Oriented Improvement Of Rice Blast Resistance Via CRISPR/Cas9 Gene Editing Technology

Rice,as a crop with the largest edible population in the world,yield is reduced every year due to the epidemic of rice blast.At present,The effective strategy to control rice blast outbreak is to use resistant varieties.In recent years,The invention of CRISPR/Cas9 gene editing technology has solved the problems of breeding years,and linkage drag in the process of traditional breeding and provides a new breeding platform for crop genetic improvement.In this study,CRISPR/Cas9 system was used for targeted editing of genes related to rice blast with Pita,Pi21 and ERF922,aim to obtain rice resistant materials with stable inheritance.The main results of this study are showed as follows:1.Pi21 gene plays an important role in resistance to rice blast in recessive gene locus,ERF922negatively regulated blast of rice resistance,and The difference of resistance of Pita to rice blast was caused by the change of amino acid position at 918.We selected the sequence of the first exon of threegenesnearbyATGasthetargetsequencetoconstructthe pC1300-2×35S::Cas9-g^Pita-g^Pi21-g^ERF922expressionvector,andwastransformedby Agrobacterium-mediated method into long-grain Japonica rice restore lines L1014.The mutation efficiency of Pita,Pi21 and ERF922 were 75%,85%and 75%,respectively in T₀ transgenic lines,most of Pita mutat genotypes were biallelic mutation?47.1%?,the frequency of homozygous genotypes was 41.2%,and no heterozygous genotypes occurred and in the types of Genetic mutations,but,two positive seedlings did not mutate at Pita targets?11.7%?,the frequency of base insertion and base deletion were 73.5%and 14.7%,respectively.however,the base with unchanged account for11.8%.The incident of The Pi21 mutant genotypes were similar to that of Pita,with maximum frequency of biallelic mutation was 58.8%,the frequency of homozygous genotype and heterozygous genotype were 35.3%and 5.9%,respectively.the mutation type of Pi21 was dominated by base deletion,with a frequency of 85.3%,and The mutation types of base insertion,replacement and base with unchanged were 8.8%,2.9%and 2.9%,respectively.The mutation types of ERF922 were relatively abundant,including insertion,deletion,replacement and simultaneous replacement deletion of bases,among which the deletion frequency of bases was nearly 50%.Pita,Pi21 and ERF922 genes were co-transformed into long grain japonica restorer line L1014 by Agrobacterium-mediated method.With the mutation of Pi21 and ERF922,Pita also tends to mutagenesis,therefore,the induction events of the three genes tend to mutation simultaneously.In this study,only Pi21 monogenic mutation and trigenic mutation of Pita,Pi21 and ERF922 were found in this transformation experiment.We obtained three homozygous mutant lines without T-DNA components by screening the T₁ isolate lines,including two kinds of Pi21 monogenic mutation and one type of trigenic mutation with Pita,Pi21 and ERF922,and Three homozygous mutant populations of T₂ lines?namely as D2101,D2102 and D0301?were constructed further.2.The resistance of three homozygous mutant lines to M.oryzae was identified at seedling stage,and rice leaf were sampled at 0h,12h,24h post inoculation.the results showed that the resistance of homozygous mutant lines was significantly improved compared with that of wild lines.The expression levels of defense-related genes in homozygous lines and wild type after inoculation were analyzed.Compared to wild type,the mutant lines enhanced the expression of genes related to the signal transduction pathways of salicylic acid,jasmonic acid and ethylene.Therefore,We hypothesized that the high expression of these defense-related genes enhanced the resistance of the homozygous mutant lines to rice blast.Meanwhile,we investigated the main agronomic traits of homozygous mutant lines and there were no significant differences between the wild type and the homozygous mutant lines in plant height,flag leaf length and width,number of productive panicles,panicle length,number of grains per panicle,seed setting percentage and thousand seed weight.Homozygous mutant lines with stable inheritance and resistance to M.oryzae were obtained by using CRISPR/Cas9 technology,which would provide good materials for improving blast resistance in rice.