Cloning And Functional Analysis Of ZmSMK9 And ZmSrl5,Two Yield-Related Trait Genes In Maize

The genetic improvement of maize kernels and stress adaptability can effectively ensure the high and stable yield of maize,and food security.The mapping and cloning of maize kernel traits and stress adaptability-related genes can provide effective gene resources for high and stable yield of maize.Two maize yield related mutants,smk9?small kernel 9?and srl5?semi-rolled leaf 5?,identified by our research group were used as study materials.Two maize yield related genes were successfully cloned through the strategies of phenotype identification,genetic analysis and map based cloning,which were named as Zm SMK9 and Zm SRL5 respectively.The function of the two genes and the mechanism of the mutated phenotypes were further elucidated.The main research results are as follows:1. Phenotypic identification of smk9.Compared with WT,the kernel size of smk9 was reduced,and the 100-kernel weight was about 45%of wild type;the endosperm and embryo development of smk9 was delayed,however,smk9 could develop into a nomal kernel with the intact structures,which germinated normally,and developed into a more compact plant.2. Mapping and cloning of Zm SMK9.Genetic analysis showed that smk9 was controlled by a single recessive nuclear gene?3:1,?²-test,p=0.56?.Zm SMK9 was mapped into a 700 kb interval using 26950 F₂ segregated kernels derived from B73 and smk9,containing six candidate genes.According to the results of resequencing and sequence alignment analysis of the candidate genes between WT and smk9,there was a single nucleotide deletion at+965 bp of GRMZM2G420723 coding sequence from initiation codon ATG,which caused frameshift mutation,while there were no differences in the sequences of the other five genes between the two materails.So GRMZM2G420723 was the key candidate gene for Zm SMK9,which encodes a p-type PPR protein containing 674amino acids,and named as Zm SMK9.3. Functional verification of Zm SMK9.Allelic mutant smk9-1 of Zm SMK9 wasanalyzed,which was caused by a single-base mutation at+718 bp of Zm SMK9 from initiation codon,resulting in the termination mutation by CAA to TAA.Selfing ears of+/smk9-1 heterozygous showed 3:1 kernel size segregation ratio??²-test,p=0.80?,which confirmed that Zm SMK9 was the functional gene controlling mazie kernel development.Zm SMK9 was a constitutively expressed gene,and Zm SMK9 protein located in mitochondria.4. Mechanism analysis of Zm SMK9.Mitochondrial gene expression and blue-nativepolyacrylamidegel-electrophoresis?BN-PAGE?analysis showed that Zm SMK9 was involved in the splicing of nad5 intron 1 and intron 4,and splicing efficiency of nad5 intron1 and intron 4 decreased in smk9 mutant;the normal assembly of mitochondrial complex I was affected in smk9,which led to complex I activity decline,the oxidative phosphorylation way block,and insufficient energy to feed kernel development,thus resulting in small kernels.5. Phenotypic identification of srl5.Compared with wild type,srl5 exhibited semi-rolled leaf,and development and yield related traits were affected to some extent;the number of bulliform cells increased significantly in srl5,and the cell wall structure of prothenchyma was abnormal,while the contents of main components?cellulose,hemicellulose,lignin and pectin?of cell wall in leaves and sheaths showed no significant difference between srl5 and wild type;stomatal density,stomatal pore size and response to ABA showed no significant difference between srl5 and wild type.6. Mapping and cloning of Zm SRL5.Genetic analysis showed that srl5 was controlled by a single recessive nuclear gene?3:1,?²-test,p=0.78?.A map based cloning strategy was uptaken by using 4,596 F₂ individules derived from B73 and srl5 to map Zm SRL5 into a 90kb interval,which contained two candidate genes,Zm00001d028159 and Zm00001d028160.Sequencing analysis showed that there was no sequence difference in Zm00001d028159 between wild type and srl5,while there was a 3745bp insertion in the first intron of Zm00001d028160 in srl5.The RNA-seq reads joints and RT-PCR results confirmed that the insertion in Zm00001d028160 resulted in truncated transcripts in srl5.So Zm00001d028160 was the key candidate gene,and named as Zm SRL5,which encodes a CASP-like protein with four transmembrane domains.7. Function verification of Zm SRL5.Two fragment deletion alleles of Zm SRL5obtained using CRISPR/Cas9,KO1 and KO2,shared similar phenotype with srl5,which verified that Zm SRL5 was the functional gene.The spatio-temporal expression analysis showed that Zm SRL5 was constitutively expressed,and had high expression in the developing leaves and low expression in the mature leaves.Subcellular localization results showed that Zm SRL5 protein located in the cell plasma membrane.8. Zm SRL5 can improve drought and salt tolerance of plants,ensure the normal growth and development of plants,and increase the yield.Compared with wild type,srl5,KO1 and KO2 showed enhanced sensitivity to drought and salt stress,and the accumulation of Na⁺in srl5 was significantly higher,indicating that Zm SRL5 was a drought and salt tolerance related gene.There was no significant difference in root related traits and root anatomical structures between wild type and srl5.The leaf cuticular permeability was significantly enhanced in srl5 and knockout lines,manifested by a faster leaf water loss rate,chlorophyll extraction rate,and lower leaf surface temperature.The epidermis of wild type was covered by platelet wax crystals densely and uniformly,while in srl5 and knockout lines,the cuticular wax crystals showed irregular and uneven distribution,almost half of which fused and stacked into blocks,and the rest were sparsely distributed.However,there was no difference in total wax content between wild type and srl5 by GC-MS?Gas Chromatography-Mass Spectrometer?analysis.A comprehensive analysis of the above experimental results further indicated that Zm SRL5 was a drought and salt tolerance related gene,which can improve drought resistance and salt tolerance by maintaining the uniform wax crystal structure and distribution,and the normal cuticular permeability of leaves,so as to ensure the normal growth and development of plants and increase the yield.9. Expression analysis of wax synthesis and transportation-related genes.The RNA-seq results showed that the expression of wax synthesis and transportation-related genes showed no difference or less difference between wild type and srl5,which were verified by q RT-PCR results.In summary,the mutation of Zm SRL5 had little influence on the wax synthesis and transport process,which might affect the cuticular permeability mainly by affecting the morphology and distribution of waxy crystals,resulting in enhanced sensitivity to drought and salt stress.