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。     

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.     

长春花生物碱生物合成途径和相关酶及其基因调控的研究进展

从长春质碱、文多灵、蛇根碱和阿吗碱及长春碱、长春新碱等的生物合成途径和催化酶及其基因的表达调控等方面概述了长春花生物碱的生物合成途径和代谢调控的研究进展,并对已经被分离和纯化或克隆的催化酶及其基因作了简要介绍。     

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。     

盐胁迫对长春花幼苗生长和生物碱含量的影响

以NaCl浓度分别为0、50、100、150、200和250 mmol·L^-1的1/2 Hoagland营养液处理长春花幼苗,7 d后测定其鲜质量、干质量、丙二醛(MDA)和叶绿素含量、色氨酸脱羧酶（TDC）和过氧化物酶（POD）活性等生理指标及文多灵、长春质碱、长春新碱和长春碱等生物碱含量.结果表明：NaCl显著地降低长春花幼苗的鲜质量和干质量,提高MDA含量;叶绿素含量在低盐浓度(50 mmol·L^-1)下与对照相比差异不显著,在高于50 mmol·L^-1时随NaCl浓度的增加而逐渐降低;在NaCl处理下,POD活性与对照相比显著上升;TDC活性在50 mmol·L^-1 NaCl处理下活性最高,而后随盐浓度的增加逐渐降低;文多灵、长春质碱、长春新碱和长春碱含量都是在50 mmol·L^-1NaCl处理下最高,分别为4.61、3.56、1.19和2.95 mg·g^-1,并显著高于对照及其他处理.盐胁迫虽然在一定程度上抑制了长春花幼苗生长,但促进了其生物碱的代谢,提高了生物碱含量;50 mmol·L^-1NaCl处理对长春花吲哚生物碱代谢的促进作用最大.     

氯化钾对长春花盛花期盐胁迫效应和生物碱含量的影响

在温室土培条件下,研究了不同浓度KCl处理对长春花盛花期盐胁迫效应以及对文多灵、长春质碱、长春碱和长春新碱等生物碱含量的影响.结果现实:(1)3‰ NaCl胁迫下,施入一定量KCl,将Na+/K+调为20:1(K2)时可显著缓解盐胁迫对长春花的危害,与不施KCl(K1)相比,长春花的鲜质量、株高、根长和相对含水量均显著提高,而长春花叶片丙二醛含量显著下降,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)活性在Na+/K+为20:1(K2)时显著高于其它处理,但当Na+/K+为10:1(K3)与5:1(K4)时,盐胁迫的危害又显著加剧.(2)Na+/K+为20:1处理的长春花盛花期文多灵、长春质碱、长春碱和长春新碱含量显著高于其它处理,分别为20.88、30.18、2.53和5.12 mg·g-1.研究表明,Na+/K+为20:1比例施用钾肥可最大限度地降低NaCl胁迫对盛花期长春花生长造成的伤害,并显著促进其生物碱的代谢,显著提高长春花盛花期4种主要生物碱的含量.     

人工种植长春花生物学性状和生物碱含量的季节动态

以人工种植的长春花（Catharanthus roseus（L.）G. Don）为材料,研究了一个生长季节内长春花的生物学性状和生物碱含量的季节动态。研究发现,长春花的株高、生物量和叶片数呈现相似的生长趋势,即前期（5~7月）缓慢增长、中期（7~9月）快速增长和后期（9~10月）增长缓慢3个明显的季节生长特征,而花总数在前期和中期也呈现相似的规律,但在后期（9~10月）快速下降。长春花叶片中3 种生物碱含量的季节变化规律明显,变化趋势相一致。3 种生物碱的含量在一个生长季节内均是先增加后降低再增加的趋势,且3 种生物碱含量均在7月下旬和10月下旬出现2个明显的高峰值。长春花叶片中长春碱含量与长春质碱和文多灵含量之间均有较强的正相关,且与文多灵含量呈显著的正相关（p<0.05）,长春花叶片中长春质碱含量与文多灵含量之间呈显著的正相关（p<0.05）。因此,生产实践中人工种植长春花的最佳采收期是10月末,并可通过改变环境因子进一步诱导长春花叶片中长春碱类物质的积累。     

弱光胁迫对大田长春花生物量分配及次生代谢的影响

以长春花（Catharanthus roseus(L.) Don.）为材料,研究了田间栽培长春花在全光和20%透光率下,经过一个生长季后生物量配置、抗氧化次生代谢产物和文朵灵、长春质碱、长春碱等目的活性物质含量的变化。研究结果表明,弱光条件显著抑制了长春花植株总生物量增长,特别是抑制了有性生殖的投入;弱光组叶片总酚和总黄酮含量显著降低,干重含量分别为对照组的62.50%和50.00%,原花青素含量则略有升高,但与全光组的差异不显著;弱光组叶片文朵灵和长春质碱含量显著高于对照组,长春碱含量略有上升但差异不显著,受生物量降低影响,3种生物碱的产量均显著下降。上述结果表明,长春花能够调控生理代谢以适应低光强环境,特别是文朵灵和长春质碱含量提升显著,林下低光强环境种植长春花可以满足土地资源充分利用和文朵灵、长春质碱优质原料的需求。     

PEG6000胁迫下长春花叶片生物碱含量及相关基因表达的分析

以长春花(Catharanthus roseus(L.)G.Don)幼苗（45 d）为材料,采用温室水培法,设定不同浓度的聚乙二醇6000(Polyethyleneglycol,PEG6000)胁迫处理,通过分析叶片脯氨酸含量和相对含水量的变化,选定35%（V/V）浓度的PEG6000模拟干旱胁迫。结果显示,干旱处理12~24 h,叶片中碱性POD活性明显增强。高效液相色谱技术分析表明,干旱处理下,叶片中文多灵和长春质碱含量逐渐升高且高于对照,然后下降;处理12~24 h,长春碱含量逐渐升高,24 h达到峰值(0.17 mg·g^-1 FW±0.003 6)。半定量RT-PCR分析表明,在胁迫处理3 h,生物碱合成途径中3个基因(Tdc、Str、Dat)表达均相对升高,相关性分析表明,PEG6000干旱胁迫下,长春花叶片中Tdc基因表达与长春质碱含量变化正相关,POD的活性与长春碱含量变化正相关(P<0.05)。     

四种主要长春花生物碱超声波法提取工艺优化研究

为探索高效、低污染的长春花中文多灵、长春质碱、长春新碱和长春碱的提取工艺,进行了超声波-甲醇法提取工艺条件的优化研究。结果表明:在单因素试验基础上,通过正交试验表明,超声波-甲醇法提取文多灵和长春质碱的最佳工艺条件为:提取溶剂:甲醇(pH4.0);料液比:1∶15;提取温度:45℃,提取时间:180 min,提取频率:59 KHz;文多灵和长春质碱平均得率为0.16%,0.08%。     

Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

ABSTRACT: BACKGROUND: As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus the establishment of an efficient genetic transformation method is required. RESULTS: To develop a genetic transformation method for C. roseus, A. tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report beta-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 muM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance mediums, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that overexpression of DAT increased the yield of vindoline in transgenic plants. CONCLUSIONS: In the present study, we report an efficient Agrobacterium-mediated transformation system for C. roseus plants with 11.11 % of transformation frequency. To our knowledge, this is the first report on the establishment of A. tumefaciens mediated transformation and regeneration of C. roseus. More importantly, the C. roseus transformation system developed in this work was confirmed in the successful transformation of C. roseus using a key gene involved in TIAs biosynthetic pathway resulting in the higher accumulation of vindoline in transgenic plants.     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis, PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V_0.3638S_0.6174C_0.0187,属于SV型。刈割使长春花的生活史型转变为V0.2847S0.6684C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（p<0.05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

刈割对栽培长春花(Catharanthus roseus)生活史型转变的影响

为了提高栽培长春花（Catharanthus roseus）的生物碱产量,应用植物生活史型的理论和方法,研究了刈割对栽培长春花生活史型转变及其生物碱代谢的影响。运用主成分分析法（Principal Component Analysis,PCA）对刈割后的长春花后生活史型变化进行定量和定性划分,发现在对照栽培环境下生长的长春花处于DE生境,定性划分结果为SV生活史型,定量划分结果为V0.3638,S0.6174C0.0187,属于SV型。刈割使长春花的生活史型转变为V0．2847S0.6544C0.0469,属于SC型。同时,对两种生活史型的长春花中长春碱及其前体文朵灵和长春质碱的含量进行了检测分析,发现刈割后的SC型长春花不同叶位叶片中的生物碱含量均显著提高（P〈O．05）,可以为提高栽培长春花生物碱含量提供科学指导,也进一步验证了生活史型理论。     

Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase

The Madagascar periwinkle (Catharanthus roseus) produces the well-known and remarkably complex anti-cancer dimeric alkaloids vinblastine and vincristine that are derived from the coupling of vindoline and catharanthine monomers. This study describes the novel application of a carborundum abrasion (CA) technique for large-scale isolation of leaf epidermis-enriched proteins in order to purify to apparent homogeneity 16-hydroxytabersonine-16-O-methyltransferase (16OMT), which catalyses the second of six steps in the conversion of tabersonine into vindoline, and to clone the gene. Functional expression and biochemical characterization of recombinant 16OMT demonstrated its very narrow substrate specificity and high affinity for 16-hydroxytabersonine. In addition to allowing the cloning of this gene, the CA technique clearly showed that 16OMT is predominantly expressed in Catharanthus leaf epidermis. The results provide compelling evidence that most of the pathway for vindoline biosynthesis, including the O-methylation of 16-hydroxytabersonine, occurs exclusively in the leaf epidermis, with subsequent steps occurring in other leaf cell types.     

A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by the Madagascar periwinkle (Catharanthus roseus). Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to either improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. A VIGS method was developed herein to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro.