Transcriptional Regulation Of Vascular Cambium Activity At Different Ages Of Ginkgo Biloba L.

The old trees can live for centuries or even millennia,which is the natural non-renewable resources and the living fossils.To date,the molecular regulation mechanisms in the long-lived perennials are still largely unknown.This is due to the distribution of regional differences of old trees,the outside damage of tree body,and the difficulties of sampling.Vascular cambium is a lateral meristematic tissue,which could continuously divide and differentiate into secondary phloem and xylem in the process of the growth of trees.Therefore,systematic research for the lifespan of old trees will has important scientific values.Ginkgo biloba L.is the one of the oldest gymnosperms in extant species,and there are many ageing over 100 year-old trees still existing in China.In this study,we used the combined method of the Illumina next-generation sequencing HiSeqTM 2500 platform and bioinformatics to perform de novo transcriptome sequencing and analysis in female G.biloba cambium at different ages.The main results are summarized as follows:1.The growth of G.biloba was evaluated and measured at different ages,such as leaf,fruit and seed germination.The results showed that the gingko trees were no obvious mechanical damage,plant diseases and insect pests in 20Y,200Y and 600Y.In addition,the chlorophyll content and seed germination rate of the gingko trees were no significant difference at three different ages.2.We performed anatomical observations of the cambial zone to identify the morphological differences in G.biloba at different ages.In VC20,the cambial zone consisted of about 8±1 layers of cells,whereas,the number of cell layer within cambium zone was decrease in VC200 and VC 600.3.We sequenced RNA samples derived from of three different ages(20-,200-,and 600-year old)using the Illumina HiSeqTM 2500 sequencer.In total,approximately 55 million raw reads were observed for each RNA-seq sample.After stringent quality checks and data cleaning,more than 51,127,258 clean reads for each sample were retained and the Q20 valued up to 98%.Then,the de novo assembly yielded 97,303 unigenes with a mean length of 1500 bp,and the N50 of unigenes valued up to 2,476bp.4.A total of 97,303 unigenes were annotated by using a BLASTx search against NCBI's NR database,Swiss-Prot,COG,KEGG,GO,NT of proteins.As a result,63,912(65.7%)unigenes were significantly similar to known proteins in the six databases.In addition,to further understand the function of the G.biloba vascular cambium unigenes,we searched the annotated sequences for genes involved in COG classifications.A total of 26,782 unigenes were categorized in 25 COG clusters.The three largest categories were 'General function prediction only','Replication,recombination and repair' and 'Transcription'.5.To detect differentially expressed genes(DEGs),those genes with a threshold |log2 fold change|?1,and with adivergence probability ?0.8 were considered to be significantly up-or down-regulated in expression.The results showed that the expression of 2,572,1,312 and 891 genes significantly changed between VC20 and VC200,VC20 and VC600,and VC200 and VC600,respectively.By the GO enrichment analysis,found that the DEGs of old trees involved in extracellular region,oxidoreductase activity,secondary cell wall biogenesis,and lignin metabolic process.In addition,the metabolic pathways and biosynthesis of secondary metabolites were the top 2 statisitics of pathway enrichment for G.biloba cambium at different ages.6.Cell division and differentiation is one of the key processes taking place in the cambium zone and is important for the growth of the vascular tissues.We investigated the genes associated with cell division and expansion.,the 21 candidate DEGs were up-regulated in 20VC,whereas these were expressed at lower levels at VC200 and VC600.In addition,we further identified genes associated with cell wall biosynthesis also were down-regulated in old trees.These results indicated that cambial activity is weaken in ancient trees than 20Y.7.We investigated the genes associated with phytohormone signaling,and identified that a large number of homologous genes responsive to auxin,gibberellin,cytokinine,abscisic acid,ethylene and brassinosteroid,which showed differential expression in the regulation of cambium activity at three different ages of G.biloba.Some genes involved in the auxin pathway and Cytokinins,which most of them were up-regulated at VC20,compared with VC200 and VC600.However,the abscisic acid receptor PYR/PYL and genes involved in ethylene biosynthesis and signaling were up-regulated in old trees.8.In cambium of G.biloba,we found that 2,814 genes were putatively identified as TFs and attached to 59 TF families.Of them,the most abundant TF family is MYB family(351,12.40%),followed by AP2-EREBP(227,8.02%),MYB-related(219,7.74%)and C3H(213,5.21%).We put the gene in count when its expression>0.3 and the distribution of each TF family seem consensus in each sample about 30%.In addition,we further analyzed the expression level of G.biloba cambium at different ages,such as GAMYB,MADS,bHLH,TCP,and zinc transporter 2-like.Compared with VC20,the total expression level of the VC200 and VC600 was down-regulated.9.To verify the expression profiles of genes identified through RNA-Seq,we further random selected twelve DEGs,which play key roles in regulating cell division,cell expansion,and cell wall biosynthesis were examined by qRT-PCR.The results showed that these genes profiles were similar to those determined by Solexa sequencing.In the present study,we used the combined method of the RNA-Seq,cytology,molecular biology and bioinformatics to study the G.biloba cambium at different ages.These results provide a fundamental basis for future study of molecular mechanisms of different ages.