Gene Isolation And Fuctional Analysis Of A Spotted-leaf Mutant Spl24 In Rice(Oryza Sativa L.)

spl24 is a novel lesion mimic mutant obtained from the IR64 mutant bank induced by ethyl methylsulfonate?EMS?.According to the previously research,the lesion mimic mutants usually as an important material to study the mechanism of programmed cell death?PCD?and disease resistance in plants.Therefore,we have carried out the characterization of spl24 mutant on different aspects which the phenotypic,physiological and biochemical characterization,disease resistance evaluation,genetic control,map-based cloning and functional analysis of the mutation,bioinformatics analysis and transcriptome sequencing.These results and analysis can provide some evidence for the spotted leaf mutant occurrence and disease resistance mechanism.The research includes the following aspects:1.The phenotype of the mutant spl24:the spot appeared at the beginning of the heading stage on the natural conditions and gradually spread from the leaves tip to the whole leaves.There are tiny and brown spot on the leaves,at last the leaves turn to brown in the late heading and filling stages.Under the field condition,the agronomic traits were measured,the mutant spl24 showed a significant decrease in plant height,seed setting rate and 1000-grain weight compared with the wild type IR64,while the effective tiller number,the lenth of the panicle,number of grains per panicle of the spl24 mutant have decreased compared with the wild type IR64.2.Physiological and biochemical abnormalities of the spotted leaf mutant spl24:There is no spots on the leaves at the tillering stage and the chlorophyll content in spl24leaves was no significant difference with the wild type IR64 except the chlorophyll a for which is lower in the mutant spl24 than IR64.At the heading stage,the contents of chlorophyll were significantly lower in the spot leaves for the mutant spl24 compared with wild type IR64 except the ratio of the chlorophyll a and b which is similar in the mutant spl24 and IR64.The results of DAB staining,the higher H₂O₂ content and the lower activity of CAT test in the leaves for spl24 compared with the WT IR64 indicated that there was H₂O₂ deposition in spl24 leaves.The activities of superoxide dismutase?SOD?and peroxidase?POD?for the active oxygen scavenging enzyme system of mutant spl24 were significantly higher than those of wild type.In addition,the PCD test results of the significantly increased content of malondialdehyde?MDA?,the broken DNA test in TUNEL experiment,the significantly decreased soluble protein and the significantly increased expression of death-related genes suggested that the spl24 mutants has PCD occurrence.Furthermore,the spots on the leaves were induced by light,and there were significant differences in the parameters of photosynthetic indexes between the mutant spl24 and the wild type IR64.3.The enhanced disease resistance of the mutant spl24:The results of inoculation with bacterial blight showed that the disease resistance of spl24 was enhanced compared with WT.The resistance-related genes including OsPR1a,OsPR1b,OsPR3,OsPR4,OsPAL3,OsPAL6,OsCHS1,OsAOS2,OsJAZ6 and OsWRKY45 genes in spl24 were expressed significantly higher than those in WT,but there was no difference in the expression of OsJAR1 between them.The results of phytohormone assay showed that the contents of SA,JA and ABA increased significantly,suggesting that the enhancement of resistance of spl24 was achieved by increasing the expression level of defense response genes and the pathways for these defense response genes.4.The spotted phenotype are controlled by a semi-dominant nuclear gene:spl24crossed with IR64 to obtain F₁,and the phenotype of F₁ showed the same spots as the mutant spl24,but the number of spots was less than spl24.The F₂ population of spl24 and IR64 have three phenotypes,first similar to the WT plants without spots?BC₂F₂-W?,second similar to F₁ plants with less spots?BC₂F₂-M?,at last similar to the mutant with serious spots?BC₂F₂-S?.A variant of the spotted plant indicates that the spotted leaf phenotype is controlled by semi-dominant gene?s?.In F₂ population of spl24/IR64,the isolation ratio of BC₂F₂-W:BC₂F₂-M:BC₂F₂-S=1:2:1(?₂^0.05=0.78<?₂^0.05=3.84),indicating that the spotted phenotype of spl24 was controlled by a semi-dominant nuclear gene.5.Single base mutation leads to the generation of spots phenotype:Map-based cloning was used to locate the gene in the 110 KB region at the long arm of chromosome11,which contains nine open reading frames.Sequencing revealed that the six ORFs as the candidate gene and one mutantion?T7695A?was dectect at the 7695 base of the DNA sequence in the fifth extron,resulting in a mutation from leucine?Leu?in the wild type to isoleucine?Ile?on the amino acid sequence.In order to verify the function of the gene,the complementary experiment and RNAi assay were performed.Tissue expression experiments showed that OsSPL24 was expressed in the roots,stems,leaves,sheaths and spikelets of rice,GUS experiments showed that OsSPL24 was a constitutive expression gene.6.OsSPL24 encodes a receptor-like cytoplasmic kinase:Analysis of OsSPL24 gene showed that OsSPL24 gene contains 7 exons and 6 introns with a full CDS length of 1791bp and encodes a protein with 596 amino acids residues.OsSPL24 encoded a receptor-like cytoplasmic kinase,which has two LysM motif and one protein kinase domain.Subcellular localization of OsSPL24 gene revealed that OsSPL24 protein was located in endoplasmic reticulum.Yeast two-hybrid experiment was performed for OsSPL24 and suggested that OsSPL24 protein probably interact with calcium binding protein or mitochondrial transcription termination factor.In summary,the lesion mimic phenotype is controlled by a semi-dominant gene,which is a DUF26 kinases that has never been reported.spl24 showed significant changes in physiological and biochemical parameters,and this may also contribute to produce strong resistance against bacterial blight in spl24.Therefore,the isolation of OsSPL24would further facilitate our studies on the mechanisms of OsSPL24 in charging of plant disease resistance.