| The timely senescence of maize leaves is an important process for their development and adaptation to the environment.It is an important factor in the redistribution of photosynthetic products and nutrients and in promoting the accumulation of dry matter in grains.It is of great significance to explore key genes that control maize leaf senescence and clarify their mechanisms of action in order to extend the functional period of leaves and increase maize yield.In this study,a maize leaf early senescence mutant els5 discovered by the research group in the breeding selection process was used as the material.Its morphological and physiological characteristics were determined by phenotype analysis and physiological index determination.Its candidate genes were identified by genetic analysis,map-based cloning,transcriptome,proteome and association analysis.The potential functional motifs and protein structures of candidate genes were explored by combining bioinformatics analysis.The main research results of this study are as follows:1.Phenotypic analysis of els5: Compared with the wild type,the mutant els5 showed a yellowing phenotype of leaves after the 5-leaf stage.The yellowing leaves occurred from bottom to top,and the yellowing speed gradually increased with the development process.The els5 plants died prematurely after the adult stage.At the 9-leaf stage,the relative content of chlorophyll(SPAD),maximum quantum efficiency of PSII photochemistry(Fv/Fm),net photosynthetic rate,stomatal conductance and transpiration rate of panicle-leaf in els5 plants decreased significantly.At the same time,oxidative stress-related malondialdehyde(MDA)and hydrogen peroxide(H2O2)accumulated,and the enzyme activities of catalase(CAT)and superoxide dismutase(SOD)that remove H2O2 also increased accordingly.At the adult stage,the plant height,panicle height,internode length,male panicle length and underground biomass of els5 were significantly reduced.2.Genetic analysis and positional cloning of els5: Genetic analysis was performed on the BC1F1 and F2 segregation populations of els5 and B73.The results showed that the ratio of normal plants to els5 phenotype plants in the segregation population was consistent with the Mendelian segregation ratio of 1:1 and 3:1,indicating that the els5 mutant was controlled by a single recessive nuclear gene.By using the BSA(Bulked Segregant Analysis)method,the candidate gene was located between the 660 In Del2 and 860 In Del markers on chromosome 8 of maize,with a physical distance of about 1.1Mb on the maize B73 reference genome.This interval contains 11 protein-coding genes,eight of which are expressed in leaves.3.Preliminary identification of candidate gene: Due to the lack of single plant crossing,it is difficult to further narrow down the mapping interval through map-based cloning,and els5-specific nucleotide variations were not detected in the coding regions of the 11 genes.To clarify whether the mutant phenotype of els5 was caused by differential gene expression,this study further analyzed the expression of these 11 genes in wild type and els5 by RNA-seq and q RT-PCR.The results showed that Zm00001eb346790,Zm00001eb346800,Zm00001eb346810 and Zm00001eb346850 had differential expression between wild type and els5.In addition,the promoter sequencing results showed that the promoter of Zm00001eb346800 in els5 specifically inserted 140 bp.The candidate gene association analysis showed that there were two SNPs significantly associated with Fv/Fm at the 3’ end of this gene(P = 2×10-4).The above results indicate that Zm00001eb346800 is a potential candidate gene for els5 and is named ZmELS5.4.Functional annotation of candidate gene: ZmELS5 encodes a methanol dehydrogenase-type β-propeller structural domain protein with a highly conserved TPM_phosphatase structural domain and one transmembrane domain,but lacks a signal peptide.This protein is a hydrophilic protein localized in chloroplasts.The analysis of cis-elements in the promoter showed that there were multiple light-responsive elements(Box 4,G-box,MRE,GTGGC-motif,I-box)and hormone-responsive elements abscisic acid(ABRE),jasmonic acid(CGTCA-motif,TGACG-motif).5.Transcriptome and proteome combined analysis of ZmELS5 regulatory network:To elucidate the potential regulatory network of ZmELS5 in regulating maize leaf senescence,transcriptome and proteome of leaves at V13 stage of els5 and its nearisogenic line Jing24 were analyzed.The results showed that the differentially accumulated proteins between the two mainly enriched in functional categories such as“ribosome”,“photosynthesis”,“porphyrin and chlorophyll metabolism”,“photosynthetic antenna protein”.The interaction regulatory network revealed that ZmELS5 was directly related to photosynthesis-related processes,involving the repair of photosystem II.The down-regulation of this gene led to the down-regulation of photosynthesis-related genes and chlorophyll degradation in leaves,once again proving ZmELS5 as a potential candidate gene.This study laid a foundation for the determination and functional study of els5 candidate genes. |
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