| Soil salinization is strong resistance to the healthy and stable production of global agriculture and forestry and the sustainable development of the ecological environment.Salinization not only reduces soil production potential and destroys plant habitat,but reduces community diversity,causes ecological chain imbalance,and leads to degradation of ecosystem function.Especially in the arid and semi-arid areas of northwest China,the climatic conditions of low precipitation and strong evaporation promote the accumulation of salt on the soil surface,aggravate the degree of salinization and accelerate land degradation.Reaumuria soongorica,a super xerophytic salt-tolerant shrub of Tamariaceae,is one of the dominant species in the desert and steppe in China.It is highly resistant,ecologically adaptable and malleable,and it also has a good effect on water and soil conservation,windbreak and sand fixation.Therefore,the study of the physiological and molecular mechanism of salt tolerance of R.soongorica helps the process of cultivating new varieties of salt-tolerant plants,improves the utilization rate of salinized soil,and provides a certain reference basis and theoretical value for the improvement of salinealkali land and the stability of the desert ecosystem.In this experiment,we firstly investigated the differences in seed germination and seedling growth characters between different seed provenances,and then we investigated the changes in growth and physiological character of R.soongorica seedlings under salt stress by using NaCl solution to simulate soil salinity,lastly,the differential expression profiles of genes and proteins in leaves of R.soongorica seedlings under salt stress were established by using RNA-seq and iTRAQ techniques.Meanwhile,to provide a theoretical basis for understanding the response mechanism of R.soongorica to salt stress,we obtained some pathways and potential candidate genes for salt tolerance in R.soongorica through transcriptome and proteome association analysis.The main results are as follows:1.Analysis of the differences between Reaumuria soongorica seeds and growth character under different provenancesIn this study,we tested R.soongorica seeds from 10 different provenances on seed quality test,germination test and seedling growth character test.The results showed that the relative conductivity of R.soongorica seeds differed significantly among the different seed provenances,while the differences in seed vigor were not significant.Among the seed provenances ZGZ,R.soongorica performed best in terms of germination potential and germination index,however,in comparison in terms of plant height and root length,R.soongorica is the highest among the WD provenances.The differences in germination potential and total leaf surface area are mainly due to the different seed provenances.The Provenance SJZ had the highest dry fresh weight of leaf and dry weight of stem,while the water content of leaf is the lowest.According to the comprehensive evaluation method of multiple character,the seed goodness ranking(growth adaptability)of R.soongorica from different provenances was as follows: SJZ > ALS > BYHT >JGZ > ZGZ > WD > XGG > SPT > LHG > SGK,these results provided a reference for material selection in subsequent experiments.2.Effects of salt stress on physiological character and comprehensive evaluation of salt tolerance of Reaumuria soongoricaIn this study,we investigated the changes in endogenous hormones,photosynthetic character and other physiological indicators in leaves of R.soongorica seedlings under salt stress and their effects on the formation of salt tolerance,and the results showed that treatment with200 mM NaCl for 3 days significantly increased SOD activity,the content of osmotic adjustment substances(proline,soluble protein),endogenous hormone content(ABA,ZR),root-toshoot ratio,and Chla/Chlb values but decreased malondialdehyde content(MDA)in the leaves of R.soongorica seedlings.The leaf water content(LRWC),net photosynthetic rate(Pn),transpiration rate(Tr),water use efficiency(WUE)and growth hormone(IAA)contents of R.soongorica seedlings were lower than those of the control under 400 and 500 mM NaCl treatments.The results of the principal component analysis indicated that the endogenous hormone content and antioxidant enzyme activity of R.soongorica seedlings could be used to comprehensively evaluate the salt tolerance of R.soongorica seedlings.The results of the comprehensive evaluation showed that the R.soongorica seedlings treated with 200 mM NaCl for 3 days were the most salt tolerant.These results provide a theoretical basis for the further investigation of the molecular regulation mechanism of salt tolerance in R.soongorica and the further exploration of salt tolerance genes.3.Transcriptomics analysis of Reaumuria soongorica under salt stressUsing RNA-seq technology to analyze the transcriptome information of R.soongorica under salt stress,we found that Clean Reads were spliced and assembled to obtain a total of79307 Unigenes greater than 300 bp in length.We used Blastx software to compare all Unigenes in Nar,GO,KEGG,eggNOG,Swiss-Prot and Pfam databases and obtained 5701 Unigenes with common annotations,based on expression difference multiplicity |log2Fold Change| > 1and significance P-value < 0.05 as the screening criteria,among them,there were 1057,3903,2169 upward,and 1334,3307,1473 downward.Gene Ontology(GO)enrichment analysis showed that genes related to salt stress were mainly enriched in the "extracellular region","cell wall","outer encapsulation structure"," oxidoreductase activity " and "peripheral cell".Kyoto Encylopeida of Genes and Genomes(KEGG)analysis indicated that leaves of R.soongorica could respond to salt stress by influencing protein translation,signal transduction,compound metabolism and modification,and antioxidant process.4.Proteomic analysis of Reaumuria soongorica under salt stressFirstly,47,177 and 136 differential proteins were screened in the 200 vs 0,500 vs 0 and500 vs 200 paired groups by iTRAQ proteome sequencing technology,based on the screening criteria of expression fold change >1.5 and <0.66 and P-value <0.05.Among them,there were36,126,67 upward and 11,51,69 downward.Secondly,29,112 and 86 differential proteins were identified in the three comparison groups that could be annotated by the COG database and could be functionally classified.By selecting protein sites with nodal connections greater than 1 in the protein interactions regulatory network and integrating the duplicated proteins in each comparison group,72 DEPs(34 upward and 38 downward)were screened,among which the most DEPs were involved in "translation,ribosome structure and biogenesis",with 17 belonging to the ribosomal protein family(RP).Lastly,the scores of each protein were calculated by Mascot search software and those with scores greater than 60 were selected as reliable proteins.The screened differential proteins were also manually grouped and a total of 21 DEPs were found to be potential targets of action for salt tolerance in R.soongorica,namely "proteins related to plant energy and metabolism"(VHA-A,VHA-E1,AHA4,CSY4,PCK1),"proteins related to photosynthesis-related proteins"(RCA,PSAF,PSAN,PSB27-1),"proteins related to plant defense and drought resistance"(P5CR,FC2,SYP71,SYP131),"proteins related to protein synthesis,processing and degradation-related proteins"(rpl2-A,RPL12 A,RPS6B,RPL7 AA,RPS2D,RPS10,RPS9,At2g47020).5.Correlation analysis on transcriptomic and proteomeThe strength of the correlation between gene and protein was explored by calculating Pearson correlation coefficients between transcriptional level and protein level data.The results showed that quantitative protein and gene expression were positively correlated in the 200 vs0,500 vs 0 and 500 vs 200 paired groups with correlation coefficients of 0.0187,0.1366 and0.0983,respectively,while the correlation coefficients for differentially expressed proteins and genes with consistent expression trends were 0.5803,0.2741 and 0.2296,respectively.In the three comparison groups,5,32 and 10 genes were found to have consistent protein and mRNA expression trends,while 0,8 and 2 genes were found to have opposite protein and mRNA expression trends,respectively.The KEGG pathway enrichment analysis revealed that the differential proteins with consistent mRNA and protein expression trends in the 500 vs 0 comparison groups were mainly involved in the "cysteine and methionine metabolism","citric acid cycle","SNARE interactions in vesicular transport" and "photosynthesis" pathways,followed by an in-depth analysis of the significantly enriched metabolic pathways,which revealed that SYP71,CS,PCC13-62,PASN,ZIFL1,LTI65 and other genes may be potential targets for salt tolerance in R.soongorica.In conclusion,R.soongorica was used as the subject to investigate the differences in phenotypes,physiological and biochemical indicators,gene and protein expression profiles of R.soongorica in response to salt stress.Also,this study has systematically investigated the mechanisms of salt stress on R.soongorica at the growth,physiological and molecular levels,providing a certain reference basis and theoretical value for the improvement of ecosystem restoration capacity and the stability of desert ecosystems. |
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