Analysis And Functional Verifcation Of Xa7 Candidate Genes In Rice

Rice Xa7 gene is a long-lasting resistance gene with high resistance to bacterial blight.The cloning of rice resistance gene Xa7 can not only enrich the theoretical knowledge about molecular mechanism of plant disease resistance.At the same time,it also provides a new source of resistance to disease resistance molecular breeding.In the earlier period,the laboratory used the hybridization constructs of Zhenhui 084 and Chenghui 448 to carry out map-based cloning.Combined with the sequencing of the BAC library of IRBB7 and IR24,the Xa7 gene was finely set within an interval of about 120kb.However,the genome sequences in the positioning interval are very different from those in conventional rice,it is impossible to further finely locate Xa7 using the chromosome exchange in the F2 population.In this study,through radiation mutagenesis of Zhenhui 084,three highly susceptible mutants were screened in 10,000 M1 strains.The PCR was used to amplify the localization interval and screened a mutant ZM2 with a large fragment deletion in the chromosome Xa7,Genetic analysis showed that F1 resistant to F1 between Zhenhui 084 and ZM2,the ratio of resistance to F2 was 3:1,and the susceptible phenotype was closely linked to the missing site.Nipponbare and ZM2 hybrid F1 were susceptible,and F2 was completely susceptible.Through high-throughput sequencing analysis of susceptible genomes,a 106-kb fragment was deleted in the Xa7 alignment region,and there was an overlap of 25-kb with the previously mapped 120-kb region.Finally Xa7 was defined as within the 25-kb region.Through bioinformatics analysis,two candidate genes were found in the 25 kb region and were expressed in IRBB7.Therefore,the constructed CRISPR/Cas9 vector knocked out the two candidate genes G1 and G2 in IRBB7.except.Four transgenic lines were obtained.After the molecular level identification and the positive strains were verified by the bacteria,it was found that all the four strains showed disease-resistant phenotypes.We speculate that the predicted G1 and G2 ORFs are not Xa7 true coding frames.Therefore,the Xa7 gene can only be cloned by functional complementation analysis of the transgene.The TAC library of rice cultivar IRBB7 was constructed using a transgene TAC vector and the TAC positive clones of the Xa7 gene mapping interval were screened by PCR.One of the positive clones was transgene.In addition,primers covering the positioning interval were designed to carry out PCR amplification of the positive single clones screened for the localization interval.The obtained fragments were loaded on the vector pCAMBIA1300,and four subclones covering the Xa7 localization interval were constructed.At present,TAC-positive clones and four sub-clone transgenic seedlings were obtained and identified as positive plants,followed by identification of large disease resistance.Thus the Xa7 gene was cloned.