Production of diosgenin by hairy root cultures ofTrigonella foenum-graecum L.

Hairy root cultures ofTrigonella foenum-graecum L. were established withAgrobacterium rhizogenes strain A₄. The hairy roots produce diosgenin, an important spirostanol for the semi-synthesis of steroid hormones. Fourteen different liquid media were investigated. The fastest growth was obtained in McCown's woody plant (WP) medium supplemented with 3% sucrose; the highest diosgenin content was observed in half-strength WP medium with 1% sucrose (0.040% dry weight), which represents almost twice the amount detected in the 8-month-old non-transformed roots (0.024%). A time-course study in WP liquid media supplemented with 3% sucrose was undertaken. In these conditions, 17 g diosgenin/g fresh weight were produced. The influence of cholesterol, medium pH and chitosan on diosgenin production was tested. The addition of 40 mg/l chitosan elevated the diosgenin content to three times that found in non-elicited hairy roots.Abbreviations MS Murashige and Skoog (1962) medium - WP McCown's woody plant medium     

黄独遗传多样性研究

采用ISSR标记技术研究了我国14个黄独样品的遗传多样性.从55条简单重复序列引物中筛选出9条多态性引物,共扩增出70条带,其中67条多态性带,多态性比率为95.71%,平均每条引物扩增出7.8条带.黄独原变种内Nei s基因多样性(h)为0.294 9,有效等位基因数(Ne)为1.491 1,Shannon多样性指数(I)为0.444 8.种水平h为0.326 3,Ne为1.552 9,I为0.488 3.基因分化系数(Gst)为0.782 1,基因流(Nm)为0.139 3.聚类分析表明来自海南省和台湾省的样品与我国内陆的样品较早分离.据此可将来自我国内陆的样品分为5组.ISSR聚类分析基本上支持依据形态特征对黄独变种的划分.同时实验结果也表明,云南可能是黄独在我国的分化中心.     

黄独块茎的甾体类成分

黄独（ＤｉｏｓｃｏｒｅａｂｕｌｂｉｆｅｒａＬ．）又名黄药子、黄药脂等,药用其块茎。主要用于治疗各种甲状腺疾病和多种癌症。对食道癌、胃癌、直肠癌的近期疗效确切,对乳腺癌、宫颈癌、膀胱癌、肺癌及肉瘤均有一定疗效［１］。关于黄独中甾体皂甙类成分的报道颇具争...     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

内生Bacillus svelezensis HBB5菌株发酵盾叶薯蓣产薯蓣皂苷元的研究

目的探讨内生Bacillus svelezensis HBB5菌株发酵宿主植物盾叶薯蓣产薯蓣皂苷元的能力。方法接种内生B.svelezensis HBB5及B.subtilis ATCC 6633菌株(0.35×10~8 CFU/mL)至含盾叶薯蓣地下茎组织的液体培养基,32℃、165~185 r/min连续发酵108 h,检测发酵液细菌、pH、淀粉、麦芽糖、葡萄糖、淀粉酶(α-amylase)及薯蓣皂苷元溶出率等指标。结果内生B.svelezensis HBB5菌株有较强的酸、碱耐受力[pH(4.8±0.2)~(8.4±0.2)],相比B.subtilis ATCC 6633[pH(5.2±0.2)~(8.7±0.2)]差异不明显;前者达峰值生长量(60×10~(8±2) CFU/mL)明显高于后者(32×10~(8±2) CFU/mL)。发酵36~60 h时,B.svelezensis HBB5、B.subtilis ATCC 6633菌株发酵液的淀粉、麦芽糖、葡萄糖浓度达峰值,分别为(37.41±3.12)、(27.83±2.14)ng/mL,(21.06±1.25)、(16.54±1.08)ng/mL,(54.33±3.12)、(36.65±2.10)ng/mL,前者均高于后者。同时,B.svelezensis HBB5菌株维持高的α-amylase酶活性及薯蓣皂苷元溶出率。结论内生B.svelezensis HBB5菌株拥有较强的耐酸碱、降解淀粉、提高薯蓣皂苷元溶出的能力,为工业生产薯蓣皂苷元提供了一个新的方法。     

葫芦巴环阿屯醇合酶基因的分离及其对薯蓣皂素合成的影响

以薯蓣皂素合成植物葫芦巴(Trigonella foenum-graecum L.)为材料,从中分离了环阿屯醇合酶基因Tf CAS,并对其序列特征、基因的表达及其对葫芦巴薯蓣皂素生物合成的影响进行了分析。结果显示,该基因全长2271 bp,共编码756个氨基酸;其氨基酸序列与蒺藜苜蓿(Medicago truncatula Gaertn.)、豌豆(Pisum sativum L.)及百脉根(Lotus japonicus L.)环阿屯醇合酶氨基酸序列的同源性分别为94%、91%和89%。利用酵母表达系统对Tf CAS蛋白的生物化学功能进行了验证,结果表明该蛋白能够催化环阿屯醇的合成。进一步利用葫芦巴发根遗传转化体系在葫芦巴中过量表达Tf CAS基因,发现该基因的过量表达大幅提高了Tf CAS的表达,且促进了葫芦巴中β-谷甾醇和薯蓣皂素的生物合成,但与对照相比差异不显著。研究结果表明Tf CAS基因参与了葫芦巴薯蓣皂素的生物合成,但其并非为该合成途径中的限速酶。     

Physical map of chloroplast DNA of aerial yam,Dioscorea bulbifera L.

Summary A physical map of chloroplast DNA (ctDNA) of aerial yam, Dioscorea bulbifera L. was constructed using three restriction endonucleases, PstI, SalI, and SmaI. In addition, a clone bank of the BamHI-digested fragments were generated, and the locations of most BamHI fragments on the map were also determined. The ctDNA of D. bulbifera was found to be a circular molecule with a total size of ca. 152 kb involving two inverted repeats of ca. 25.5 kb, and small and large single copy regions of ca. 18.5 and 83.4 kb, respectively. The genes for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) and the ATP-synthase subunits and (atpB/atpE) were mapped.Contribution from the Plant Germ-plasm Institute and the Laboratory of Genetics (No. 504), Faculty of Agriculture, Kyoto University, Japan. The work was supported in part by a Grant-in-Aid (No. 60400005) from the Ministry of Education, Science and Culture, Japan     

In vitro evaluation of some high yield potato (Solanum tuberosum L.) cultivars under imposition of salinity at the cellular and organ levels

Salinity and drought stress, which combines a lack of water and sodium toxicity, are more of the problems faced by plants and agricultural crops in newly reclaimed lands. Therefore, the direction of our research is to produce salinity-tolerant plants to increase the productivity of crops under conditions of salt stress. Potato callus was studied using different concentrations of NaCl (0.0, 50, 75, 100, 125, 150 and 200 mM). Shoot induction was obtained from callus treated with MS medium containing 4.0 and 5.0 mg l^?1 TDZ + 0.5 mg l^?1 GA3 with NaCl up to 125 mM and 150 mM for Rosetta and Victoria, respectively. When plantlets were cultured on MS medium containing 3.0 mg l^?1 kinetin and 1.0 mg l^-1paclobutrazol (PBZ) with 80 or 90 g l^?1 sucrose after two months gave a good microtuber per explant of Rosetta and Victoria cultivar which gave number of microtuber/plantlet (1.85) and (2.40) when plantlets treated with 125 mM and 150 mM NaCl of Rosetta and Victoria cultivar, respectively. In general, the results were shown in each treatment of NaCl and that amounts of proline at 125 and 150 mMNaCl were significantly more than 0.0, 50, 75 and 100 mM NaCl. This result is related to the role of proline in the osmotic adjustment of a higher concentration of salinity. The results showed that the amounts of sodium increased with increasing the salt concentration, but the amount of potassium decreased and also increased the Na+/K+ ratio with increasing the salt concentration. This research is important for in vitro potato plant regeneration, which requires optimization before genetic transformation can be achieved.     

黄独脱毒苗叶片和茎段再生体系的建立

以黄独茎尖再生苗为试材,研究不同因素对黄独脱毒苗叶片和茎段再生体系的影响,以期对黄独脱毒苗的再生体系进行优化。结果表明,叶片和茎段诱导愈伤组织的最佳培养基是MS+KT 2 mg·L^-1+2,4-D 2 mg·L^-1;叶片和茎段诱导愈伤组织的最佳蔗糖浓度分别为30和50 g·L^-1;叶片和茎段在黑暗中较容易诱导出愈伤组织;叶片和茎段愈伤组织分化的最佳培养基是MS+KT 4 mg·L^-1+NAA 0.1 mg·L^-1;继代2次的叶片和茎段愈伤组织较容易分化;黄独不定芽生根的最佳培养基是1/2MS+IBA 0.1 mg·L^-1+NAA 0.5 mg·L^-1+PP₃₃₃ 1 mg·L^-1。本实验成功建立了黄独脱毒苗叶片和茎段的再生体系,为黄独脱毒苗的工厂化生产奠定了技术基础。     

黄独(Dioscorea bulbifera L.)不同居群叶表皮微形态特征的比较观察

应用扫描电子显微镜(SEM)对15个黄独(Dioscorea bulbifera L.)居群(11个野生居群及4个栽培居群)植株的叶表皮微形态特征(包括气孔器、表皮毛和气孔周围表皮细胞的特征)进行了观察和测量,并据此编制了15个黄独居群的检索表.观察结果表明:15个黄独居群植株叶片在气孔器外拱盖内缘类型,气孔器旋转方向,气孔长轴长度及密度和深度,叶片表皮毛的有无及类型,叶肉腺毛的长、短轴长度及密度,气孔周围表皮细胞垂周壁和平周壁的形态以及平周壁表面颗粒形状等特征上均有明显差异.气孔器外拱盖内缘有光滑和浅波状2种类型;气孔器的旋转方向分为不定向旋转、左旋和右旋3种方式;气孔长轴长度和密度的变化幅度分别为18.59～31.93 μm和86～356 mm-2.叶片表皮毛均仅存在于下表皮,可分为乳突和多细胞头单细胞柄腺毛2种类型,乳突主要分布于主脉,腺毛主要分布于二级脉和叶肉;叶肉腺毛的长、短轴长度的变化幅度分别为25.00～55.00和24.86～44.29μm,大部分居群腺毛密度的变化幅度为6～29 mm-2.叶片气孔周围表皮细胞垂周壁的形状有平直、平直隆起、平直脊状隆起、弯曲、弯曲脊状隆起和弯曲拱状隆起6种类型;表皮细胞平周壁的表面纹饰有具瘤条纹和光滑条纹2种形态,其扩散方式也有2种形式:一种为不环绕气孔且四方扩散,另一种为环绕气孔2～3周后扩散但扩散方向不定;平周壁上的颗粒形状有簇晶状、屑状、密集粉状、粉状、粒状和片状6种类型.比较分析结果显示:黄独不同居群叶表皮微形态特征的多态性较为丰富,地理分布相近的居群微形态特征的相似性较高,这些特征与黄独的各种下分类群相对应.     

Ion accumulation in lucerne plants regenerated from salt-adapted suspension cultures compared with recultured cells from these plants

Lucerne (Medicago media cv. Rambler) plants, regenerated from cell suspension cultures adapted to 200 mm NaCl, and cell cultures derived from the leaflets of regenerated plants showed enhanced Na⁺ exclusion compared with the unselected plants and cell cultures. There was a decrease in the K⁺ concentration in response to NaCl treatment but the selected plants and cell cultures maintained a significantly higher K⁺ concentration and showed much lower Na⁺/K⁺ ratios than unselected plants and cell cultures. The proline concentration increased in the shoots and cell cultures in response to NaCl stress but the selected plants and cell cultures showed a significantly higher proline concentration than unselected plants and cell cultures. Received: 10 December 1996 / Revision received: 28 February 1997 / Accepted: 25 August 1997     

Stimulated production of diosgenin in Dioscorea galeottiana cell suspension cultures by abiotic and biotic factors

Dioscorea deltoidea cell suspension cultures were established in modified Murashige and Skoog medium. The diosgenin production increased from 0.10 g^–1 to 3.98 g^–1 dry cell weight when cells were cultivated in the light and in a growth medium limited in phosphate and sucrose. The addition of 1.3 g of autoclaved fungal mycelium of Alternaria tenuis per litre of cell culture growing in the dark induced the production of 0.04 mg diosgenin g^–1 dry cell weight. In both cases, the production of diosgenin was preceded by a transient induction of isopentenyl diphosphate isomerase activity.     

黄独微型块茎低温离体保存的GC/MS代谢组学分析

GC/MS检测方法采用初步探明黄独低温离体保存微型块茎的差异代谢物。与黄独微型块茎25℃离体保存相比较,黄独微型块茎4℃离体保存的差异性代谢物有丙氨酸（Alanine）、儿茶素（Catechin）、N,N-双（2-羟乙基）甲胺（N,N-Di-（2-Hydroxyethyl）-methanamine）、水杨酸（Salicylic acid）、柠檬酸（Citric acid）和山梨糖（Sorbose）等。在黄独微型块茎4℃离体保存中,丙氨酸（Alanine）参与氰基氨基酸代谢;儿茶素（Catechin）参与次生代谢产物生物合成、黄酮类化合物的生物合成和苯丙素的生物合成;水杨酸（Salicylic acid）参与多环芳烃降解、微生物在不同环境中的代谢、植物激素信号转导、次生代谢产物生物合成、二恶英降解、苯丙氨酸代谢、芳烃降解、植物激素生物合成、铁载体组非核糖体肽合成和苯丙素的生物合成等。柠檬酸（Citric acid）参与来自鸟氨酸、赖氨酸和烟酸的生物碱生物合成、组氨酸和嘌呤的生物碱生物合成、微生物在不同环境中的代谢、植物次生代谢产物的生物合成、2-氧代羧酸代谢、萜类和类固醇的生物合成、原核生物固碳途径、次生代谢产物生物合成、来自莽草酸途径的生物碱生物合成、来自萜类化合物和聚酮的生物碱生物合成、柠檬酸循环（TCA循环）、植物激素生物合成、乙醛酸和二羧酸代谢、双组分系统、苯丙素的生物合成以及来自鸟氨酸,赖氨酸和烟酸的生物碱生物合成等。黄独低温离体保存微型块茎差异代谢物的初步发现为进一步了解其低温离体保存的分子机制奠定了基础,也为低温离体保存黄独微型块茎的破除休眠以及其后续萌发提供了理论依据。     

Isolation and characterization of diosgenin from in vitro cultured tissues of Helicteres isora L

The objective of the present study was to isolate, characterize, quantify and compare the accumulation of bioactive secondary metabolite—diosgenin from in vitro cultured cells of Helicteres isora and plant parts. The levels of this secondary compound were examined by using various biochemical techniques. The result showed that maximum diosgenin was obtained from in vitro cultured cells as compared to the plant parts. The fallout of this study is important, since levels of diosgenin detected in the in vitro cultured cells were more than in the plant parts. In vitro cultured cells accumulate comparatively higher amount of diosgenin making Helicteres isora a potentially new and indigenous source of diosgenin.     

Isolation and characterization of an intermediate steroid metabolite in diosgenin biosynthesis in suspension cultures of Dioscorea deltoidea cells.

The aglycon form of the steroidal sapogenin furost -5-ene-3 beta, 22,26-triol, 3 beta- chacotrioside 26 beta-D-glucopyranoside was isolated from cell suspension cultures of Dioscorea deltoidea and its molecular structure was determined by mass spectrometry and 1H and 13C n.m.r. spectroscopy. From kinetic studies and incorporation experiments with [1-14C]acetate it was concluded that the steroidal compound (in the glycoside form) is an intermediate in vivo in diosgenin biosynthesis. It accumulated in growing cells of D. deltoidea and was metabolized to diosgenin (in the glycoside form, i.e. dioscin ) in non-dividing cells.     

黄独微型块茎鲨烯合酶的基因克隆与序列分析

通过黄独微型块茎转录组数据库筛选到SQS基因的核心片段,利用RT-PCR技术获得SQS基因保守片段,采用RACE技术获得SQS基因的3′及5′末端序列,并采用生物信息学方法进行序列分析。结果表明,黄独微型块茎SQS基因编码序列长1548 bp,编码415 bp的氨基酸序列,理论分子量为46786.38 D,等电点(pI)为5.97。SQS蛋白为疏水性蛋白,无信号肽,含有SQS所必需的功能结构域,属于Isoprenoid_Biosym_C1 superfami?ly。黄独SQS蛋白与其他植物的SQS蛋白同源性较高,其中与盾叶薯蓣的SQS蛋白氨基酸相似性为96.4%。本试验结果从黄独微型块茎中首次获得SQS基因cDNA全长序列,该基因具有SQS同源基因的典型特征,为进一步研究黄独微型块茎SQS基因结构、基因表达和基因突变提供了基础,并为薯蓣属植物三萜合成通路关键酶SQS的正选择位点与功能的关联性分析提供了数据支持。     

Genetic fidelity of in vitro regenerants,encapsulation of shoot tips and high diosgenin content in <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L., a potential alternative source of diosgenin

Dioscorea bulbifera L. containing the pharmaceutically important compound, diosgenin, was regenerated in vitro through nodal segments on supplemented Murashige and Skoog medium (MS). Diosgenin was at 12 mg g⁻¹dry wt in 12-week-old plantlets raised on MS with various growth hormones. Random amplified polymorphic DNA (RAPD) analysis showed genetic fidelity of regenerants. Encapsulation of shoot tips in 3% (w/v) calcium alginate for storage and germplasm exchange was achieved.     

Bafoudiosbulbin H,a new clerodane diterpene from the flowers of Dioscorea bulbifera L. var sativa

A new clerodane diterpenoid, bafoudiosbulbin H (1), was isolated from the flowers of Dioscorea bulbifera L. var sativa. Its acetylation using acetic anhydride-pyridine and catalytic amount of 4-DMAP at 60 °C yielded bafoudiosbulbin H acetate (2) together with a clerodane with an unprecedented acylation pattern (bafoudiosbulbin H1, 3). The reaction of the known bafoudiosbulbin G (4) in the same conditions yielded demethylbafoudiosbulbin G (5). Structural elucidation of 5 led to the revision of the stereochemistry previously assigned to 4. Structures were elucidated using spectroscopic techniques, including 1D and 2D NMR (¹H, ¹³C, HSQC, COSY, HMBC, ROESY, NOESY) and mass spectrometry (HRESIMS).     

Bafoudiosbulbins A, and B, two anti-salmonellal clerodane diterpenoids from Dioscorea bulbifera L. var sativa

Two clerodane diterpenoids, Bafoudiosbulbins A 1, and B 2, together with five known compounds: tetracosanoic acid, 1-(tetracosanoyl)-glycerol, trans-tetracosanylferulate, beta-sitosterol and 3-O-beta-D-glucopyranosyl-beta-sitosterol were isolated from the tubers of Dioscorea bulbifera L. var sativa. Their structures were established by spectroscopic methods (1D and 2D-NMR, MS) and X-ray crystallographic diffraction analysis of compound 1. The CH2Cl2-soluble portion of the crude extract and the two clerodanes were screened for anti-bacterial activity using both agar diffusion and broth dilution techniques against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, Salmonella paratyphi A and Salmonella paratyphi B. They both showed significant activities against P. aeruginosa, S. typhi, S. paratyphi A and S. paratyphi B.