Cloning And Functional Studies Of Pc(S)-THBO Gene From Phellodendron Chinense Schneid

Phellodendron chinense Schneid.is a perennial woody herb belong to Phellodendron genus of Rutaceae,its dry bark have berberine,phellodendine,palmatine,the content of berberine is the highest.However,the study of key genes and biological on the berberine biosynthesis are little.Tetrahydroberberine oxidase（THBO）is an important oxidoreductase,and play key role during the process of berberine biosynthesis in Phellodendron chinense Schneid.The physiological function of Pc（S）-THBO gene in the process of berberine biosynthesis from Phellodendron chinense Schneid is studed and analyzed,and provides theoretical basis support for genetic breeding and comprehensive utilization.In this paper,the full-length sequence of Pc（S）-THBO gene of Phellodendron chinense Schneid.was cloned and obtained using RACE-PCR technology,the characteristics of nucleic acid sequence and protein sequence were analyzed by bioinformatics method,the activity of oxidoreductase was assayed by prokaryotic expression and purification method,the expression pattern of Pc（S）-THBO gene in root,stem and leaf was detected by fluorescent quantitative real time polymerase chain reaction（qRT-PCR）technology,the physiological function of Pc（S）-THBO gene in the process of berberine synthesis of Phellodendron chinense Schneid.was studied using genetic transformation technology.The main results are as follows:1.The Pc（S）-THBO of Phellodendron chinense Schneid.gene was cloned and obtained using RACE-PCR technology,its full-length sequence（mRNA）of Pc（S）-THBO gene was 1042 bp.Bioinformatics analysis showed that the protein Pc（S）-THBO was composed of 253 amino acid sequences encoding by 762 bp,and its relative molecular mass was 27 kDa,no signal peptide,no transmembrane domain,belongs to amphoteric protein which was a triose phosphate isomerase existing in the cytosol.2.The expression levels of Pc（S）-THBO gene were detected by qRT-PCR method.The results showed that the expression of Pc（S）-THBO gene of Phellodendron chinense Schneid.seedlings culturing for one year was root>stem>leaf.All different concentration exogenous Gibberellin3（GA3）significantly increased the expression levels of Pc（S）-THBO gene in root tissues,all different concentration exogenous 6-Benzylaminopurine（6-BA）significantly enhanced the expression levels of Pc（S）-THBO gene in root tissues,but 20 mg·L^-1 6-BA significantly enhanced the expression levels of Pc（S）-THBO gene in stem tissues.The 10 mg·L^-1 and 20 mg·L^-1 naphthlcetic acid（NAA）significantly improved the expression levels of Pc（S）-THBO gene in root tissues.3.The prokaryotic expression vector pGEX-4T-1-Pc（S）-THBO was constructed and obtained the fusion protein GST-Pc（S）-THBO through expression and purification vitro for identification of its oxidoreductase activity.4.The overexpression vector of pCAMBIA3300GFP-Pc（S）-THBO was constructed,and infected callus of Phellodendron chinense Schneid using EHA105 strain.The results showed that the expression level of Pc（S）-THBO in infected callus was enhanced,and the content of berberine was increased,indicated that Pc（S）-THBO promoted berberine biosynthesis of Phellodendron chinense Schneid..