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

通过在甘草毛状根上引入细胞色素 P-450 基因生产甘草甜素

• 批准号: 07672377
• 负责人: HIROTANI Masao
• 金额: $ 1.41万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07672377/
• 关键词: Glycyrrhiza uralensis; cytochrome P-450; glycyrrhizin; hairy root culture; polymerase chain reaction; チトクローム P-450; hairy root culture; グリチルリトン酸

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.

为了进行项目的研究，我们进行了两个实验。其中之一是建立了以毛状根培养为外源基因导入系统的菌根植物。另一个是在水培条件下从乌拉尔甘草根中分离到P-450基因，用发根农杆菌（Agrobacteriumrhizogenes）直接接种6种乌拉尔甘草的无菌苗，在萌发后2 ~ 3周诱导出毛状根。在含有500 mg/L克拉福纶的Murashige &amp; Skoog（MS）琼脂培养基上消除产生的不定根尖中的细菌<-1>。去除处理后，将不定根在YEB琼脂培养基上培养以确认细菌的去除。结果表明，从6种根茎植物中获得了27株不定根。在27株不定根中，有19株用常规方法检测到了冠瘿碱的存在。用oligo（dT）_ -M4引物逆转录总RNA中Poly（A）^+mRNA<20>，PCR扩增。在含有1x TBE缓冲液的2%琼脂糖凝胶上分析RT-PCR产物。将扩增的DNA连接到pCR^<TM>II中并转化到One Shot^<TM>感受态细胞中。使用M13正向和反向引物进行菌落PCR，确定插入片段的存在。从含有约500 bp插入片段的殖民地中，用碱-SDS法分离质粒，用双脱氧法测定插入片段的序列。不幸的是，包含保守的F-GR-C-G P-450序列的克隆直到现在还没有被检测到。

项目成果

Zhou Y., Hirotani M., Yoshikawa T.and Furuya T.: "Flavonoids and phenylethanoids from hairy root cultures of Scutellaria baicalensis." Phytochemistry. 44 (1). 83-87 (1997)

Zhou Y.、Hirotani M.、Yoshikawa T. 和 Furuya T.：“来自黄芩毛状根培养物的黄酮类化合物和苯乙醇类化合物。”

K.Kawaguchi et al.: "Biotransfor mation of digitoxigenin by cultured ginssug cells" Phytochemistry. 42. 667-669 (1996)

K.Kawaguchi 等人：“培养的人参细胞对洋地黄毒苷的生物转化”植物化学。

K.Kawaguchi et al.: "Biofransformation of digitox : geuin by cultured ginsong cells" Phytochomistry. 42. 667-669 (1996)

K.Kawaguchi 等人：“洋地黄毒的生物转化：培养人参细胞的 geuin”植物化学学。

Kawaguchi, K., Watanabe T., Hirotani M.and Furuya T.: "Biotransformation of digitoxigenin by cultured ginseng cells." Phytochemistry. 42 (3). 667-669 (1966)

Kawaguchi, K.、Watanabe T.、Hirotani M. 和 Furuya T.：“培养的人参细胞对洋地黄毒苷的生物转化。”

Y.Zhou et al.: "Flavonoids and phenylethanoids from hairg root cultures of S.baialensis" Phytochemistry. 44. 83-87 (1997)

Y.Zhou 等人：“来自 S.baialensis 发根培养物的黄酮类化合物和苯乙醇类化合物”植物化学。