| Maize,as one of the primary staple crops,plays a vitally important role in global food security and energy demands.Mutants have proven to be valuable tools in uncovering the function of maize genes,and the creation of a mutant library serves as a crucial platform for identifying and utilizing superior maize genes.Drought is a significant environmental factor that negatively impacts maize yield.By leveraging the mutant library to investigate drought-resistant genes and their molecular mechanisms,we can provide essential theoretical support for breeding new drought-resistant maize varieties.This effort is of immense significance in addressing food security concerns.In this study,we utilized the Ac/Ds transposon system to create a mutant library with the genetic background of the elite inbred line K17 in China.From this library,we identified a drought-associated gene,Zm C2H2-149.Subsequently,we employed molecular biology techniques to unravel the molecular regulatory mechanisms of this gene under drought stress.The main research findings are as follows:1.We utilized the Ac/Ds transposon system to generate a mutant library consisting of 3,191 lines with the genetic background of K17.Through field phenotypic surveys,we identified 22 distinct mutant phenotypes encompassing various traits such as grain development,plant height,leaf morphology,and reproductive organ development.Notably,the highest frequency of mutant phenotypes was observed in grain development,accounting for approximately 5.98%of the entire library.This was followed by leaf development and plant height mutants,which accounted for 2.43%and 1.74%of the library,respectively.2.To precisely determine the Ds insertion sites of K17 genome and the mutants,we utilized Pac Bio SMRT and Illumina sequencing technologies,resulting in a 2.24 Gb genome sequence with a scaffold N50 length of 33.57 Mb.By leveraging high-quality maize GBS pan-genome genetic maps,we accurately anchored 2.21 Gb of the sequence to 10 chromosomes,covering 98.7%of the assembled genome size.Through the integration of transcriptome data,ab initio prediction,and homologous prediction,we identified 42,776 genes.The sequencing data aligned to the K17 genome achieved alignment and coverage rates exceeding 98%.Furthermore,BUSCO assessment revealed that 97.1%of complete genes were identified in the K17 genome,surpassing the 96%of W22v2 and B73v4.These findings highlight the high quality of the assembled K17 genome,positioning it as a valuable reference for accurately identifying Ds elements.3.To determine the quantity and distribution of Ds transposon elements within the K17 genome,we utilized BLAST and bowtie2 software to align the conserved 75 bp sequences located at the ends of Ds elements（Ds2 type）with the K17 reference genome.Our analysis revealed the presence of 29 Ds transposon elements spread across 10chromosomes.Further examination of the amplified Ds elements and their adjacent sequences in the mutant lineage,K17,and W22 genomes indicated that,with the exception of the 4th chromosome,9 Ds elements on the remaining 9 chromosomes were susceptible to transposition by the Ac transposase,indicating that they may serve as donor sites for Ac/Ds transposition events.4.Using TEAseq technology,21,046 Ds loci were identified from 3,191 mutant lines.By setting the sequencing abundance LPM4250 of Ds loci as the threshold,11,707 high-quality germinal Ds insertions were determined in 3,098 mutant lines.Whole-genome distribution revealed a high frequency of mutant lines and Ds insertions near 9 Ds donor sites in the K17 genome.Gene structure analysis showed that approximately 60%of Ds loci were distributed within gene regions,marking 5,651genes.Comparison with Ds-tagged genes in the Ac Ds＿Vollbrecht and Ac Ds＿Dooner mutant libraries revealed 4,194 unique Ds-tagged genes in this study,accounting for9.8%of the total genes.The total number of Ds-tagged genes were increased from the original 7,198 to 11,392,covering 26.63%of the entire gene set.Additionally,integration of data from the maize Ds and Mu transposon mutant libraries revealed that28,216 genes were tagged by these two types of transposons,covering 65.96%of the genes.5.Following the exposure of 150 randomly selected Ac/Ds mutants to drought treatment,a significantly drought-resistant mutant line,Dm116-2,was identified.PCR validation of 9 Ds loci located within gene regions in this line confirmed 4 Ds insertion mutant genes.Seedling drought experiments showed that the drought resistance of the Zm K17＿10g12870.1 mutant was notably higher than that of the wild type and the other3 mutants.The Zm K17＿10g12870.1 gene encodes a C2H2 zinc finger protein,and was therefore named Zm C2H2-149.Subcellular localization results indicated that this gene was localized in the cell nucleus,and transcriptional activity analysis revealed that Zm C2H2-149 exhibited transcriptional repression activity.These findings suggested that Zm C2H2-149 was a drought-responsive transcription factor with transcriptional repression activity.6.The seedling drought experiment revealed that the zmc2h2-149 displayed significant drought resistance,with elevated activities of antioxidant enzymes such as POD,SOD,CAT,and higher levels of proline and soluble proteins compared to the wild type.Overexpression of the Zm C2H2-149 gene were observed to be more susceptible to drought,exhibiting lower leaf relative water content and antioxidant enzyme activities compared to wild-type plants.Field drought experiments indicated that the zmc2h2-149 had a lesser impact on traits such as plant height,ear length,and yield under drought conditions,while overexpressing Zm C2H2-149 plants showed inhibited growth,reduced plant height,smaller ears,and a significant decrease in yield under drought stress.These findings suggested Zm C2H2-149 was a negative regulator of drought stress response.7.RNA-seq analysis was conducted on both the WT and zmc2h2-149 mutant to uncover 794 differentially expressed genes in response to drought stress.GO enrichment analysis indicated a notable enrichment of biological processes associated with abiotic stress response and plant hormone response.DAP-seq analysis identified1,097 potential target genes that were directly regulated by Zm C2H2-149.GO enrichment analysis further revealed that these genes were enriched in biological processes related to stress response,redox reactions,and hormone response.By integrating the findings from both analyses,we pinpointed 23 downstream target genes likely under the direct regulation of Zm C2H2-149.Notably,the expression of Zm00001d007345,which encodes an 11-beta-hydroxysteroid dehydrogenase 1B named Zm HSD1,was significantly upregulated under drought conditions among these downstream targets.8.Yeast one-hybrid experiments revealed the binding of Zm C2H2-149 to the promoter of the Zm HSD1 gene.Dual-luciferase assays confirmed that Zm C2H2-149bound to the promoter of Zm HSD1,leading to the inhibition of downstream gene expression.RT-q PCR analysis demonstrated a significant increase in Zm HSD1expression in zmc2h2-149 mutant,while a notable decrease was observed in Zm C2H2-149-OE.Seedling and field drought experiments indicated that the drought resistance of zmhsd1-Dsand zmhsd1-crispr mutants was lower compared to the wild type.These findings suggested that Zm C2H2-149 negatively modulated maize drought tolerance by suppressing the expression of the Zm HSD1 gene. |
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