Molecular Mechanism Of Oxytropis Glacialis Adaptation To Extreme Environment In Qinghai Tibet Plateau Based On RNA-seq

Natural selection leads to the biological adaptive evolution has been regarded as an important mode of the generation of biodiversity in nature.At present,the adaptive evolutionary mechanism is still the central issue of evolutionary biology.The Qinghai-Tibet Plateau is known as the "third pole",has a variety of extreme environments such as strong ultraviolet radiation,low temperature,salinization,low oxygen,and so on.and It is also one of the hotspots of the world's biodiversity and a natural laboratory for exploring the adaptive evolution of organisms in extreme environments.Oxytropis glacialis is an endemic species in the Qinghai-Tibet Plateau.It can be widely distributed under multiple environmental stress conditions.It has strong resistance to stress.At the same time,Oxytropis glacialis is considered to be the most serious poisonous grass which endangers the grassland animal husbandry because of its toxic whole grass.In this study,17 samples of Oxytropis glacialis from 6 populations(Yangbajing YBJ,Cuoqin CQ,Zabuye ZBY,Sapu SP,Shuanghu SH and Everest ZF)were sequenced,combined with population environmental detection and bioinformatics analysis,explored the molecular mechanism of the Qinghai-Tibet Plateau Oxytropis glacialis adapting to the plateau extreme environment.This study draws the following conclusions:(1)RNA-seq results showed that 9,569,217,88 raw reads were obtained from 17 RNA samples of 6 populations,9,509,381,26 clean reads were obtained after quality control,Q20(%)was greater than 98%,3,017,38 unigenes were obtained through optimized assembly,5,403,084,00 clean reads were obtained after assembly and filtration,and 3,389,857,38 of them were compared to the assembly transcripts,the comparison rate was 62.7%.GO annotation of unigenes showed that most of unigenes were annotated to cell process,metabolic process,cell,cell part,binding and catalytic activity;GO enrichment of unigenes showed that the regulation of cell shape,intra-Golgi vesicle-mediated transport,pigment biosynthesis process,abscisic acid-activated signaling pathway,peptide-serine phosphorylation and peptide-serine modification were the main enrichment terms.KEGG annotation of unigenes showed that,of all the metabolic pathways,most unigenes were annotated to carbohydrate metabolism,amino acid metabolism,energy metabolism,lipid metabolism,translation,signal transduction,transportation and catabolism,environmental adaptation.KEGG enrichment of unigenes showed that most of the unigenes were enriched to photosynthesis,carotenoid biosynthesis,MAPK signaling pathway-plant,plant hormone signal transduction and plant-pathogen interaction.In this study,the RNA-seq analysis of Oxytropis glacialis showed that the genes related to cell shape regulation,pigment biosynthesis process,abscisic acid activation signal pathway,MAPK signaling pathway-plant,plant hormone signal transduction,photosynthesis,carotenoid biosynthesis were enriched,compared with the previous studies,this preliminary revealed that Oxytropis glacialis may respond to stimulation by activating multiple signals,it can regulate the expression of related genes by signal transduction,then regulate the metabolism of related products and the cell process,metabolism process,so as to adapt to the extreme environment of the Qinghai Tibet Plateau and ensure normal life activities of Oxytropis glacialis.These functional genes may be helpful for Oxytropis glacialis to adapt to the extreme environment of low temperature,strong radiation and salinization in the Qinghai Tibet Plateau,it also provides a basis for further exploring its adaptive molecular mechanism and poison weed control.(2)RNA-seq data of Oxytropis glacialis at different altitudes combined with weighted gene coexpression network analysis(WGCNA)showed that,MEyellow(cor=0.662,p= 0.00397)was the most correlated with the altitude.The GO enrichment analysis of MEyellow module showed that most genes were enriched in amino biosynthesis process,translation,peptide biosynthesis process,peptide metabolism process,DNA repair,cellular amino metabolism process,DNA metabolism,DNA integration,lipid metabolism process,nucleobase-containing compound metabolism process,heterocycle metabolism process,nucleic acid metabolism process,organic cyclic compound metabolic process,cellular macromolecule metabolism process,nitrogen compound metabolism process,macromolecule metabolism process,cellular nitrogen compound metabolism process,cellular aromatics metabolism process,biology metabolism and metabolism.In this study,the genes related to DNA repair,DNA metabolism and DNA integration are: BRCA2,DMAP1,G6PD1,SCC1,ERCC2,INOC1,SMC6 A,STIL3,IRAK4,CAPG,TOP2,DICER1,MYBP,CAPG,RGP1,CNBP,STR9,GIS2,CYP716 A,IRAK4,POLX,XYL1,TF21,which indicates that the cells may be vulnerable to damage at strong radiation,genes related to DNA repair,DNA metabolism,DNA integration were enriched,which is beneficial to the survival of Oxytropis glacialis in strong radiation of plateau.Compared with the previous studies,the enrichment of these genes related to basic metabolism indicates that Oxytropis glacialis is active in the expression of basic metabolism,and the active metabolism may be conducive to the adaptation of Oxytropis glacialis to the plateau environment.The radiation intensity increases with the elevation,and the radiation intensity is high in the high altitude,and the effect of strong radiation on photosynthesis is the greatest.The MEyellow module was the highest correlation with altitude,we obtained 10 genes related to photosynthesis by KEGG annotation,psaA,psaH,psaN,psaO,psbA,psbE,psbP,psbQ,petC and petF.These genes had the lowest expression in ZF populations with the highest elevation,the expression in the other five populations was positively correlated with altitude.Unigenes KEGG annotation obtained 5 genes related to the photoprotective substances flavonoids synthesis,HCT,CYP73 A,CCOAMT,CA4 H and C4 H.The expression of these genes was lowest in the highest ZF populations,and increased with elevation in the other five populations.The Qinghai Tibet Plateau has not only strong radiation,but also low temperature all year round,and the temperature is negatively correlated with the altitude.8 genes related to low temperature response were obtained by annotation,including BSK,RPL?21,KCS,IRAK4,CRPM2,TGS1,SRC2 and cold-acclimation specific protein 15.The expression of related genes of ZF,SH and SP populations at higher altitudes was higher than that of ZBY,CQ and YBJ populations at lower altitudes.(3)Soil conductivity can reflect the level of soil salt concentration,in this study,the measurement results of the soil conductivity of the sampling points showed that ZBY(saline environment,conductivity was 0.3 ms/cm),conductivity of YBJ was 0.14 ms/cm,ZF was 0.13 ms/cm,CQ was 0.09 ms/cm,SH was 0.05 ms/cm,SP was 0.01 ms/cm.YBJ,CQ,SP,SH,ZF were non-saline environments.Using RNA-seq data combined with WGCNA,it was found that the most significant correlation with soil conductivity was MEpurple(cor=0.589,p=0.00797),and 23 genes related to salt stress were screened out by annotation.According to the products encoded by these genes,these genes can be divided into the following four categories.First,genes related to antioxidant enzymes: CCPR,pep N,NDUFS4,msrA,mtnD,GPX,trxA,SOD2,PTA14,PRDX5.Second,genes related to osmotic regulators: RFS,ACLY,metK,metE.Thirdly,genes related to hormone: SAUR,AX10 A.Fourth,other genes: HRR25,PSMB7,MYO1,ATPeV1 D,RAB1A,RAN,EEF1 A.These genes related to salt stress were highly expressed in ZBY populations(saline environment),indicating that under the salt stress,oxytropis glacialis adapted to the saline environment by increasing the expression of salt stress-related genes.(4)We obtained the metabolic pathways of Oxytropis glacialis and pathogens interaction by KEGG annotation of unigenes.When Oxytropis glacialis is infected by pathogens,it will activate the corr esponding genes to respond to the signal stimulation,regulate the expression of downstream genes and transcription factors(RAR1,SG T1,HSP90,WRKY25,WRKY29,WRKY1,NHO1,PR1),Among them,the expressions of RAR1,SGT1 and HSP90 will cause hypers ensitive reponse;WRKY1 induces the expression of defense-related genes and programmed cell death;the expression of WRKY25 and WRKY29 will lead to the expression of defense-related genes NHO-1 and PR1,thus causing phytoalexin accumulation and miRNA production to ensure the normal life activity of Glacier Oxytropis.