Transcriptional Regulation Of Vascular Cambium Activity At Different Ages Of Paulownia Tomentosa

Wood is produced by the secondary growth of perennial woody plants.The activity of the vascular cambium is essential for the production of high-quality wood.In order to understand the regulation of vascular cambium activities on plant growth,people have invested a lot of energy.However,the study of transcriptional regulation in the age-related vascular cambium is still largely unknown.In this experiment,we will use Paulownia tomentosa,one of the important fast-growing tree species in China,as the research material.We respectively constructed cDNA libraries of yrl and yr20 paulownia vascular cambium,and used the Illumina HiSeqTM2000 sequencer to sequence two RNA samples of different ages.In this study,we used a variety of biological methods to conduct a detailed study on the vascular cambium of Paulownia tomentosa.The specific experimental results are summarized as follows:1.We performed anatomical observation of samples containing phloem,vascular cambium,and xylem through semi-thin section technology to identify the morphological differences of Paulownia tomentosa at different ages.In yrl,the vascular cambium had about 7-8 layers of cells,whereas,only 3-4 layers were observed in the vascular cambium in yr20.The number of cell layers in the vascular cambium was significantly reduced.2.We used the Illumina HiSeqTM2000 sequencer to sequence two RNA samples of different ages.24185502 and 23148014 raw readings were observed in the two RNA sequence samples,respectively.After the deletion of low-quality sequences,sequences containing connectors,and sequences with N ratio greater than 10%,we obtained 23533462 and 21501826 clean reads,and the percentages of Q20 were 98.36%and 96.66%respectively.Then,we used Trinity software to perform de novo assembly.A total of 70695 unigenes were obtained,with a mean length of 1282bp,and the N50 of unigenes valued up to 1815bp.3.We used BLASTx search to annotate 70695 unigenes into seven databases of Nr,Nt,Pfam,KOG,Swiss-Prot,KEGG and GO.As a result,41426 unigenes were annotated in the Nr database,accounting for 58.59%of the total number of unigenes.In addition 47995,36258 and 39149 unigenes were significantly similar to known proteins in Nt、Pfam and Swiss-Prot databases,respectively,accounted for 67.89%,51.28%and 55.37%of the total.In order to better understand the function of unigenes in vascular cambium for Paulownia tomentosa,we conducted further research on the unigenes annotated in the KOG database.A total of 14829 unigenes were classified into 25 clusters of KOG respectively.The three clusters with the largest number of unigenes were ’posttranslational modification,protein turnover,chaperones’,’general function prediction only’,and ’translation,ribosomal structure and biogenesis’.4.In order to identify differentially expressed genes（DEGs）,it was considered that genes with a threshold |log2（fold change）|>1 and q-value<0.05 are significantly up-regulated or down-regulated.The results showed that a total of 3386 differentially expressed genes were obtained between the two samples,of which 1830 were up-regulated and 1556 were down-regulated.5.Through GO enrichment analysis of differentially expressed genes,it was found that in biological processes,differentially expressed genes were mainly enriched in functional groups such as’metabolic processes’,’single organism metabolic processes’,and ’redox processes’.In the cell composition,the ’extracellular region’,’thylakoid’ and ’thylakoid part’ were the most annotated.Among the molecular functions,the most annotated groups were ’catalytic activity’,’oxidoreductase activity’,and‘cofactor binding’function groups.In addition,through KEGG enrichment analysis,we found that the differentially expressed genes were classified into 20 different pathways.The differentially expressed genes were significantly enriched in ’starch and sucrose metabolism’,’phenylpropane biosynthesis’,and’galactose metabolism’ pathways.6.In order to further verify the expression profiles of genes,we randomly selected nine differentially expressed genes and performed qRT-PCR verification.The results showed that the expression trends were consistent with the results of RNA-seq,and the difference multiples were similar too.The result on the one hand proved the reliability of the sequencing result,on the other hand,they also indicated that these genes may be involved in the transcriptional regulation of paulownia vascular cambium of different agesIn summary,this study has conducted in-depth studies on the vascular cambium of Paulownia tomentosa in different ages through transcriptome sequencing technology combined with experimental techniques such as cytology,molecular biology,and bioinformatics.This result will provide a scientific theoretical basis for explaining the influence of tree age on vascular cambium activity.