Identification And Characterization Of Rice Blast Resistance Genes In The Elite Rice Restorer Line R2115

Rice is one of the three most important food crops in the world.More than half of China’s population depends on rice as a staple food,so the high and stable yield of rice is an important guarantee for food security in China and the world.Rice blast,a fungal disease caused by Magnaporthe oryzae,is one of the three most serious diseases affecting rice yield in the world,it is the most economical and environmentally friendly way to control rice blast that using rice blast resistance gene to breed resistant varieties now.Using rice blast resistance restorer lines to select varieties is the most lasting and effective method.Therefore,disease resistance breeding needs to continuously seek for rich germplasm resources and find new broad-spectrum disease resistance genes,so as to overcome rice blast and ensure food security.R2115 is an excellent restorer line selected by the institute of ecology of Sichuan agricultural university,which has been widely used in cross breeding.After about 10 years of field identification,R2115 showed resistance to rice blast.At present,it has been confirmed that Pi2 in Yahui2115 is the main R gene resistant to rice blast,but it has not been reported whether R2115 contains other R genes or new genes resistant to rice blast.For this reason,this study used the technical means of gene localization,gene expression analysis of disease resistance and sequencing comparison to identify and verify the disease resistance genes.Transcriptome sequencing was used to discover new genes resistant to rice blast,and the functional analysis and functional mechanism of new genes were investigated by using transgenic technology,rice blast resistance analysis,ubiquitination proteome sequencing and other biological technologies.The main results are as follows:1.The blast resistance R gene Pib and Pid2 are present in R2115Through the extreme group-recessive population method,the gene that enforces R2115 resistance to the strain M14Ga089 was located at 35,104,642-35,104,668bp nearby of chromosome 2,and named it Pir2115-1（t）.The R gene of rice blast resistance cloned near Pir2115-1（t）of chromosome 2 was Pib,and the expression level of Pib-R2115 in R2115was high.Meanwhile,no mutation in amino acid sequence was found between Pib-R2115and the reported Pib,indicating that Pir2115-1（t）was Pib.By analyzing the expression levels of samples at different time points after inoculation,it was found that the expression of Pid2 was up-regulated in R2115.The amino acid sequence of Pid2-R2115 showed that the amino acid sequence of Pid2-R2115 is consistent with the reported Pid2-Y1B amino acid sequence without mutation,indicating that Pid2 is present in R2115.2.Candidate gene Os03g0401951 positively regulated rice resistance to rice blastThe candidate gene Os03g0401951 was used to construct the overexpression vector and obtain the transgenic positive lines.After the identification of rice blast inoculation,it was found that in the over-expression lines,the gene expression of Os03g0401951 increased significantly compared with the control TP309,leaf disease spot area significantly decreased,the relative biomass of M.oryzae decreased significantly,and the expression level of defense-related genes also increased,indicating that Os03g0401951 increased rice resistance to rice blast.3.The overexpressed lines of new gene Os UMP1 are more resistant to rice blastInoculated Os UMP1 transgenic overexpressed lines with M.oryzae,it was found that compared with wild-type TP309,the leaf spots area of Os UMP1 overexpressed lines decreased,and the biomass of M.oryzae significantly decreased,showing certain disease resistance,indicating that Os UMP1 gene enhanced rice resistance to rice blast.At the same time,the co-isolation and identification of T1 generation of overexpressed lines were carried out,and the inoculation of M.oryzae in leaves showed that positive plants were more resistant to M.oryzae than negative plants.The leaf sheaths of overexpressed lines were inoculated with rice blast fungus fused with green fluorescent protein to observe the appressorium germination and mycelial growth.The result showed that Os UMP1overexpressed lines inhibited the infection process of rice blast fungus by inhibiting the spore germination and mycelia growth,which further indicated that Os UMP1 gene enhanced resistance to rice blast fungus in rice.Inoculated Os UMP1 overexpressed lines with rice blast fungus and a series of defense reactions were analyzed.The results showed that the transcription levels of defense-related genes KS4 and PR10b were significantly up-regulated in Os UMP1 overexpressed strains.And they produced a lot of H₂O₂,the enzyme activities of catalase（CAT）and peroxidase（POD）that decompose H₂O₂were determined,and it was found that the enzyme activities of catalase（CAT）and peroxidase（POD）in Os UMP1 overexpressed material were reduced compared with those of the wild type,thus reducing the decomposition of H₂O₂,which proved that Os UMP1 gene positively regulated the resistance of rice to rice blast.The knockout mutant ump1 of Os UMP1 gene was inoculated with M.oryzae,and it was found that compared with the control TP309,the leaf disease spot area of the knockout mutant was significantly increased and the relative bacterial biomass was also increased,indicating that ump1 gene negatively regulated the resistance to rice blast.4.Os UMP1 regulates the accumulation and enzyme activity of rice proteasomeThe proteasome content and the enzyme activity of proteasome were detected after Os UMP1 overexpressed material was inoculated with rice blast fungus.The results showed that the content and enzyme activity of 26S proteasome in Os UMP1 overexpressed material were higher than that of wild-type TP309 at the background and 12h after inoculation with M.oryzae,indicating that Os UMP1 gene positively regulated the content of 26S proteasome and the enzyme activity of proteasome.Through ubiquitination sequencing and proteomic sequencing,analysis of these data showed that,Os UMP1 may enhance the resistance of rice to multiple strains of M.oryzae or different pathogens by regulating the ubiquitin-protein degradation pathway mediated by proteasome.5.Os UMP1 positively regulated resistance to bacterial blight and rice sheath blightThe lesion length of bacterial blight and sheath blight in the leaves of Os UMP1overexpressed material were found to less than that of wild-type TP309,indicating that Os UMP1 positively regulated the resistance to bacterial blight and sheath blight,which indicated that Os UMP1 gene is a broad-spectrum disease resistance gene to a variety of pathogens.6.Os UMP1 did not affect the agronomic traits and yield of riceThrough phenotypic identification and seed examination of Os UMP1 overexpressed material,it was found that there were no significant differences between the overexpressed material and wild-type TP309 in plant height,tillers,seed setting rate,1000-grain weight and yield per plant,indicating that Os UMP1 gene did not affect agronomic traits and yield of rice.