岷江百合花青苷合成调控基因LrMYB15启动子克隆及活性分析OA

[Objective]This study aimed to clone the promoter of R2R3-MYB transcription factor MYB15 gene from Lilium regale(LrMYB15)and to detect its transcriptional activity,thereby providing reference for elucidating the regula-tory mechanism of LrMYB15 gene in flower color formation and environmental responses in Lilium regale.[Method]Ge-nomic DNA and RNA were extracted from the outer tepals of Lilium regale on the day of flowering.The promoter of Lr-MYB15 gene was cloned using chromosome walking method.Cis-acting elements in the promoter sequence were pre-dicted using PlantCARE database.A pBI121-LrMYB15-pro:GUS fusion expression vector was constructed and transiently transformed into leaves of Nicotiana benthamiana via Agrobacterium tumefaciens strain GV3101.Promoter activity was assessed under different light conditions and exogenous hormones[auxin(IAA),abscisic acid(ABA),and methyl jasmo-nate(MeJA)]using GUS histochemical staining,and activity of the promoter was further examined through real-time fluorescence quantitative PCR.A promoter vector with deleted light-responsive elements,pBI121-ΔLrMYB15-pro:GUS,was constructed to verify the function of light-responsive elements in the LrMYB15 gene promoter sequence.Expression levels of LrMYB15 gene in Lilium regale tepals under the three exogenous hormone and light treatments were detected by real-time fluorescence quantitative PCR,and anthocyanin content was measured.[Result]A 2269-bp promoter of LrMYB15 gene was successfully cloned.The promoter region contained a total of 97 cis-acting elements.In addition to core cis-acting promoter elements such as TATA-box and CAAT-box,it included three categories of cis-acting elements associated with distinct biological functions:growth and development-related elements,hormone-responsive elements,and abiotic stress-responsive elements.GUS histochemical staining demonstrated that the LrMYB15 gene promoter exhibited obvious transcriptional activity,driving expression of downstream GUS gene.Its activity was extremely low under dark condi-tions.Promoter activity of LrMYB15 gene was induced by light and enhanced by exogenous application of the three plant hormones(IAA,ABA,and MeJA).Following infiltration with the pBI121-ΔLrMYB15-pro:GUS vector lacking light-responsive elements,only extremely weak and sparse blue spots were observed in tobacco leaves under continuous dark treatment,indicating that deletion of light-responsive elements greatly reduced the light-induced activity of LrMYB15 gene promoter.The GUS gene relative expression levels in transiently transformed tobacco leaves detected by real-time fluorescence quantitative PCR were consistent with the GUS histochemical staining results.The relative expression of Lr-MYB15 gene in Lilium regale tepals was consistent with the GUS staining results,and changes in anthocyanin content in the tepals showed a consistent trend with the relative expression of LrMYB15 gene.[Conclusion]This study has success-fully cloned and identified the promoter of LrMYB15 gene from Lilium regale,confirming that it is synergistically regu-lated by light and hormones,with light-responsive elements playing a critical role in its light-induced activity.Lr-MYB15 gene positively regulates anthocyanin synthesis in Lilium regale tepals.The promoter of LrMYB15 gene can be uti-lized to drive expression of target genes in plants.