Identification Of TIFY Gene Family In Poplar And The PtWRKY36 Gene Deletion Using The Technique Of CRISPR/Cas9 Gene Editing

The plant-specific TIFY proteins are widely present in land plants and play the important roles in the regulation of plant stress-responses.In this study,we carried out a bioinformatics analysis of TIFY genes in poplar.The 24 TIFY genes were identified and classified into four subfamilies(ZML,JAZ,PPD and TIFY).We identified 17 collinear TIFY gene pairs in the poplar genome,ten paralogous gene pairs were involved in largescale interchromosomal segmental duplication events.Numerous abiotic stress ciselements were widely found in the promoter regions.Analysis of the Ka/Ks ratios indicated that the paralogs of the PtTIFY family principally underwent purifying selection.Microarray data and qRT-PCR analysis revealed that 24 PtTIFY genes were differentially expressed in various tissues.Quantitative real-time RT-PCR analysis of TIFY genes expression in response to salt,JA hormones and low-temperature stress revealed their stress-responses profiles.The results of this study provided valuable information for further exploration of the TIFY gene family in poplar.CRISPR/Cas9 gene editing technology has been widely used in many species,and has achieved very good results,strongly demonstrate the universal adaptability of CRISPR/Cas9 mechanism.In this study,poplar PtWRKY36 was used as a candidate gene for exon and intron structure analysis to assess target off-target effects and select appropriate knockout targets.The recombinant knockout vector PtWRKY36-sgRNA+Cas9 was constructed using the pP1 C.4 vector as templates.Hygromycin gradient screening experiments confirmed that the callus of the poplar leaves formed a hygromycin selective pressure of 30mg/L.The knockout PtWRKY36 gene transformant plant was obtained using Agrobacterium-mediated genetic transformation to provide material and technical support for further research on the application of CRISPR/Cas9 systems in woody plants.