Salt Stress Response Mechanism Of Stellera Chamaejasme And Characterization Analysis Of ScPNP-A Gene

Stellera chamaejasme Linn.is a perennial herb of the Stellera in Thymelaeaceae.The whole plant is toxic,and its main toxic component is chamaejasmin,which can poison and kill cattle,sheep,and other livestock.S.chamaejasme is used as a medicinal plant in the"Divine Framer’s Classic of Materia Medica",and is also recorded in many Pharmacopoeias such as"Chinese Materia Medica".In the classic series of Tibetan medicine"Four-Volume Medical Code"and"Desirable Precious Tree",it is recorded that S.chamaejasme has the pecial effects in external application for the treatment of swelling,recalcitrant and hemorrhoids,and internal treatment for rickets,inflammation and edema.Existing studies have also shown that various active ingredients in S.chamaejasme have antibacterial,antiviral and antitumor activities.At present,the research on S.chamaejasme is mainly focused on the separation and identification of chemical components,active ingredient analysis,etc.Their growth habits and physiological characteristics in natural habitats,as well as molecular mechanisms for adapting to harsh environments,are rarely reported.Compared with other grassland plants,S.chamaejasme is more suitable for harsh environments such as high salinity,drought and cold.In recent years,the rapid expansion of S.chamaejasme has severely damaged grassland ecological equilibrium and,consequently,seriously endangered the development of animal husbandry.In this thesis,in order to understand the basic survival characteristics of S.chamaejasme,its physiological and photosynthetic characteristics in natural habitats were studied.Second generation high-throughput sequencing was also used to establish a genetic database of S.chamaejasme in response to salt stress.On this basis,bioinformatics,molecular biology and physiological and biochemical methods were used to reveal the mechanism of salt tolerance in S.chamaejasme at the physiological and molecular levels,which would provide a theoretical basis for the effective prevention and control,and sustainable development and utilization of S.chamaejasme.The main findings are shown as follows:（1）During the flourishing flowering period of S.chamaejasme at its natural habitat,the photosynthesis curve was measured by LI-6400XT and the results showed that the net photosynthetic rate curve of S chamaejasme was double-peaked,which indicated there was a typical midday break.By fitting the light response curves using different mathematical models,results showed that the right-angled hyperbolic correction model was the most suitable because of the higher R² and the accuracy to fit the inhibitory data produced by high light intensity.The photosynthetic characteristics,chlorophyll fluorescence characteristics,physiological and biochemical indexes and chloroplast pigment content of S.chamaejasme began to decline gradually at different stages after the full bloom period.（2）We performed RNA-seq on 15 S.chamaejasme samples from five different stages（0 h、3 h、12 h、24 h and 72 h）under salt treatment using the Illumina Hiseq 4000 platform,and obtained 115.74 GB clean data.Sequence assembly produced 121,781 unigenes,of which64,561（53.01%）were annotated against different databases.Organism similarity comparison showed that the top five matching species were Theobroma cacao,Gossypium arboreum,Vitis vinifera,Citrus sinensis and Jatropha curcas.A total of 5,888 differentially expressed genes（DEGs）were generated by gene expression analysis.Gene Ontology（GO）analysis showed that these DEGs mainly functioned in response to salt stress,response to light stimulation,hormone-mediated signal transduction,and response to Ca²⁺.The results of KEGG enrichment indicated that the synthesis of terpenoids,plant hormone signal transduction and zeatin biosynthesis may play an important role in the salt stress response of S.chamaejasme.（3）On the basis of an established local transcriptome database of S.chamaejasme using the transcriptome data,ten internal reference gene（RG）candidates,e.g.18S,60S,CYP,GAPCP1,GAPDH2,EF1B,MDH,SAND,TUA1 and TUA6,were selected using the BLAST program.Afterwards,the gene expression stabilities of those candidates in gene normalization analysis under three abiotic stresses（drought,cold and salt）and three hormones（ABA,GA and ETH）were evaluated by following geNorm,NormFinder and BestKeeper procedures.Results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses,whereas TUA6 and SAND,TUA1 and CYP,GAPDH2 and 60S were the best choices for ABA,GA,and ETH treatments,respectively.Moreover,GAPCP1and 60S were assessed to be the best combination across all the samples,and 18S was the least stable RG for use as an internal control in all of the experimental subsets.The expression patterns of two target genes（P5CS2 and GI）further verified that the RGs that we selected were suitable for gene expression normalization.In addition,GAPCP1 and EF1B were used as internal reference genes to standardize the expression levels of six randomly selected genes in the salt-stressed DEGs.The results were consistent with the transcriptome sequencing data,which confirmed that the reliability of the transcriptome sequencing results.（4）Using the FPKM values of the DEGs obtained from the transcriptome data of 15 samples under salt stress of S.chamaejasme as the original data,weighted gene co-expression network analysis（WGCNA）was employed to further analyze the potential regulatory genes and signaling pathways involved in the stress response of S.chamaejasme.The result showed that 2803 of 5,888 DEGs were divided into six modules through WGCNA.In each module,the top five genes with the highest connectivity were identified by the weight coefficients,and 27 genes directly involved in salt stress response were chosen through GO annotations.To explore the role of the module in response to salt stress in S.chamaejasme,the hub gene ScGH3.1 in the blue module was used for further functional analysis and the results showed that overexpression of ScGH3.1 significantly enhanced the salt stress tolerance of Arabidopsis thaliana,and preliminarily validated the potential positive activity of blue module under salt stress.（5）In this study,a S.chamaejasme plant natriuretic peptide A gene（ScPNP-A）was identified and characterized for the first time.ScPNP-A belongs to a member of the DPBB₁（double-psi beta-barrel）superfamily and is a DPBB₁ domain containing protein.Subcellular localization in the epidermis of leaves indicated that ScPNP-A was localized in the extracellular（secreted）space.In S.chamaejasme,the expression of ScPNP-A was significantly up-regulated by salt,drought,and cold stress.The physiological and biochemical indicator detection and resistance-related genes expression indicated that overexpression of ScPNP-A can significantly improve salt,drought and freezing tolerance in Arabidopsis.In addition,ScPNP-A overexpression can enhance the resistance to pathogens by enhancing systemic acquired resistance（SAR）in Arabidopsis.These results demonstrated that ScPNP-A could function as a positive regulator in plant response to abiotic stresses and biotic stresses.