Cloning And Functional Identification Of Transcription Factor PeWRKY31 In Populus × Euramericana

WRKY transcription factor,which specifically binds cis-acting elements in plants to regulate the expression of downstream genes.It is an important transcription factor in plants.It not only participates in the processes of plant growth,development and metabolism,but also regulates the responses of plants to biological and abiotic stresses.At present,in-depth research on WRKY transcription factors is focused on Arabidopsis and rice,while the research in other plants is limited.In this study,a new WRKY transcription factor,PeWRKY31 gene,was cloned from（Populus×euramericana）.In this paper,the bioinformatics analysis,the sub-cell location analysis and transcriptional group analysis of transcription factor PeWRKY31 were carried out,and the over-expression vector was further constructed to transform into the tobacco,and the salt tolerance test and the feeding test of the transgenic lines were carried out.This thesis aims to identify the function of transcription factor PeWRKY31.The main findings are as follows:（1）Cloning and bioinformatics analysis of PeWRKY31 gene.Specific primers were designed according to the reference sequence of PeWRKY31 gene obtained from the transcriptome sequencing data,and c DNA was obtained by reverse transcription of RNA from Populus×euramericana.The transcription factor was named PeWRKY31.The ORF region of the gene is 1842 bp and encodes 613 amino acids.The PeWRKY31protein is an acidic and unstable hydrophilic protein,and the phosphorylation modification of the PeWRKY31 protein is mainly serine,and the threonine is supplemented.The homologous sequence comparison of the PeWRKY31 encoded protein with other WRKY proteins showed that PeWRKY31 was a transcription factor of type II WRKY of zinc finger structure C₂H₂ type.Phylogenetic tree analysis showed that PeWRKY31 was closely related to the same genus of Populus trichocarpa.（2）Subcellular localization analysis.Subcellular localization vector 35S:GFP:PeWRKY31 was constructed for transient transformation of tobacco and observed under fluorescence microscope.The results showed that the fluorescent signal of 35S:GFP in the control group was distributed in the nucleus and cell membrane,while the35S:GFP:PeWRKY31 was just located in the nucleus.These results suggest that PeWRKY31 transcription factor is a nuclear localization protein.（3）Construction of over-expression PeWRKY31 gene vector and genetic transformation of tobacco.The over-expression vector was constructed by the traditional enzyme cutting connection method and transformed into tobacco by Agrobacterium-mediated method.The transgenic lines were obtained by Kan rooting screening and extraction of the tobacco DNA for further PCR detection.Subsequently,transcriptome sequencing was performed,the analysis of KEGG enrichment show that the PeWRKY31 gene can be enriched to many important pathways related to salt tolerance and insect resistance.Then the growth and physiological indexes of transgenic tobacco lines planted in greenhouse were measured.The results showed that the transgenic PeWRKY31 gene had no significant effect on the growth and physiology of the plants.（4）Salt tolerance analysis of transgenic tobacco with PeWRKY31 gene.Using Na Cl as the salt stress factor,the salt concentrations were 0‰,4‰and 7‰,respectively.After salt stress,the growth indexes,electrical conductivity,chlorophyll content,MDA content,POD activity and SOD activity of and transgenic tobacco lines and wild-type tobacco were measured to compare their salt tolerance.The plant height,ground diameter and chlorophyll content of transgenic tobacco lines and wild-type tobacco decreased with the increase of salt concentration,and there was no significant difference between transgenic tobaccos and wild-type tobacco at the same concentration.When the salt concentration reached 7‰,the electrical conductivity,MDA content,POD activity and SOD activity of transgenic tobacco lines were significantly better than those of wild type tobacco.The results indicated that the transgenic PeWRKY31 gene could improve the salt tolerance of plants to a certain extent.（5）Insect resistance analysis of transgenic tobacco with PeWRKY31 gene.When the leaves of transgenic and wild-type tobacco lines with the same leaf area were placed in the same petri dish or placed separately to raise the larvae of Helicoverpa armigera,the loss of leaf area of transgenic tobacco lines was significantly less than that of wild-type tobacco.The results showed that the transgenic PeWRKY31 gene could improve the insect resistance of plants.