Elicitation of camptothecin production in cell cultures ofCamptotheca acuminata

Campothecin production was increased with elicitors, methyl jasmonate, jasmonic acid, yeast extract elicitor, and ferulic acid in suspension cultures ofCamptotheca acuminata. jasmonic acid was found to be the most efficient elicitor. Camptothecin production increased 11 times by using the optimum dosing concentration of jasmonic acid which was 50 μM. The kinetics of camptothecin accumulation in response to the treatment with jasmonic acid showed that the camptothecin accumulation reached the maximum value at 4 days after jasmonic acid dosing and then a rapid decrease in camptothecin accumulation was observed.     

喜树原形成层到形成层转化的研究

观察了枣树茎中原形成层到形成层的转化过程。距茎端0．5mm处,节间开始伸长之前,横切面上4—5个原形成束及束间的分生组织组成原形成层环。径向切面观,原形成层环呈现出较均一的结构。随着节间开始伸长,由于原形成层细胞发生假横向分裂,出现了长短两类细胞,长细胞多数端壁倾斜,短细胞多数端壁平截。以后,长细胞发育为纺缍状原始细胞,短细胞发育为射线原始细胞,部分射线原始细胞可以伸长井侵入生长而转化为纺缍状原始细胞。在节间伸长将停止时,此种转化基本完成。喜树为非叠生形成层,纺缍状原始细胞和射线原始细胞都有多核现象发生。     

喜树的分泌结构及其与喜树碱积累的关系

为了揭示喜树碱(camptothecin,CPT)在喜树(Camptotheca acuminata Decne.)中的积累部位及积累规律,运用组织化学技术和高效液相色谱技术对喜树茎、叶中喜树碱的积累部位和含量进行了相关性分析.结果发现两类分泌结构与喜树碱积累密切相关:一类是分布于幼茎和幼叶表面的单细胞腺毛;另一类是分布于喜树幼茎和幼叶中的由1～2层细胞包围而成的分泌道.由此推断,喜树中的分泌结构为喜树碱的主要积累部位.     

喜树内生真菌的分离及其抗肿瘤活性代谢产物的筛选方法

从喜树(Camptotheca acuminata Decne.)的根、枝条、叶和果实中分离纯化了48株内生真菌,通过对各个菌株的少量发酵培养,HPLC分析结合色谱峰紫外扫描检测方法,以紫外扫描图谱的相似性为依据,对喜树内生真菌产生喜树碱结构类似物进行初步筛选,并进一步以抑瘤实验确证其抗肿瘤活性.结果证明,以该方法筛选到的10个内生菌株中有7个菌株发酵液对HL-60细胞增殖具有显著的抑制活性.相对于常规的生物活性筛选,高效液相色谱结合色谱峰紫外光谱的方法,在药用植物内生真菌活性次生代谢产物筛选研究中具有快速、高效的特点.     

Molecular cloning, expression profiling and functional analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata

As the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis, DXP reductoisomerase (DXR, EC: 1.1.1.267) catalyzes a committed step of the MEP pathway for camptothecin (CPT) biosynthesis. In order to understand more about the role of DXR involved in the CPT biosynthesis at the molecular level, the full-length DXR cDNA sequence (designated as CaDXR) was isolated and characterized for the first time from a medicinal Nyssaceae plant species, Camptotheca acuminata. The full-length cDNA of CaDXR was 1823 bp containing a 1416 bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that CaDXR was more ancient than other plant DXRs. Tissue expression pattern analysis revealed that CaDXR expressed strongly in stem, weak in leaf and root. CaDXR was found to be an elicitor-responsive gene, which could be induced by exogenous elicitor of methyl jasmonate. The functional color complementation assay indicated that CaDXR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that DXP reductoisomerase plays an influential step in isoprenoid biosynthesis.     

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

接种AM真菌对喜树幼苗生长及光合特性的影响

 喜树(Camptotheca acuminata)是我国特有的多年生亚热带落叶阔叶树种, 因其次生代谢产物喜树碱具有良好的抗肿瘤活性而备受关注。通过温室盆栽接种试验, 观察了3属6种丛枝菌根(AM)真菌木薯球囊霉(Glomus manihot)、地表球囊霉(G. versiforme)、透光球囊霉(G. diaphanum)、蜜色无梗囊霉(Acaulospora mellea)、光壁无梗囊霉(A. laevis)和弯丝硬囊霉(Sclerocystis sinuosa)对喜树幼苗生长及光合特性的影响。结果表明, 除地表球囊霉外, 其余菌根幼苗生物量显著高于无菌根幼苗, 蜜色无梗囊霉、弯丝硬囊霉和透光球囊霉的菌根幼苗生物量分别达到无菌根幼苗的1.6倍、1.4倍和1.3倍。与无菌根幼苗相比, 蜜色无梗囊霉菌根幼苗叶片的净光合速率(P_n)、气孔导度(G_s)和蒸腾速率(T_r)均有显著提高, 而胞间CO₂浓度(C_i)与气孔限制值(L_s)则变化不明显。接种透光球囊霉、蜜色无梗囊霉、光壁无梗囊霉和弯丝硬囊霉的喜树幼苗叶片叶绿素a含量、总叶绿素含量、叶绿素a/b和类胡萝卜素含量均显著高于无菌根幼苗, 而叶绿素b含量只有木薯球囊霉和弯丝硬囊霉菌根幼苗显著高于无菌根幼苗。接种AM真菌对喜树幼苗叶片叶绿素荧光参数影响较小, 只有透光球囊霉菌根幼苗叶片的最大光能转换效率(F_v/F_m)显著高于无菌根幼苗, 接种木薯球囊霉和弯丝硬囊霉的喜树幼苗的PSⅡ有效光化学量子产量(EQY)显著高于无菌根幼苗, 弯丝硬囊霉菌根幼苗的光化学淬灭(qP)显著高于无菌根幼苗, 非光化学淬灭(NPQ)则显著低于无菌根幼苗。     

Drip‐tips are Associated with Intensity of Precipitation in the Amazon Rain Forest

Drip‐tips are a common feature of the leaves of rain forest trees, but their functional significance remains contested. The most widely accepted hypothesis is that drip‐tips assist drainage of the lamina thereby aiding drying of the leaf surface and reducing the rate of colonization and abundance of epiphyllic organisms. The drying action of drip‐tips may also enhance transpiration and reduce the need for investment in support structures. Furthermore, drip‐tips may help prevent splash erosion around the base of the tree. Data from 130 forest Amazonian plots are used to investigate the abundance and distribution of drip‐tips and, through regression methods that incorporate spatial autocorrelation, seek to identify associations between the frequency of drip‐tips and a range of climatic variables. The average frequency of species and trees with drip‐tips across all plots was 32 and 33 percent, respectively. Trees and species with drip‐tips were significantly more prevalent in the Central‐East Amazon than the other regions. Drip‐tips were also associated with tree species that have smaller maximum heights and with trees with smaller trunk diameters. The proportion of species and individuals with drip‐tips was more strongly correlated with precipitation of the wettest trimester than with total annual precipitation or length of the dry season. Our results extend and provide support for both existing hypotheses for the functional benefit of possessing a drip‐tip. Moreover, the currently unrecognized macrogeographic association between the frequency of drip‐tips in trees of the tropical forest understory and areas of heavy precipitation suggests a new function for this trait.     

南方红豆杉水浸提液对喜树种子发芽和幼苗生长的化感作用

化感作用是影响人工混交林种间关系的重要因素之一。已有研究表明：喜树南方红豆杉混交对喜树生长有明显促进作用,并从混交改善混交林地微气候角度解释了这种促进作用,但未从种间化感作用角度探讨这种作用。为了探究南方红豆杉是否对喜树具有潜在的化感促进作用,从而更全面了解喜树南方红豆杉混交林种间关系,考察了不同浓度（25、50 g/L和100 g/L）的南方红豆杉（Taxus chinensis var.mairei）鲜叶、凋落叶、枝和根水浸提液对喜树（Camptotheca acuminata）种子发芽和幼苗生长的影响。结果表明：（1）南方红豆杉凋落叶浸提液对喜树种子发芽和幼苗生长无显著影响（P>0.05）,而鲜叶、枝和根浸提液对喜树发芽和幼苗生长均具有不同程度的促进作用（P<0.05）,且作用强度大体与浓度呈正相关;（2）100 g/L的南方红豆杉鲜叶、枝和根浸提液浸泡的喜树种子,其发芽率比对照组分别提高8.1%、14.9%和25.6%;（3）南方红豆杉鲜叶浸提液只有在高浓度（100 g/L）时,对喜树幼苗基径生长具有促进作用（P<0.05）,而对其株高、全株干重和净光合速率无显著影响;南方红豆杉根和枝浸提液对喜树幼苗株高、基茎、干重和净光合速率均具有促进作用（P<0.05）,与对照组相比,100 g/L的根和枝浸提液浇灌喜树幼苗,可以使喜树幼苗株高分别提高14.2%和8.4%,基径分别提高19.0%和15.3%,干重分别提高23.1%和15.9%,净光合速率分别提高11.6%和6.1%。研究结果表明,在喜树南方红豆杉混交林中,南方红豆杉对喜树的正向化感作用可能是促进喜树生长的重要因素之一。     

喜树内生菌与喜树碱的关系

通过对喜树幼苗中喜树碱含量的分析,发现不同生长期、不同器官中喜树碱的含量不同,幼叶和根中喜树碱的含量较高。虽然喜树中含有对真核细胞具有毒性作用的喜树碱,但仍有12种内生菌从喜树的不同器官中分离出来。内生菌对喜树碱的敏感性实验表明,10μg／mL喜树碱对2种内生菌的生长几乎没有抑制作用,即是100μg／mL浓度的喜树碱对它们的生长抑制也是有限的。