Construction Of Rice WRKY28 Mutants Based On CRISPR/Cas9 Technology And Exploration Of The Gene Functions In Disease Resistance

Rice is an important food crop that support more than half of the population in the world.Bacterial blight,caused by Xanthomonas oryzae pv.oryzae(Xoo),affects the rice yield and grain quality seriously.WRKY transcription factors play crucial roles in plant defense responses.Exploring the functions of WRKY transcription factors can not only helps us to understand the mechanism of plant defense responses but also provide gene resources and related theory guidance for traditional breeding and genetic engineering breeding.We previously screened a WRKY transcription factor gene,OsWRKY28,which was related to disease resistance.In addition,recently,the CRISPR/Cas9 system has been successfully applied to a variety of species because of high efficiency in gene editing.Here,we report that the gene editing efficiency of optimized CRISPR/Cas9 and the function analysis of OsWRKY28.The main results are showed below:1.Optimization of CRISPR/Cas9 construction method and efficiency test withD3 geneThe CRISPR/Cas9 vector system was optimized in order to construct gene editing constructors in two steps.With optimized CRISPR/Cas9 system,three genome editing constructors targeting different sites of D3 gene in rice(Oryza sativa L.spp.Japonica cv.Nipponbare)were generated,and the transgenic plants were successfully produced by Agrobacterium-mediated transformation.The sequencing results of To transgenic plants showed that the mutation rate of target site DDRC1 was 35.48%,and five mutation types were detected;the mutation rate of target site DDRC2 was 25.00%,and also with five mutation types;the mutation rate of target site DDRC3 was 20.00%,and only one mutation type was detected.The phenotype,height and tiller,together with the genotypes of homozygous mutants were investigated,and the gene editing efficiency of the optimized CRISPR/Cas9 system along with the position effect and mutation patterns were also analyzed and discussed.In summary,the findings provided important information for further developing and utilizing of rice germplasm on variety agronomic trait with the optimized CRISPR/Cas9 system.2.Construction of WRKY28 mutants based on optimized CRISPR/Cas9We built the gene editing vectors targeting OsWRKY28 using optimized CRISPR/Cas9 and got the transgenic plants by Agrobacteriunm-mediated transformation.The sequencing results of To generation transgenic plants showed that the mutation rate of target site was 38.46%.We obtained four homozygous mutations in T1 generation transgenic plants,including Mutant1 with deleting four bases(CCAA),Mutant2 with inserting a base(C),Mutant3/4a with inserting a base(T),Mutant4b with inserting a base(A).3.The expression pattern of Os WRKY28The results of subcellular localization showed that OsWRKY28-sGFP N-termina fusion proteins localized to the nucleus in plasma cells or tobacco leaf cells and OsWRKY28-sGFP C-termina fusion proteins localized to the nucleus and chloroplast in plasma cells or tobacco leaf cells.Yeast auto-activation assay demonstrated that the OsWRKY28 full-length protein fused with the GAL4-BD domain did not activate downstream report gene expression in yeast.Therefore,OsWRKY28 had no self-activation activityTo investigate the tissue expression patterns of OsWRKY28,RT-PCR was performed.The results showed that OsWRKY28 had a relative low expression level in leaf,flag leaf and floret,and had a relative high expression level in root and stem.To investigate the expression pattern of OsWRKY28 under biological and abiotic stress,wild type plants were treated with different plant growth regulators?H2O2,PEG,NaCl and Xoo.The results showed that the expression level of OsWRKY28 in leaf and root were up-regulated in different degree treated with JA,KT,6-BA,NAA,GA and Xoo.And expression level of OsWRKY28 in leaf were up-regulated treated with SA,IAA,H2O2,NaCl and PEG.We speculate that OsWRKY28 may participate in the disease resistance signal pathway of hormone.4.Acquisition overexpression material of OsWRKY28 and preliminary analysisWe built the overexpression vectors of OsWRKY28 and got the transgenic plants by Agrobacterium-mediated transformation.We identified the hygromycin and analyzed the expression level of OsWRKY28 of T,generation transgenic plants.The results showed that OsWRKY28 were overexpressed in T,generation transgenic plants compared with wild type.And we analyzed the expression level of marker gene(PR1b,PR5,PRIOa)of T1 generation overexpressed transgenic plants.The results showed marker gene were overexpressed in part of the T1 generation overexpressed transgenic plants compared with wild type.In addition,positive T1 generation transgenic plants were used for bacterial inoculation.Lesion lengths in OsWRKY28 overexpression plants were shorter than wild type?The results suggest that OsWRKY28 is likely involved in the response of the rice-Xoo by inducing the expression of PR gene.