Genome-wide Identification,Expressional And Functional Analysis Of AGO Gene Family During Early Somatic Embryogenesis In Dimocarpus Longan Lour

Longan（Dimocarpus longan Lour.）is a tropical and subtropical evergreen fruit tree with important economic value in the south of China.It was found that the quality and yield of longan fruit were closely related to its embryo development status.Plant somatic embryo and zygote embryo were highly similar,so plant somatic embryogenesis could be an excellent alternative system for studying plant embryogenesis mechanism.Studies have shown that plant somatic embryogenesis is regulated by specific genes and epigenetic regulation,especially DNA methylation.A certain level of DNA methylation is necessary for the development of normal somatic embryos.Micro RNAs can mediated the DNA methylation and the process is affected by the proteins which involved in the micro RNAs process and synthesis.AGOs are the key proteins in the process which involved in mediating DNA methylation,so AGO genes may regulate the plant somatic embryogenesis,but the specific mechanism is unclear.At present,little is known about the expression regulation and function mechanism of AGO gene in longan.Therefore,the longan AGO genes family were identified from the third generation of longan genome data,and the expression patterns of Dl AGOs in embryogenesis,different tissues and organs,hormones and abiotic stress treatments were analyzed.Meanwhile,the function of Dl AGO genes and promoters were studied by means of ectopic integration.Finally,transcriptome analysis of demethylation in longan embryo callus was carried out treated with 5-azac to explore the effect of methylation level on the formation process of early embryo of longan,and the expression pattern of AGO genes in the treatment process by q PCR.This study provides scientific base for exploring the methylation regulation mechanism of AGO during the early stage of somatic embryogenesis in longan.Main results are as follows:1 Genome-wide identification,bioinformatics analysis of AGOs family in longan early somatic embryogenesis,and cloning of Dl AGO4,Dl AGO7,Dl AGO10,Dl AGOMEL110 AGO genes were identified from the third generetion of longan genome database and named according to the member annotated into Arabidopsis thaliana or rice.Dl AGO genes were distributed in 1,4,8,10,12,13,14,15 chromosomes,encoded from 884 to 1064 amino acids,contained PAZ and Piwi domain,while the Dl AGO proteins were hydrophilic alkaline protein.Phylogenetic tree analysis showed that the plant AGO could be divided into four branches,and longan AGOs are the closest relative to Citrus sinensis AGOs.The cDNA sequences of Dl AGO4,Dl AGO7,Dl AGO10 and Dl AGOMEL1 genes by cloning were 3425 bp,3418 bp,3775 bp and3289 bp,respectively.The homology of amino acid sequences of Dl AGOs was low.2 Cloning and bioinformatics analysis on the promoters of Dl AGOs in longan ECThe lengths of 5’flanking sequences of AGO genes were 1437 bp、1809 bp、1514 bp、1807 bp、1707 bp、1722 bp、1784 bp、1746 bp、1794 bp and 1512 bp,respectively.The bioinformation analysis showed that the promoter of Dl AGO1,Dl AGO4 and Dl AGO5-2 had Cp G island.Based on the analysis of online software Plant Care,the result showed that all the 5’flanking sequences of Dl AGOs contained amounts of TATA-box,CAAT-box core elements and a lot of putative elements responded to hormone,light,and stress.3 Expression patterns analysis of Dl AGOs familyThe expression profiles of Dl AGOs in different tissues and embryo,showed that Dl AGO genes had relatively high expression levels in seeds,flowers,young fruits,stems,and in the early and middle of embryo development,so they were involved in the regulation of early and middle embryogenesis and the formation of seeds,stems,young fruits,and flower buds.Dl AGO genes could response to the treatment with different hormones（2,4-D,KT,Me JA,SA）and abiotic stresses（light treatment,high and low temperature,salt,osmotic,drought,ABA）,but different members had different expression patterns,indicated the diversity of gene function between family members.4 The functional verification of Dl AGOs promotersExpressional vectors of pro AGOs were constructed to transform tobacco leaves and longan EC,through histochemical staining,transient expression of GUS gene and promoter hormone response analysis,the results showed that all the promoters of Dl AGOs were able to drive GUS expression and the transcriptional activity of Dl AGO genes promoters could be induced by ABA,some of which are induced by Me JA,SA and GA₃,suggesting that the promoters of Dl AGOs were hormone-induced promoters.5 Subcellular localization of Dl AGO7,Dl AGO10 and Dl AGOMEL1proteinIn order to explore the specific function of proteins encoded by Dl AGO7,Dl AGO10 and Dl AGOMEL1,the fusion expression vectors were constructed,and instantaneous transformed in onion epidermal cells.Subcellular localization assay indicated that Dl AGO7 mainly located in the cell nucleus,Dl AGO10 and Dl AGOMEL1 in the cell cytoplasm.6 Effects of 5-azac on early somatic embryogenesis and expression of Dl AGOs in longanTo analyze the effects of 2,4-D and 5-azac on early somatic embryogenesis and expression of Dl AGOs in longan,different concentration of 2,4-D and 5-azac were used to culture longan EC under dark conditions for 20 days.Microscopic observation results showed that globular embryos were generated in the medium of 2,4-D_0.1mg/Lwith control group and added with 5 uM and 25 uM 5-azac,while in the medium of 2,4-D_0.5mg/Lonly treated with 5 uM 5-azac had globular embryos,but the other treatment groups did not appear,and the high concentration of 5-azac could lead to cell metamorphosis;the quantitative results showed that different concentrations of 2,4-D and 5-azac could affect the expression of Dl AGOs,and the 5 uM 5-azac treatment could improve the expression of Dl AGO4.Based on the above experimental results,embryogenic callus cultured 1,3,6,9 and 12 d treated with 5-azac concentration of 0,2.5 and5 uM.Microscopic observation showed that there were no globular embryos in the control group at cultured 9 d,but the treatment groups had;the quantitative results showed that Dl AGO4 was up-regulated when treated with 5-azac for 9 d.7 Transcriptome analysis of longan early somatic embryogenesis treated with 5-azacWe used high-throughput RNA-Seq technology to investigate the transcriptome of longan EC treated with 2.5 uM 5-azac for 9 d without5-azac as the control group.Transcriptome sequencing results showed that there were 1617 genes differentially expressed including 960up-regulated genes and 657 down-regulated genes in the pairwise comparison of CK-vs-T.KEGG pathway analysis showed that the differentially expressed genes（DEGs）were mainly enriched in plant hormone signal transduction,plant MAPK signaling pathway,starch and sucrose metabolism.In addition,lots of DEGs were enriched in the butyrate metabolism,C5-branched diacid metabolism,sulfur metabolism,selenic compound metabolism,taurine and taurine metabolism.After5-azac treatment,the longan DNA methylation level decreased,promoting the up-regulated expression of genes related to longan somatic embryogenesis（FUS3,ABI3,FT1,GABA-T3,CAS,GLP,etc.）and AGO4gene,thus promoting the process of early somatic embryogenesis of longan.Therefore,5-azac treatment promoted the expression of Dl AGO4,and Dl AGO4 combined with 24-nt lmi RNA inhibited DNA methylation and promoted somatic embryogenesis in longan.Meanwhile,5-azac inhibited histone acetylation and promoted somatic embrygenesis of longan.Histone butyrylation was facilitated by butyrate and promote somatic embryogenesis in longan.