| Sophora alopecuroides L.as a plant resource in northwest China,which has important ecological and medicinal value.In this study,based on the transcriptome sequencing analysis of S.alopecuroides after salt stress in the laboratory,a quantitative proteomics method using tandem mass spectrometry(Tandem Mass TagTM,TMT)was used to analyze the proteome of leaves under 150mmol·L-1 NaCl stress with the growth of 50d,match and identifydifferentially expressed proteins and analyze the function from the perspective of bioinformatics.Combined analysis of proteome and transcriptomedata was conducted to explore the molecular mechanism of S.alopecuroides responded to salt stress and alkaloid synthesis of secondary metabolites.Lysinedecarboxylase gene(SaLDC)is a key enzyme gene for the synthesis of MA and OMA in S.alopecuroides,and it also played an important role in coping with salt and low temperature stress.Based on the transgenic Arabidopsis with SaLDC,this study explored the SaLDC to NaCl and low temperature stress,and further analyzed the function of SaLDC.The main research results are as follows:(1)The grown of 50d S.alopecuroides were treated with 0,50,100,150,200mmol·L-1 NaCl solution.The activities of SOD,POD and CAT,and the contents of Pro and MDA upward with the increase of NaCl concentration;the content of Na+ in leaves was increased,while the content of K+decreased,it indicated that S.alopecuroides responded to salt stress by regulating the content of physiological indexes related to stress resistance and improved salt tolerance.(2)Under 0,50,100,150,200mmol·L-1 NaCl stress,the relative expression of SaLDC in leaves was higher than that in the control at 3d,and the highest was at 150mmol·L-1;But when the stress for 7d,the relative expression of SaLDC in the leaves increased first and thendecreased,and when the stress exceeded 100mmol·L-1,the relative expression of SaLDC continued todecrease and was lower than the control.After 3d and 7d of stress,the content of MA in roots,stems and leaves were significantly reduced;the content of OMA in roots and leaves were the highest than that in the control at 3d,butdecreased at 7d,indicated that NaCl stress can affect the biosynthesis of MA and OMA to a certain extent by afected the expression of SaLDC in S.alopecuroides.(3)TMT proteomics was used to analyzed the leaves of S.alopecuroides under 150 mmo1·L-1 NaCl stress for 0d,3d and 7d,and a total of 5 505 proteins were identified.By comparing the protein expression levels indifferent stressdays,127(3d vs 0d),247(7d vs 0d)and 60(3d vs 7d)differential proteins were obtained.GO enrichment analysis showed that thedifferential proteins were mainly involved in Catalytic activity,Binding and Transporter activity;KEGG metabolic pathway indicates that the enriched metabolic pathways were mainly cAMP signal pathway,phosphatidylinositol signal system,amino acid metabolism and biosynthesis of secondary metabolite alkaloids.The combined analysis of proteomics and transcriptomics showed that short-term(3d)salt stress mainly affected the pathways and proteins related to signal transduction,and long-term(7d)salt stress could respond to regulate the expression of proteins related to the synthesis of flavonoids and alkaloids from secondary metabolites.The results of PRM and qPCR showed that TMT proteomics omics had high accuracy and reliability.(4)The selected three T2 transgenic Arabidopsis seeds with high SaLDC expression were subjected to stress with 0,50,100,150,200mmol·L-1 NaCl solution.With the increased of NaCl concentration,the germination rate,germination potential and root length of transgenic and WT seedsdecreased,and the relative saltdamage rate increased.The root length of transgenic was longer than that of WT,but the root length of transgenic and WT was 0 under 200mmol·L-1 NaCl stress.The MDA content of the three transgenic lines increased in a short period of time,and at 48 h and 72 h was lower than that in WT with the same time.The relative expression of SaLDC of transgenic lines was significantly higher than that of WT.It indicated that SaLDC was involved in salt stress.(5)The seeds of three T2 transgenic Arabidopsis lines with high SaLDC expression were subjected to low temperature stress at 4?,it was found that there was no significantdifference in germination rate and germination potential between the transgenic lines and WT,while the root length was longer than that of WT,and extremely significant;Under low temperature stress,it was found that with the extension of stress time,the MDA content and SaLDC relative expression in three transgenic lines were basically consistent with salt stress,and the relative expression of SaLDC increased to the highest at 48h,it indicated that SaLDC was also involved in low temperature stress. |
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