长春花萜类吲哚生物碱含量测定及相关基因的表达分析

采用反相高效液相色谱法(RP-HPLC)对23个长春花品种中的重要萜类吲哚生物碱文多灵、长春质碱和长春碱含量进行了测定,发现这3种生物碱的含量在不同品种中存在较大差异。相关性分析表明,文多灵、长春质碱和长春碱这3种生物碱中,文多灵含量和长春碱含量有极显著的相关性(P0.01)。利用RT-PCR分析了香叶醇10-脱羧酶基因(g10h)和异胡豆苷合成酶基因(str)在萜类吲哚生物碱含量有显著差异的品种之间的表达水平差异,并结合生物碱含量数据结果,发现g10h和str的表达水平和文多灵和长春质碱的总含量有显著的正相关性(P0.05),说明g10h和str基因可以作为长春花中文多灵和长春质碱含量的参考基因标记。该研究对为选育高萜类吲哚生物碱含量长春花品种及长春花萜类吲哚生物碱代谢工程具有重要意义。     

外源乙烯对长春花生理水平和生物碱积累的影响

药用植物长春花中含有100多种萜类吲哚生物碱（TIAs）,其中具有抗肿瘤功效的长春碱和长春新碱受到关注。为了研究外源乙烯处理对长春花生长情况、生理状态和萜类吲哚生物碱合成的整体影响,本文以长春花幼苗为实验材料,使用外源乙烯处理后对比了不同生长条件下长春花的生物量积累、根茎伸长、光合参数以及生物碱含量等指标,分析了生物碱合成与其他指标之间的相关性。结果表明,外源乙烯处理使长春花乙稀释放量上升,乙烯信号响应因子erf基因表达量提高。乙烯利抑制长春花幼苗生物量积累、根纵向生长,促进茎秆横向加粗生长,由非气孔因素导致净光合速率（P_n）和气孔导度（G_s）下降。外源乙烯促进异胡豆苷（STR）、长春质碱（CAT）、文多灵（VIN）和长春碱（VINB）积累,并且促进长春碱合成途径中关键酶基因str和CrPRX上调表达。相关性分析结果表明,次生代谢产物的积累、生长指标、光合参数之间存在明显的相关性;长春质碱、文多灵、长春碱与茎秆直径（SD）显著正相关（P < 0.05）,与生物量（B）、株高（H）、根长（RL）、净光合速率（P_n）呈显著负相关（P < 0.05）。本文为研究外源乙烯调控长春花生物碱积累的机制提供理论基础。     

植物生长调节物质对长春花细胞中吲哚生物碱积累的影响

适当浓度的脱落酸(ABA)、乙烯利和乙酰水杨酸(ASA)均对长春花悬浮细胞中吲哚生物碱的积累有较明显的促进作用.2mg·L-1ABA对吲哚生物碱的诱导效果最好,乙烯利的最适剂量为1 g·L-1,1 mg·L-1ASA有利于阿玛碱积累,而2 mg·L-1ASA则有利于长春质碱积累.     

土壤农杆菌转化的长春花冠瘿细胞培养

以土壤农杆菌C₅₈诱导的长春花冠瘿组织与从长春花茎、叶外植体诱导的愈伤组织进行比较,发现冠瘿组织在生长、总吲哚生物碱含量及药用成份阿吗碱含量等方面都优于愈伤组织。测定了光照、温度、蔗糖浓度及外加L－色氨酸前体等,对长春花冠瘿细胞的生长、总生物碱及阿吗碱含量的影响,为长春花冠瘿细胞培养生产吲哚生物碱的实际应用研究提供理论依据。     

浙江富阳和海南文昌人工种植长春花生物碱积累组织特异性对比

药用植物长春花的人工栽培分布区主要在我国海南省,目前已引种扩大到浙江、四川等地。为了研究不同栽培区长春花生物碱积累的区域性差异和组织特异性分布特点,本文以浙江富阳和海南文昌两地所产的同苗龄长春花为原料,对其不同部位的长春碱、文朵灵和长春质碱的含量进行对比,分析3种生物碱在不同部位中积累的相关性。研究结果表明,海南文昌所产长春花植株的叶片中3种生物碱含量均高于浙江富阳。但分枝中长春碱含量特点是浙江富阳要显著高于海南文昌;不同部位生物碱之间相关性分析表明,叶片和主茎中文朵灵和长春质碱含量呈显著正相关,而分枝中长春碱与其前体长春质碱成极显著负相关。本文为长春花人工栽培区分布评估提供了理论指导。     

色氨酸对海水处理下长春花生物碱含量的影响

以长春花为(Catharanthus roseus(L.)G.Don)材料进行温室实验,研究了不同浓度色氨酸对20%海水处理下长春花幼苗生物碱含量的影响,发现20%海水中加入色氨酸,长春花中色氨酸脱羧酶(TDC)活性提高,吲哚生物总碱以及长春碱、长春质碱、文多灵、长春新碱含量显著增加.其中加入500 mg/L的色氨酸最有利于生物碱含量的增加.     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系（C20D）筛选出一种激素完全适应型的细胞系（C20hi),考察了两种细胞生长、阿玛碱合成和引吲哚生物碱生物合成相关的酶的活性,结果表明：在生长培养基上二生长无显差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍。通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和long牛儿醇-10-脱氢酶活性,发明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的。     

剩余培养基对长春花愈伤组织和细胞培养的影响(简报)

剩余培养基对愈伤组织和悬浮细胞的生长及积累吲哚生物碱均有促进作用。     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系(C20D)筛选出一种激素完全适应型的细胞系(C20hi),考察了两种细胞生长、阿玛碱合成和与吲哚生物碱生物合成相关的酶的活性,结果表明:在生长培养基上二者生长无显著差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍.通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和牛儿醇-10-脱氢酶活性,发现在生产培养基中培养细胞的3种酶活性均显著高于生长培养基,但与阿玛碱积累没有密切关系.研究结果还表明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的.     

外源刺激物对长春花冠瘿细胞生长和吲哚生物碱含量的影响

所谓外源刺激物（ｅｌｉｃｉｔｏｒｓ）是指一些生物（多为真菌提取物）的或非生物的分子,它们能够通过信号传导途径,刺激植物发生防御反应,诱导特定的次级代谢产物的形成和积累^[1,5]。目前,有关elicitors对植物细胞次级代谢的影响和机制的研究引起了人们的普遍关注。我们曾经报道了以土壤农杆菌感染,Ti质粒转化得到的长春花冠瘿细胞,无论在生长还是在具有药用价值的次级代谢产物吲哚生物碱含量方面,都优于一般细胞培养常用的愈细胞^[2].本文研究了深红酵母匀浆物和果胶降解物作为外源刺激物,对长春花冠瘿细胞生长和吲哚生物碱含量的影响。     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres

Summary Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secolaganin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase. Offprint requests to: P. J. Facchini     

Selection of fungal elicitors to increase indole alkaloid accumulation in catharanthus roseus suspension cell culture

Various fungal elicitors derived from 12 fungi were tested to improve indole alkaloid production in Catharanthus roseus cell suspension cultures. Results show that different fungal mycelium homogenates stimulate different kinds of indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which ranged from 2- to 5-fold higher than the control. Some fungal culture filtrates also efficiently elicited the biosynthesis of different indole alkaloids. The optimal elicitor addition and exposure time for the maximal alkaloid production were on day 7 after subculture and for 3 days of treatment but different fungal elicitors showed the different optimal treatment dosages. Additions of elicitor at the doses ranging from 5 mg/l to 30 mg/l of carbon hydrate equivalent resulted in varieous amounts and kinds of indole alkaloid accumulation. Exposed to a same fungal elicitor, several different cell lines generated the different responses regarding as growth rate, culture color and alkaloid production.     

Effect of plant growth regulators on the biosynthesis of vinblastine,vindoline and catharanthine in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Catharanthuse roseus is a well-known medicinal plant for its two valuable anticancer compounds: vinblastine and vincristine, which belongs to terpenoid indole alkaloids. Great efforts have been made to study the principles of its secondary metabolic pathways to regulate the alkaloids biosynthesis. In this article, different plant growth regulators were shortly applied to Catharanthus roseus plants during the blooming period to study their effects on the biosynthesis of vinblastine, vindoline and catharanthine. Salicylic acid and ethylene (ethephon) treatments resulted in a significant increase of vinblastine, vindoline and catharanthine while abscisic acid and gibberellic acid had a strongly negative influence on the accumulation of the three important alkaloids. Methyl jasmonate showed no great effect on the production of these valuable alkaloids. Chlormequat chloride highly enhanced the accumulation of vinblastine but greatly decreased the contents of vindoline and catharanthine.     

Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors

Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l(-1)) and an improved catharanthine accumulation (17 mg l(-1)). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l(-1)) and a high ajmalicine accumulation (41 mg l(-1)) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l(-1), 32 mg l(-1) and 22 mg l(-1) catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway.     

Selection of protoclones for high yields of indole alkaloids from suspension cultures ofCatharanthus roseus and the qualitative analysis of the compounds by LC-MS

Summary To produce economically important indole alkaloids by cell culture, we have selected protoclones ofCatharanthus roseus for high yields of catharanthine and ajmalicine. Protoplasts were enzymatically isolated from suspension-cultured cells. Protoclone VPC-10 produced catharanthine at 5.9 μg/g fresh wt of cells after 10 days of culture, although the original cell line did not produce it at a level detectable by HPLC. Under the same conditions, protoclone VPC-15 produced ajmalicine at 133.6 μg/g, which was about 3 times the productivity of the original cell line. In addition, the indole alkaloids were qualitatively confirmed by LC-MS.     

Indole alkaloid production by hairy root cultures of Catharanthus roseus

Hairy root cultures of Catharanthus roseus were established by infection with six different Agrobacterium rhizogenes strains. Two plant varieties were used and found to exhibit significantly different responses to infection. Forty-seven hairy root clones derived from normal plants and two derived from the flowerless variety were screened for their growth and indole alkaloid production. The growth rate and morphological appearance showed wide variations between the clones. The alkaloid spectra observed were qualitatively but not quantitatively very similar to that of the corresponding normal plant roots. No vindoline or deacetyltransferase activity could be detected in any of the cultures studied. O-acetylval-lesamine, an alkaloid which has not been previously observed in C. roseus was identified from extracts of hairy root clone No. 8. Two root clones were examined for their growth and alkaloid accumulation during a 26-day culture period. Alkaloid accumulation parallelled growth in both clones with ca. 2 mg ajmalicine and catharanthine per g dry weight being observed.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Regulation of secondary metabolism in higher plants. Effect of alkaloids on a cytochrome P-450 dependent monooxygenase.

An end-product indole alkaloid, catharanthine, inhibits a membrane-bound cytochrome P-450 dependent monooxygenase of the higher plant, $\text{Vinca rosea}$. Kinetic analysis revealed the alkaloid to be a reversible, linear, non-competitive inhibitor (K_i=1 mM) with respect to its substrates, geraniol and NADPH. Comparable inhibition of the solubilized monooxygenase by catharanthine tends to exclude a mechanism based upon disruption of membrane organization. On the basis of its inhibition of solubilized hydroxylase in the presence and absence of sodium cholate, it is also unlikely that catharanthine competes for putative phospholipid binding site(s). Two additional end-product alkaloids, vinblastine and vindoline were less inhibitory. Since the hydroxylase catalyzes one of the first committed steps in the biosynthesis of indole alkaloids, these observations suggest feedback control of the pathway by catharanthine.     

长春花(Catharanthus roseus)中吲哚类生物碱含量的比较

建立了反相高效液相法测定长春花中吲哚类生物碱文多灵、长春质碱和阿玛碱含量的方法,色谱柱为HiQ sil C18色谱柱（250 mm×4.6 mm,5 μm）;流动相为1%二乙胺水溶液(磷酸调pH=7.2)—甲醇—乙腈/2:1:1(V/V);流速为1 mL·min^-1;检测波长为215 nm;柱温为40℃。并采用此方法对长春花根、茎、叶、花和种子以及不同产地的长春花中的这3种生物碱进行了检测,结果表明文多灵和长春质碱主要存在于在根、茎、叶、花中,阿玛碱主要存在于种子中;并且随着地理位置的北移,长春花中的文多灵和长春质碱的含量逐渐降低,温室中人为控制长春花中的栽培条件能提高二者的含量。