Bupleurum 3 UGT Gene Expression Characteristics And Prokaryotic Expression And Protein Purification

Objective: Using qRT-PCR method, the expression characterizations of BcUGT1、BcUGT3and BcUGT6of Bupleurum chinense DC. after methyl jasmonate (MeJA) inductionand in different plant tissues were investigated. The target protein was successfully expressedand purified. The present work will be helpful for follow-up bio-function analysis ofBcUGT1、BcUGT3and BcUGT6.Methods: The RNA in adventitious roots of Bupleurum chinense DC. after methyljasmonate (MeJA) induction and untreated were extracted. And the RNA in root, stem, leaf,flower and fruit of Bupleurum chinense DC. were extracted too. Using a key enzyme genes,beta-AS gene as reference in qRT-PCR method, the expression characterizations of BcUGT1、BcUGT3and BcUGT6of Bupleurum chinense DC. after methyl jasmonate (MeJA) inductionand in different plant tissues were investigated. The ORF sequence of glycosyltransferase geneBcUGT1、BcUGT3and BcUGT6cloned from Bupleurum chinense DC. was analyzed. Afterenzyme digestion, respectively inserted into the pET-28a (+), and pET-30a (+), to construct theprokaryotic expression vector. Thereafter, the recombinant vectors of BcUGT1、BcUGT3andBcUGT6were constructed for its expression in E. coli., BL21(DE3) plysS, BL21CodonPlus(DE3) and Rosetta-gamiB (DE3) plysS, by different temperature and IPTG concentration. UsePrepEase His-tagged protein purification kit, to purify the target protein.Results: Using a key enzyme genes, beta-AS gene as reference in qRT-PCR method, theexpression characterizations of BcUGT1、BcUGT3and BcUGT6of Bupleurum chinense DC.after methyl jasmonate (MeJA) induction and in different plant tissues were investigated.The results of the expression characterizations of BcUGT1、BcUGT3and BcUGT6ofBupleurum chinense DC. after methyl jasmonate (MeJA) induction showed that the increasingcontent of bupleurum saponin a correspond with the increasing content of BcUGT1geneexpression. The BcUGT3and BcUGT6expression level increased after methyl jasmonate(MeJA) induction. The expression of BcUGT3was increasing with the extension of time. Thefourth day, the expression of BcUGT3wasabout seven times.The expression level of BcUGT6was about2times.The results of in vitro expression in E. Coli showed that in the condition of0.5mM or1 mM IPTG,16°C,20h, BcUGT1and BcUGT3target protein expressed in BL21-CodonPlus(DE3)–RIPL with pET-28a (+) or pET-30a (+) as vector, and in the condition of0.5mM or1mM IPTG,20°C,24h, BcUGT6target protein expressed in Rosetta-gamiB（DE3）plysS withpET-28a (+) as vector.Using PrepEase His-tagged protein purification kit, the target proteinwas purified.Conclusion: The gene expression and characteristics analysis of BcUGT1, BcUGT3andBcUGT6showed that the three genes may be involved in bupleurum saponins biosynthesis. Thepresent work will be helpful for follow-up bio-function analysis of BcUGT1、BcUGT3andBcUGT6.