人参毛状根生物合成熊果苷的分离与鉴定

熊果苷(arbutin),化学名称为对-羟基苯-β-D-吡喃葡萄糖苷,能够竞争性抑制酪氨酸酶的活性从而抑制黑色素的形成,被国际公认为高效祛斑美白剂,是化妆品中理想的添加成分.人参(Panax ginseng C. A. Mey.)自古以来就是名贵药材,由于人参在栽培过程中存在着栽培困难、周期过长、地域限制等难题,人参的组织培养受到了广泛的重视.本实验室已建立了人参细胞大量培养体系[1]和人参毛状根培养体系[2],并把熊果苷与人参细胞配伍应用到化妆品生产中,产品深受广大消费者青睐.用植物培养物对外源底物进行生物转化,从而对其结构进行修饰,以获得更有意义的产物的研究报道很多[3～9],也是当今研究的热点.本实验室已对人参生物转化熊果苷的基本条件进行了初步探讨[10],本文在此基础上,对转化产物进行了分离鉴定.     

Ginsenoside production in different phenotypes of Panax ginseng transformed roots

Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A. Meyer with Agrobacterium rhizogenes A4. The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium. Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching. The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A. rhizogenes transformation conditioned a higher biomass formation. When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype. The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions. This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots. The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group. From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.     

Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis

To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A. Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples. 27 putative ginsenosides were detected in ginseng hairy roots. Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV. Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production. We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside. Some of the hairy root lines produce only a single ginsenoside in relatively high amounts. These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.     

Characterization of RNase-like major storage protein from the ginseng root by proteomic approach

The most abundant root proteins of ginseng (Panax ginseng) have been detected and identified by comparative proteome analysis with cultured hairy root of ginseng. Four abundant proteins (28, 26, 21 and 20 kDa) of P. ginseng had isoforms with different pl values on two-dimensional gel electrophoresis (2DE). The results of N-terminal and internal amino acid sequencing, however, showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the expressed sequence tag database of P. ginseng and found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicates that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had no RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in ginseng main root were not expressed in cultured hairy roots of ginseng. 2DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root-specific RNase-like proteins, which function as vegetative storage proteins of ginseng for survival in the natural environment.     

人参的遗传改良*

遗传改良是人参育种的重要手段之一,而遗传转化和再生体系的建立是开展人参遗传改良工作的前提和基础。人参植株再生可以通过器官发生和体细胞胚发生,间接体细胞胚发生是人参植株再生的主要途径,从不同外植体,不同碳源,体细胞胚优化和无激素再生等方面进行了综述。在人参遗传转化方面,发根农杆菌和根癌农杆菌对人参的遗传转化均已成功,人参皂苷合成途径中的关键酶基因和抗除草剂基因也已陆续导入人参,得到了遗传改良的转化人参。发根培养系统可用于大量生产人参皂苷,讨论了rolC基因对人参发根诱导的作用,发根植株再生能力及生物反应器培养,最后指出了人参基因工程研究中存在的问题。     

人参发根的诱导及其适宜培养条件的研究

利用发根农杆菌A₄菌株在人参根外植体上直接诱导产生发根。在1/2MS固体培养基上建立起发根离体培养系,经连续多代的培养,发根仍保持旺盛生长状态。PCR扩增结果表明,发根农杆菌R_I质粒的rolC基因已在人参发根基因组中整合并得到表达。液体培养基中发根生长速度约为固体培养的2倍。经对发根中人参皂苷含量及比生长速率的测定,筛选出高产发根系R9923。利用HPLC法测定了R9923发根系中单体皂苷Rg1、Re、Rf、Rb1、Rc、Rb2和Rd的含量,人参总皂苷含量达15.2mg/g。确定1/2MS培养液（30g/L蔗糖）、摇床转速110r/min、每２周更换一次培养液、继代培养时间4周,为人参发根生长适宜条件。探讨了培养容积、发根初始接种量以及分级放大培养工艺对发根大规模生产过程中生物产量和皂苷含量的影响。     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

Saponin production by hairy root cultures ofPanax ginseng CA meyer: Influence of PGR and polyamines

A culture of hairy roots ofPanax ginseng C.A. Meyer was set up in order to investigate the possibility of producing ginseng saponin. Roots cultured in 1/2 MS medium in the presence of 2 mg/L IAA and 0.1 mM spermidine showed the maximal growth rate, whereas other polyamines increased the growth of hairy roots only slightly or not at all. High saponin root contents were obtained in culture media supplemented with 0.5 mg/L GA and 1 mM putrescine.     

Saponin production by cultures of Panax ginseng transformed with Agrobacterium rhizogenes

Hairy root culture of Ginseng (Panax ginseng) was established after roots were induced on callus following infection with Agrobacterium rhizogenes. The transformed cultures of ginseng could be subcultured as an axenic root culture in the absence of phytohormones, and grew with extensive lateral branches more rapidly than the ordinary cultured roots induced by hormonal control from ginseng callus. The hairy roots synthesized the same saponins, ginsenosides, as those of the native root, up to about 2.4 times in the quantity, and up to about 2 times in comparison with that of the ordinary cultured roots, on dry weight basis.Abbreviations Ms medium Murashige and Skoog's medium - 2,4-D 2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - K kinetin This paper is Part 52 in the series "Studies on Plant Tissue Culture". For Part 51, see Ayabe S, Udagawa A, Furuya T (1987).     

Production of transgenic plants via Agrobacterium rhizogenes-mediated transformation of Panax ginseng

 Hairy roots of Panax ginseng were obtained after root disks were infected with wild-type strain Agrobacterium rhizogenes 15834. Three lines of hairy roots with different pigmentation were selected. Embryogenic callus was induced on Murashige and Skoog medium containing 1.0 mg/l 2,4-D. The frequency of embryogenic callus formation was 37.4% in a line with red pigmentation. Somatic embryo development from embryogenic callus was efficiently achieved by lowering the concentration of 2,4-D (0.5 mg/l). After the germination of somatic embryos on medium with 10 mg/l GA₃, the embryos were transferred to 1/2-MS medium without GA₃. The transformed ginseng plantlets had an actively growing root system with abundant lateral roots. The phenotypical alteration of transformed ginseng plants might be valuable character for increasing root yield. Received: 27 March 1999 / Revision received: 18 May 1999 / Accepted 8 July 1999     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Auxin levels and auxin binding protein availability inrolB transformedBeta vulgaris cells

The final biological effect of auxin depends both on free auxin levels and on auxin perception capacity.RolB transformedBeta vulgaris L. hairy roots provide a system for studying both factors. Highly purified plasma membrane fractions were prepared with aqueous two-phase partitioning. Individual hairy root clones were assessed for the binding activities of plasma membrane-bound auxin binding proteins and for their free intracellular indole-3-acetic acid levels. The presence of a high affinity auxin binding protein with a dissociation constant of 9.07 x 10^?7 M was detected in the plasma membrane fractions isolated from non-transformed seedling roots and the six clones ofrolB transformed hairy roots. However, the levels of specific IAA binding considerably varied among different hairy root clones and between transformed and non-transformed roots. The levels of the detectable polypeptide in immunoblotting with an antibody against maize 22-kD auxin binding protein subunit were in good agreement to the levels that were detected in auxin binding assays. Differences in the indole-3-acetic acid levels were found between transformed and non-transformed roots and also between different transformed hairy root clones. A negative correlation was observed between free intracellular IAA levels and its specific binding to the plasma membrane-bound auxin binding proteins. A latency study indicated that the binding site for auxin may be located on the exterior face of the plasma membrane     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.     

The production of ginsenosides in hairy root cultures of American Ginseng, Panax quinquefolium L. and their antimicrobial activity

Panax quinquefolium, American ginseng, is valued for its triterpene saponins, known as ginsenosides. These constituents possess a number of pharmacological properties and hairy root cultures can synthesize similar saponins to those of field-cultivated roots. The antibacterial activity of extracts from three hairy root clones of P. quinquefolium L. was tested against a range of standard bacterial and yeast strains. The agar diffusion method was used to evaluate inhibition of microbial growth at various extract concentrations. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains. Susceptibility testing to antibiotics was also tested using the disk diffusion method. The minimal inhibitory concentration values of the extracts, obtained by agar diffusion, ranged from 0.8 to 1.4 mg/ml. The results showed that extracts from hairy root cultures inhibited the growth of bacteria and yeast strains and suggest that they may be useful in the treatment of infections caused by pathogenic microorganisms.     

Amyloplast distribution in hairy roots induced by infection with Agrobacterium rhizogenes.

To elucidate the rapid and plagiotropic growth of hairy root induced by A. rhizogenes, a root apex was investigated with respect to it's amyloplast deposition, activity of alpha-amylase and glucose content. The amyloplasts distributed in the hairy roots were fewer than those of the adventitious root. Since auxin availability is enhanced in hairy roots, it could affect the statolith degradation by elevating alpha-amylase activity so that the energy requirement for rapid growth could be fulfilled as represented of glucose content. Consequently, it is suggested the overall decrease of starch grains could result in the lack of gravi-response in hairy roots.