Production of glycyrrhizin by introduction of a cytochrome P-450 gene on a Licorice hairy root

点击翻译按钮获取中文摘要

We performed two experiments for proceeding of our project. One of them is the establishment of the hairy root culture as an introduction system of foreign genes for Glycyrrhiza species. The other is the isolation of P-450 gene from the root of G.uralensis on hydroponics.Hairy roots of six species of Glycyrrhiza were induced by direct inoculation with Agrobacterium rhizogenes on sterile seedlings from 2 to 3 weeks after germination. The bacteria in the generated adventitious root tips were eliminated on Murashige & Skoog's (MS) agar medium containing 500mgL^<-1> claforan. After elimination treatment, adventitious roots were cultured on YEB agar medium o confirm elimination of bacteria. In the result, 27 strains of adventitious roots were established from 6 species of Glycyrrhiza plant. Opine was detected by usual manner in 19 of the 27 strains of adventitious roots.In the next experiment total RNA were isolated from the root of G.uralesis on hydroponics by SDS-Phenol method. Poly (A)^+mRNA in total RNA were reverse transcribed using oligo (dT)_<20>-M4 primer and PCR amplified. RT-PCR pruducts were analyzed on 2% agarose gels containing 1x TBE buffer. The amplified DNA was ligated into pCR^<TM> II and transformed into One Shot^<TM> competent cells. The determination on the presence of insert was carried out colony PCR using M13 forward and reverse primers. From the colony which include a insert size approximately 500bp, plasmid was isolated by alkali-SDS method and the sequence of the insert ws determined by dideoxy method. Unfortunately, clones containing the conserved F--GR-C-G P-450 sequence were not detected until now.