Study On The Responses Of Ota To Saline-alkali Stress Based On Transcriptome And Metabolome Joint Analysis

Soil salinization is a worldwide environmental and ecological problem,which leads to the decrease of cultivated land area and poses a great threat to food security.It has become one of the hot topics in the utilization of saline-alkali land to deeply analyze the mechanism of plant saline-alkali tolerance,dig out the functional genes of saline-alkali tolerance,and carry out saline-alkali tolerance crop breeding.Oat（Avena satiba L.）is an excellent forage crop,widely cultivated in the world,and is considered as one of the improved alternative crops in saline-alkali land.Therefore,it is of great significance to further study the saline-alkali resistance mechanism of oat for improving and exploiting saline-alkali land resources.In this study,the saline-alkali tolerance of different oat varieties was firstly identified,and then the representative resistant and sensitive varieties were selected for saline-alkali stress treatment,and the saline-alkali tolerance mechanism was revealed by analyzing the physiological and biochemical indexes,transcriptome and metabolome of oat in response to saline-alkali stress.The main conclusions are as follows:1.Identification and selection of saline-alkali tolerant genetic resources of oatUnder 6 different saline-alkali stress conditions,63 oat varieties（lines）from 7 different regions were used as test materials to screen the saline-alkali tolerance of oat bud stage.According to the results of curve fitting by Boltzmann,[Na⁺]150 mmol·L^-1 p H 9.8 was determined to be the highest concentration for the identification of saline-alkali tolerance of each variety（line）.Then under N150p9.8 salinization and alkali stress,The indexes of germination,bud length,root length,overground fresh weight and fresh weight per plant of 63 oat varieties（lines）were tested.Then the clustering analysis was carried out,the 63 varieties（lines）tested were divided into 4 categories.According to the comprehensive index and practical value,BY as saline-alkali resistant variety and YZY as sensitive variety were determined for the next study.2.Effects of saline-alkali stress on growth and physiological and biochemical indexes of oat seedlingsUnder the saline-alkali stress the plant height,dry weight and photosynthetic pigment content decreased with the increase of stress concentration,but the effect on BY was less than that on YZY.MDA content in oat root increased with the increase of saline-alkali stress concentration,and the increase of YZY was higher than BY.Under saline-alkali stress,the contents of proline,soluble sugar and soluble protein of the two varieties increased.The content of betaine increased significantly in leaves and roots of BY and increased significantly in YZY leaves,but not in roots.The protective enzymes were changed,all of which showed a trend of first increasing and then decreasing with the increase of saline-alkali concentration,and the increase of BY was higher than that of YZY.Among them,POD and APX are more prominent.Under saline-alkali stress,oats maintained their K⁺/Na⁺by choosing absorption Na⁺or increasing absorption K⁺.BY root can maintain a stable K⁺/Na⁺value.But it was stable under low or middle saline-alkali stress,and significantly increased at high stress in YZY root.The values of K⁺/Na⁺in the leaves showed the same rule that they did not change at low concentration but decreased significantly at middle or high saline-alkali stress.3.Transcriptomic and metabolic analysis of oat in response to saline-alkali stressA total of 44,591 Unigene were obtained by oat transcriptional sequencing assembly,and31,365 Unigene were annotated.A total of 23,423 Unigene received 5,709 GO items annotation;and 12,085 unigene annotated by KEGG.The gene expression of oat was significantly changed after saline-alkali stress.Comparison YZY and BY without saline-alkali stress showed that there were only 64 different genes,among which 39 were up-regulated and 25 were down-regulated.But after stress,the differential genes were 1,700,of which 923 were up-regulated and 777 were down-regulated.Through GO functional enrichment analysis of DEGs,it was found that the number of DEGs in the following terms increased significantly under the saline-alkali stress,such as belongs to the biological process（BP）of protein phosphorylation,secondary metabolite biosynthetic process,flavonoid biosynthetic process,and belongs to the molecular function（MF）of protein serine/threonine kinase activity,DNA binding,ATP binding,heme binding,and belongs to the cell components（CC）of plastid,mitochondrion,plasma membrane,cell wallThrough KEGG functional enrichment analysis There were 7 metabolic pathways and 83differentially expressed genes between the two oat after stress.Among them,the most genes are distributed in Phenylpropanoid biosynthesis pathway,followed by Starch and sucrose metabolism pathway,ABC transporters pathway,Inositol phosphate metabolism pathway,Glycerophospholipid metabolism pathway,etc.Some of these genes in GO and KOG have been annotated with their peroxidase activity,monooxygenase activity,secondary metabolites biosynthetic transport and catabolism.So the significantly enriched genes in these pathways may be responsible for the difference in salinity and alkali tolerance between the two oat varieties.Transcription factor prediction revealed there are 925 genes annotated,belonged to 64transcription factor families.Under the response of saline-alkali stress,78 transcription factor genes belonging to the 31 transcription factor family were differentially expressed in oat root,In the WRKY,b HLH,AP2/ERF-ERF and b ZIP family,the number of differential expression ranked in the forefront successively.Moreover,a large number of genes of transcription factor families have been confirmed to be involved in the response to saline-alkali stress.A total of 752 metabolites were detected from 12 oat samples based on a Widely Targeted Metabolomics analysis.10 and 6 metabolites were induced by saline-alkali stress and detected in BY and YZY,respectively.These metabolites belong to organic acids and derivatives and flavonoids.Compared with each treatment group,the amount of different metabolites ranged from 96 to 157.By analyzing the pathway of differential metabolites,The numbers of differential metabolites in Metabolic pathways,Biosynthesis of secondary metabolites pathway were large.Analysis of differential metabolites in the pathway revealed that the response of BY to saline-alkali stress by betaine,proline and rosmarinic acid was the most prominent.On the other hand,YZY was the most prominent response to saline-alkali stress by accumulation of4-Hydroxybenzoic acid,Citric acid,Cis-Aconitic acid and Isoliquiritigenin.The results showed that the metabolic responses of two oat varieties to saline-alkali stress were not identical.These metabolites can be regarded as characteristic metabolites of saline-alkali tolerance of oat.4.Multi-omics joint analysis of oat in response to saline-alkali stress and quantitative asssy of target genesThere were 53 DEGs associated with 60 DAMs in BY,and 20 DEGs associated with 22DAMs in YZY.KEGG co-enrichment analysis revealed that 6 metabolic pathways were significantly enriched in the two oat varieties,and these pathways were all related to the biosynthesis and metabolism of amino acids,and these reflect the common characteristics of oat in response to saline-alkali stress.There were a total of 27 metabolic pathways co-enriched in BY,among which 12 were related to amino acid metabolism,and the rest included biosynthesis of phenylpropanol.However,the metabolic pathway co-enriched only in YZY is closely related to the metabolism of sugar,which reflects that the main characteristic of YZY variety in response to saline-alkali stress is related to sugar.The differential genes or metabolites in these metabolic pathways may be responsible for the differences in the response to saline-alkali stress between BY varieties and YZY.In this study,32 genes were selected for q-PCR asssy,and the results were consistent with the transcriptional sequencing results.Most of the genes bringed out were associated with the response to oat saline-alkali stress,including genes involved in Pyruvate dehydrogenase E1,ABC transporter,delta-1-pyrroline-5-carboxylate synthetase,peroxidase,polyamine oxidase-like,beta-glucosidase,sucrose synthase,etc.These genes will be the main candidate genes to further explore the molecular mechanism of oat in response to saline-alkali stress.In this study,through germplasm selection,physiological and biochemical indexes determination and omics analysis of saline-alkali stress of oat,the main mechanism of action of oat in response to saline-alkali stress was identified,which provided theoretical basis for saline-alkali resistance breeding and cultivation of oat.