Genome-Wide Analysis Of MRNAs,miRNAs And ALDH Gene Family In Soybean Response To Drought Stress

Soybean[Glycine max(L.)Merr.]is an economically and ecologically important crop that not only provides protein and oil for food and feed,but also serves as a key source of green manure in agro-ecosystems due to its highest capacity of biological nitrogen fixation(BNF)observed among leguminous crops.Soybean is considered as a sensitive crop to water shortage environment.Drought is one of the major factors limiting soybean yield,and is projected to be more intense with global climate change and the reduction of water resources.Little progress has been made on the research of candidate genes and regulatory mechanism about drought-tolerance in soybean.Therefore,identification of the candidate genes and uncover the regulatory network of soybean in response to drought stress are very important for providing gene resources and theoretical basis for soybean drought-tolerance breeding program.In this study,two soybean varieties including PI(PI595843,drought-tolerant)and NT(Nan tong xiao yuan dou,drought-sensitive)were selected based on the previously screening of soybean drought-tolerant germplasm.Differently expressed genes(DEGs)and differently expressed miRNAs(DEMs)of drought-tolerant(PI)and drought-sensitive(NT)soybean varieties in response to drought stress were identified using high-throughput Illumina sequencing technology,and candidate genes and pathways for soybean drought-tolerance was proposed and discussed.In addition,a genome-wide analysis of aldehyde dehydrogenases(ALDHs)family in soybean genome was conducted.The main results were as follows:1.Phenotypic and physiological responses of two soybean varities in response to drought stressPI(drought-tolerant)and NT(drought-sensitive)were selected and subjected to drought stress for studying the effects of drought stress on morphological,physiological and biochemical of different drought-tolerant soybean cultivars.The relative water content(RWC)of leaves decreased slowly and leaf wilting appeared later in PI compared with NT as the drought treatment progressed.Under the same degree of drought stress,the accumulation of H2O2 in PI leaves was lower,which resulted in less damage to the cell membrane as reflected by lower relative electrical conductivity and lower malondialdehyde(MDA)content,and the survival rate of PI were higher than that of NT.In addition,drought severely inhibited the net photosynthetic rate,stomatal conductance and biomass accumulation of soybean,but more significantly in NT than PI.Stomatal aperture in PI leaves was significantly smaller than those of NT after exogenous ABA treatment,suggesting that PI might reduce the rate of water loss by adjusting the stomatal closure,leading to improved leaf relative water content and drought tolerance.2.Comparative transcriptome profiling analysis of drought-tolerant and drought-sensitive soybean varieties in response to drought stress.The mRNA expression profiling of PI and NT leaves under drought treatment for 14 days and control was investigated using RNA-seq by Illumina HiSeqTM 2500.Using the threshold of False Discovery Rate(FDR)≤0.05 and |log2 FC(Fold Change)|≥1 to identify the differently expressed genes(DEGs).Under drought stress,298 and 253 drought-responsive DEGs(drought vs control)were identified in PI and NT,respectively.Through comparative analysis between two varieties,a total of 98 DEGs were identified as drought-tolerance candidate genes in PI,which were significantly enriched in the Gene Ontology(GO)terms of transferring acyl groups and transferring glycosyl groups(including cellulose synthase and UDP-Glycosyltransferase).In addition,a weighted gene co-expression network analysis(WGCNA)was performed and revealed that ’lightskyblue’module including 1660 genes was significantly correlated with RWC and MDA.Candidate hub genes such as two cellulose synthase and two UDP-Glycosyltransferase genes were identified based on the intergenic connectivity,which coincides with the GO enrichment analysis of drought-tolerance candidate genes.QRT-PCR was used to confirm the relative expression patterns of 10 genes,and the correlation between mRNA-Seq and qRT-PCR was high(r2=0.809).Finally,the celloluse content in the leaves and stems of PI and NT under drought treatment for 14 days was measured,and the results showed that drought stress induced accumulation of the cellulose content and more accumulation was observed in drought-tolerant varivity(PI),which indicates that the cellulose content might affect the drought tolerance in soybean.3.Comparative miRNA profiling analysis of drought-tolerant and drought-sensitive soybean varieties in response to drought stress.Four small RNAs(sRNAs)libraries including control and drought treatment for 14 days were constructed using the leaves of PI and NT.Following high throughput sequencing and filtering,616 known and 182 novel miRNAs were identified in the four sRNAs libraries.Using the threshold of p_value≤0.01 and |log2FC(Fold Change)|≥1 to identify the differently expressed miRNAs(DEMs).A total of 299 miRNAs were found to be down-regulated and 75 miRNAs were up-regulated in PI in response to drought stress;while 24 down-regulated miRNAs and 24 up-regulated miRNAs were found in NT response to drought stress.Three previously published soybean degradome sequencing datasets(PRJNA304685、GSE50063、GSE34433)were used to predict the targets of DEMs.As a result,we found 584 and 105 target genes were predicted to be putatively regulated by 173 known and 35 novel DEMs in PI,repectively;while 77 and 53 target genes were predicted to be putatively regulated by 21 known and 15 novel DEMs in NT,repectively.MapMan functional classification of target genes of DEMs suggested that a higher proportion of target genes participated in stress and hormone metabolism in PI.AgriGO enrichment analysis showed that the target genes of DEMs were enriched in the process of signal transmission,regulation of transcription and response to endogenous stimulus terms in PI;while the target genes of DEMs were enriched in response to organic substance,response to endogenous stimulus and response to hormone stimulus in NT.In addition,the integrated analysis of mRNA-seq and sRNA-seq found that 9 DEGs were putatively regulated by 13 DEMs in PI,including eleven negatively and two positively correlated miRNA-target modules in expression levels,while 6 DEGs were putatively regulated by 7 DEMs in NT,including three negatively and four positively correlated miRNA-target modules.4.Genome-wide identification and bioinformatic analysis of ALDH genes in soybeanAldehyde dehydrogenases(ALDHs)play an essential role in the detoxification of aldehyde molecules by facilitating the irreversible oxidation of a series of endogenous and exogenous aromatic and aliphatic aldehydes into their corresponding non-toxic carboxylic acids.A more comprehensive analysis of the ALDH gene superfamily in soybean,however,is lacking,and its role in abiotic stress response is unknown.Using the Hidden Markov Model(HMM)profile of the ALDH domain(PF00171),53 GmALDHs were identified in soybean by HMMER 3.0 software,and grouped into 10 ALDH families according to the ALDH Gene Nomenclature Committee and phylogenetic analysis.Soybean ALDH superfamily expanded mainly by whole genome duplication or segmental duplications.Gene network analysis identified 1146 putative co-functional genes of 51 GmALDHs based on the SoyNet databases.Gene Ontology(GO)enrichment analysis suggested the co-functional genes of these 51 GmALDHs were enriched in the process of lipid metabolism,photosynthesis,and proline catabolism.In addition,22 co-functional genes of GmALDHs related to plant response to water deprivation or watert ransport were identified.In addition,the occurrence frequencies of three drought-responsive cis-elements(ABRE,CRT/DRE,and GTGCnTGC/G)were compared in GmALDHs that were up-,down-,or non-regulated by water deficit Higher frequency of these three cis-elements was observed for the group of up-regulated GmALDHs as compared to the group of down-or non-regulated GmALDHs by drought stress.Three GmALDHs which were highly induced by drought stress were identified using qRT-PCR.Our study provides a foundation for future investigation of GmALDHs function in soybean.