Identification And Functional Analysis Of Key Gene Related To Mature Kernels 5-foymyltetrahydrofolate In Maize

Folate is a class of water-soluble B vitamin essential for humans.It is involved in many important biology processes.In many underdeveloped countries and area,humans take up folates maily from staple crops.Most of the staple crops contain low levels of folates,and folate deficiency develops into a serious worldwide nutrient health problem.Maize is one of the largest food production crops on the world,providing main daily calories for more than 300,000,000 human people in the worldwide.Identifying key genes in maize kernels for folate variation by genetic methods can improve folate levels in mature maize kernels and solve folate deficiency nutrient health problem.Gene significantly associated with 5-F-THF concentration main in maize kernels was identified by genome wide association study in this paper.Poential functional allele variations in this gene were also identified by candidate gene association study,and gene function was demonstrated by linkage mapping,expression analysis,haplotype analysis,phylogenetic analysis,transgenic research and subcellular localization.These findings provide genetic foundation for folate metabolism and provide new ideas for improving nutriential quality in maize.The main results were as follows:1.Folate levels of mature maize kernels in 531 maize inbred lines of association population were analyzed in three environments.The results show that 5-F-THF is the main folate derivatives in mature maize kernels and there was abundant phenotypic variations in mature maize kernels.BLUP data was obtained from integrating phenotype data in three environments.BLUP data indicates that the concentration of 5-F-THF ranges from 0.58 to 8.18 nmol/g DW with a mean of 1.66 nmol/g DW.ANOVA analysis show the concentration of 5-F-THF significantly difference between various genotypes.Broad-sense heritability was calculated using 5-F-THF concentration in three environments with heritability 0.79.It is indicated that 5-F-THF concentration variations were main controlled by genetic factor.We can locate the genetic factor controlling the variations of phenotype using genetic mapping methods.2.Genome wide association study was conducted using 5-F-THF concentration as phenotype and 558529 SNPs as genotype.36 loci identified by GWAS were mapped to 9genes distributing on 5 chromosomes.A locus located on chromosome 5 was predicted to code a putative glutamate formiminotransferase I,and gene ID is GRMZM2G124863.Two complete linkage disequilibrum sites Chr5.s₁9676906 and Chr5.s₁9676907 were shown most significantly associated with 5-F-THF concentration,and both P values were1.46×10^-21.Variations on two sites can lead to amino acid substitution between Asn and Gly.This change can expain 27.6% phenotypic variation.3.Resequence of ZmGFT1 in 155 maize inbred line were carried out,and 80polymorphism sites significantly associated with 5-F-THF were identified by candidate gene association study.In 80 polymorphism sites,24 sites were in 5’UTR,8 sites were in exons,and 48 sites were in introns.Chr5.s₁9676906 and Chr5.s₁9676907 were identified again.Four InDels S422,S637,S649 and S696 were also identified in 5’UTR.The results of expression analysis indicate that 4 InDel sites wer also putative functional sites influencing 5-F-THF concentration.4.Normalized expression in seeds 15 days after pollination from RNA-seq as phenotype and 558529 SNPs as genotype,the result shows that an expression QTL(P=7.15×10^-13)was mapping to the location of Zm GFT1.This indicates there may be exist cis-regulate mechanism in ZmGFT1.QTL mapping was carried out using a F_2:3segregation population from GEMS31 and Dan3130.A QTL locus was mapped to the region which ZmGFT1 was located in.This QTL can explain 14.9% of 5-F-THF concentration variation.5.The results of haplotype analysis using 6 polyphism sites display that the genotype of Chr5.s₁9676906 was G in Hap1,Hap4 and Hap7,and Hap1,Hap4 and Hap7 have significantly high levels of 5-F-THF than other haplotypes.Hap1 contain all favour alleles variation of 6 sites,and has most high level of 5-F-THF,up to 2.12 nmol/g DW.9 haplotypes can explain 40%variation of 5-F-THF concentration.5-F-THF concentration of Qi319-GFT1 overexpression Arabidopsis seeds was significantly increase than col wildtype.However,5-F-THF concentration of B73-GFT1overexpression Arabidopsis seeds was as much as that in col wildtype.5-M-THF concentration of Qi319-GFT1 and B73-GFT1 overexpression Arabidopsis seeds were significantly decrease than col wildtype.5-F-THF concentration of B73-GFT1overexpression maize leaves was significantly increase than control group.In contrast,5-F-THF concentration of RNAi maize leaves was significantly decrease than control group.A three-dimensional protein structure of ZmGFT1 was predicted using Swiss-Model using homology modeling.It was observed that the binding pocker of ZmGFT1 located at the interface of two protomers.The Asn-to-Gly change at S2069occurred at the interface,which might change the spatial structure of the binding pocket and then influence the function of the protein.The subcellular localization of ZmGFT1was investigated.The results show that ZmGFT1 protein was co-localization with nuleus and endoplasmic reticulum,but not to mitochondria,peroxisomes or Golgi apparatus.