Functional Analysis Of ZmNup58 In Response To Drought And Salt Stress In Maize

Maize(Zea Mays L.)is not only an important industrial raw material and feed crop,but also the world’s most productive crop.The increase of corn yield and quality is an important measure to ensure food security in China.However,maize breeding has been faced with a series of problems such as poor maize germplasm resources.Therefore,the construction of maize Ds mutant library by using the Activator-Dissociation System(Ac/Ds)mutant System can effectively enrich the germplasm resources of maize in the world,and the stress-related genes are screened out from the Ds mutant library for phenotype identification and functional verification.Therefore,the response mechanism of the gene to stress was further clarified,which provided a theoretical basis for maize breeding and production of high yield and stress resistance.Based on the maize Ds mutant library constructed earlier by our research group,100 families with background of K17 were randomly selected from the mutant library.Adapter-PCR technology was used to amplify Ds flanking sequences,and the obtained Ds flanking sequences were sequenced by Illumina.Finally,the insertion position of Ds in maize genome was preliminarily determined based on the analysis of sequencing results,and the possible insertion genes were screened out.A gene related to stress response was selected for preliminary functional analysis,namely phenotype identification,physiological index and transcriptome analysis of mutants and overexpressed plants.Specific research results are as follows:1.By analyzing all Ds insertion sites in 100 mutant families,about 3644 inheritable Ds insertion sites were identified;These loci were distributed on 10 chromosomes,of which the maximum number of loci on chromosome 10 was 581,the minimum number of loci on chromosome 6 was 127,and the range of loci on other chromosomes was 218-515,indicating that the Ac/Ds system of maize could be used to construct a Ds mutant library covering the whole genome of maize.2.Among 3644 Ds insertion sites,a total of 1342 maize genes were covered,among which 186 genes had two or more insertions.According to the online functional annotation of Gramene,70 genes are involved in "response to stress(GO:0006950)".These genes are distributed on 10 chromosomes,mainly involved in the response to oxidative stress,hormone,water shortage,salt and temperature,etc.3.The mutant Zm00001d005856 was selected as the material for phenotype identification and Ds insertion site verification under adversity stress.The analysis of Ds insertion site by designing genome/genome and genome/Ds primers showed that the insertion site was correct.The mutant was more sensitive to drought and salt stress after 2 days of treatment with 20% PEG 6000 and 1.5% Na Cl,respectively.Zm00001d005856 is a Nuclear pore complex protein NUP58(named ZmNup58),encoding 470 amino acids.The results of subcellular localization showed that ZmNup58 was localized in the nucleus,and its function was probably mainly in the nucleus.4.In order to further verify whether the gene is responsive to drought and salt stress,an overexpression vector ZmNup58-5941 was constructed.Combined with the genetic transformation of maize,the overexpressed transgenic lines were obtained.The results showed that after 2 days of treatment,the wild-type plants showed more severe leaf wilting and crimping than the over-expressed ones.These results further proved that the over-expression of ZmNup58 improved the drought and salt tolerance of the plants.5.RNA-seq technique was used to analyze the expression of overexpressed plants and wild-type plants under drought stress.Transcriptome data before and after drought treatment showed that compared with wild-type plants,there were 38 Differential expression genes(DEGs)in overexpressed plants before drought treatment,among which 3 DEGs were down-regulated and 35 DEGs were up-regulated.After drought treatment,there were 1203 differentially expressed genes in the overexpressed plants compared with the wild-type plants,among which 931 DEGs were down-regulated and 272 DEGs were up-regulated.These DEGs involve a total of 94 pathways,and the most significantly enriched ones are mainly photosynthesis,organic metabolism,plant hormone signal transduction,REDOX reaction,cellulose synthesis,defense process under stress,linoleic acid metabolism,aquaporin synthesis,glucose metabolism,lipid metabolism,amino acid metabolism,etc.Endopeptidase activity and so on.The results of this study will help to further understand the molecular mechanism of maize response to drought stress and provide a new idea for breeding new maize varieties with drought resistance.