Screening of taxol-producing endophytic fungi from Taxus chinensis var. mairei

A total of 38 endophytic fungus strains were isolated from Taxus chinensis var. mairei by aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of Taxus taxadiene synthase (TS) gene, a rate-limiting enzyme gene in the taxol biosynthetic pathway. Twelve out of 38 isolated endophytic fungus strains showed PCR positive for the ts gene. Subsequently, taxol and its related compounds were extracted from culture filtrates and mycelia of the PCR positive strains, separated by column chromatography and analyzed by High Performance Liquid Chromatography and Mass Spectrum. The analysis result showed that 3 strains could produce taxol and its related compounds at the detectible level. This study indicates that molecular detection of the ts gene is an efficient method for primary screening of taxol or its related compounds-producing endophytic fungi which can improve prominently screening efficiency.     

Isolation of an endophytic fungus producing baccatin III from Taxus wallichiana var. mairei

The objective of this study was to isolate endophytic fungi producing baccatin III from yew for the purpose of baccatin III and paclitaxel manufacture. Surface sterilized bark of Taxus wallichiana var. mairei was used as source material with potato dextrose agar culture medium for isolation of endophytic fungi. Fungal cultures were extracted with a mixture of chloroform/methanol (1:1, v/v) and the baccatin III in the extracts was determined and authenticated with LC–MS. An endophytic fungus that produced baccatin III was identified by ITS rDNA and 26S D1/D2 rDNA sequencing. A total of 192 endophytic fungal strains were isolated from T. wallichiana var. mairei. Only one of the 192 strains produced baccatin III and it was identified as Diaporthe phaseolorum. The productivity of this strain cultured in PDA culture medium was 0.219 mg/l. The isolated endophytic fungus produced baccatin III at a relatively high level and shows promise as a producing strain for baccatin III and paclitaxel manufacture after strain improvement.     

南方红豆杉内生真菌的抗肿瘤产物震颤毒素的研究

从红豆杉内生真菌青霉菌Penicillum sp. H⑥.1发酵产物中自然析出一种化合物,经图谱检测和文献比对,鉴定为震颤毒素Penitrem A。四唑盐（MTT）比色法研究表明Penitrem A对恶性黑色素瘤细胞A375有一定的抑制作用,且抑制作用随浓度的增大而增强,IC₅₀为27.24μg/mL。本研究是首次从红豆杉内生真菌发酵产物中分离得到震颤毒素Penitrem A,为抗肿瘤药物开发提供了理论参考。     

南方红豆杉内生真菌的分离鉴定及其细胞毒活性研究

采用平板培养法对采自广东省的南方红豆杉进行内生真菌的分离,并通过形态学和分子生物学方法进行分类鉴定;共分离鉴定内生真菌22株,主要为刺盘孢属(Colletotrichum)、球座菌属(Guignardia)、座腔菌属(Botryosphaeria)、节菱孢属(Arthrinium)、炭角菌属(Xylaria)、毛壳菌属(Chaetomium)、交链孢属(Alternaria)、拟茎点霉属(Phomopsis)、长蠕孢属(Helminthosporium)等属。还采用MTT法测定了这些内生真菌发酵粗提物的细胞毒活性,活性研究发现有6个菌株的粗提物在作用浓度为100μg/mL时,对至少1种受试肿瘤细胞株的抑制率在50%以上,其中菌株A223和A240对人乳腺癌细胞MCF-7的抑制率在90%以上。表明南方红豆杉内生真菌多样性丰富,有些菌株具有良好的细胞毒活性,值得进一步深入研究。     

Taxol from Tubercularia sp. strain TF5, an endophytic fungus of Taxus mairei

The diterpenoid taxol is an important anticancer agent used widely in the clinic. The purpose of this work was to identify a taxol-producing endophytic fungus (strain TF5) isolated from Taxus mairei and study its anticancer activities. Strain TF5 was identified as a Tubercularia sp. according to the morphology of the fungal culture, the mechanism of spore production and the characteristics of the spores. Strain TF5 produced taxol, when grown in potato dextrose liquid medium and analyzed by thin layer chromatography, high performance liquid chromatography, ultraviolet and mass spectrometry. The fungal taxol, which was isolated from the organic extract of the TF5 culture, had strong cytotoxic activity towards KB and P388 cancer cells in vitro, tested by the MTT assay. Observed with immunofluorescence and electron microscopy, the fungal taxol enhanced microtubule stability and bundling in culture cells and induced tubulin polymerization in vitro similar to the authentic taxol.     

南方红豆杉天然林群落结构特征研究

本文对福建三明陈大镇南方红豆杉天然林群落进行研究,测定了该群落11种主要树种的重要值,分析了该群落的种类组成、物种多样性、群落结构及其动态。结果表明,人为干扰较轻的南方红豆杉天然林群落比较稳定,天然更新良好,处于旺盛生长阶段。     

南方红豆杉丛枝菌根(AM)的研究

研究了南方红豆杉根部丛枝菌根真菌(AMF)侵染情况、菌根形态结构以及根际土中AMF孢子的种类与数量.结果显示:南方红豆杉可与AMF形成典型的丛枝-泡囊型菌根,侵染率在71.2%~94.4%,但是历山、蟒河自然保护区的侵染强度优于人工栽培区;在南方红豆杉根际土中共分离鉴定出5种AMF,无梗囊霉属1种、球囊霉属4种,分别是:光壁无梗囊霉、地表球囊霉、地球囊霉、缩球囊霉、明球囊霉,其中光壁无梗囊霉为优势种;南方红豆杉的根由表皮、外皮层、内皮层、中柱组成,AMF只侵染表皮层、内皮层,不能侵染中柱.这为将来利用AMF接种技术进行南方红豆杉的繁殖、移植栽培以及紫杉醇的积累等研究提供了理论依据.     

南方红豆杉的ISSR遗传多样性分析

采用ISSR标记技术对南方红豆杉迁地保护小种群及其衍生自然种群5个小斑块(小居群)的遗传多样性进行了分析.从90条引物中共筛选出10个多态性引物,获得ISSR谱带102条,其中多态性谱带74条,占72.54%.结果显示,南方红豆杉迁地保护小种群和迁地保护衍生自然种群的Nei's基因多样性指数(H)分别为0.220 9和0.254 8,Shannon信息指数(I)分别为0.322 4和0.376 2.5个小斑块居群的H分别为0.190 7、0.210 4、0.194 4、0.160 5和0.187 3,I分别为0.281 0、0.309 8、0.288 0、0.235 5和0.273 5.迁地保护衍生自然种群的遗传多样性高于迁地保护小种群,但各小生境下的的小斑块居群遗传多样性低于迁地保护小种群.聚类分析结果表明:南方红豆杉迁地保护各自然小居群间的遗传距离与这些居群的地理生境有关,而与地理距离并没有显著相关性.     

南方红豆杉无性系种群结构和动态研究

研究了元宝山南方红豆杉克隆种群的结构和动态．结果表明,种群S1～S6级个体均存在,S1、S2级个体占总个体数的64％．从元宝山南方红豆杉种群的大小级结构图看,结构呈较规则的金字塔形;南方红豆杉无性系幼树幼苗的贮备较为丰富,种群的年龄结构呈较规则的金字塔形,存活曲线近于倒J形;种群大小级动态指数Vpi=38．26％,考虑外部干扰时,Vpi′：6．38％>0,表现为稳定型种群．南方红豆杉种群更新的主要途径是无性繁殖,种群的数量动态主要受克隆内部调节机制以及外部环境的影响．生存分析结果表明,种群幼树幼苗的生存状态不稳定,死亡的可能性很大,而成年个体的生存状态则较为稳定．     

红豆杉种质资源遗传多样性的AFLP分析

目的：通过对5份红豆杉种质资源的AFLP分析,探求各种质间的遗传多样性。方法：采用扩增片段长度多态性（AFLP）标记,在DNA水平上进行遗传多样性研究,筛选了32对选择性扩增引物,将扩增出的条带作为原始矩阵,用NTSYS-PC软件计算并分析了红豆杉种质间的相似度,构建了遗传系统进化树。结果：（1）SDS法提取的红豆杉基因组DNA质量较佳,能够满足AFLP分析的要求;（2）从32对选择性扩增引物中,筛选出10对多态性较强、带型较好、分辨率较高的组合;（3）构建了红豆杉AFLP指纹图谱,将5个红豆杉种质全部区分开来;（4）通过构建进化树,把5个种质分成3类。结论：红豆杉种质资源有丰富的遗传多样性。     

RAPD Analysis on High-taxane-contained Accessions of Taxus chinensis var. mairei

Taxus chinensis var. mairei (kemee et Levi. ) Cheng et L.K. Fu is one of the resource plants in China. The taxot contents from different localities are variable. RAPD and HPLC analysis were carried out with 24 samples from 12_localities. A total of 256 loci were amplified among which 224 loci were polymorphic (87.8%). Based on the HPLC and morphological analysis, the relationship among RAPD markers, morphology and habitat was explored. According to the results, high-taxane-contained accessions are present in three localities. This study might be of benefit to the resource conservation and species improvement.     

南方红豆杉盆栽扦插育苗技术研究

南方红豆杉(Taxus chinensis var. mairei)是中国特有的国家一级保护植物。2009-2010年采用2年龄实生苗为采条母株进行室内盆栽扦插试验研究,结果表明:珍珠岩和河沙两种基质相比较,珍珠岩上生根率显著高于河沙,是更佳选择;用市售的"花语牌"快速促根剂配制成5、10、15、2 0mg/mL 4个浓度分别处理插穗基部并以蒸馏水作对照发现,10 mg/mL、15 mg/mL处理有利于生长,15 mg/mL处理后生根率显著高于5 mg/mL、10 mg/mL、20 mg/mL及对照。再以同一浓度生根粉分别浸泡4 h、8 h、10 h作对比发现,8 h和10 h处理后生长效果较好,10 h的生根率显著高于4 h、8 h。     

南方红豆杉产紫杉醇内生真菌的分离鉴定

从南方红豆杉(Taxus chinensisvar.mairei)植株分离到内生真菌63株,其中24株经鉴定分属于19个种(属).真子囊菌纲的2个属2株,半知菌纲的14个属22株.真子囊菌纲的无毛毛壳属(Achaetomiumsp.)的1株为新发现种.半知菌纲的葡萄孢属(Botrytissp.)1株和无孢菌群(Mycelia sterlia)2株的培养液,经浓缩萃取,薄层层析检测,Rf值分别为0.404、0.404、0.401,与紫杉醇对照品Rf值一致,为南方红豆杉产紫杉醇内生真菌.     

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l^–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.     

不同栽培条件下南方红豆杉生长特性研究

以4～6年生南方红豆杉为材料,对其不同栽培条件下地径、冠幅、株高、当年抽高、当年侧枝数及总侧枝数等生长特性进行了研究。结果表明:南方红豆杉4～6年生幼林生长量随年龄增大而增加,但其增加的趋势变缓,生长速度变慢;耕作农田中南方红豆杉生长最好,土壤板结未耕作农田生长较差,去除上层20cm耕作土的农田最差;南方红豆杉纯林生长好于落叶阔叶林下(林分郁闭度0.6～0.7),且种植密度越大差异越明显;南方红豆杉落叶阔叶林下套种及纯林生长量均随栽培密度减小而增大,不同栽培密度相差越大,生长量差异越明显。     

南方红豆杉小孢子发生与雄配子体发育

采用石蜡切片法,对南方红豆杉小孢子发生及雄配子体发育过程进行了系统地观察。结果表明：南方红豆杉小孢子叶球于7月下旬分化,9月中旬形成造孢细胞,11月初形成小孢子母细胞;同一小孢子叶球中的小孢子母细胞表现出发育不同步现象;11中旬,进入减数分裂时期,形成游离小孢子后休眠越冬,于翌年1月下旬逐渐成熟,成熟花粉粒为单核;2月中下旬开始散粉,散粉时间持续15 d 左右。花粉落入胚珠后,经过3次分裂形成管细胞、柄细胞和2个精子;管细胞和柄细胞最终退化解体,未见花粉败育现象。认为南方红豆杉小孢子发生与雄配子体发育正常,不是致其濒危的主要原因。     

濒危植物南方红豆杉遗传多样性的RAPD分析

采用RAPD技术检测了山西南部南方红豆杉8个种群的遗传多样性。利用21个10聚寡核苷酸引物共检测出134个位点,其中多态性位点123个,占91.79%,8个种群的遗传多态位点百分率分别为67.16%(红豆峡)、67.91%(凤凰谷)、66.42%(小梯河)、66.42%(蟒河)、50.75%(历山西峡)、43.28%(云蒙山)、78.36%(长治宾馆)、50.75%(磨河)。南方红豆杉的遗传多样性分析结果显示：Shannon指数为2.180,其中31.7%的遗传多样性来自种群间,68.3%来自种群内;Nei指数为0.571,种群间的遗传分化系数(Gst)为0.181。8个种群间的遗传相似性分析结果显示：壶关红豆峡和陵川凤凰谷种群间的遗传距离最小(0.109 2),壶关红豆峡和陵川蟒河种群间遗传距离最大(0.55)。本研究结果揭示,南方红豆杉自然种群具有较高的遗传多样性,其遗传多样性不是导致该种群濒危的主要原因,导致南方红豆杉种群濒危的原因可能与南方红豆杉自然种群及群落所在生境的直接破坏及其本身生物学和生态学特性所导致的自然更新不良有着密切的联系。     

红豆杉内生真菌次生代谢物的分离鉴定及其抗菌活性 分析

以产紫杉醇类化合物的云南红豆杉内生真菌12.3.2为目标菌株,对其次生代谢物进行分离鉴定和抗菌活性研究。通过硅胶柱层析,共分离得到3个主要成分,红外光谱、质谱、核磁共振氢谱等光谱解析鉴定其分别为松柏烯、邻苯二甲酸二异辛酯、油酸乙酯。生物活性试验表明3种化合物对金黄色葡萄球菌、枯草杆菌、大肠杆菌、绿脓杆菌和白色链球菌这5种致病菌都有一定的抑制作用。其中松柏烯对金黄色葡球菌、枯草杆菌和白色念珠菌有较强的抑制作用。松柏烯是首次报道从植物内生真菌次生代谢物中分离得到,这为松柏烯的来源开辟了一条重要的途径,同时也为松柏烯的进一步开发利用奠定了基础。     

Study on Enhanced Production of Taxol from Taxus chinensis var. mairei in Biphasic-liquid Culture

The effects of precursor feeding and mixed sugar supplements on the growth and taxol production of Taxus chinensis var. mairei Cheng et Fu in the biphasic-liquid culture were studied. Precursor feeding (phenylanine, benzamide and sodium acetate) and mixed sugars (maltose and sucrose) resulted in significant increase of taxol production not only in the monophasic-liquid culture but also in the biphasic-liquid culture of T. chinensis var. mairei. The synergistic function of precursor feeding on day 10 and mixed sugars on day 11 in the biphasic-liquid culture revealed more significant increase of taxol production, which was four times that of the control (the monophasic-liquid culture without precursors or mixed sugars).     

南方红豆杉内生细菌分离鉴定及系统发育分析

目的:了解南方红豆杉内生细菌类群及其系统发育关系;方法:对分离菌株进行形态学、生理生化和分子鉴定,并做系统发育分析.结果:从南方红豆杉茎、皮和叶中共分离到内生细菌52株.对12株内生细菌16S rDNA进行PCR扩增,扩增产物大小约为1 400bp,对11株内生细菌16S rDNA测序结果,用BLAST软件进行相似性比对,发现TB02株为Enterobacter属,相似性99％;9株为Bacillus属,相似性99％～ 100％:TB13株为Lysinibacillus,相似性97％.通过构建系统发育树发现这11株内生细菌明显聚为两大支.结论:11株内生细菌分别鉴定为肠杆菌、芽孢杆菌和Lysinibacillus,芽孢杆菌为南方红豆杉内生细菌优势菌属.芽孢杆菌和Lysinibacillus亲缘关系较近,二者和肠杆菌之间亲缘关系较远.