Gene Mapping Of A Space Mutagenesis Closed Glume Mutant Maize And Functional Analysis Of Key Candidate Genes In Leaf Color Mutants At Seedling Stage

The closed glume 1（cg1）is a mutant progeny obtained by spatial mutagenesis of the inbred line RP125.The mutant phenotype is that the male flower glume does not crack.Etiolated-albino leaf 1（eal1）at seedling stage is a polymeric descendant of ms39 obtained by spatial mutagenesis.Previous studies supposed Zm00001d049160（ZmSig2A）in molecular marker location region as the key candidate gene.eal1 compared to the control,ZmSig2A showed a level of amino acid valine（Val）residue deletion,the eal1’s ZmSig2A was named ZmSig2A^ΔVal480.In this study,the genetic analysis and molecular marker localization of mutant cg1 were performed.Functional analysis and verification of eal1’s key candidate gene ZmSig2A were performed on the basis of previous studies.The main research results are as follows:1.The cg1 mutant traits in Yunnan and Sichuan are stable and heritable.The mutant cg1 hybridized with the inbred lines B73 and Mo17,and the F₁ glume was cracked,and the pollen was released.The F₂ population showed trait separation.The ratio of the released pollen plants and the mutants was close to 3:1,indicating that the mutant traits were controlled by a pair of recessive nuclear genes.The cg1 gene was located near the long arm centromere of chromosome 9,located between IDP74592930 and umc1271,with a physical distance of 15.6Mb.The genetic distance between cg1’s mutation site and IDP66919480 was 0.18c M,and that between cg1’s mutation site and umc1271 was0.35c M,respectively.2.The expression of ZmSig2A^ΔVal480 and ZmSig2A in the first leaves and the second leaves of the mutant eal1 and Wt were examined by q RT-PCR.The results showed that the expression of ZmSig2A^ΔVal480 in the untransformed first leaves and the second leaves was significantly higher in the mutant than the expression of ZmSig2A in the wild type.Furthermore,using the second leaf as a material,the expressions of ZmSig2A^ΔVal480 and ZmSig2A in eal1 and Wt after greening and greening were compared.It was found that the expression of ZmSig2A^ΔVal480 and ZmSig2A genes decreased with leaf development.At the same time,the expression of ZmSig2A^ΔVal480 in eal1 before greening was significantly higher than that in ZmSig2A in Wt,but there was no significant difference in expression between the two after greening,indicating that there was a certain relationship between the difference of phenotype and the difference in expression.3.The ZmSig2A protein was localized to the chloroplast by constructing a ZmSig2A and EGFP fusion expression vector for transient expression in tobacco epidermal cells.Furthermore,the expressions of some photosynthetic pigment synthesis related genes,chloroplast development related genes and photosynthesis-related nuclear genes between mutant and wild type were compared and analyzed.The expression of some photosynthetic pigment synthesis genes and chloroplast development related genes in eal1 mutants was significantly affected,and the expression of some photosynthesis-related nuclear genes was significantly different compared with the control.4.Based on the previous ZmSig2A transformation of Arabidopsis thaliana sig2 mutant,12 T₃ generation single copy transgenic lines were identified by resistance screening and genomic PCR amplification.At the same time,a mutant ZmSig2A^ΔVal480 over expression vector was constructed to transform Arabidopsis sig2 mutants,and 10 T₃ generation single copy transgenic lines were screened and identified.The results of transgenic phenotype showed that all T₃ generation single copy transgenic lines heterologously expressing ZmSig2A could restore the Arabidopsis sig2 mutant phenotype,indicating that ZmSig2A not only has similar sequence to SIG2,but also has similar protein function.However,the T₃ generation single copy transgenic line heterologously expressing ZmSig2A^ΔVal480 can only partially restore the sig2 mutant phenotype,indicating that the deletion of the Val residue in the mutant ZmSig2A^ΔVal480has an effect on the protein function of ZmSig2A;A partial sig2 mutant phenotype was restored in the transgenic lines,presumably because the ZmSig2A^ΔVal480expression level in this line was higher than that of other lines that could not restore the mutant phenotype.5.An over expression vector of ZmSig2A gene was constructed,and maize inbred line B104 was used as a transgenic receptor material.An over expression vector of ZmSig2A gene was constructed,and maize inbred line B104 was used as a transgenic receptor material.Then,through agrobacterium-mediated genetic transformation of B104 immature embryos,30 T₁transgenic ears were obtained,and the selected T₁ transgenic plants were mixed with mutant eal1 to form F₁,and the transgene fragments were introduced into eal1mutant.We will obtain F₂ isolates through F₁ self-cross,and complete functional complementary verification of ZmSig2A gene through the combined comparative analysis of genotype and phenotype in the isolates.