Cloning Of The Promoter Of The Sucrose Transporter Protein Gene PsSUT2 And Screening Of Its Reciprocal Transcription Factors In The Peony

Peony(Paeonia suffruticosa)is a deciduous perennial shrub,which is popular for its rich flower shape,large corolla and bright flower color.However,after planting in pots,peony plants are often dwarfed and the flower buds are defeated,which seriously affects their ornamental value and has become a bottleneck in the industrial development of potted peonies.The group found that sucrose plays an important role in the growth and development of potted peony,and cloned the full-length c DNA of PsSUT2 gene,which is related to sucrose transport in peony,and studied its function.In this study,the promoter sequence of PsSUT2 gene was cloned from the petals of ’Luoyang Red’ peony in full bloom,and its cis-acting element was analyzed.GUS histochemical staining and GUS real-time quantitative expression analysis were used to detect the activity of the promoter of PsSUT2 gene,and the core key region of the promoter was inferred,based on The cis-acting elements on the promoter were combined with WGCNA to construct a co-expression network to predict potential transcription factors regulating PsSUT2,and then the potential transcription factors were analyzed and cloned for expression,after which the transcription factors interacting with the target promoter were screened by yeast single-hybrid and dual-luciferase assays,and bioinformatics analysis was performed to initially predict their structural and functional characteristics.The results are as follows:(1)The promoter sequence of the sucrose transporter protein gene PsSUT2,with a total length of 1,819 bp,was cloned from the petals of ’Luoyang Red’ peony at the flowering stage,and the promoter sequence was analyzed bioinformatically.The online database Plant CARE was used to analyze the possible cis-acting elements on this promoter,including the core elements TATA-box,CAAT-box and cis-acting elements involved in abscisic acid response,light response,drought induction,low temperature response,gibberellin response and methyl jasmonate response.(2)The PsSUT2 gene promoter was deleted from 5’ by 4 segments according to the location of the cis-acting element,with the lengths of 1 255 bp,641 bp,260 bp and109 bp,and fused to the p BI121-35s-GUS vector.The PsSUT2 promoter was proved to be active by GUS histochemical staining and GUS real-time quantitative expression analysis,and the core region was presumed to be located between-260 bp and-109 bp.(3)The gene co-expression module was constructed by WGCNA analysis,and PsSUT2 gene was identified to be located in the yellow module,in which 9 transcription factors were closest to PsSUT2 expression pattern.Combining with the information of cis-acting element prediction results,6 potential regulatory transcription factors were initially identified,namely: PsCCCH30,PsMYB20,PsMADS9,PsWRKY20,Psb HLH3,PsBIM1.(4)The expression of the six potential regulatory transcription factors screened and PsSUT2 gene were determined by real-time fluorescence quantitative assay,and the expression patterns of the six transcription factors and PsSUT2 gene were similar,and the median relative expression correlation coefficient was 0.86,which was basically consistent with the predicted results.After that,PsCCCH30,length 2,151 bp;PsMYB20,length 891 bp;PsMADS9,length 603 bp;PsWRKY20,length 1,677 bp;Psb HLH3,length 1,731 bp;PsBIM1,length 1,539 bp were successfully cloned.(5)Successfully constructed PsSUT2 promoter with p Lac Zi recombinant expression vector and PsCCCH30,PsMYB20,PsMADS9,PsWRKY20,Psb HLH3,PsBIM1 of p B42 AD recombinant expression vector,transformed yeast receptor state,yeast single hybrid results show that PsMADS9 and PsMYB20 and PsSUT2 gene The promoter had a reciprocal relationship and was able to activate the expression of PsSUT2 gene;the plant expression vector of PsSUT2 with p Green II0800-LUC and the p Green II62-SK plant expression vector of PsCCCH30,PsMYB20,PsMADS9,PsWRKY20,Psb HLH3,PsBIM1 were successfully constructed and transiently transformed tobacco,and the ability of PsMADS9 and PsMYB20 to bind to the promoter sequence of PsSUT2 gene and positively regulate the expression of PsSUT2 was verified again by dual luciferase assay.(6)Bioinformatics analysis revealed that PsMYB20 protein has an isoelectric point of 8.48 and an instability coefficient of 49.70,which is an unstable protein with no transmembrane structure and is hydrophilic,and its subcellular localization predicts that PsMYB20 protein is located in the nucleus and chloroplast;PsMADS9 has an isoelectric point of 9.43 and an instability coefficient of 32.00,which is a stable protein with no transmembrane structure PsMADS9 is a hydrophilic protein and its subcellular localization predicts that PsMADS9 protein is located in the nucleus.