| Paulownia is a deciduous tree belonging to Paulownia Sieb.It is an important fast-growing timber,farmland shelterbelt and landscape tree species in China.It has important value in timber supply,food security and ecological environment improvement.Drought is the main threat to Paulownia in the whole life cycle,which seriously affects the growth and development of Paulownia.Plant secondary metabolism refers to the process of synthesizing secondary metabolites that are not necessary for life activities.Its metabolites are the result of long-term adaptation of plants to the environment and evolution.They can be used as transcription regulators and signal transduction compounds,which can strengthen the defense ability against biotic stress invasion and the adaptation process to abiotic stress,so it provides a new direction for us to explore the response mechanism of Paulownia to drought stress from the secondary metabolism process.In this study,the response mechanism of secondary metabolism of Paulownia fortumei under drought stress was studied by using Nanopore sequencing technology and sRNA sequencing technology.The main results of this study are as follows:1.For the first time,the full-length transcriptome analysis of the secondary metabolic gene expression Paulownia fortumei under drought stress was carried out,and six genes related to secondary metabolism in response to drought stress were identified.In this study,Nanopore three-generation sequencing technology was used to perform full-length transcriptome sequencing on a total of six samples of the Paulownia.fortunei drought stress treatment group(dsPF1,dsPF2,dsPF3)and the control group(PF1,PF2,PF3).E ach sample was sequenced to produce Clean Data of 10.76Gb,and 65,450 transcripts were finally obtained after de redundant analysis.After screening,a total of 6085 DEGs were identified,of which 2972 were up-regulated differentially expressed genes and 3113 differentially expressed genes with down-regulated expression.Through the enrichment analysis of GO and KEGG,we found that the differentially expressed genes play a regulatory role in 9 pathways related to secondary metabolism,including the flavonoid biosynthesis,tryptophan metabolismsand plant hormone signal transduction related to sesquiterpene ABA.Through the analysis of these pathways,we identified 6 genes related to drought stress,such as ABA receptor and ethylene responsive transcription factor,which regulated the gene of ethylene responsive transcription factor 1 as a response gene for drought stress,and played a positive role in regulating ABA signaling.By controlling the coumaroylquinate(coumaroylshikimate)3’-monooxygenase,the lignin was synthesized under drought stress to enhance the drought tolerance of Paulownia.2.For the first time,the expression of secondary metabolism related genes of Paulownia fortunei under drought stress was analyzed by sRNA technology,and 54 closely related differentially expressed miRNAs were found.By using Illumina sequencing platform,85.41 M Clean Reads were obtained from the sRNA sequencing of the drought stress treatment group(dsPF1,dsPF2,dsPF3)and the control group(PF1,PF2,PF3).278 miRNAs were obtained in six samples,including 17 known miRNAs,261 novel miRNAs,.Among the identified miRNAs,239 miRNAs predicted 3197 target genes.After screening,54 differentially expressed miRNAs were identified,of which 24 miRNAs were up-regulated and 30 miRNAs were down-regulated.The enrichment analysis results of GO and KEGG of these differentially expressed miRNAs show that they are mainly involved in the biosynthesis of aromatic secondary metabolites in phenols,the biosynthesis of nitrogen-containing secondary metabolites,and plant hormone signal transduction related to sesquiterpene ABA.4 pathways related to secondary metabolism.6 miRNAs were randomly selected,and the results of verifying the expression level of miRNA in the sRNA sequencing by qPCR were reliable.3.Correlation analysis of full-length transcriptome sequencing data and sRNA sequencing identified three drought stress response genes closely related to secondary metabolism.Correlation analysis of differentially expressed miRNA and mRNA.Through enrichment analysis of differentially expressed mRNA regulated by differentially expressed miRNA for GO and KEGG,6 pathways related to secondary metabolism were found,which were related to phenol secondary metabolite biosynthesis,nitrogen-containing compound secondary metabolite biosynthesis and sesquiterpene ABA-related plant hormone signal transduction.Through analysis of differentially expressed mRNA enriched in 6 metabolic pathways,4 drought stress-responsive genes,including genes that regulate coumaroylquinate(coumaroylshikimate)3’-monooxygenase related to lignin synthesis,were identified.Differentially expressed mRNA and miRNA correlation analysis,through GO and KEGG enrichment analysis of differentially expressed mRNA,to find 6 pathways related to secondary metabolism.After the analysis of metabolic pathway,3 drought stress response genes,such as the gene regulating ethylene response transcription factor 1,were identified.The expression of these genes makes Paulownia produce related secondary metabolites under drought stress,changes the physiological and biochemical processes in vivo,and improves the drought tolerance of Paulownia.In this paper,we analyzed and identified the genes responding to drought stress in the process of secondary metabolism by full-length transcriptome and sRNA.We found that Paulownia fortunei can activate related genes and regulate stomatal switch to reduce water transpiration in Paulownia through sesquiterpene ABA pathway,eliminate the damage caused by high concentration of reactive oxygen species and increase the expression level of lignin.These results provide theoretical support for elucidating the drought resistance mechanism of Paulownia. |
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