镰刀菌诱导子对盾叶薯蓣离体培养物中薯蓣皂素合成的影响

研究了镰刀菌(Fusarium solani)诱导子对盾叶薯蓣离体培养物中薯蓣皂素合成的影响。结果表明:当培养基中诱导子糖含量达到30 mg GE/L时能使薯蓣皂素的含量明显提高;薯蓣皂素含量的最大值出现在镰刀菌诱导子处理后的第35 d,达到对照的2倍左右;盾叶薯蓣离体培养物PPO、SOD活性以及可溶性蛋白含量在添加诱导子后呈现一定的规律性变化。     

菊叶薯蓣DcPMK基因克隆及互作蛋白筛选

以菊叶薯蓣为研究材料,采用RT-PCR技术克隆获得DcPMK基因,进行生物信息学、组织特异性和诱导表达分析,并构建DcPMK的诱饵载体以筛选拟南芥酵母文库中互作蛋白,为深入研究菊叶薯蓣萜类物质的合成积累提供一定理论基础。结果表明：DcPMK基因开放阅读框大小为1 536 bp（GenBank登录号MZ171241）,编码511个氨基酸。蛋白质序列比对分析发现DcPMK与近源种基因序列一致性达88.85%,具有1个保守的ATP结合位点Gly-X-Gly-XX-Ala。N-J进化树显示DcPMK与海枣（Phoenix dactylifera）等单子叶植物的遗传距离较近。HPLC结果显示菊叶薯蓣皂素主要在根茎中积累,qRT-PCR结果表明DcPMK基因在各组织中均有表达,在老茎中表达量最高,根茎中表达量最低;水杨酸诱导后皂素含量的变化与DcPMK表达量的变化趋势吻合：随着叶片中皂素含量提高,DcPMK上调表达。与阴性对照相比,DcPMK基因没有自激活性和细胞毒性,并筛选到27个互作明显的拟南芥基因,如生长发育相关基因AtKCR1（AT1G67730）、AtRPS9M（AT3G49080）、AtASY4（AT2G33793）,非生物与生物逆境相关基因AtVDAC2（AT5G67500）、AtVDAC3（AT5G15090）、AtRH8（AT4G00660）及花色素苷积累相关基因AtPHR2（AT2G47590）等。以上结果说明DcPMK能够参与菊叶薯蓣的萜类物质合成,并通过蛋白质-蛋白质相互作用的方式广泛参与其生长发育和胁迫响应等代谢途径。     

薯蓣皂素的节水生产工艺研究

盾叶薯蓣是我国特有的甾体激素类药源植物,其根茎所含的薯蓣皂素是合成多种甾体激素的前体物质。本文在预发酵酸水解的基础上探讨不同盐酸浓度、不同加水量以及不同中和试剂对薯蓣皂素提取率的影响,旨在降低生产过程中的耗水量。实验结果表明:HCL浓度为2.5 mol/L时,薯蓣皂素提取率最大,为2.408%;皂素含量随加水量的减少而减少;最佳的中和试剂为CaO。     

生物合成薯蓣皂素的途径设计及关键酶分析

薯蓣皂素是一种天然甾体皂苷元,可作为数百种类固醇药物的前体,具有重要药用价值。目前工业生产薯蓣皂素主要依赖化学提取法,因此该法依赖植物材料和耕地且对环境有害。随着代谢工程和合成生物学的发展,生物合成法受到广泛关注。文中综述了生物合成薯蓣皂素的代谢途径和关键酶,并在酿酒酵母Saccharomyces cerevisiae中设计其异源合成途径,提出改造策略,以期为全生物合成薯蓣皂素提供有价值的参考。     

薯蓣皂素对小麦幼苗抗旱性的影响

用不同浓度的薯蓣皂素喷施处理小麦幼苗叶片,研究薯蓣皂素在干旱胁迫下对小麦幼苗影响。结果表明,喷施一定浓度的外源薯蓣皂素,在干旱胁迫下可保持小麦幼苗叶片相对含水量,提高SOD和POD活性,降低MDA含量,对小麦幼苗抗旱有一定的作用。且薯蓣皂素最佳质量浓度为0.5 mg.L-1。     

盾叶薯蓣元素组成特点及其与土壤营养元素的关系研究

盾叶薯蓣(Dioscorea zingiberensis Wright)根状茎与地上部分各元素的含量明显不同,根状茎中含镍、铁、磷、铝量远高于地上部分,镁、钙、锰量远低于地上部分;盾叶磐蓣对钾、锌、铁等元素的吸收量大于农作物;根状拳与地上部分的比值(R2／U2)较大;大量元素在根状茎中的含量(A)及地上部分含量(B)的大小排序差别较大,微量元素含量A、B值的大小排序完全一致。盾叶薯蓣根状茎的皂素平均含量为2．87％,皂素含量与土壤有机质、全氮、全磷、全钾、有效铁、有效锌含量关系密切。薯蓣皂素含量高的盾叶薯蓣集中分布在大巴山北坡东段化龙山脉一线。     

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。     

盾叶薯蓣的快速繁殖

对盾叶薯蓣（Ｄｉｏｓｃｏｒｅａ ｚｉｎｇｉｂｅｒｅｎｓｉｓ Ｗｒｉｇｈｔ）的外植体进行组织培养,筛选到诱导芽和诱导根的优化培养基和培养条件,建立了无性快速繁殖培养系,获得完整的培养植株并移栽成功。并用分光光度法初步分析了该诱导植株生长根的薯蓣皂素含量。     

光强对盾叶薯蓣生长发育及皂素含量的影响

以生长1年的安姜3号盾叶薯蓣(Dioscorea zingiberensis)为研究材料,用盆栽方法对其进行不同遮阴处理,研究其生长发育、根茎产量和皂素含量等的变化特征.结果显示:(1)全光照下薯蓣植株生长较弱,20%自然光下生长茂盛;随着光照强度的减弱,株高、茎粗、叶片厚度和鲜叶质量都呈先升后降的趋势;自由水含量总体上呈上升趋势,自由水与束缚水的比值呈先升后降趋势;中等遮阴条件下茎分支数最多,叶面积较小,叶宽与叶长的比值也较小;(2)中度遮阴下总叶绿素和叶绿素a含量最大,而叶绿素b随着光强的减弱大致呈先降后升的趋势.(3)随着光照强度的减弱,根鲜重大致呈增加的趋势,根部芽头数量呈先升后降趋势,根部含水量呈先降后升趋势,皂素含量则与之相反.研究发现,不同光照条件下薯蓣根茎鲜重无显著差异,但光照条件对其地上部分和地下部分生物量的分配却有显著影响;安姜3号盾叶薯蓣在40%自然光强下生长发育最好且皂素含量最高.     

药源植物盾叶薯蓣甾体皂苷及皂苷元的研究进展

盾叶薯蓣是重要的甾体激素类药源植物,其根茎中薯蓣皂苷元含量居薯蓣属植物之冠,为我国的特有种。为了寻找高含量的资源、筛选新的生理活性成分,多年来我国学者做了大量的研究工作。主要概括了盾叶薯蓣的资源分布、薯蓣皂苷元的提取工艺、化学成分、药理、含量测定等方面的研究。     

菊叶薯蓣的快速繁殖研究

我国野生的薯蓣皂甙类植物由于盲目采集已日渐枯绝,甚至面临绝种的危险。原产于墨西哥的菊叶薯蓣不仅产量高、含薯蓣皂甙也高,因此在我国有很大的开发利用潜力。本文的工作是对菊叶薯蓣的外植体进行组织培养,筛选出了诱导芽和诱导根的优化培养基与培养条件,建立了无性快速繁殖培养系,获得了完整的培养植株并移栽成功。     

Expression analysis of steroid pathway genes revealed positive correlation with diosgenin biosynthesis in <Emphasis Type="Italic">Trillium govanianum</Emphasis>

Trillium govanianum Wall. ex D. Don, popularly known as Nagchhatri, is an important medicinal plant of northwestern Himalayas. No data exist on molecular characteristics of diosgenin biosynthesis, the main chemical component of T. govanianum. HPLC analysis revealed diosgenin content of 2.4 and 0.7% in hydrolyzed rhizome extracts of Chamba and Lahaul and Spiti, respectively. Quantitative real-time PCR analysis of five genes of steroid pathway in the rhizomes of two locations, differing for diosgenin content showed up to 3.2-fold increase in expression level of genes viz. hydroxymethylglutaryl Co-A reductase (HMGR), farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), 26-O-beta-glucosidase (BETA) and cycloartenol synthase (CAS) in rhizomes of Chamba (2.4%) as compared to Lahaul and Spiti (0.7%), thus inferring their role in diosgenin biosynthesis. The present study delivers the first report where an elite chemotype of T. govanianum for diosgenin content was identified and partial sequences of five genes of steroid pathway were cloned and investigated for their potential role in diosgenin biosynthesis in T. govanianum. The result of this study has prospective applications in the genetic improvement of this medicinally important plant species.     

盾叶薯蓣环阿屯醇合酶全长基因的克隆与分析

环阿屯醇合酶(cycloartenol synthase,CAS)是薯蓣皂甙元生物合成途径中的第一个关键酶.以基因组DNA为模板,利用染色体步行和长距离PCR方法首次克隆了盾叶薯蓣CAS全长基因.序列分析比较结果表明,盾叶薯蓣CAS全长基因为7 192 bp,由18个外显子和17个内含子组成.外显子总长为2 280 bp,编码759个氨基酸,最长的外显子为198 bp,最短的为47 bp;内含子总长4 912 bp,最长的内含子为1 551 bp,最短的为68 bp.Southern blot杂交分析表明,CAS基因在盾叶薯蓣基因组中为单拷贝.     

黄姜中薯蓣皂甙元的提取及定性定量分析

对黄姜中提取的薯蓣皂甙元进行红外和紫外可见波谱扫描,与标准品谱图对比分析,可知提取出的皂甙元与薯蓣皂甙元标准品有相同的结构。用分光光度法测定黄姜中薯蓣皂甙元的含量,以香荚兰醛-高氯酸-冰醋酸的加入顺序,在80℃水浴中加热15min,显色30min后,在542nm处测定吸收值能达到最佳值,最后得出皂甙元含量为2.27%,RSD为1.1%,回收率在97.2%~98.8%之间。     

穿龙薯蓣中薯蓣皂苷元含量的动态分析

目的：对穿龙薯蓣中薯蓣皂苷元的含量进行动态分析。方法：超临界CO2萃取技术提取,高效液相色谱法测定含量。结果：穿龙薯蓣根茎中花期薯蓣皂苷元含量最高为1．35％,枯萎期含量最低为1．03％。穿龙薯蓣地上部分不同生长时期均不合薯蓣皂苷元。结论：穿龙薯蓣根茎中花期薯蓣皂苷元含量最高,枯萎期含量最低。穿龙薯蓣地上部分不同生长时期均不合薯蓣皂苷元。     

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.     

Novel effects of diosgenin on skin aging

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor α, estrogen receptor β or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor α, estrogen receptor β or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17β-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17β-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.     

The Quantitative Analysis and Identification of Steroidal Sapogenins of the Dioscorea Plant

The results for the quantitative analysis and identification of steroidal sapo-genins of 16 species of Dioscorea rhizomes are given in Table 1. The amount of steroidal sapogenin varies from 0.073%–5.93%, its highest content of diosgenin is 5.93% in D. zingiberensis C. H. Wright. 2. The results observed from paper chromatography, thin layer chromatography and gas-liquid chromatography revealed that except the sapogenin of D. chingii Pr. and Br. is similar to tokorogenin, all other sapogenins after recrystallization yield a spot identical to diosgenin or yamogenin. Meanwhile, in most Dioscorea sample, a spot identical to △ 3, 5-diene-25D-spirostane was shown by using thin layer chromatography and gas-liquid chromatography. (Table 2). 3. The results observed from infrared spectrum and gas-liquid chromatography revealed that the sapogenins of D. collettii, Hook. f; D. collettii. Hook, f. var. hypoglauca, Pei and Ting. and D. tokoro Makino. contained 25L-spirostane. (A900≤ A920 cm-1).