Gene Mapping And Complementary Verification Of A Lesion Mimic Semi-dominant Mutant Als1 In Rice

In the long-term process of evolution,plants have developed fine defense system to prevent the growth and spread of pathogens.Plant diseases could directly affect plant nutrient absorption,photosynthesis,growth and metabolic activities to affect plant yield and quality traits.Most lesion mimic mutants demonstrate enhanced resistance to pathogens,which is extremely important for plant breeding and the study of disease resistance mechanism of plants.In this study,a lesion mimic mutant als1（apoptosis leaf and sheath 1）was identified from ethylmethane sulfonate（EMS）treated rice three-line restorer,Jinhui10.Als1 showed lesion mimics on both leaf blade and sheath.This study conducted phenotypic analysis,cytological observation,expression analysis of defense-related genes,histochemical analysis,genetic analysis,physiological index determination,gene expression site analysis,light-induced analysis and photosynthetic pigment content determination,etc.Using molecular marker-assisted breeding,genetic linkage analysis and fine mapping of candidate gene was carried out and also complementary verification of the candidate gene.This study laid the foundation for the study of disease resistance mechanism.The main studies are as follows:1 Phenotypic analysis of mutant als1The phenotype of als1 was consistent with the wild-type in the seedling stage.From the four-leaf stage,brown spots appeared on the forth leaf blade and leaf sheath from top to bottom.As plant growth,brown spots increased gradually until all the leaves and sheaths were filled,the phenotype maintained in the whole life stage.The leaves gradually withered from the tip of the blade to the bottom part of the blade.Compared with the wild-type,the plant height,primary branch number,secondary branch number,ear length and 1000-grain weight of als1 were significantly decreased.In addition,the number of effective panicles,grains per panicle,and grains per panicle were extremely significantly decreased.Thus,the seed setting rate was significantly reduced.2 sunlight induced reaction of als1To observe whether the appearance of rust spots were induced by light,the leaves of wild-type（WT）and mutant als1 without rust spots were shielded with tin foil paper at the tillering stage.The results showed that no rust was observed on leaves of the shading part of both WT and als1;rust spots were regenerated on light-shielding parts of leaves in als1 after restored to light for several days and no rust spots were observed in the WT.3 Photosynthetic pigment content analysisThe determination of photosynthetic pigments content showed that carotenoids（car）,total chlorophyll（Total Chl）,chlorophyll a（Chla）,and chlorophyll b（Chlb）showed significant reduction in the mutant compared with the wild-type.4 Cytological observationThe cross-cutting of the leaves was observed by fluorescence microscopy.Under white light,the wild-type mesophyll cells showed a tender green color.Under ultraviolet light,the cells were arranged neatly and almost no enamel accumulation;while mesophyll cells of als1 showed brown color under the white light and the cell arrangement is slack and severely damaged and a large amount of enamel deposition in the ultraviolet light.It is indicated that a large number of mesophyll cell structures are damaged in the mutant.5 Histochemical analysisLeaves of the wild-type and mutant als1 in the tillering stage were subjected to diaminobenzidine（3,3,-Diaminobenzidine,DAB）and trypan blue staining.The results of DAB staining showed that there were a large number of dark brown aggregates precipitated in and around the rust spots of als1,while almost no dark brown aggregates in the wild-type leaves.After trypan blue staining,the rust spots and surrounding area of als1 leaves appeared dark blue,and the wild type leaves show a uniform light blue distribution.The results indicated that there was a large amount of reactive oxygen species（ROS）accumulation around the rust of ALS1 mutant,and cell death occurred in als1 leaves.6 Determination of physiological indicatorsCompared with the wild-type,the enzyme activities of protective enzyme system catalase（CAT）,superoxide dismutase（SOD）,and peroxidase（POD）showed a significant reduction in the first,the second and the third leaves of mutant als1 from top to bottom.Except that the content of H₂O₂ showed almost no difference between the wild-type and the mutant in the third leaf,the content of·OH and O₂^-increased significantly in the all three tested leaves.At the same time,the content of H₂O₂increased significantly in the first and the second leaves from top to bottom.The above results indicated that there was a large amount of active oxygen outbreak in als1.7 Defense response-related gene expressionIt has been reported that plant defense responses in disease-like mutants are often activated.The quantitative analysis of related genes involved in defense response was carried out.The results indicated that the oxygen stress-related protein genes POX22.3and POC1,plant secondary metabolite pathway-associated protein gene OsPAL4,and acquired immune-related protein gene NH1 were significantly raised.In the leaves and sheaths of rice,the expression levels of pathogenic protein genes PR1a,PR1b,PR2 and PR10 were significantly up-regulated.It was shown that the resistance reaction was activated in the mutant als1.8 Genetic analysisThe mutant als1 was obtained by EMS mutagenesis.The F₁ generation were generated by crossing homozyous als1 with restorer line,Xiong 1A.All the F₁population showed the lesion mimic phenotype of als1,and the traits of self-crossed F2generation were isolated,including 2198 plants of lesion-like mutants and 688 normal plants,whose genetic separation ratio is in accordance with the 3:1 of Mendel’s law of inheritance.It was indicated that the mutant als1 was controlled by a pair of dominant nuclear genes.9 Gene mapping and complementatary verificationThe F₂ generation of normal phenotype was used to genetically mapping the candidate gene.Finally,the target gene was located between the marker DD-1 and DD2-1 on the 7th chromosome with a physical distance of 242 kb.There were 11candidate genes in this interval.By gene cloning and genome and CDS sequencing,it was found that only one base mutation occurred in Os07g0488400.To further determine the candidate gene Os07g0488400 is our target gene,ALS1.The coding frame of the mutant Os07g0488400 was overexpressed in the wild-type by genetic engineering technology.The transgenic T₀ generation plant showed the lesion mimic phenotype of als1,and the gene sequencing mutation site was bimodal.This result confirmed that Os07g0488400 is the target gene ALS1.10 ALS1 gene expression analysisQuantitative analysis of the roots,stems,leaves,sheaths and ears of the mutant als1 at booting stage revealed that the ALS1 gene was mainly expressed in leaves and sheaths.In addition,quantitative analysis of mutant leaves with different lesions revealed that the deeper the lesion,the higher the level of expression of ALS1.11 Rice blast resistance analysis and hormone determinationTotal salicylic acid（SA）and jasmonic acid（JA）contents in wild-type and mutant were determined by HPLC.Compared with the wild-type,the SA content in the mutant als1 was significantly increased,and the JA content was extremely significantly increased.This indicates that the ALS1 gene is involved in SA and JA-mediated defense responses.In order to determine whether als1 showed the enhanced resistance to rice blast,als1 and the wild-type were inoculated with two stains of rice blast:ZA31 and ZB15 in the five-leaf stage.Als1 was found to exhibit disease resistance to both strains,and the total area of rice blast lesions was significantly less than that of the wild-type.