High Throughput Mining Saline-alkaline Tolerance-related Genes By Integrating Multi-omic Data Of Alfalfa

Alfalfa(Medicago sativa L.),also known as“King of forages”,is a perennial flowering plant belonging to the legume family Fabaceae,and is the most widely cultivated forage crop in the world.It has a high protein content and is easily digested by livestock.Alfalfa possesses a certain resistance to salt stress and can therefore grow well in mildly saline soils.However,at soil salt concentrations greater than 0.3%and p H greater than 9.5,growth and development of alfalfa becomes significantly inhibited,adversely affecting the overall yield and quality of the crop.Currently,the mechanisms underlying the saline–alkaline tolerance of alfalfa are not fully understood.Alfalfa cultivars(Medicago sativa L.cv.Zhaodong)with excellent saline–alkaline tolerance were used for small RNA sequencing,degradome sequencing,and proteome sequencing.Gene network construction and other bioinformatics techniques were used to analyze the data.Using the obtained transcriptome sequencing data,combined with the three types of sequencing data obtained in this study,the predicted miRNA-target gene regulatory network and miRNA-target gene regulatory network based on degradome sequencing were constructed,and integrative analysis of transcriptome and proteome was performed.The graph theory method was then used to analyze the network.Mining the functional subnets in the network,genome annotation,screening of regulatory genes,q RT-PCR technology,physiological index measurement,and transgenic technology were used to accurately screen the key genes of saline–alkaline stress tolerance of alfalfa at the transcriptome,post-transcriptional,and proteome levels.The main results are as follows:1.Construction of a miRNA-target gene regulatory network for small RNA sequencing analysis and predictionThis study identified 149 conserved and 11 putative novel miRNAs that were differentially expressed.The results of target gene prediction of differentially expressed miRNAs and GO analysis of the targets revealed that the functions of many target genes of these miRNAs may be relevant to the saline–alkaline tolerance of alfalfa.We constructed based on miRNA target prediction and negatively correlated miRNA targets.In plants,24 nucleotide small interfering RNAs(24-nt siRNAs)are mainly located in the repetitive region of the chromosomes.These siRNAs are widely distributed in the upstream and downstream 3 kb regions of the transcriptional region,but less distributed in the transcriptional region and upstream and downstream 500 bp regions.Gene Ontology(GO)analysis was performed on the genes corresponding to differentially expressed siRNAs in the 1000–2000 bp upstream and downstream regions of the transcriptional regions with the greatest distribution.The functions of these genes were mainly concentrated in“oxidoreductase,“protein dimerization“ion binding”and“regulation It is speculated that these genes may play important roles in regulating gene expression and stress resistance.2.Sequencing analysis of degradome and construction of an miRNA-target gene regulatory network based on the degradomeIn this study,degradome sequencing was performed on alfalfa under 1 and 7 days of saline–alkaline stress using high-throughput sequencing.Data analysis showed that a total of 230 miRNA-target gene pairs of alfalfa under saline–alkaline stress were obtained.By constructing the miRNA-target gene regulatory network of degradome data,the results showed that the constructed network consisted of 353 nodes,containing128 conserved miRNAs,8 predicted miRNAs,and 217 target genes.The results of differential expression analysis revealed 31 differentially expressed miRNAs and 8differentially expressed m RNAs in the network.GO analysis showed that the most annotated items of target genes in the degradome included“regulation of cellular process”and“DNA binding”.3.Proteomic sequencing and combined analysis of transcriptome and proteomeIn this study,proteome sequencing was performed on alfalfa under 1 and 7 days of saline–alkaline stress using high-throughput sequencing.A total of 103,1900,and1922 differentially expressed proteins(DEPs)were identified in 1 d vs.CK,7 d vs.CK,and 7 d vs.1 d,respectively.It was found that these differentially expressed proteins are involved in a variety of cellular functions,such as photosynthesis,signal transduction,transport,and carbohydrate metabolism.The proteome and transcriptome data previously obtained by integrative analysis,as well as the number of DEGs in 1 d vs.CK,and 7 d vs.CK,were much higher than those of DEPs.They are also involved in several biological functions,such as carbohydrate metabolism,photosynthesis,signal transduction,and redox,which are important for the tolerance of alfalfa to saline–alkaline stress.The key proteins were verified by enzyme assays.It was found that SOD,CAT,chitinase,and LOX activity,as well as soluble sugar and sucrose content,were increased.4.Construction and analysis of an integrated saline–alkaline stress regulatory networkIn this study,high-throughput sequencing of the transcriptome,small RNA,degradome,and proteome were integrated.Based on this,a saline–alkaline stress regulation network,as well as the first multi-omics alfalfa saline–alkaline stress regulation network,were constructed.The network was divided into eight modules,and five candidate subnets of the saline–alkaline stress response were excavated.Six candidate miRNAs and 30 candidate key regulatory genes for the saline–alkaline stress response were predicted from the network.Most of these genes were annotated to functions related to redox and signal transduction,indicating that redox and signal transduction were the key functions of alfalfa tolerance to saline–alkaline stress.5.Verification of saline-alkaline resistance of transgenic tobacco with key gene Ms FTLIn this study,the function of the key gene Ms FTL screened by multi-omics integration was verified.7-day-old tobacco seedlings were treated with saline-alkaline stress on MS for 6 days.We found that the transgenic plants with Ms FTL gene had longer root length and higher fresh weight.When the domesticated tobacco seedlings were treated with saline-alkaline stress,the leaves of WT tobacco wilted seriously,and the growth state of transgenic tobacco with Ms FTL gene was significantly better than that of WT.Then,the physiological indexes of 8-week-old tobacco treated with saline-alkaline stress for 7 days were detected.The results showed that the accumulation of H2O2 and O2~-in transgenic plants with Ms FTL gene was significantly lower than that in WT.At the same time,under saline-alkaline stress,the activities of antioxidant enzymes(POD,SOD,CAT)of transgenic plants with Ms FTL gene were higher than those of WT.Based on the above results,the transgenic plants with Ms FTL gene had stronger resistance to saline-alkaline stress.