Molecular Cloning And Function Analysis Of HPPR Gene Promotor In Perilla Frutescens

Rosmarinic acid is one of the natural multifunctional phenolic acid compounds,and it also is a kind of secondary metabolites with a variety of activities in Perilla frutescens.With the understanding of nutrients and active substances in Perilla frutescens,the Rosmarinic acid content is as high as 1.29%.In this paper,the promotor of Hydroxyphenylpyruvate reductase gene(HPPR)was firstly cloned from the leaves of Perilla frutescens.Due to the substrate of HPPR was 4-hydroxyphenylpyruvate,which was a precursor of rosmarinic acid and homogentisate.HPPR was considered as the first key enzyme in rosmarinic acid synthesis pathway.So analysing the cis-regulatory elements in HPPR gene promoter could lay a foundation for the further function verification in stress response.This study also laid the theoretical basis for genetic breeding of Perilla frutescens.(1)The cDNA sequence of HPPR gene of Perilla frutescens was obtained from datebase(Accession Number:HM587131.1).The primers according to the cDNA sequence of HPPR gene were designed and the sequence of DNA was obtained.It was 2525bp.HPPR gene promoter was obtained from genomic DNA by genomic walking.It showed that the HPPR promoter length was 2216bp.Plant Care datebase showed that the HPPR promoter region contained several kinds of elements,including some typical elements TATA-box,CAAT-box.It also involved in some elements which responsed to light,such as Box I,G-box,GT1-motif,MNF1,SPI.Gibberellin(G-box),salicylic acid(TCA-element),Methyl Jasmonate(CGTCA-motif),abscisic acid(ABRE),auxin(TGA-element)et al were also included in this promotor.Besides,it included some elements responsing to abiotic stresses,such as Fungal elicitor response element(Box W1),MYBHvl bonding site(CCAAT-box),heat stress(HSE),Low temperature response(LTR),drought induced binding sites(MBS)et al.These characteristics fully showed the efficiency and the complexity of gene promoter regulation at the transcription level.(2)Primers were designed to amplify 5' successive promoter truncations fragment of HPPR promoter.The target fragments with the restriction sites were obtained,and connected them to PB1121 binary expression vector which was cut off the CaMV 35S promotor.Then we could obtain the recombined plasmids Pl1 P2 and P3.These vectors were transferred into tobacco with the help of Ti-plasmid in Agrobacterium tumefaciens.At last we selected the positive colonies by kanamycin.Infected by Agrobacterium,the tobacco was cultivated for three days.Then tobacco leaves and stems were stained through GUS histochemical.This study determined the position of the core promoter sequences according to the result of dyeing,which laid a foundation for further research about tissue-specific expression and function analysis of HPPR promoter.