Impact of rolC Gene on Ginsenosides Content in Ginseng Hairy Roots

摘要

According to Bulgakov's studies, the rolC gene is in the ORF12 of T-DNA of R_i plasmid. A pair of primers were designed and synthesized according to the analytical results of RiA_4T_L-DNA sequence by Slightom et al. Both of 0.8 kb PCR product was obtained by PCR using the DNA of the plasmid R_i of A_4 and the genome DNA of hairy root R9923 as template. The PCR product was ligased into pGEM-T vector and sequenced. The result of sequencing showed that the PCR product was same as the sequence of rolC of ORF12. The vector pGEM-T-rolG digested by restriction enzyme SacI and vector pUC-19 digested by Sma I restriction enzyme was recombined into the pUC19-rolC plasmid that was screened by blue-white selection. Both of pUC19-rolC and pBI121 were degested by the restriction enzyme Xba I and Sac I, the rolC gene was inserted into pBI121 to construct an expression vector pBI-rolC. Hairy root was expressed by cotyledon of Panax ginseng that was transformed by engineering bacteria LBA4404 (pBI-rolC) that constructed with pBI-rolC. Transformation was confirmed by PCR amplification of rolC genes from the hairy roots of P. ginseng. The content of monomer ginsenoside of Rg1, Re, Rf, Rb1, Rc, Rb2 and Rd in hairy root was determined by the HPLC. The total ginsenosides content in the hairy toot came up to 18.55 mg/g.