Cloning And Functional Analysis Of Rice Lesion Mimic Gene SPL36 In Rice

Lesion mimic mutant are spontaneous necrotic spots that are generated in the absence of biotic or abiotic stress.The study of disease-like mutants can analyze the regulatory mechanisms of programmed cell death and plant defense response-related pathways.In this study,we used EMS chemical mutagenesis to treat the rice stalk rice variety Yundao,and a mutant spl36 that could stabilize the hereditary disease-like phenotype was selected from the mutant library of mutations.Investigate how the gene SPL36 regulates programmed cell death?PCD?through experimental methods such as physiology,biochemistry,and cytology.SPL36 was mapped by map-based cloning,and SPL36 mutation sites were found by sequencing.In addition,related experiments such as functional complementation,tissue expression analysis,and subcellular localization were performed for the identified candidate genes.The effects of SPL36 loss of function on plant resistance were further explored through inoculation experiments and analysis of expression levels of defense-related genes.Finally,the function of the protein encoded by SPL36 was analyzed,and a series of salt stress experiments were conducted to explore whether SPL36 participates in salt stress response..The results showed that spl36 mutants had programmed cell death and premature senescence of leaves.SPL36 was involved in programmed cell death and induced defense responses in spl36 mutants by upregulating defense-related genes.At the same time,rice SPL36 may negatively regulate the response to salt stress.Based on experimental data,the new disease-like mutant spl36 will help elucidate the regulatory pathways of programmed cell death and disease resistance and the mechanism of rice salt tolerance.The detailed research results are shown as follows:?1?No significant changes in spl36 leaves and wild type?WT?leaves before tillering stage.In the tillering stage,the disease-like spots appeared from the leaf tips.From tillering to heading,these necrotic spots start spread and gradually spread to the entire leaf.Cover the mutant spl36 leaves with 2-3 cm aluminum foil during the tillering season,and use the uncovered mutant leaves as controls.7 days later,it was observed that there was no spread of disease spots in the covered area of the shaded leaves,while disease-like spots spread on the unshielded control leaves.This shows that the disease-like phenotype of the mutant spl36 is induced by light.At the same time,the main agronomic traits of the mutant spl36 such as plant height,grains per ear and thousand-grain weight were significantly reduced.?2?Chlorophyll a and chlorophyll b of mutant spl36 decreased significantly compared with wild type.Further determination of photosynthetic rate of plants in this period showed that the net photosynthetic rate of mutant was significantly reduced.Using transmission electron microscopy to observe the ultrastructure of chloroplasts,it was found that the chloroplasts of the mutant spl36 had shrunk,the chloroplasts became smaller,and the sheet structure inside the chloroplasts was disordered.?3?TdT-mediated dUTP Nick-End Labeling?TUNEL?showed that strong and randomly distributed of TUNEL signals in the nucleus of mutant spl36,while only weaker TUNEL signals were detected in wild type.In addition,the content of H₂O₂ and malondialdehyde?MDA?in the mutant spl36 was significantly higher than that of the wild type,and the activities of peroxidase?POD?and superoxide dismutase?SOD?decreased significantly.?4?The wild type and mutant spl36 at the tillering stage were inoculated,and the leaf blight strain HM73 was inoculated by the leaf cutting method.Observe the change of the inoculation site and measure the length of the lesions 5 days and 10days after the inoculation.After 5 days of inoculation,the wild-type leaf tips showed obvious blemishes,but the mutants did not show obvious lesions.After 10 days of inoculation,the length of the wild-type lesions was significantly longer than that of the mutants.The expression of defense-related genes in wild-type and mutants at tillering stage was examined by qRT-PCR.The results showed that the expression levels of defense genes were significantly increased,such as MPK12,WRKX53,BIMK2,AOS2,ASP90,LYP6,PR2,PR1a,PR1b.?5?Genetic analysis revealed that the spl36 phenotype is controlled by a single recessive nuclear gene.SPL36 was mapped to a 60 kb region between the Indel markers InDel1 and InDel2 by map-based cloning.A website query?http://rice.plantbiology.msu.edu/?predicts that there are 11 open reading frames?ORFs?in this region,including 7 expressing proteins and 4 functional proteins.A mutation was found in the gene LOC?Os12g08180 by sequencing and comparison.The nucleotide C of the 1462 of the coding region of the gene was replaced with T,which caused the encoded amino acid to change from arginine to cysteine.The gene encodes a receptor-like protein Kinase precursor.?6?Functional complementation experiments confirmed that the SPL36mutation is responsible for the appearance of the mutant spl36 phenotype.The SPL36overexpression vector?pUbi::SPL36?was further introduced into the spl36 mutant,and the phenotype in the positive transgenic line completely returned to normal.Transgenic plants carrying the SPL36 promoter-GUS fusion construct were used to detect SPL36 tissue expression patterns.The results showed that the GUS signal was detected in roots,stems,leaves,sheaths and young ears,which was consistent with the results of real-time quantitative PCR detection of SPL36 gene expression in various tissues.In addition,the full-length coding sequence of SPL36 was fused to the N-terminus of green fluorescent protein?GFP?,and transiently expressed in rice protoplasts and tobacco leaves,the GFP signal appeared on the plasma membrane.?7?The wild-type and mutant mutants were subjected to salt stress in the plate and salt stress in the hydroponic seedling stage,respectively.In the plate experiment,there was no significant difference in the germination rate of the mutant spl36 and the wild type without salt treatment,and the length of the root part was also not significantly different.After salt treatment,the germination rate of both mutants and wild-types decreased significantly,while the germination rate of wild-types was also significantly lower than that of mutants,and the root length of the mutants was significantly lower than that of wild-types.After salt stress in the hydroponic seedling stage,statistical analysis was performed on the fresh weight,electrical conductivity,and ultimate survival rate of the plants before and after treatment.The results showed that the mutant spl36 was more sensitive to salt treatment,suggesting that SPL36 is involved in the response to rice salt stress.