| Maize(Zea mays)plays an important role in world economic development and food security because that it is a vital feed crop and raw materials for food,chemical industry,fuel and pharmaceuticals industry.With the continuous deterioration of the global climate and water scarcity,drought has become an important abiotic stress affecting crops growth and yield.MicroRNA(miRNA)is a type of non-coding RNA that is widely found in eukaryotes and plays an important regulation role in organ construction,growth and development,signal transduction and stress response.In this study,we used the root tissues from the drought-tolerant inbred line AC7643,the drought-sensitive inbred line AC7729/TZSRW and 14 recombinant inbred lines(RILs)under well water and water stress treatments for small RNA sequencing and RNA-Seq.The sequencing data was applied for known miRNA identification and discovering novel miRNA,exploring the drought-response miRNA and the expression regulatory pattern between miRNAs and their target genes in maize root.Besides,we initially identified which biological processes that candidate genes regulated by miRNAs may be involved in drought response.The main results are as follows:1.We identified 284 known miRNAs and 91 novel miRNAs by analysis of small RNA sequencing data from the root tissues of 16 maize inbred lines with different drought sensitivity under well water and water stress treatments.The identified known miRNAs were mainly from zma-MIR169,zma-MIR395,zma-MIR171 and zma-MIR166 families.And the length distribution of the identified miRNAs was between 18 nt and 23 nt,the miRNA with the length of 21 nt was the most,accounting for 70.13%.In addition,more than 86% of miRNAs were expressed in both parents and RILs,and 3.74% of miRNAs were specific in drought-tolerant or drought-sensitive under two treatments.2.By analyzing the expression levels of miRNAs,we found that the expression levels of miRNAs from zma-MIR168,zma-MIR159 and zma-MIR166 families were higher under the two treatments in each material,while miRNAs from zma-MIR399,zma-MIR2218 and zma-MIR2275 families have low expression levels.Moreover,the expression specificity of zma-MIR169 and zma-MIR2275 families were higher than that of zma-MIR408 and zma-MIR398 families in different maize inbred lines.Furthermore,over 50% of miRNAs which were both expressed in parents and RILs was expressed lower in RILs compared to parents.3.By predicting the target genes and target mimicry of miRNAs,a total of 2846 pairs of miRNA/target and 4910 pairs of miRNA/target mimicry were obtained.Further correlation analysis between the expression levels of miRNAs and target genes revealed that 228 pairs of miRNA/target were significantly positively correlated,while 161 pairs of miRNA/target were significantly negatively correlated.In addition,the level of miRNA expression variation in the positive correlation group was significantly lower than that in the negative correlation group.However,the expression of miRNA target mimicry in positive correlation group was significantly higher than that in negative correlation group.Furthermore,the analysis of miRNA binding sites on target genes shown that miRNAs mainly bound in the untranslated regions(UTRs)and exon regions and there was no significant difference between positive correlation group and negative correlation group.Combined with the results of real-time quantitative verification,the results showed that the variation trend of expression between some miRNA and target genes was different in different inbred lines,indicating that the response of expression regulation mediated by miRNA to drought stress was different in different drought tolerant inbred lines.On the other hand,by designing the overexpression vector of miRNA,two the fluorescent report vector with miRNA target and target mimicry binding site sequence respectively,the tobacco transient expression analysis shown that miRNA target mimicry can be indirectly involved in the regulation between miRNA and target genes by locking miRNAs.4.Compared the change of miRNA expression levels in the two treatments,the results revealed that 50 miRNAs changed significantly after water stress(FDR≤0.05),which were mainly from zma-MIR156,zma-MIR160 and zma-MIR166 families.Comparing the miRNA expression levels of drought-sensitive and drought-tolerant inbred lines,it was found that the miRNA expression levels of the two groups were significantly different under the two treatments,and the miRNA expression levels of the two groups were also significantly different after drought stress.The miRNA expression levels of drought-tolerant materials were more severe than those of drought-sensitive materials.5.Three miRNAs,miR168b-3p,miR397b-5p and miR528a-5p,which were significantly responsive to drought stress,were screened out.And the target genes Zm00001d047683,Zm00001d038371 and Zm00001d012101 which significantly related to the expression levels of the three miRNAs were selected as candidate genes.Homozygotes of Arabidopsis mutants hsr8,lac12 and rglg2,which were homologous genes of candidate genes,were identified by three primer or two primer based-method.Phenotypic identification was carried out under drought stress and salt stress.Under drought stress and salt stress,mutants and wild type showed significant differences in root length,root surface area and other traits(t test,P<0.05),and showed different tolerance under stress.It was speculated that Zm00001d047683,Zm00001d038371 and Zm00001d012101 these three candidate genes may respond to drought stress and salt stress,and were related to root growth and development under stress in plants.6.Combined drought tolerance identification data and the mixed liner model(MLM)to analyze the association of miRNA-mediated candidate genes,we found 15 SNP marker loci significantly associated with plant mortality under drought stress(P<0.005),including nine genes,three transcription factors such as MYB3R-3 and PCF8,two phospholipid-related proteins such as phospholipase D family protein,and several functional proteins such as NOI.Besides,we used candidate gene-related SNP marker loci and drought stress-related traits for haplotype analysis.As a result,we got 60 SNP marker loci were significantly correlated with drought stress-related traits at P<0.01 level,including five groups of linked SNP markers. |
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