Functional Analysis Of The Promoter Of Cp1-SST,the Key Enzyme Gene Of Fructan Synthesis,and Screening Of Transcription Factors Related To Polysaccharide Synthesis In Codonopsis Radix

Objective:Codonopsis Radix is produced in Shangdang Basin.It is a native medicine in Shanxi and has the effects of replenishing qi,strengthening spleen and lungs.Sucrose:sucrose 1-fructosyl transferase?Cp1-SST?was previously cloned from Codonopsis Radix by our group.The gene responds to drought stress and plays an important role in fructan synthesis.Research related to the gene in Codonopsis Radix has been initiated,but there is no research on the transcription regulation of fructan biosynthesis pathway in Codonopsis Radix.Based on the subcellular localization of Cp1-SST,this paper explored the response mechanism of Cp1-SST gene to drought stress and related sugar metabolism,cloned the promoter of Cp1-SST gene and performed functional analysis of its sequence and its regulating effect;The Codonopsis Radix transcriptome database was analysed to screen for transcription factors related to fructan synthesis,in order to clarify the regulation of fructan synthesis.It provides the basis for the molecular mechanism and lays the theoretical foundation for exploring the geoherbalism of Codonopsis Radix.Methods:1.Cp1-SST gene was cloned into pHBT-GFP-NOS vector by seamless cloning technology to obtain 1-SST-GFP plant transient expression fusion vector.The protoplasts of Arabidopsis thaliana were transformed by PEG-Ca²⁺-mediated method,and the protein subcellular localization was observed by fluorescence confocal microscope.2.The genomic DNA of Codonopsis Radix was extracted with CTAB method.Using Codonopsis Radix genomic DNA as a template,the sequence of Cp1-SST gene promoter?p1-SST?was cloned using Tail-PCR technology,and the cis-acting elements in the promoter were comprehensively analyzed using online analysis software PLACE and PlantCARE.The full length of p1-SST was cloned,and the recombinant vector pCAMBIA1381-p1-SST was constructed,transformed into Agrobacterium tumefaciens GV3101,and the tobacco leaves were infected by the bacterial liquid vacuum infusion method.After co-cultivation,GUS chemical tissue staining was performed to observe the pattern of the promoter activity.3.Previously,our research group sequenced the Codonopsis Radix transcriptome,and selected genes with significant differences in relative expression through data analysis.Collect the roots of Codonopsis Radix in four different growth periods,including the vine growth period,flower bell period,blooming period,and harvest period,and the different development parts of roots,stems,leaves,flowers,and fruits in the flowering period of Codonopsis Radix,to extract RNA.And reverse transcription into cDNA,as a template for transcription factor screening.Through fluorescence quantitative PCR analysis,the genes related to polysaccharide synthesis were screened out.Results:1.Construct the recombinant plasmid of pHBT-1-SST-GFP,express the 1-SST-GFP fusion protein,and observe the subcellular localization of the protein by fluorescence confocal microscopy.The results show that Cp1-SST is located in the chloroplast.2.Using Codonopsis Radix genomic DNA as a template,the sequence of Cp1-SST gene promoter was cloned by Tail-PCR technology to be 747 bp long.Comprehensive analysis using online analysis software PLACE and PlantCARE.In addition to the promoter conservative sequences TATA-box,CAAT-box,enhancer,the Cp1-SST gene promoter sequence also includes MYB binding site,dehydration response element,low temperature response element And many other components.The recombinant vector pCAMBIA1381-p1-SST was constructed and transformed into Agrobacterium tumefaciens GV3101,and the tobacco leaves were infested by vacuum infusion.The results of chemical tissue staining showed that the tobacco leaves of 35S::GUS?positive control?could detect the GUS staining reaction;GUS staining reaction was not detected in tobacco leaves of::GUS?negative control?;GUS staining could be detected in tobacco leaves of p1-SST::GUS Reaction.3.The genetic data of the Codonopsis Radix transcriptome after sequencing were preliminarily screened.The roots of Codonopsis Radix at different developmental stages and different tissues at the same growth stage were used as materials for quantitative PCR analysis to screen out genes related to polysaccharide synthesis.The results showed that after fluorescence quantitative PCR screening,among them,Unigene13082_All,CL1353.contig1_All,Unigene12984_All,CL906.Contig2_All,CL2094.Contig2_All had the highest relative expression.Conclusion:1.Cp1-SST subcellular localization in the chloroplast.Combined with the previous research results of the research group,it is speculated that the Cp1-SST gene is expressed in the root,and then it may be transferred to the main site of sucrose synthesis-chloroplast to play a role in preparation for further synthesis of glycans.This article provides directions for the functional mechanism of Cp1-SST function,and also lays a foundation for the systematic study of mechanisms of plant formation,growth and development,and even stress tolerance of Codonopsis Radix.2.Using Tail-PCR technology to clone the Cp1-SST gene promoter sequence is747 bp long,PLACE and PlantCARE comprehensive analysis,p1-SST has multiple promoter conservative sequences,and a variety of cis-acting elements.Construction of the expression vector pCAMBIA1381-p1-SST,infecting tobacco for transient expression analysis,the tobacco leaf of p1-SST::GUS detected GUS staining reaction,indicating that p1-SST can initiate downstream GUS gene expression in tobacco,and also shows p1-SST possesses promoter activity and can initiate the expression of downstream gene Cp1-SST.3.After fluorescent quantitative PCR analysis and screening,the genes Unigene12984_All,CL906.Contig2_All,CL2094.Contig2_All may be inversely related to the synthesis of Codonopsis pilosula polysaccharides;while the gene CL1353.contig1_All is related to the synthesis of polysaccharides.According to the annotation of the above genes in the Codonopsis Radix transcriptome data,there may be an interaction relationship with p1-SST.