation and protein modification.However,studies on lncRNAs in plant embryogenesis have not been reported yet.In this study,longan was identified by high-throughput sequencing system using embryonic callus(EC),Incomplete embryonic compact structure(ICpEC),and globular embryo(GE)as experimental materials.The experiment systematically identified three stages of lncRNAs in the early longan SE.Screening and expression analysis were performed by differentially significant analysis of lncRNAs.Construction of regulatory networks for lncRNAs related to auxin,abscisic acid and ethylene response factors by KEGG enrichment analysis.The expression analysis of early SE in longan and the expression analysis under the corresponding hormone treatment were carried out.The family members related to the ERF of the lncRNAs target gene were identified.To predict the relationship between lncRNAs and miRNAs,through miRNA overexpression and inhibition expression,and Quantitative Real-time PCR(qRT-PCR)was used to verify the expression of related lncRNAs and mRNAs.Overexpression vectors of lncRNAs were constructed and transformed into longan protoplasts and EC by PEG transformation and Agrobacterium-mediated transformation.The main contents of the research are as follows: 1 Identification of early lncRNAs prediction of target genes in longan somatic embryosHigh-throughput sequencing of three samples(EC,ICpEC and GE)in longan using ?umina HiSeq sequencing technology.By comparing the differential expression of lncRNAs between samples,more specific lncRNAs exist in the GE stage,the role is mainly in the ICpEC to GE stage.The function of lncRNAs is mainly achieved by cis or trans acting on target genes.KEGG enrichment analysis of target genes with significant differences in lncRNAs revealed that the "plant-pathogen interaction" and "phytohormone signal transduction" pathways were most abundant in EC vs.GE and ICpEC vs.GE.2 Screening and expression analysis of lncRNAs and their target genes in early longan SETo validate the expression of lncRNAs in early SE of longan,A total of 20 lncRNAs with significant differences expression in RNA-Seq and 5 lncRNAs associated with plant hormone signaling were selected for qRTPCR validation.Except for LTCONS-00027337,the remaining 24 lncRNAs were detected in EC,ICpEC and GE.The results showed that the RNA-Seq of 16 lncRNAs in 24 lncRNAs was consistent with the expression trend of qRT-PCR.This study systematically analyzed the ERF family of target genes for lncRNAs,and further analysis of 5 ERF genes enriched in the early SE process of longan found that,with the significant changes in endogenous hormones in early SE of longan,more DlERF genes are needed to promote the normal development of embryos.By comparing the results of qRT-PCR and RNA-Seq expression of 5 DlERF genes which were significantly differentially expressed in the early longan SE,it was found that most of the expressions in the early longan SE were consistent,and 3 D1 ERF gene showed the highest expression in longan GE stage.It can be seen that more ERF genes are needed to participate in the maintenance of the GE stage from the EC to the GE stage,which further indicates that in the early stage of somatic embryogenesis,cell division and differentiation,and resistance are gradually enhanced.Using different concentrations of exogenous hormones to treat longan EC,it can be seen from the complex network of relationships that lncRNAs,which are plant hormones,play a crucial role in the longan SE.3 The relationship between lncRNAs and miRNAs in the early longan SE.In addition to interacting with encoded proteins,lncRNAs may also interact with miRNAs.This experiment predicted longan lncRNAs as precursors,target genes and endogenous target mimic(eTM)of miRNAs.To further validate the relationship between lncRNA,miRNA and mRNA during early development of longan SE,qRT-PCR validation was performed on the predicted networks of Dlo-miR172 a,Dlo-miR159 a.1 and Dlo-miR398 a.By analyzing the expression levels of overexpression and inhibition of expression of Dlo-miR172 a,Dlo-miR159 a.1 and DlomiR398 a,we found that Dlo-miR172 a and its associated lncRNA and mRNA during the early longan SE have the same expression trend as predicted.However,the regulatory relationship between Dlo-miR398 a,Dlo-miR159 a.1 and its associated mRNA and lncRNA is complex and requires further exploration.4 Overexpression vector construction and transient expression of lncRNAs in the early longan SEThis experiment constructed an overexpression vector of LTCONS-00042843 and LTCONS-00006334.Overexpression of LTSONS-00042843 and LTCONS-00006334 in longan callus and protoplasts was achieved by Agrobacterium-mediated transformation and PEG transformation.From the qRT-PCR results in protoplasts,LTCONS-00042843 and LTCONS-00006334 and their target genes showed significant increases,further confirming that LTCONS-00042843 and LTCONS-00006334 have a positive regulatory relationship with their target genes.LTCONS-00042843 acts as an eTM of miR172 a,which has a negative regulatory effect.However,when the longan was transferred to the longan,the target genes of LTCONS-00042843 and LTCONS-00006334 showed opposite trends,which were different from the regulation in SE and the tendency to transfer into longan protoplasts.It is speculated that it may be due to the test.Different materials lead to differences in their regulatory networks or regulatory preferences.