Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

Development of an efficient transformation system for Catharanthus roseus cell cultures using particle bombardment

We have developed an efficient direct DNA transfer procedure for the facile engineering of Catharanthus roseus cell cultures. Particle bombardment of callus derived from leaf material permitted rapid selection and establishment of transgenic cell lines. Transgenic callus were recovered at a frequency of between 60–80% of total callus bombarded with a single plasmid. Bombardment using two separate plasmids resulted in a 25–60% frequency of transgenic callus recovered, up to 90% containing both input plasmids. Between 10–20 g FW of transgenic material was produced within 3 months of bombardment, providing sufficient material for molecular and biochemical analyses. We developed two complementary systems allowing selection on either hygromycin or kanamycin to permit re-transformation using plasmids carrying additional genes of interest. Use of leaf tissue as explant for transformation avoids time-consuming and labor intensive procedures involving suspension cultures. We provide molecular data on integration and expression of selected and non selected transgenes in a number of transgenic callus lines. Transgene integration events for co-transformed plasmids were relatively simple, occurring at one or two sites in the genome for most of the lines we analysed. Molecular analysis of callus resulting from co-transformation experiments using two different plasmids revealed that in nine of 10 putative transgenic lines we selected for analysis both plasmids had integrated into the genome. RNA gel-blot analysis and histochemical staining showed that an unselected transgene, gusA, was expressed in seven of the ten lines we analysed.     

Annuario della Società Botanica Italiana 1932

A sandy culture experiment was conducted to investigate the effects of exogenous CaCl₂ on the indole alkaloid accumulation in Catharanthus roseus under salt stress. One-month seedlings of C. roseus were treated with the different concentrations of NaCl (0, 50, and 100 mmol l^? 1) and 7.5 mmol l^? 1 CaCl₂. The plant samples were analyzed after 7 days of the treatments. The NaCl-stressed plants showed decrease of fresh and dry weight and increase of malondialdehyde (MDA) content compared to control. Tryptophan decarboxylase (TDC) activity increased significantly under 50 mmol l^? 1 NaCl without CaCl₂ addition, 50 mmol l^? 1 NaCl with 7.5 mmol l^? 1 CaCl₂, and CaCl₂ treatment without NaCl addition. There was a significant increase in peroxidase activity under NaCl stress compared to control. The vindoline, catharanthine, vincristine, and vinblastine contents increased under salt stress (especially with 50 mmol l^? 1 NaCl treatment with or without CaCl₂). Addition of CaCl₂ to NaCl-stressed plants increased biomass, TDC activity, vindoline, and catharanthine contents and lowered MDA and vincirstine contents compared to the plants without CaCl₂. The plants treated with CaCl₂ alone showed higher TDC activity, vindoline, catharanthine, and vinblastine content when compared to control. The results showed that exogenous CaCl₂ could promote the indole alkaloid metabolism under salt stress.     

Synthesis and Biological Evaluation with Plant Cells of New Fosmidomycin Analogues Containing a Benzoxazolone or Oxazolopyridinone Ring

Fosmidomycin, 3-(N-formyl-N-hydroxyamido) propylphosphonic acid sodium salt, is an efficient inhibitor of 1-deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme of the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway notably present in Plasmodium species. We have synthesized a new series of analogues of fosmidomycin, containing a benzoxazolone, benzoxazolethione or oxazolopyridinone ring. As the MEP pathway is involved in the biosynthesis of all isoprenoids, accumulation of ajmalicine in Catharanthus roseus cells was chosen as a marker of monoterpenoid indole alkaloid (MIA) production. None of the twelve studied phosphonic esters 3 and phosphonic acids 4 affected periwinkle cell growth, but some of them (3c, 3e, 3g and 3h) showed a significant inhibition of ajmalicine accumulation: 45–85% at 125?μM. Surprisingly, this effect disappeared by conversion of 3c and 3g into the corresponding acids 4c and 4g, respectively.     

Metabolic Discrimination of Catharanthus roseus Calli According to Their Relative Locations Using 1H-NMR and Principal Component Analysis

Creating a plant-cell suspension culture involves first transferring the callus into liquid media, but there are no objective criteria for selecting the location of the callus to be transferred. In this study, inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by ¹H NMR (nuclear magnetic resonance) spectrometry and principal component analysis (PCA). It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to determine the separation of major metabolites on score plots. The levels of valine, lactic acid, threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, lactose, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas 2-oxoglutaric acid, oxalacetic acid, sucrose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by ¹H-NMR-based metabolomic analysis.     

新疆阿勒泰地区粉红椋鸟的繁殖生态及雏鸟食性研究

研究了新疆阿勒泰地区粉红椋鸟的繁殖生态学及雏鸟的食性.粉红椋鸟每窝产卵数2～6枚不等,育雏高峰在每日的9:30～13:30,孵化率为96.45%±18.95%,雏鸟成活率为89.38%±14.99%,觅食范围在4 km半径范围内,雏鸟多以直翅目(蝗虫)为食(占94.07%),也有少量鳞翅目和鞘翅目昆虫.阿勒泰地区粉红椋鸟的产卵日期晚于其他地区,孵化率及雏鸟成活率高于其他地区.建议在草原上设置砖堆招引粉红椋鸟,其效果高于石堆巢.     

弱光对长春花（Catharanthus roseus）幼苗中可溶性糖、生物碱及激素含量的影响

阐述了低光照强度和正常光照条件下长春花（Catharanthus roseus）幼苗叶片中可溶性糖、生物碱及内源激素等几种生理活性物质含量的动态变化,以揭示弱光胁迫对长春花生理代谢的影响及上述生理代谢对植物适应弱光环境的生态学意义。结果显示,弱光培养提高了长春花在自身支撑结构的投入,如叶柄长和节间长都显著增加,同时抑制了有性生殖。在弱光培养的第1周,长春花叶片中果糖（fructose,Fru）、葡萄糖（glucose,Glc）和蔗糖（sucrose,Suc）的含量分别由处理前的（0．62±0．01）,（1．86±0．12）,（0．24±0．01）mg／g FW下降为（0．38±0．02）,（0．60±0．03）,（0．17±0．02）mg／g FW,均显著低于对照（P〈0．01）,表明碳同化水平下降。长春花叶片中文朵灵（vindoline,VIN）和长春质碱（catharanthine,CAT）含量在弱光条件下呈显著增加趋势,处理结束时是对照的3倍左右,而它们的耦合产物长春碱（vinblastine,VLB）合成和积累受到抑制,其含量在第3周时仅为对照的50％。弱光条件处理前2周促进了长春花叶片中脱落酸（abscisic acid,ABA）、赤霉素（gibberelline,GA3）和吲哚乙酸（indole-3-acetic acid,IAA）含量水平的积累。这些结果表明,在弱光条件下长春花幼苗的上述生理代谢都发生着显著的变化,可能在植物适应弱光胁迫过程中发挥着积极的调控作用。     

硝态氮对海水胁迫下长春花幼苗光合特性及离子含量的影响

采用砂培试验研究了硝态氮对海水处理下长春花幼苗光合特性和离子分布的影响.结果表明：（1）高浓度海水显著抑制长春花幼苗的生长,提高硝态氮浓度能显著缓解海水胁迫对长春花幼苗生长发育的抑制作用,促进地上、地下部生物量积累.（2）随着海水浓度的增加,长春花幼苗叶片净光合速率、蒸腾速率、气孔导度和水分利用效率较对照均显著降低,细胞间隙CO2浓度显著增加;增加硝态氮浓度可显著提高长春花幼苗的净光合速率、蒸腾速率、气孔导度和水分利用效率,显著降低细胞间隙CO2浓度.（3）随着海水浓度的增加,长春花幼苗体内K＋含量较对照显著降低,而Na^＋、Cl^-含量显著增加,提高硝态氮浓度有利于长春花幼苗对K＋的吸收和向上运输,维持地上部较高的K^＋/Na^＋.研究发现,增加硝态氮能维持高浓度海水胁迫下长春花幼苗体内养分平衡,缓解盐胁迫对其光合生理和生长造成的伤害.