Transcriptome Analysis Of Early Somatic Embryogenesis And Effects Of Cle Peptides On Embryonic Tissue Development In Cinnamomum Camphora L.

Camphor tree(Cinnamomum camphora L.),is a tall and everygreen tree belongs to the genus Cinnamomum of Lauraceae.It has important ornamental and economic values,and is cultivated in many countries.However,genome resources are lacking in camphor tree.It is difficult to study gene functions and structures,which makes genetic improvement of this tree restricted.Somatic embryogenesis is a notable illustration of cell totipotency,by which somatic embryos differentiates into a new plantlet via a process similar to zygotic embryogenesis.The mechanisms of somatic embryogenesis are far from clear.Our previous work demonstrated that the pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree.Frequency of somatic embryogenesis was significantly improved from16.29% to 93.27%.This is the most effective way reported so far for somatic embryo induction in camphor tree.To better understand the molecular basis of somatic embryogenesis induced by osmotic stress,de novo transcriptome sequencing of three tissues of camphor tree(immature zygotic embryos,sucrose-pretreated immature zygotic embryos,and somatic embryos induced from sucrose pre-treated zygotic embryos)were conducted using RNA＿Seq technology.Somatic embryogenesis-related genes were analyzed,and expression of candidate genes was verified by quantitative Real-Time PCR.Based on the transcriptome data,the effects of CLE peptides on embryogenic callus development is investigated.The main results were as follows:1.A total of 30.70 G high quality clean reads were obtained from c DNA libraries of the three samples.The overall de novo assembly of c DNA sequence data generated205,592 transcripts with an average length of 998 bp,and 114,229 unigenes with an average length of 680 bp.Gene descriptions,gene ontology terms or metabolic pathways of 38020 unigenes(33.28% of all unigenes)were annotated by Nr,Nt,Swiss Prot,GO,KOG,KEGG and PFAM databases based on BLAST search.A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were indentified.Phylogenetic analysis of the 897 genes and Arabidopsis homologous genes were conducted.2.Comparative transriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples.The differentially expressed genes were divided into six groups based on K-means clustering.GO enrichment analysis showed significantly differential expression of genes responding to stress and stimulus.Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and q RT-PCR data.3.A total of 19 CLE genes and 14 WOX genes previously annotated were identified based on our transcriptome data.Expression level analysis of CLE genes in different tissues of camphor tree show they were differentially expressed in immature zygotic embryos,sucrose-pretreated immature zygotic embryos,and somatic embryos induced from surose-pretreated zygotic embryos.17 CLE peptides were indentified using Soft Berry and Bio Edit.Sequence and phylogenetic analysis of the 19 CLE genes and Arabidopsis homologous genes were conducted.Embryonic tissues were treated with different CLE concentrations and different culture conditions.Expression level of WOX genes in different tissues of camphor tree by was analysed a Real-Time PCR technology.WOX genes showed low expression level in embryonic callus when treated with of two of the CLE peptides.WOX5 showed high expression in embryonic callus when treated with TDIF peptide,but the expression of WOX4 were down-regulated.