Effects Of PfWRKY6 On Transcriptome And MiRNA Expression Changes In Populus Trichocarpa

WRKY,one of the largest transcription factor families in plants,is widely involved in regulating plant growth and development,and in response to biotic and abiotic stresses.Paulownias are an important deciduous fast-growing and greening tree species,which have high ecological and economic value.However,the Paulownia witches’ broom caused by phytoplasma infection has seriously hindered the development of the paulownia industry.In our previous study,PfWRKY6 was the partner of Paulownia witches’ broom phytoplasma effector Pa WB-SAP54,and it had a high homology with AtWRKY6.In order to verify the biological function of PfWRKY6,we transformed it into Populus trichocarpa using agrobacterium-mediated genetic transformation,and performed transcriptome and small RNA sequencing to reveal the effect of PfWRKY6 on gene expression changes in Populus trichocarpa.The main conclusions are as follows:1.Obtain the PfWRKY6 transgenic P.trichocarpa plants.The plant morphology identification,PCR and q RT-PCR verification showed that the target gene fragment had been successfully integrated into the Populus trichocarpa genome,and a transgenic plant was successfully obtained.2.Construction the transcriptome expression profile of PfWRKY6 transgenic Populus trichocarpa.Transcriptome sequencing analysis of PfWRKY6 transgenic Populus trichocarpa showed the average data output of per sample was 6.72 G,and a total of 3,249 differentially expressed genes were obtained,of which 1,483 were up-regulated and 1,766 were down-regulated.These differentially expressed genes were mainly involved in plant hormone signal transduction,plant-pathogen interaction,phenylpropanoid biosynthesis,and plant MAPK signaling pathway.Transcription factor prediction analysis found that these differentially expressed genes mainly included ERF,MYB,WRKY,bHLH,and NAC families.3.Construction the miRNA expression profile of PfWRKY6 transgenic Populus trichocarpa.The six samples produced an average of 33.6 M of data,and a total of 360 miRNAs were identified.Of which,61 were differentially expressed,which have290 target genes.KEGG enrichment analysis of these target genes showed that the number of genes significantly enriched in the glycolysis/gluconeogenesis and carbon metabolism.4.Elucidation the regulatory relationship between genes and miRNAs in PfWRKY6 transgenic Populus trichocarpa.Correlation Analysis of differentially expressed miRNAs and differentially expressed genes showed a total of 56 miRNA-target gene relationship pairs were found between 21 differentially expressed miRNAs and 35 differentially expressed genes.The regulatory targeting relationship analysis suggested that one miRNA can target one or multiple genes,and multiple miRNAs can common regulate one target gene.Finally,a total of 44 key genes in response to PfWRKY6 were screened,including genes related to regulate plant morphology,such as Lac(7478550),RKF1(18103112),and genes in response to various biotic and abiotic stresses,such as MYB82(7459879),NRT1(7487987),NAC21/22(7492113)and GSO1(7484428).