Functional Analysis Of Blast Resistance Associated Gene Pi-G3

Rice blast is caused by pathogenic fungus Magnaporthe oryzae,and is one of the three major diseases in rice.Analysis and utilization of rice blast resistance gene function may help reveal the mechanism of rice resistance to rice blast and accelerate rice disease resistance breeding.Gene pi-G3 which is related to rice blast resistance and encodes oxidoreductase,was obtained by GWAS analysis in the early stage of our laboratory.We preliminarily studied the function of this gene was in this paper.CRISPR/Cas9 knockout vector and overexpression vector of this gene were constructed respectively,and transgenic plants were obtained through agrobacterium-mediated transformation.Such punch inoculation and ROS production measurement assay were utilized to identify the phenotype and analysis function of transgenic mutant.At the same time,a GFP localization vector was constructed to subcellularly localize pi-G3.We preliminarily proved that pi-G3 is involved in the rice blast resistance.The results of the study are as follows:1.Select the editing target of pi-G3 gene to construct CRISPR-Cas9 vector(PYB02-piG3)and simultaneously PCR-amplify the full-length cDNA of pi-G3 to construct overexpression vector(pROXs-HA-pi-G3).Bacillus transformed to obtain transgenic mutants.2.Real-time quantitative PCR and high-throughput sequencing of transgenic materials identified the corresponding CRISPR mutants and overexpression mutants,obtained CRISPR mutants: 43090-7-4,43090-5-5,wild type 43090-9-1;obtained overexpression plant 43090-12-5,43090-12-13.3.Identification of the phenotype of Punch inoculation and ROS production measurement assay showed that the gene pi-G3 was associated with resistance to blast disease in rice;agronomic traits investigations of CRISPR mutants pi-G3 revealed that knockout the gene is no significant different compared to wild type.4.Gene pi-G3 was observed in rice chloroplasts under confocal microscope through subcellular localization.6.The results of the prokaryotic expression laboratory showed that pi-G3 could be induced under the condition of adding IPTG,which laid the foundation for the next step of the protein level experiment of this gene;A novel gene pi-G3 related to rice blast resistance was discovered by GWAS in this study,we preliminary analysed the resistance phenotype and agronomic traits phenotype of the gene,which provided new resources for rice blast resistance breeding.