Screening And Identification Of Target Genes Regulating Root Development In Populus Ussuriensis PuZFP1

Trees are the key resources to maintain the stability of forest ecosystem.Drought is one of the most serious environmental constraints to tree growth and development worldwide.Therefore,it is of great significance to cultivate drought-resistant tree species and understand the molecular response mechanism of trees to drought stress for sustainable forest yield increase.When trees respond to drought stress,the roots of trees adjust their morphological characteristics,physiological,biochemical and molecular changes to complete the response to drought stress.In current studies,zinc finger protein transcription factors play different regulatory roles in plant growth and development,morphogenesis and stress response,but studies on their drought resistance mechanism are mostly focused on the above-ground part,and less on the underground part.In this study,the target genes of PuZFP1 regulating root response to drought stress was identified.(1)Based on the results of previous studies,this study reconstructed the EAR suppressive domain in PuZFP1,constructed two overexpression vectors(p BI121-PuZFP1m-GFP and p BI121-PuZFP1d-GFP)with EAR mutation and deletion,respectively,and carried out genetic transformation of Populus ussuriensis.The root traits of PuZFP1m-OE and PuZFP1d-OE lines were highly similar to those of PuZFP1 inhibited expression lines.Compared with WT,the number of lateral roots increased,while the adventitious root length did not change significantly.These results indicated that mutation or deletion of EAR motif in PuZFP1 would lose its original transcriptional inhibition function,and further proved that PuZFP1 could affect root development.(2)Transcriptome sequencing(RNA-seq)was used to GO enrichment and classification of PuZFP1 differential genes.It was found that inhibition of PuZFP1 expression could affect the expression of many genes related to root development and ABA signaling pathway.The directly binding sequence of PuZFP1 transcription factor was identified by DNA affinity purification sequencing(DAP-seq).Two candidate target genes Pu WRKY46 and Pu PP2C34 with the highest matching degree were screened by RNA-seq and DAP-seq co-analysis,and they were related to root development or ABA signaling pathway.q RT-PCR was used to verify RNA-seq and DAP-seq data,and the results showed that Pu WRKY46 and Pu PP2C34 showed a significant up-regulation trend in PuZFP1 inhibition lines,which was consistent with RNA-seq and DAP-seq analysis results.(3)In order to further explore the candidate target gene of PuZFP1 in response to drought stress,we verified PuZFP1 candidate target genes related to ABA pathway by the double molecular fluorescence complementary assay(LUC),and found that PuZFP1 could bind to the promoter region of Pu WRKY46 and Pu PP2C34 genes.Further gel(EMSA)migration experiment,the experimental results show that PuZFP1 can and Pu WRKY46 gene promoter region "CAGTGG" element and Pu PP2C34 gene promoter region "TTTTGTTTT" sequence specificity,thus directly regulating Pu WRKY46 and Pu PP2C34 expression.In conclusion,PuZFP1 can affect the root development of Populus ussuriensis.Through screening and identification of target genes that PuZFP1 may regulate under drought stress,the PuZFP1 binding element was found,and it was confirmed that PuZFP1 regulates gene expression by directly binding to the promoter region element of Pu WRKY46 and Pu PP2C34 genes.