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

为了推进我们的项目，我们进行了两次实验。其中之一是建立毛状根培养作为甘草种外源基因的引种体系。另一种方法是通过水培法从乌拉尔根中分离到P-450基因。用发根农杆菌直接接种6种甘草，在萌发后2 ~ 3周的不育苗上诱导出毛状根。产生的不定根尖中的细菌在含有500mgL^<-1>克拉福兰的MS琼脂培养基上消灭。消除处理后，不定根在YEB琼脂培养基上培养，以确认细菌的消除。结果：从6种甘草植物中培养出27株不定根。27株不定根中，有19株用常规方法检出了碱。第二步，采用sds -苯酚法从水培根中分离总RNA。用oligo (dT)_<20>-M4引物逆转录总RNA中的Poly (A)^+mRNA，并进行PCR扩增。RT-PCR产物在含有1倍TBE缓冲液的2%琼脂糖凝胶上分析。将扩增的DNA连接到pCR^<TM> II中，转化为One Shot^<TM>感受态细胞。利用M13正向和反向引物进行菌落PCR检测插入物的存在。从菌落中分离出约500bp的插入片段，用碱法sds分离质粒，用双脱氧法确定插入片段的序列。不幸的是，包含保守的F- GR-C-G -450序列的克隆直到现在才被检测到。

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.