Cloning And Breeding Practice Of A Broad Spectrum Of Rice Blast Resistance Gene Pi2-2

Rice blast is a fungal disease of rice,which seriously affects the yield and quality of rice.The main methods that prevent and control rice blast are to spray chemical reagents in field,but using chemical reagents for a long time will make the Magnaporthe grisea resistant to reagents which lose resistance ability,what's worse,it will endanger the balance of the ecosystem and affect the health of human body and mind.The planting of broad-spectrum resistant rice varieties can not only reduce the cost of rice production,but also promote the sustainable development of the environment.Therefore,mining and identification of rice blast resistance gene,isolate and clone broad-spectrum and durable blast resistance genes have very important significance to research the resistance mechanisms as well as the interaction mechanism between pathogen and resistance genes and the development of broad-spectrum blast resistant rice varieties.Jefferson,a long-grain tropical japonica cultivar grown in the southern U.S.,confers broad-spectrum blast resistance to 42 blast isolates.A dominant broad-spectrum blast resistance gene Pi2-2 which has been finely mapped within 0.17 cM between SSR marker RM19817 and AP5659-3 at the Pi2/9 locus of chromosome 6.Other broad-spectrum blast resistance genes such as Pi2?Pi9?Pigm and Piz-t were also localized at the same locus.By constructing BAC libraries in the genomic region and fine-mapped the gene within a region approximately 270 kb,two positive BAC clones whose contig cover the whole Pi2/9 locus were selected.Our main task is further to clone the gene Pi2-2 and analysis its'evolution characteristics so that it can lay the foundation for exploring the molecular mechanism of broad-spectrum resistance genes,at the same time,using Pi2-2 gene to preform breeding practice through marker-assisted selection technology.Progress and achievements are as follows:1.The sequence of Pi2-2 candidate gene was isolated and it's protein structure is analysised.The complete sequence of Pi2-2 candidate gene was isolated and cloned by using a method of homologous cloning.Bioinformatics analysis showed that the encode region DNA sequence of Pi2-2 candidate gene are 8601 bp in length,including 3 exons and 2introns;it's full-length cDNA sequence are 3099 bp and it encodes a CC-NBS-LRR type protein with 1032 amino acids.Protein molecular formula is C₅₁₈₄H₈₄₀₂N₁₄₂₈O₁₅₃₆S₄₄,which is a hydrophilic protein with 116721.02 Da molecular weight and isoelectric point 6.95.2.Completed the molecular evolution analysis of Pi2-2 candidate genes.Homology analysis of Pi2-2 and Pi9 shows that the similarity and homology of Pi2-2 candidate gene reached 99%.17 SNP mutations present in the second exons of the coding region comparison with the Pi9 gene,including 2 synonymous mutations and 15 nonsynonymous mutations.Through the analysis of homologous protein in Pi2/9 locus with Pi9,Pi2,Piz-t,Pi50,and Pigm,the amino acid difference mainly occur in the LRR region;phylogenetic analysis indicated that Pi2-2 and Pi9 are in the same cluster,suggesting that the Pi2-2 gene has a closer genetic relationship and higher homology with Pi9 in evolution.There is a difference of 44 amino acids,12 amino acid differences,48 amino acids difference,41amino acid differences between Pi2-2 and Pi2,Pi9,Piz-t,Pigm,Pi50,respectively.It also provides a new basis for further analysis of the molecular mechanism and molecular evolution of broad-spectrum resistance to rice blast.3.Gene editing of target gene was completed and two targets were successfully designed.12 transgenic T₀ generation plants under Jefferson background were obtained by Agrobacterium mediated transformation.Among them,6 plants were positive,including 5kinds of mutation with 3 homozygous and 3 heterozygous mutations and the target gene editing rate was 50%.The first type of mutations lead to 1 amino acids deleted without causing frameshift mutations;the second mutation type,the third mutation type and the fourth type of mutations result in early termination of encoding sequence,only encoding1022 amino acids;the fifth mutation type is non-synonymous mutations,and other amino acids do not change.At present,transgenic plant of a target is obtained,which will be used for subsequent analysis of blast resistance and to verify candidate gene function.4.Using Pi2-2 gene to carry out molecular breeding practice to improve rice blast resistance.A co-dominant molecular marker Pi2-2 Pro.1 was developed to improve rice blast resistance of 5 rice accessions or lines?E32,He85,CO1403,Shuhui527,TAO1B?through combining with the traditional backcross breeding method and molecular marker assisted selection technology.At present,the BC1F1 population of the E32×Jefferson,He85×Jefferson,CO1403×Jefferson,Shuhui527×Jefferson,TAO1B×Jefferson obtained Lays the foundation for the further cultivation of novely resistant riceblast rice lines.A clear and stable 701 bp band were amplified in Jefferson genome and about 450 bp band in other 95rice lines.It's rate was 98.96%.The assisted-selection efficiency is 100%in shuhui527×Jefferson BC₁F₁ groups,which showed that the molecular marker Pi2-2 Pro.1 can be effectively used to carry out to improve the blast resistance based on Pi2-2 gene.