Identification,Gene Mapping And Cloning Of Leaf Vein Mutants In Maize

Maize is the largest food and feeding grain in the world,which plays a significant role in agricultural industry.As an important C₄ plant,photosynthesis contribute 90%of the grain yield.The veins are important tissues of the leaves providing water and inorganic salts,and outputing photosynthetic products.Besides,they also support the leaves,so that they can extend into the space and ensure the physiological function of the leaves smoothly.Therefore,cloning the genes associated with vein formation,and analyzing their function,which has great theoretical significance and application value in maize.Ethylmethylsulfone（EMS）is a chemical mutagen,which can mutate G/C to A/T resulting the mutations in the genome and showing the different phenotypes due to the mutation of functional genes.The mature pollens of inbred line RP125 were treated with EMS,which is the EMS mutant library.Based on our previous screening of the library,the leaf veins mutant named veins network 1（ven1）was identified.The agronomic characters of this mutant were collected and investigated,and its leaves were observed at a cellular level.The physiological and biochemical indicators were determined as well.At the same time,segregation population with different backgrounds were constructed and genetic analysis was conducted.The mutated gene was obtained by map-based cloning method.The mutated sequence was conducted with bioinformatics analysis.Expression pattern analysis was performed using different tissues.Four allelic mutants were identified by constructing a double-stranded material and sequencing of the mutation site.The results were as following:1.The main veins of mutant are discontinuous with the green clour distributed unevenly accompaning by a sudden phenotype of small lines intermittent light transmission phenomenon.The phenotype of ven1 appeared at the stage of six leaves.The phenotype appeared on both sides of the main veins of the leaves at the sixth leaf stage,and gradually weakened from the bottom to the top,and the phenotype will keep in the whole growth process.Comparing with wild type,The characteristic of ven1including plant height,root system,area of leaf conducting photosynthesis,grain yield were decreased.The determination of chlorophyll content demonstrated that the content of chlorophyll A,B and total chlorophyll in mutants were significantly lower than those in wild type.Blade cytology observation showed that vascular bundles in the mutant leaves are disordered and the number of chloroplasts is locally reduced.2.Based on the genetic analysis of F₁ and F₂ population crossed ven1 with RP125,B73 and Mo17,respectively,which illustrated that this mutation was controlled by single recessive gene.Two bulked DNA pools were created using 20 mutant phenotype and wild type phenotype individual plants in the F₂ population with the crossing combination B73and ven1.The result showed that umc2350 and bnlg1074 on chromosome 10 appeared to be co-separated with mutant phenotype using more than 200 SSR markers.Combineing the genotyping of single plant,we could verify that this gene located on chromosome 10.At the same time,Using the BSRseq analysis strategy with 60 recessive single strains and 60normal single strains in the F₂ population of B73/ven1,which showed that there was an obvious peak on chromosome 10 of maize,indicating the location of the mutated.Some pleomorphic In Dels markers were explored around umc2350 and bnlg1074 on the basis of genome information of B73 and sequencing data of RP125,thus analyzing genotype of these markers in 1196 recessive plants.As a result,the target gene was narrowed the region of 620 kb,between indel-9 and indel-10.This region contained 5 ORF based on the annotation information of reference genome.Primers and genes were designed and amplified between the wild type and mutant plants.Sequencing alignment revealed that there is a SNP in the Zm Ven1 gene that is mutated from G to A,and the physical position is located at 64,865,144.This site is located at the last base of intron6-7,and it is speculated that this mutation will result in variable Zm Ven1 Pre-m RNA alternative splicing.Sequenceing analysis showed that the mutation site was homozygous mutation in the pool,indicating that the mutation at the site was co-segregated with the phenotype,confirmed the truth of this locus.3.To further clarify the accuracy of cloning genes,we screened another 4phenotypic mutants in the mutant library,named ven1-1,ven1-2,ven1-3,and ven1-4.These materials were hybridized with ven1,and the leaf phenotype of F1 was investigated in the field.The results demonstrated that the F1 of the five materials showed the similar phenotype with ven1,which indicated that the five materials were allelic.At the same time,we designed the full-length amplification primers of Zm Ven1gene and amplified 4 alleles.Sequencing analysis showed that:The mutation of ven1-1occurs at the first base of intron25-26,from G to A,and the physical position is at64,827,976.The mutation result in variable Zm Ven1 Pre-m RNA alternative splicing;The mutation of ven1-2 occurs in the 7th exon,the G mutation is A,and the physical position is 64,865,126.The mutation causes the 422 amino acid of the protein encoded by the gene to be mutated from arginine to tryptophan;The mutation of ven1-3 also occurs in the 7th exon,the G mutation is A,and the physical position is 64,865,066.The mutation causes the 442 amino acid of the protein encoded by the gene to be mutated from arginine to tryptophan;The mutation of ven1-4 occurs in the fifth base of intron3-4,which is mutated from G to A,and the physical position is at 64,871,588.The mutation result in variable Zm Ven1 Pre-m RNA alternative splicing.To further determine the authenticity of the above mutations,we amplified the c DNA of Zm Ven1 by specific PCR amplification,and combined with agarose gel electrophoresis experiments and sequencing analysis,it was proved that the mutation caused Zm Ven1 mutation at the m RNA level.In summary,we initially determined that the located and cloned gene Zm Ven1 is a gene accociated with the mutant phenotype.4.Based on the information of maize genome database,the Zm Ven1 length is 57,694bp in the genome with longest c DNA 4429 bp,including 33 exon and 32 intron,encoding1341 amino acid.Through the protein domain site,it was found that the gene contained three domains:p-loop＿ntpase,PHD and Helicase＿C＿4,code RING/FYVE/PHDzinc finger superfamily protein.Phylogenetic tree analysis showed that compared with other species,the gene had higher homology and closer evolution in maize and sorghum.5.Q-Teller and q RT-PCR was done to analyze the expression level of Zm Ven1 gene in different tissues,and the results showed that Zm Ven1 gene was expressed in many tissues in maize.And the expression level was the highest in the germ.This indicates that the gene may be related to the growth and development of maize.6.Combining with the phenotypic identification of mutants,it is speculated that the partial or complete loss of RING/FYVE/PHD zinc finger superfamily protein activity is affected by the formation of vascular bundles in maize,most notably the disorder of the veins,and also the inclusion of corn.The phenotype of other tissues and organs of the vascular bundle changes,eventually resulting in a decline in corn yield.The candidate gene Zm Ven1 is very likely to be a new gene related to the morphogenesis of vascular tissue.This gene also has important significance and research value in the overall growth and development of plants.