Identification And Gene Cloning Of Mutant Qk2 Of Maize Grain Filling Defect

Maize is an important food crop.Its planting area and output rank first in China.It is the main feed and the main food source of some areas in China.At present,it has become a crop for both food and feed.In recent years,the supply of corn is gradually threatened,and its yield is affected by various environmental factors and the reduction of cultivated land area.As the main organ of yield traits,the research on the excavation and function identification of the genes related to the development of grain is of great significance to the analysis of the genetic mechanism of the formation of yield traits and the cultivation of high-yield and high-quality corn varieties.Grain mutants are the basis of genetic research on Yield Traits in maize.In this study,a gap mutant named qk2（quekou-2）with starch deficiency at the top of corn grain was obtained by EMS（Ethyl methanesulfonate）mutagenesis of pollen of inbred line RP125.The agronomic characters of qk2 were investigated,the related physiological and biochemical indexes were measured,the map cloning and allelic verification of ZmQK2 gene were carried out,and the biological functions of ZmQK2 were preliminarily analyzed by combining the bioinformatics analysis of candidate gene,quantitative analysis of tissue and subcellular location.The researchresult are as follows:1.The phenotypic characteristics of qk2 were as follows:the grain length,grain width,ear weight,axle weight,100 grain weight,ear weight,ear length,ear diameter and axle diameter of the mutant were significantly smaller than those of the wild type;the leaf length,leaf width,leaf area,plant height,ear height,internode length and wild type were not significantly different;the starch synthesis and accumulation defects of qk2 began to appear 15 days after pollination,and increased with the filling time The addition gap phenotype is more and more obvious.2.Genetic analysis:the B73×qk2 genetic population was constructed.F₁hybrids had no gap separation.The number of seeds with gap phenotype separation appeared on F₁selfing ears.The number of normal and gap seeds was investigated.Statistical analysis showed that the separation ratio of normal and gap was 3:1,indicating that the mutation was controlled by recessive single gene.3.Fine mapping of ZmQK2:combined with BSA mapping strategy,20 recessive and 20 normal individuals in F₂population of B73×qk2 were pooled.More than 200pairs of polymorphic SSR markers were used for genotype analysis.It was found that the SSR marker bnlg2231 on chromosome 6 was separated from the mutant phenotype,and the mutant gene was located on chromosome 6.Then 10 polymorphic indels markers were developed,and the genetic exchange rate was calculated by using the genotype data of 742 recessive individual plants.Finally,the candidate genes were precisely located between indel-9 and indel-10,with a physical distance of 6.7 Kb.There was only one open reading frame in this segment,and the annotated gene was Hexokinase7（HXK7）,with a total length of 3,032 bp,7 exons,encoding 459 amino acids.4.Cloning and allelic verification of ZmQK2:using the fragment of HXK7 gene to design the amplification primer of the full-length sequence,cloning the genome of the mutant and the wild type respectively.The sequencingresult show that there is a base mutation from G to a on the fifth exon of this gene,which leads to the mutation of the amino acid encoded from glycine（G）to glutamate（E）;combining with the mapping population All recessive single plants were sequenced in a mixed pool,and the mutation was homozygous,which was initially identified as a candidate gene.In addition,another similar phenotypic material qk2-1 in the mutant library was hybridized with qk2,and the F₁Seed also showed a gap phenotype.Further sequencing showed that qk2-1 had a base mutation from G to A on the fifth exon of HXK7 gene,and the amino acid encoded was from glycine Theresult showed that the HXK7 encoded by ZmQK2 was the gene controlling the gap phenotype.5.Determination of physiological and biochemical indicators:the soluble sugar content of qk2 in mature grains was slightly higher than that of wild type;the amount of enzymes in grains did not change much at different time points after pollination between mutants and wild-type materials,but the enzyme activity in mutants decreased significantly.There are significant differences between 6 DAP and 12 DAP grains compared to wild type,and there are extremely significant differences between8 DAP,15 DAP,and 20 DAP grains.6.Bioinformatics analysis of ZmQK2:phylogenetic tree analysis showed that zmqk2 had eight family homologous genes in maize;Sb HXK7（Sorbi-g069800）gene in sorghum had the highest homology with ZmQK2;sequence alignment showed that ZmQK2 gene had three conserved domains in different species,and the mutation sites of qk2 and qk2-1 were all in the conserved domain;however,these loci were all in the conserved domain The point mutation did not change the three-dimensional conformation of the protein.7.Tissue expression characteristics:ZmQK2 was expressed in different tissues and grains at different stages,and the highest expression was found in grains 8 days after pollination.In addition,the eight families of HXK homologous genes in maize were expressed in the grains at different time after pollination.The expressions of ZmHXK4,ZmHXK5,ZmHXK8,ZmHXK9 and ZmHXK10 were mainly concentrated in the period from the end of pollination to the early stage of grain filling,i.e.the early stage of grain development;the expression of ZmHXK6 was mainly concentrated in the grain filling stage;while the expressions of ZmHXK3a and ZmHXK3b were mainly concentrated in the early stage and filling stage of grain development High expression.It is suggested that HXK family genes play an important role in the process of corn grain development and yield formation.8.Subcellular localization of ZmQK2:a primer with ecor1 site was designed to amplify the longest transcripts of ZmQK2 gene.The transcripts were connected with pM999-35s:GFP vector by homologous recombinase,and the recombinant plasmids were constructed and transferred into maize protoplasts for subcellular localization.Fluorescence confocal microscopy showed that ZmQK2 was located in the cytoplasm.9.The mutation caused qk2 to weaken the perception of external sugar signals:the seeds germinated by qk2 and RP125 were placed on MS medium containing different concentrations of glucose.After germination,they were cultured in the dark for 7 days and the growth of the seedlings was observed.When the glucose concentration is lower than 4%,there is no significant difference in the growth of the seedling hypocotyl,but at 4%glucose concentration,the growth of wild-type seedling hypocotyl growth is significantly higher than that of the mutant,when the glucose concentration is 5%-6%The performance is extremely significant,indicating that the mutation of ZmQK2 reduces qk2’s perception of external sugar.10.The possible mechanism by which ZmQK2 regulates grain filling:Further analysis revealed that the amino acid residue encoded at the mutation site was the target of myristoylation modification.The mutation caused the loss of the myristoylation site of HXK7.The process of myristoylation modification affects its normal physiological function;at the same time,the activity of superoxide dismutase in the mutant 12 DAP grain is significantly increased.It is speculated that the mutation of the ZmQK2 gene may have an effect on the process of removing active oxygen from the grain cell,resulting in endosperm cells Apoptosis prematurely affects the filling and ultimately leads to the phenotype of qk2.However,further research is needed on the relevant regulatory mechanisms.