磷甘霉素和洛伐它汀处理对中国红豆杉悬浮培养细胞生物合成紫杉醇的影响

采用非甲羟戊酸途径抑制剂磷甘霉素和甲羟戊酸途径抑制剂洛伐它汀对中国红豆杉悬浮细胞培养物进行处理.在添加和未添加茉莉酸甲酯诱导的情况下,前者使紫杉醇产量减少了2/5和1/5,后者使紫杉醇产量减少了1/6和1/10,表明两种途径对紫杉醇的生物合成都具有贡献,其中非甲羟戊酸途径贡献较大;通过定量PCR技术分别检测两条途径的关键酶5-磷酸脱氧木酮糖还原异构酶(DXR)和3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)mRNA水平的变化,发现两种抑制剂都能够激活hmgr和dxr的转录,表明两种代谢途径之间存在协同作用,共同为紫杉醇的生物合成提供前体.     

中国红豆杉TcNPR1基因的克隆与功能研究

水杨酸（SA）可诱导红豆杉细胞中紫杉醇的合成,病程相关基因非表达子基因（NPR1）是SA介导的植物系统获得性抗性（SAR）信号途径中的关键信号分子。本研究从中国红豆杉（Taxus chinesis）细胞中克隆了TcNPR1基因,该基因完整的开放阅读框大小为1857 bp,编码蛋白序列由619个氨基酸组成,含有典型的NPR1保守结构域、锌指结构域和锚蛋白结构域。进化树分析发现,TcNPR1蛋白与拟南芥AtNPR3和AtNPR4类蛋白的亲源关系较近。TcNPR1基因表达响应SA、干旱和NaCl胁迫处理,且随着处理时间增加,TcNPR1基因表达量逐渐增大。超表达TcNPR1的转基因烟草植株对干旱的耐受力增强,叶片内可溶性糖含量达43.8 mg/g（FW）,比对照高出2倍左右,说明TcNPR1基因具有表达功能,此结果为探究中国红豆杉细胞响应SA诱导大量合成紫杉醇的信号途径提供了依据。     

Enhanced production of taxol in suspension cultures of Taxus chinensis by controlling inoculum size

Inoculum size (1.5-6.0g dry weight/l) significantly affected cell growth and accumulation of intracellular and extracellular taxol in Taxus chinensis. A shorter cultivation time and a higher biomass productivity were achieved using inoculum size of 6.0g DW/l. Both the intracellular content and total production of taxol were increased almost 30% with an increase of inoculum size from 1.5 to 3.0g DW/l, while an even higher inoculum size decreased taxol formation. The extracellular taxol concentration was relatively higher (0.091mg/l) at low inoculum sizes of 1.5 and 2.0g DW/l; and in various cases it was less than 25% of the total amount of taxol produced.     

Effects of inoculum size and age on biomass growth and paclitaxel production of elicitor-treated Taxus yunnanensis cell cultures

Suspension cultures of Taxus yunnanensis cells were inoculated with cells of different culture ages (12-24 days) at various densities [50-250 g fresh weight (fw)/l], and treated (on day 7) with a mixture of elicitors, including Ag(+), chitosan and methyl jasmonate. The biomass productivity (during the production stage) increased dramatically with inoculum size, but decreased with inoculum age over 16 days. The volumetric yield and productivity of taxol (paclitaxel) also increased with inoculum size, while the specific taxol yield (per cell) was mainly dependent on inoculum age, with an optimum of 20 days, during the early stationary phase. The highest taxol yield and productivity, 39.8 mg/l and 1.9 mg/l per day, respectively, were obtained with a 20-day-old inoculum at 200 g fw/l. Taxol excretion by the cells increased with inoculum age but decreased with inoculum size. The elicitor-induced activities of catalase (CAT) and phenylalanine ammonia-lyase (PAL) also depended mainly on inoculum age; higher PAL activity and lower CAT activity were obtained with an older inoculum, corresponding to a higher taxol yield. The results show that both inoculum size and age are important variables for taxol production, though the latter more profoundly influences elicitor-induced taxol biosynthesis of the cells. Inoculum size and age are also interrelated and should be optimized together in a two-stage culture process.     

内生真菌紫杉醇生物合成的研究现状与展望

紫杉醇是重要的抗癌药物之一,已经证明其对多种癌症具有显著疗效。目前,人们主要是从红豆杉的树皮中提取、分离和纯化紫杉醇,但由于红豆杉为生长缓慢、散生、濒危的珍稀植物,且随着紫杉醇临床用途的不断拓宽,市场需求的稳定增长,单纯依靠从红豆杉树皮中提取紫杉醇已经无法满足日益增长的市场需求。为了解决紫杉醇的药源不足,科学家已把目光从红豆杉树分离提取紫杉醇转向了其他替代方法,如化学全合成、半合成、组织培养与细胞培养、微生物发酵法生产紫杉醇等。因此,了解内生真菌紫杉醇生物合成的分子基础和遗传调控机制,对解析内生真菌紫杉醇生物合成机制、构建高产紫杉醇基因工程菌株和早日实现内生真菌紫杉醇工业化生产具有重要的科学意义和现实意义。结合本课题组多年来的科研工作,概述了红豆杉细胞紫杉醇生物合成途径、内生真菌发酵生产紫杉醇的优势、产紫杉醇内生菌的分离研究现状和生物多样性及紫杉醇生物合成相关基因的研究现状。内生真菌生物发酵合成紫杉醇是可以无限生产、大量获取紫杉醇、解决紫杉醇药源短缺问题的很有前景的方法之一。     

补料培养与溶氧控制联合应用对红豆杉细胞培养的影响

为进一步提高红豆杉 (Taxuschinensis (Pilg.)Rehd .)细胞培养过程中紫杉醇的产量 ,采用细胞悬浮培养方法研究了补料培养与溶氧控制联合应用对紫杉醇产量的影响。 5L反应器中补料培养研究表明 ,培养过程中第 16天添加含 2 0g/L蔗糖的补料培养液有利于细胞的生长及紫杉醇的合成。 2 0L反应器中补料培养的研究结果表明 :2 0 %饱和度培养时紫杉醇含量最高 (0 .98mg/gDW) ,但 4 0 %～ 6 0 %溶氧饱和度能提高紫杉醇的产量。进一步研究表明 ,细胞在 6 0 %溶氧饱和度培养 2 0d后转入 2 0 %溶氧饱和度继续培养 12d ,能显著提高紫杉醇产量。补料培养与溶氧控制联合应用时 ,2 0L反应器中红豆杉细胞培养紫杉醇产量可达 18.7mg/L。     

Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound,methyl jasmonate and in situ solvent extraction

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.     

过表达Bapt基因提高中国红豆杉细胞的紫杉醇产量

过表达紫杉醇合成关键酶基因是提高红豆杉细胞紫杉醇产量的有效方法.本文通过构建C-l3苯丙素侧链CoA-乙酰转移酶 (C-13 phenylpropanoid side chain CoA acetyltransferase,BAPT) 基因Bapt的表达载体p1303-SbaptN, 采用根癌农杆菌介导法转化中国红豆杉细胞,经潮霉素抗性筛选获得转基因细胞系.转基因细胞的PCR和Southern印迹分析结果表明,T-DNA及其包含的基因与宿主细胞染色体成功整合;Western印迹结果表明,转基因细胞中报告基因GusA-mgfp5正常表达;QRT PCR结果显示,转基因细胞的Bapt mRNA表达量是未转化细胞的1.26 倍;HPLC检测结果显示,转基因细胞的紫杉醇产量为37.4 μg/g,是未转化细胞的1.87倍. 本研究结果表明,过表达Bapt基因提高了中国红豆杉细胞的紫杉醇产量     

红豆杉细胞培养产物提取策略及其对紫杉醇的影响

研究了硫酸铈铵及原位提取对红豆杉细胞悬浮培养过程中细胞生长、紫杉醇合成及释放的影响。红豆杉细胞悬浮培养过程中培养第12d添加2mg/L硫酸铈铵能获得最大紫杉醇产量8．3mg/L,其中2．4mg/L释放到细胞外,分别为对照组的4倍及12倍。同时添加2mg/L硫酸铈铵、5％油酸(v/v)时胞外紫杉醇产量达到9mg/L,为对照组的45倍。将硫酸铈铵及原位提取与补料培养相结合,最高紫杉醇产量可达24．5mg/L,其中60％释放到胞外。     

Hydrogen peroxide from the oxidative burst is not involved in the induction of taxol biosynthesis in Taxus chinensis cells

In cell suspension cultures of Taxus chinensis, 40 mg/l fungal elicitor from Aspergillus niger and 20 microM HgCl2 elicited 5.7 and 3.6 mg/l taxol, which was a 9-fold and 5-fold increase vs. compared with the control, respectively. The fungal elicitor induced hydrogen peroxide (H2O2) accumulation but HgCl2 did not, indicating that H2O2 was not necessary for enhancement of taxol induced by elicitor. Compared with the treatment with fungal elicitor alone, exogenous catalase, ascorbic acid, diphenylene iodonium and superoxide dismutase induced a 0.45, 0.4, 0.7 and 1.4-fold H2O2, but elicited taxol production, which was 0.98, 1.2, 1.1 and 0.9-fold, respectively, vs. non-treated cells Elicitor-induced taxol production was not accorded with the amount of H2O2 production.     

Taxol content in the bark of Himalayan Yew in relation to tree age and sex

Taxol content in the bark of Taxus baccata trees growing in a homogenous (uniform) environment at Jageshwar, District Almora in Central Himalaya has been quantified. The average taxol concentration in the bark of sampled trees was 0.0558+/-0.008% (of dry wt.) and was about 64% higher for male plants (averaged across tree age) in comparison to female trees. Maximum taxol content was recorded in the bark samples collected from trees of >110 yrs age. ANOVA indicates a significant difference in the taxol content of bark from trees of different ages, however, differences were not significant between sexes. Taxol was quantified by HPLC using a standard curve prepared with authentic taxol; the identification of bark taxol was confirmed by UV and mass spectrometry. The total taxol content of the bark of Taxus trees across an age series was found to range between 0.064 to 8.032 g/tree, and a tree of about 100 yrs age can yield 5.74 kg dry bark.     

聚乙二醇对东北红豆杉培养细胞的

在改良的B     

东北红豆杉细胞培养生产紫杉醇的调控研究

研究了诱导子、前体及抑制剂的协调作用对东北红平杉生产紫杉醇的影响。结果表明,向培养基中加入80mg/L水到、80mg/L茉莉酸甲酯、0.5mmol/L乙酸钠、2mmol/L苯丙氨酸、0.5mmol/L丝氨酸、0.1mmol/L甘氨酸、10mg/L肉桂酸、0.5mmol/L苯甲酸钠、5mmol/L丙酮酸钠、10mg/L氯化氯胆碱、和1mg/L赤霉酸可使紫杉醇含量提高368.65%。并且证明交互作用对紫杉醇合成有显著作用。     

过表达Dbtnbt基因提高中国红豆杉细胞的紫杉醇含量

本文通过克隆3′-N-去苯甲酰紫杉醇N-苯甲酰转移酶（3′-N-debenzoyltaxol-N-benzoyltransferase,DBTNBT）基因Dbtnbt,构建其表达载体p1303-SDbtnbtN,转化中国红豆杉细胞,经潮霉素抗性筛选获得转基因细胞系. 转基因细胞分析结果表明,T-DNA及其包含的基因与宿主细胞染色体成功整合;转基因细胞中报告基因GusA-mgfp5正常表达;转基因细胞的Dbtnbt mRNA表达量是未转化细胞的1.33 倍;转基因细胞的紫杉醇产量约为27.3 μg/g,是未转化细胞的1.37倍. 本研究结果表明,过表达Dbtnbt基因将中国红豆杉细胞的紫杉醇产量提高约37%.     

溶氧水平对红豆杉细胞悬浮培养的影响研究

紫杉醇 (Ｔａｘｏｌ)是源自红豆杉提取物的一种高度衍生化的二萜类化合物 ,临床实验结果表明紫杉醇对于卵巢癌、乳腺癌、胃肠道癌等具有明显的抗肿瘤活性[1] ,因而受到世界各国的广泛关注 ,并已被美国食品与药品管理局 (ＦＤＡ)批准用于卵巢癌与乳腺癌的治疗[2 ] 。到目前为止紫杉醇仍然主要从树皮中提取 ,但由于红豆杉生长缓慢 ,天然资源非常有限 ,加快其替代来源的研究势在必行。利用植物细胞悬浮培养生产紫杉醇作为一种可行的选择 ,近年来取得了较大的进展[3 ,4 ] 。本文研究了摇瓶及 2 0 Ｌ反应器培养过程的溶氧水平对细胞生长及紫杉醇…     

代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Growth and rutin production in hairy root cultures of buckwheat (Fagopyrum esculentum M.)

Buckwheat (Fagopyrum esculentum Moench.) is a potentially important source of rutin, a natural flavonoid with antihyperglycemic, antihypertensive, and antioxidative properties. To examine in vitro production of rutin, we established a hairy root culture of buckwheat by infecting leaf explants with Agrobacterium rhizogenes R1000, and tested the growth conditions and rutin production rates of these cultures. Ten hairy root clones were established; their growth and rutin production rates ranged from 233 to 312 (mg dry wt per 30 mL flask, and 0.8 to 1.2 (mg/g dry wt), respectively. Clone H8, which had high growth and rutin production rates (312 mg dry wt per 30 mL flask and 1.2 mg/g dry wt, respectively), was selected for further experiments. H8 showed maximal growth and rutin content at 30 days in culture in MS medium. Of four tested culture media, half-strength MS medium was found to induce the highest levels of growth (378 mg dry wt per 30 mL flask) and rutin production (1.4 mg/g dry wt) by clone H8. In contrast, supplementation with auxins (0.1-1 mg/l IAA, IBA and NAA) increased the growth rate, but had no significant effect on rutin production by H8. Collectively, these findings indicate that hairy root cultures of buckwheat culture could be a valuable alternative approach for rutin production.     

产紫杉醇内生真菌Z58的分离和鉴定

本研究从曼地亚红豆杉（Taxus x media）树皮内表皮分离得到一株产紫杉醇的内生真菌Z58,通过高效液相色谱法、质谱法和核磁共振波谱法对其紫杉醇提取物进行了分析. 结果表明,内生真菌Z58的紫杉醇提取物具有和紫杉醇标准品相近的色谱特征峰,其保留时间为10.2 min;也与紫杉醇标准品具有相同的质谱特征峰（（M+Na）+=876）和1H-NMR谱带.并通过形态学特征分析和18S rDNA序列分析,将内生真菌Z58初步鉴定为肉座菌属（Hypocrea sp.）真菌.肉座菌Z58的紫杉醇产量约为2.5～3.0 μg/g（紫杉醇/菌丝干重）,是一株具有潜在应用价值的产紫杉醇内生真菌.