Effect Of Betulin On NO Metabolism Of Betula Platyphylla And Cloning Of BpbZIP25 In Response To NO

Nitric oxide(NO)is a key signaling molecule that mediates plant growth and development,secondary metabolite regulation and stress resistance.Protein S-nitrosylation is an important way for NO to play its signal transduction role.Our previous study revealed that NO and protein S-nitrosylation promoted the accumulation of triterpenoids such as betulin in suspension cells of birch.NO,protein S-nitrosylation and betulin are all factors that the plant adapts to the bad environment,but the relationship among them is still unclear.It has not been reported whether triterpenoids such as betulin affect the growth and NO metabolism of birch suspension cells.In addition,the target protein of S-nitrosylation is not yet clear.Therefore,on the basis of reviewing the literature,this paper analyzed the effects of exogenous addition of betulin on the growth and NO metabolism of birch suspension cells.The third-generation full-length transcriptome data and reported birch genomic data were used to identify the birch bZIP gene family,and screen the birch bZIP genes in response to NO.On this basis,bZIP genes related to triterpenoids such as betulin were further screened by correlation analysis.The main results are as follows:1?Effects of exogenous betulin on suspension cell growth and NO metabolism of Betula platyphyllaThe phenotype,cell viability,fresh weight,pH value,conductivity and malondialdehyde of birch suspension cells treated with 0.1 ?mol/L?5?mol/L betulin were all not significantly different from those of the control.After treatment with 0.1?mol/L?5?mol/L betulin for 7 days,the NO content,reduced glutathione(GSH)content and nitrosothiol(SNO)content in the birch suspension cells all showed an increasing trend.Respectively reached the maximum value under the treatment of 5p.mol/L,0.5?mol/L and 0.5?mol/L betulin,which were increased by 178.95%,45.10%and 6.87%compared with the control.Similarly,the activity of nitric oxide synthase,glutathione reductase and nitrosylated glutathione reductase and the change trend of their gene expression levels were consistent with the above changes in the content of corresponding substrates or products.However,the activity change of nitrate reductase,one of the key enzymes in NO synthesis,was opposite to the increase of NO content,and the reason needs further study.2.Identification and expression pattern analysis of BpbZIP family genes of Betula platyphyllaBased on the three-generation full-length transcriptome data obtained from the PacBio Sequel ? sequencing platform combined with the second-generation transcriptome data and the Betula genome data,a total of 57 birch BpbZIP genes were obtained,and a total of 10 conserved motifs existed.With reference to the classification of the Arabidopsis bZIP gene family,57 Betula platyphylla BpbZIP genes were divided into 13 subfamilies.Chromosome mapping results showed that 46 BpbZIP genes were successfully located on 13 chromosomes,mainly distributed at the ends of the chromosomes.After 1?mol/L NO treatment for 30 days of birch seedlings and GSNOR gene silencing birch,the expression trends of 57 BpbZIP genes were different.The top 7 genes in terms of expression levels were BpbZIP26>BpbZIP25>BpbZIP22>BpbZIP24>BpbZIP23>BpbZIP14>BpbZIP48.The expression levels of BpbZIP26 and BpbZIP25 were 10.30 times and 7.49 times of the control group,respectively.3?Cloning and bio informatics analysis of BpbZIP gene of Betula platyphylla in response to NOThe full length of seven BpbZIP gene sequences in response to NO was obtained by PCR,and bioinformatics analysis showed that the seven BpbZIP genes encoded proteins that were non-secretory hydrophilic proteins.The prediction results of nitrosation sites showed that nitrosation sites were detected in 5 BpbZIP proteins,which were ranked according to score as BpbZIP22>BpbZIP48>BpbZIP25>BpbZIP24>BpbZIP14.score as BpbZIP22>BpbZIP48>BpbZIP25>BpbZIP24>BpbZIP14.Phylogenetic analysis showed that BpbZIP14,BpbZIP23,BpbZIP24 and BpbZIP26 had high homology with bZIPs involved in the regulation of terpene synthesis in Artemisia annua,rice and dandelion,respectively.According to the published sequencing data of birch,the expression trends of the cloned 7 BpbZIP genes were different in the 7 layers of bark tissue of birch,among which BpbZIP25 and BpbZIP26 were both highly expressed in the non-conductive phloem and the conductive phloem.4?Subcellular localization analysis of BpbZIP25The correlation analysis between the expression of 57 BpbZIP genes and the content of birch triterpenoids under NO treatment showed that the top three genes highly correlated with the content of birch triterpenoids were BpbZIP25,BpbZIP24 and BpbZIP26.Their correlation coefficients were 1.00,0.99 and 0.96,respectively.Therefore,it is speculated that BpbZIP25 mediates NO to promote the synthesis of birch triterpenoids.To verify its function,we constructed interference vectors and subcellular localization vectors of BpbZIP25 gene,and subcellular localization results showed that the protein encoded by BpbZIP25 was localized in the nucleus.The above results lay a foundation for further study of NO mediated by BpbZIP25 to promote the synthesis of birch triterpenoids.