Characterization And Gene Cloning Of Senescene Mutant Els-d1 In Rice

Rice is one of the most important edible crops for human beings,the premature senescence of rice leaves leads to a series of effects on the growth and development of rice,finally,it will cause the reduction of rice yield and quality.Thence,it is very important to research the premature senescence of rice because it is an important factor that restricts the yield and quality of rice.In this study,we obtained a rice early senescence mutant els-d1 by EMS chemical mutagenesis.We observed the phenotype of mutant els-d1,we performed phenotypic observation,agronomic traits identification,chlorophyll content determination,trypan blue staining and DAB staining,and constructed a genetic analysis population and a gene localizing population,respectively.The candidate genes controlling the phenotype were identified by map cloning.The main results are as follows:1.Phenotypic characterization: 10 days after sowing,the mutant els-d1 plants began to yellow from leaf tip and leaf edge,then curled,and the plant height was significantly higher than the wild-type,the angle between mutant els-d1’s leaf was also bigger than the wild-type.2.Investigation of agronomic traits: By looking at the agronomic traits of the wild-type DG and the mutant els-d1 in the mature period,we found that the spike length,1000-grain weight,plant height and yield of the mutant els-d1 were significantly lower than those of the wild type.3.Photosynthetic pigment content determination: The photosynthetic pigment content in the leaves of the mutant els-d1 was measured during the three periods-before senescence,at the beginning of senescence and obviously at the time of senescence.The contents of chlorophyll a,chlorophyll b and chlorophyll in mutant els-d1 leaves in three times respectively showed downward trends,and all of them were significantly lower than those in the wild type.4.MDA content and soluble protein content determination: The contents of MDA and soluble protein in the leaves of the mutant els-d1 were measured before senescence,at the beginning of senescence and obviously at the time of senescence.The content of MDA in the leaves of the mutant els-d1 was higher than that in the wild-type,but the change trend of the soluble protein was opposite.5.Trypan blue and DAB staining: Trypan blue staining was carried out on the leaves of the same part of the mutant els-d1 and the wild type,and the leaves of the mutant els-d1 with premature senescence phenotype showed a large number of dark blue spots after staining,indicating cell death,In the wild type,there are almost no spots.After DAB staining,the mutant els-d1 leaves had a large number of reddish brown spots,indicating a large amount of hydrogen peroxide accumulation,while wild type leaves had almost no reddish brown spots.6.Genetic analysis: We performed reciprocal crosses between the mutants and rice 02428.We found that all of the F1 plants displayed normal phenotype.In contrast,the F2 generation had segregation of character.els-d1×02428 appeared segregation with wild type and mutation phenotypes.The segregation ratios match the value of 3:1 under X2-test.These results suggest that the els-d1 mutant phenotype is controlled by a recessive single nuclear gene.7.Gene mapping: Using SSR and Indel markers to analyze the F2 populations,the mutant els-d1 was located on the long arm of chromosome 3 of rice,and the physical distance between labeled In Del76 and In Del79 was 84 kb.The region contained 13 genes,of which 7 genes had functional annotations.Els-d1^candidate-2 was selected as a possible candidate gene by sequencing comparison.8.vector construction:In order to verify whether the gene Els-d1^candidate-2 is the target gene,we constructed a recovery vector p CAMIBA1300-Els-d1^candidate-2.