| Agropyron mongolicum is a diploid perennial forage of the Gramineae wheat family.It is a drought-resistant dominant grass species growing in desert grasslands,has precious gene resources for genetic improvement of Poaceae crops,and has important feeding,ecological,and breeding values.Micro RNA(mi RNA)is a non-coding RNA commonly present in plants that is involved in regulating various stress responses such as drought,salt,and cold stress.It is one of the key regulatory factors for m RNA expression.However,the regulatory mechanisms of drought tolerance-related mi RNAs and their target genes in A.mongolicum need to be investigated.This project explored and identified in depth the mi RNAs and target genes related to drought tolerance in A.mongolicum based on Small RNA,transcriptome,and degradome sequencing and analyzed the expression patterns of mi RNAs and target genes.The mi RNA-m RNA co-expression regulatory network was also constructed based on WGCNA to analyze the regulatory relationship between hub genes and corresponding mi RNAs,and the hub gene AmMADS47 was functionally verified.The main results are as follows:1.A total of 1,104 mi RNAs were identified by Small RNA sequencing,containing895 known mi RNAs and 209 novel mi RNAs.Known mi RNAs were derived from 71 mi RNA families,and conservativeness analyses indicated the presence of precursor sequences of A.mongolicum mi RNAs in 46 species.A total of 101 differentially expressed mi RNAs were obtained,including 89 known mi RNAs and 12 novel mi RNAs.2.A total of 41,792 Unigenes were obtained by transcriptome sequencing,with a total of 1,166 differentially expressed genes(DEGs)in the seven comparison groups.The number of DEGs increased with drought duration,indicating that drought activates the expression of drought-responsive m RNAs.The GO analysis of DEGs mainly enriches GO terms such as chloroplast,oxidation-reduction process,and response to water deprivation.KEGG mainly enriches pathways such as starch and sucrose metabolism,plant hormone signal transduction,phenylpropanoid biosynthesis,indicating that these GO terms and KEGG pathways are involved in the regulation of drought stress in A.mongolicum.3.A total of 99 differentially expressed mi RNAs negatively regulating 1,474 differentially expressed target genes were identified in the integrated analysis of transcriptome-small RNA-degradome,which contained 91 differentially expressed mi RNAs negatively regulating 1,438 target genes and eight novel mi RNAs negatively regulating 36 target genes,with the 36 target genes functionally annotated as uncharacterized oxidoreductase,NADP-thioredoxin reductase C precursor,chloroplast ferredoxin-dependent glutamate synthase,and others.The mi RNA-target gene regulatory network was mapped,and multiple regulatory relationships were found between mi RNA and target genes.4.Thirty-nine co-expression network modules were constructed by WGCNA technology,four drought-specific modules and five hub genes were screened,and the regulatory network of mi RNA-hub genes was mapped.Five hub genes respond to drought stress through genes that regulate functions such as abscisic acid,response to water deprivation,chloroplast and others.Bioinformatics studies were carried out on five drought-resistant candidate hub genes;their PI values ranged from 5.35 to 8.58 and the aliphatc index ranged from 69.59 to 113.13.Amcar C,AmPAO2,and AmCCX1 proteins are localized extracellularly,and AmMADS47 and AmHOX24 proteins are localized in the nucleus by subcellular localization prediction.The secondary and tertiary structures of the proteins consisted mainly of α-helixes and random coils.Four pairs of mi RNA-drought-resistant candidate hub genes were found to exhibit negative regulation and one pair to exhibit positive regulation by q RT-PCR analysis.5.AmMADS47 protein is localized to the nucleus by a tobacco subcellular localization and belongs to the nuclear protein genes.Overexpression of AmMADS47 reduced the drought tolerance of rice by observing and detecting the phenotypes,relative gene expression levels,drought-related physiological indices,and ROS staining of AmMADS47 overexpression rice under drought stress.A total of 21,521 DEGs were obtained by transcriptome sequencing.Phenylpropanoid biosynthesis and glutathione metabolism were the shared pathways with the highest and second highest number of enriched genes,and photosynthesis-antenna proteins was the shared pathway with the highest enrichment score and significance by analyzing the Top20 enriched pathways in the DEGs of the two comparison groups(OE vs WT and OED vs WTD).These three pathways served as the focus of subsequent analyses.Down-regulated expression of most genes in overexpressed AmMADS47 rice(OE)reduced the synthesis of key enzymes such as PAL,4CL,GST,and others compared to wild-type rice(WT).The regulation mode of genes in drought was analyzed in the three pathways.The genes were up-regulated in overexpressed AmMADS47 rice under drought stress(OED),which are some drought-sensitive genes in wild-type rice under drought stress(WTD).Most genes positively regulating drought in WTD plants were down-regulated in OED plants.It was shown that AmMADS47 affected the synthesis of key enzymes and the expression mode of drought-responsive genes in three candidate pathways in rice,thereby reducing the drought resistance of trans-AmMADS47 rice. |
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