| Bacterial blight（BB）caused by Xanthomonas oryzae pv.oryzae（Xoo）is one of the most widely distributed and endangered bacterial diseases in the world,which seriously affect the rice yield and rice quality.Planting resistant varieties has been proven to be the most economical and effective approach to control BB.In recent years,sequence-specific nucleases（SSNs）have been demonstrated to be powerful tools for the improvement of crops traits via gene-specific genome editing,and CRISPR/Cas9 is thought to be the most convenient and effective method.In this study,we use the CRSIPR/Cas9 system multi-targets editing a Xoo induced gene Os11g39100 to improve rice bacterial blight resistance and gene function research,.The main results obtained in our study are as follows:1.qRT-PCR was used to analyze the relative expression level of Os11g39100 gene in indica variety IR24 and wild rice gene introgression line W6023.The results showed that the gene was highly expressed in IR24 while it was almost not expressed in W6023,and the expression level of this gene in IR24 showed a tendency to be induced by Xoo infection.The results are consistent with our pervious RNA-seq data and this gene was tentatively named as Xig1（Xoo induced gene）.2.The Xig1 gene was cloned by PCR.The DNA sequence of Xig1 gene in IR24 and W6023 are both 612 bp and only contains one exon.There are 3 SNPs differences in the nucleotide sequence,and 2 amino acid changes in the protein level.The promoter region of this gene in IR24 was identical to the sequence of indica rice Shuhui 498,and the cis-acting elements in the promoter region were analyzed.3.The transient expression of YFP-XIG1 fused protein in tobacco leaves was located on the cell membrane and nucleus,and the result indicated that Xig1 can be expressed as a protein in plant cells.4.A 3 targets CRISPR/Cas9 vector targeting Xig1 in IR24 were constructed,and 31 T0 transgenic positive plants were obtained.After inbreeding and molecular identification,17 lines were obtained in the T2 which were modified at the target sits.8 transgenic lines were selected,which without T-DNA in genome,for identification of bacterial blight resistance in the T3 generation.The results showed that compared with the wild-type controls,the lesion lengths of the 8 gene-editing lines at 1 and 2 weeks after inoculation were significantly shortened and the shortening length was between 40%and 50%.The main agronomic characteristics of the above 8 strains were not significantly different from the wild type.In summary,we obtained a bacterial blight susceptible-associated gene,and 8 materials which resistance to rice bacterial blight were significantly increased,in this study. |
