Cloning And Functional Analysis Of Spotted Leaf Gene SPL37 In Rice

Spotted leaf refers to the phenomenon that plants spontaneously produce typical necrotic lesions without being invaded by external pathogens.It is a common harmful variation in rice,which can lead to poor rice quality and reduced yield.Current studies have found that programmed cell death（PCD）and activation of plant defense responses are the main causes of the spotted leaf phenotype,and spotted leaves play a role in enhancing plant disease resistance.In this study,a new spotted leaf mutant was obtained by chemical mutagenesis of japonica rice variety Taipei 309 by EMS,and named it spl37.This study plans to conduct a series of assay experiments and analyses on spl37spotted leaf mutants,including phenotypic characteristics analysis,physiological and biochemical index determination,disease resistance analysis,genetic characteristic analysis,target gene cloning and functional analysis,and bioinformatics analysis.,to understand the occurrence mechanism and disease resistance mechanism of spotted leaf mutant spl37,aiming to provide theoretical reference for the study of spotted leaf mutants and technical reference for the mining of spotted leaf genes.This research mainly includes the following aspects:（1）Phenotypic analysis of spotted leaf mutant spl37:Under natural growth conditions,the phenotype of mutant spl37 was similar to that of the wild type（WT）until the tiller stage.At the beginning of the tillering stage,some leaves at the bottom of the mutants began to have reddish-brown spots.At the peak tillering stage,spots appeared from the lower leaves to the higher leaves.At the heading stage,these spots spread rapidly and became more serious.And gradually spread over the entire leaf,and many of the bottom leaves begin to wither.After the plants grew and matured,the field agronomic traits were investigated.Compared with wild-type Taipei 309,spl37 had extremely significant reductions in plant height,ear length,number of grains per ear,and number of secondary branches.There is also a decrease in the number of branches.（2）Physiological and biochemical analysis of spotted leaf mutant spl37:At the peak tillering stage,reddish-brown spots appeared in spl37.At this stage,the content of chlorophyll b in mutant spl37 decreased significantly,while the content of chlorophyll a and carotenoids increased significantly;the contents of chlorophyll a and b in mutant spl37 were significantly decreased,carotenoid contents increased significantly,and the ratio of chlorophyll a and chlorophyll b contents increased,and the relative chlorophyll content SPAD value of mutant spl37 also decreased significantly.At the same time,through the shading experiment,it was found that light can induce the phenotype of the mutant spl37spotted leaf,and the net photosynthetic rate of mutant spl37 was also significantly lower compared to wild-type Taipei 309.By measuring senescence-related biochemical indicators,it was found that compared with wild-type Taipei 309,the hydrogen peroxide content of mutant spl37 was significantly increased,the activity of catalase（CAT）enzyme was significantly decreased,and the enzyme scavenging reactive oxygen species in mutant spl37 leaves.The activities of superoxide dismutase（SOD）and superoxide dismutase（POD）in the system were significantly increased.According to the above detection results,it was found that there was excessive accumulation of reactive oxygen species in the leaves of mutant spl37.In addition,the content of malondialdehyde（MDA）in the leaves of mutant spl37was also significantly increased compared to wild-type Taipei 309,and the number of DNA fragments detected by TUNEL experiment was also increased,proving that the mutant spl37 leaves in There are cases of cell death.Resistance analysis of spotted leaf mutant SPL37:Compared with the wild-type Taibei 309,the resistance of the mutant SPL37 to Xanthomonas oryzae was significantly enhanced.Meanwhile,qRT-PCR showed that the expression levels of related defense genes:OsMAPK12,OsAOS2,OsNPR2,OsPR1a,OsPR1b,OsWRKY53 were significantly increased,which may have a positive regulation effect on the enhancement of disease resistance of mutants.（3）The traits of the spotted leaf mutant spl37 are controlled by a single of recessive gene:artificial crosses were performed with spl37 as the female parent and 9311 as the male parent.The F₁ phenotype was normal,and no individuals with spotted leaf mutation appeared.The ratio of individuals with normal phenotype and spotted leaf phenotype was about 3:1,which was in line with Mendel’s law of segregation at a segregation ratio of 3:1,indicating that a pair of recessive genes controlled the spotted leaf trait.（4）Mutations in the SPL37 gene result in a speckled phenotype in leaves:SPL37 was mapped to a 246.4 kb region on chromosome 5 between the In Del1 and In Del2 markers by map-based cloning.Querying the Rice Genome Database（http://rice.plantbiology.msu.edu/）found that there are 27 open reading frames（ORFs）in this region,including 5 proteins of unknown function,17 transposon proteins,and 3 heat shocks protein and 2 functional proteins.Through sequencing and alignment,we found that one of the candidate genes was mutated.The 1066th nucleotide in the coding region of the gene was replaced by C,resulting in the encoded amino acid changing from lysine to glutamine.We named this gene as SPL37 candidate genes.（5）Through the website prediction analysis,it was found that the expression of SPL37 gene decreased significantly under the stress of drought and high salt.The salt tolerance of wild-type Taipei 309 and mutant spl37 were compared by the salt stress experiment of plate dish and hydroponic culture.The results showed that the mutant spl37 is more sensitive to salt treatment.In conclusion,spl37 is a spotted leaf mutant controlled by a recessive gene.The mutation of the gene SPL37 leads to obvious changes in the physiological and biochemical characteristics of the mutant,which affects the growth and development of the mutant,and also makes the mutant to some white leaves.The resistance to leaf blight was significantly enhanced.Therefore,further study of SPL37 is of great help to reveal the molecular mechanism of salt tolerance and disease resistance of rice,and can lay a foundation for breeding high-quality rice varieties with high resistance.