Phenotypic Identification Of Four Rice Dwarf And Susceptible Allelic Mutants And Its Gene Function Study

Plant height and disease resistance,as two key factors affecting rice yield,play important roles in rice breeding.The application of dwarf resources and disease-resistant resources in breeding has greatly increased the yield of rice.However,in traditional breeding,a single genetic background will reduce the resistance of bred varieties.Therefore,it has become a hot spot for breeders to explore dwarf and disease-resistant resources.Four dwarf and susceptible rice sheath blight mutants were identified in Xinong 1B mutant library treated by EMS mutation.The main conclusions of histochemical section analysis,scanning electron microscope analysis,photosynthetic pigment content and photosynthetic parameters analysis,expression pattern analysis and genetic analysis of these four mutants are as follows:1.Phenotypic analysis and agronomic traits investigation of dssb1 and allelic mutants.Compared with wild type,the internodes of dssb1 and allelic mutants are shorter in the whole growth period.And the leaf color of dssb1 and allelic mutants deepened.In the field planting investigation,it was found that the incidence of sheath blight of these four mutants was more serious than that of wild type.2.Determination of photosynthesis and photosynthetic pigment content of dssb1 and allelic mutants.The photosynthetic characteristics of dssb1 and allelic mutants were analyzed.Except dssb1-2,the net photosynthetic rates of dssb1,dssb1-1 and dssb1-3 were higher than those of wild type,and the stomatal conductance,intercellular carbon dioxide content and transpiration rate of these four allelic mutants were also higher than those of wild type.The results of determination of photosynthetic pigment content showed that the contents of chlorophyll a and chlorophyll b of dssb1,dssb1-1,dssb1-2and dssb1-3 were higher than those of wild type,and the difference reached a very extremely significant level,which was also the reason for the deepening of leaf color of mutant.3.Histochemical section analysis of dssb1 and allelic mutants.The middle leaves of dssb1 and allelic mutant the first leaf were analyzed by frozen section,and the main and lobule veins of leaves were statistically analyzed.It was found that the number of main veins of dssb1-1,dssb1-2 and dssb1-3 was less than that of wild type except dssb1,while the number of lobule veins of dssb1-1 was more than that of wild type.Moreover,frozen sections showed that the size of leaf epidermal silicified cells increased significantly,and the number was significantly higher than that of wild type.And the leaf edges of mutants were abnormal.At the same time,we made paraffin sections on the leaves of dssb1 and allelic mutants,and found that the vascular bundle development of dssb1,dssb1-1,dssb1-2 and dssb1-3 mutants was inconsistent with that of wild type,and all of them were abnormal.Meanwhile,paraffin sections showed that the aerenchyma of leaves was also abnormal.4.Determination of cell wall components in dssb1 and allelic mutants.The cell wall components of wild type and allelic mutant leaves were determined.The results showed that lignin and hemicellulose content of allelic mutant decreased,while cellulose content increased.5.Disease resistance analysis of dssb1 and allelic mutants.When planted in the field,it was found that all the four allelic mutants were susceptible to sheath blight.After artificial inoculation with sheath blight and bacterial blight,the investigation and statistics showed that both sheath blight and bacterial blight were seriously affected 14 days after inoculation.At the same time,the expression of genes NPR1,PR1a,PR10,and WRKY45 related to the course of the disease were significantly reduced in the mutant,and the difference reached a extremly significant level.6.Genetic analysis and gene mapping of dssb1 and allelic mutants.The genetic analysis of F₂population constructed by crossing these four allelic mutants with Jinhui 10 showed that the mutant phenotype of these four mutants were controlled by a pair of recessive nuclear genes,and the target gene was finally locked between indel3-2 and indel3-3 primers on chromosome 3 by fine mapping,with the physical distance of about 78Kb.Through the comparison of PCR amplification and sequencing of the genes in the interval,it was found that these four mutants were mutated at different positions in the coding frame of LOC＿Os03g04680.7.Expression analysis of DSSB1.Real-Time PCR analysis showed that DSSB1 gene is expressed in all parts of rice,but its expression in panicles is significantly higher than that in other parts,followed by leaf sheaths and leaves,and low expression in roots and stems.Compared with the wild type,in these four mutants,the expression of the DSSB1 was significantly up-regulated,and the difference reached an extremly significant level.8.Protein analysis and functional identification of DSSB1.The full-length coding frame of DSSB1 gene is 1617bp,which encodes a cytochrome P450monooxygenase Os CYP96B4 containing 538 amino acids.The theoretical molecular weight of the protein is 60434.32Da and the theoretical isoelectric point is 9.19.Subcellular localization showed that DSSB1 was an endoplasmic reticulum protein.Yeast self-activation test showed that DSSB1 protein had no self-activation activity.