Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and I
《蜂胶》国际标准的英文
《蜂胶》国际标准的英文International Standard for PropolisPropolis is a natural substance that has been used for centuries due to its wide range of beneficial properties. It is derived from the resinous substance collected by bees from various plants, mixed with beeswax, enzymes, and pollen. Propolis has been widely recognized for its antimicrobial, antioxidant, and anti-inflammatory effects, making it a valuable ingredient in food, cosmetics, and medicinal products. As the global demand for propolis grows, the establishment of an international standard for propolis becomes crucialto ensure its quality and safety. In this article, we will explore the importance of an international standard for propolis and its key components.I. Definition and ScopeThe international standard for propolis aims to provide a concise and comprehensive definition of propolis and its key components. It covers the types of propolis commonly found worldwide, including the resinous content, beeswax ratio, and pollen composition. The standard outlines the acceptable quality parameters for propolis, such as its color, odor, and purity. Additionally, it defines the specific tests and analytical methods to determine the propolis content accurately.II. Chemical Composition AnalysisTo establish an international standard for propolis, a thorough analysis of its chemical composition is essential. The standard should include detailed instructions on the identification and quantification of the major classes of compounds found in propolis, such as polyphenols, flavonoids, andterpenoids. Gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) are some of the advanced analytical techniques used to determine the chemical profile of propolis accurately.III. Quality Control and Safety AssessmentEnsuring the quality and safety of propolis is of utmost importance to protect consumers and promote international trade. The international standard should incorporate guidelines for quality control, including the establishment of maximum limits for contaminants, such as heavy metals, pesticides, and microbial impurities. Moreover, the standard should outline the safety assessment procedures, including acute and chronic toxicity tests, to determine the harmful effects of propolis consumption.IV. Labeling and Packaging RequirementsIn order to facilitate international trade and enhance consumer awareness, the international standard for propolis should also cover labeling and packaging requirements. This includes clear and accurate labeling of the propolis content, origin, and batch number. Additionally, the standard should specify the recommended storage conditions and shelf life to maintain the quality and efficacy of propolis products.V. International Harmonization and RecognitionTo ensure global acceptance and harmonization of the international standard for propolis, collaborations and mutual recognition agreements are essential. International organizations and regulatory bodies should work together to establish a unified standard that can be adopted by differentcountries. This will streamline the import-export procedures and facilitate the trade of propolis products while maintaining consistent quality and safety standards worldwide.ConclusionThe establishment of an international standard for propolis will play a crucial role in promoting the quality, safety, and trade of propolis products globally. With a comprehensive definition, accurate chemical composition analysis, robust quality control measures, and harmonization efforts, the international standard will serve as a benchmark for propolis producers, manufacturers, and consumers. By adhering to the standard, the industry can ensure the availability of high-quality propolis products that deliver the expected therapeutic benefits while building trust among consumers worldwide.。
深海真菌DiaporthephaseolorumFS431次级代谢产物的分离鉴定
郭珩１ꎬ３ ꎬ刘洪新３ ꎬ陈玉婵３ ꎬ李赛妮３ ꎬ李浩华３ ꎬ陈闪冲２ꎬ３ ꎬ章卫民３ ꎬ高晓霞１
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中草药微量活性物质识别与获取关键技术与应用
项目名称：中草药微量活性物质识别与获取关键技术与应用申报奖种：科学技术进步奖完成单位：中国医学科学院药物研究所,北京科莱博医药开发有限责任公司完成人：庾石山,石建功,张东明,于德泉,陈晓光,张建军,王珂,申竹芳,马双刚, 屈晶,林生,徐嵩,李勇,吕海宁,李创军项目简介: 寻找中草药有效成分一直是现代中草药研究的核心。
我们发现，某些中药的个别微量成分显示出显著的药理活性与独特的化学结构。
然而，这些微量成分是否是有效成分、能否代表该中药的特定功效呢？为此，我们在国家首批973计划项目、国家“重大新药创制”重大专项、国家自然科学基金重点项目等5项课题支持下，历经13年，围绕中药微量药效成分的寻找、获取、评价、确定等关键问题开展研究，取得了以下研究成果：1. 建立了中草药微量活性物质识别、评价与获取的系列关键技术建立了中草药大规模样品快速制备技术，组合闪式色谱快速剔除杂质和常量成分、富集微量成分技术，微量活性物质识别、锁定与获取技术，实现了中草药微量活性成分的高效识别、快速锁定和定向获取，为揭示中草药微量活性物质在中药（复方）中的关键药效作用，以及进一步全面、系统地认识中草药药效物质提供了强有力的技术支撑。
2.获得了50余种中草药的微量成分及其组分，包括400余个微量化合物，其中活性化合物100余个，活性组分5个。
有6个微量活性化合物被国际权威期刊Nat.Prod.Rep.(IF:10.1)遴选为热点报道，其入选代表本领域的国际领先水平。
3.发现了10种中草药的20余种（类）微量关键药效物质。
如天麻，过去一直认为其镇静催眠有效成分为天麻素（含量千分之二），但我们发现，其关键药效物质为微量成分天麻腺苷，含量虽仅为百万分之四，但药效是天麻素的1000倍；单刀根散瘀消肿的药效物质不清楚。
我们从中获得的微量间苯三酚类新结构化合物Lysidisides A和B显示了强的血管内皮保护作用，并呈现良好的量效关系，为创制新型的治疗心脑血管疾病药物提供新的源头分子。
胡萝卜素的分离和鉴定实验流程
胡萝卜素的分离和鉴定实验流程英文回答：Separation and Identification of Carotenoids: Experimental Procedure.Carotenoids are a group of pigments found in plants, algae, and some bacteria. They are responsible for the yellow, orange, and red colors of many fruits and vegetables. Carotenoids are important for human health because they are precursors to vitamin A and have antioxidant properties.The separation and identification of carotenoids is a complex process that requires the use of specialized equipment and techniques. The following is a general overview of the experimental procedure:1. Extraction: Carotenoids are typically extracted from plant material using a solvent such as acetone or methanol.The extraction process can be carried out using a varietyof methods, including maceration, soxhlet extraction, and ultrasound-assisted extraction.2. Filtration: The extract is filtered to remove any particulate matter.3. Chromatography: The extract is then subjected to chromatography to separate the different carotenoids. Chromatography is a technique that separates compoundsbased on their different physical and chemical properties. The most common type of chromatography used for carotenoid separation is high-performance liquid chromatography (HPLC).4. Spectral analysis: The separated carotenoids arethen identified using spectral analysis. Spectral analysis involves measuring the absorption or emission of light bythe carotenoids. The most common type of spectral analysis used for carotenoid identification is ultraviolet-visible (UV-Vis) spectrophotometry.5. Confirmation: The identity of the carotenoids can beconfirmed using additional techniques, such as mass spectrometry or nuclear magnetic resonance (NMR) spectroscopy.中文回答：类胡萝卜素的分离和鉴定实验流程。
4种沉香树叶片挥发油化学成分GC-MS分析
热带作物学报2022, 43(1): 196 206 Chinese Journal of Tropical Crops收稿日期 2021-07-06；修回日期 2021-08-19基金项目 2019年海南省重点研发计划科技合作方向项目（No. ZDYF2019203）；2020年中央财政林业技术科技推广示范资金项目（琼[2020]TG05）。
作者简介 刘欣怡（1997—），女，硕士研究生，研究方向：珍贵树种研究。
*通信作者（Corresponding author ）：戴好富（DAIHaofu ），E-mail ：*****************.cn ；王 军（WANG Jun ），E-mail ：****************.cn 。
4种沉香树叶片挥发油化学成分GC-MS 分析刘欣怡1，王雅丽2,3，王 昊2,3，王露露2，梅文莉2,3，戴好富2,3*，王 军2,3*1. 海南大学林学院，海南海口 570228；2. 中国热带农业科学院热带生物技术研究所，海南海口 571101；3. 海南沉香工程技术研究中心/海南省黎药资源天然产物研究与利用重点实验室，海南海口 571101摘 要：比较3个白木香良种‘热科1号’（Aquilaria sinensis ‘Reke 1’）、‘热科2号’（A. sinensis ‘Reke 2’）、‘热科3号’（A. sinensis ‘Reke 3’）及国外引种的印度沉香（A. agallocha Roxb.）植物叶片挥发油成分的异同，旨在为其进一步开发利用提供依据。
采用水蒸气蒸馏法提取4种叶片中的挥发油，用气相色谱-质谱联用（GC-MS ）技术对其成分进行分析获得总离子流图，通过Date Analysis 化学工作站，结合Nist2005和Wiley275质谱库及前人鉴定方法等对检测到的色谱峰进行检索及人工解析；利用峰面积归一化法测定其化学成分的相对百分含量，统计数据后进行对比分析。
RAPD法结合TLC鉴别中药牛蒡子及其混淆品
阶段,一定部位的突起呈新月形,并且由于进一步的顶端生长和边缘生长,结果最后包住茎尖(图3-6)。
在苗端的下面可见纵行排列的初生分生组织,初生分生组织一般由长方形细胞组成,2～3行纵向排列,染色很深;初生分生组织以向顶的形式进行分化(图3-6)。
叶基具鞘的植物顶端分生组织最初在局部地方有细胞分裂,再想侧面扩张,结果形成了半月形的原基,这种原基不呈背腹对称,而是在开始发生的地方最厚,两侧逐渐变细。
原基开始垂直地生长,细胞分裂也进一步围绕着顶端扩张,因而促使向上形成一圈组织包围茎端,它比起原来的生长地点稍短。
这种包围生长的一个结果是顶端叶原基的中央纵切面也经过顶端相对一侧的叶鞘[3](图3-5)。
叶鞘的发育不同于叶片,它的生长主要是通过边缘分生组织的活动及它所衍生出来的细胞的长大。
叶片的分化比叶鞘早,因此,叶的分生组织的活动越来越受到叶鞘基部的限制[2]。
3 讨论当植物生长到生殖阶段时,营养顶端会全部或部分变成生殖顶端。
当生殖阶段开始发生时,最容易看到的是体轴的突然迅速生长,伸长的轴上会产生单个花或一花序[1]。
湖北麦冬在生殖阶段会长出花葶,花葶上着生由许多小穗状花序组成复穗状总状花序。
开花后期,湖北麦冬的小穗状花序顶端上会长出叶芽,这些叶芽与花簇生于苞片叶腋内。
在自然界中,有一些植物亦有类似湖北麦冬植株上长出小植株的现象,但其发生部位不同。
如叶状沼兰Malax is p aludosa的顶部,经过细胞分裂活动,可以产生许多胚状结构,这种结构叫胚状体,胚状体可萌发,形成小植株[4]。
甘薯、蒲公英、榆、刺槐等在根上发芽[5],这些根芽常常像侧根那样内生的起源(侧根原基一般在中柱鞘形成,有些植物的内皮层也参加了它们的形成)[3],这种根芽是由于侧根偶然接近土壤表面,受光的影响控制,发育成芽[6]。
水稻、小麦在结果时,如遇上适宜的条件,胚直接萌发长出小植株。
根据解剖学观察结果可知,湖北麦冬开花后长出小芽并非由表层薄壁细胞组织恢复分生能力发育而来,而是由于营养顶端部分分化成生殖顶端。
制马钱子的功效与作用
制马钱子的功效与作用制马钱子的功效与作用马钱子，又称大戟、大马钱子，是一种多年生草本植物，属于大戟科马钱子属。
马钱子的药用价值在我国早有记载，并广泛应用于中医药领域。
在此篇文章中，我们将详细介绍马钱子的功效与作用，既包括其药用方面的作用，也包括其其他应用领域的作用。
一、马钱子的药用功效1. 治疗风湿病：马钱子具有散寒除湿、行气活血的作用，可用于治疗风湿病引起的关节疼痛、肢体酸痛等症状。
马钱子内含有丰富的生物碱和黄酮类化合物，这些活性物质可以促进血液循环，减轻炎症反应，从而起到缓解风湿病的作用。
2. 治疗湿疹和皮肤病：马钱子具有清热解毒、消肿止痒的特点，可用于治疗湿疹、皮肤病等症状。
马钱子中的黄酮类化合物具有抗炎、抗过敏的作用，可以减少过敏原对皮肤的刺激，提高皮肤的抵抗力，从而减轻或消除湿疹和皮肤病的症状。
3. 抗肿瘤作用：近年来的研究表明，马钱子中的一些活性成分具有抗肿瘤的作用。
其中，所含的倍半萜类化合物可以干扰肿瘤细胞的生理活动，抑制肿瘤细胞的生长和扩散。
此外，马钱子中的黄酮类化合物还可以调节免疫系统，增强机体的抵抗力，抑制肿瘤的发展。
4. 清热解毒：马钱子具有清热解毒的功效，可以用于治疗热病、急性感冒、喉咙肿痛等症状。
马钱子中的生物碱类物质可以中和体内的自由基，减轻炎症反应，降低体温，从而起到清热解毒的作用。
5. 改善消化功能：马钱子含有大量的黄酮类成分，可以促进胃肠蠕动，增加胃液的分泌，改善消化功能。
马钱子还可以刺激胃黏膜的分泌，增加食欲，缓解消化不良、胃胀等不适症状。
二、马钱子的其他应用领域1. 植物保护剂：由于马钱子具有一定的杀菌、杀虫作用，可用于制作植物保护剂。
将马钱子的叶子、茎、根煮沸后，过滤提取液，制成植物保护剂，可用于预防和治疗农作物常见的病虫害，如炭疽病、白粉病等。
2. 纺织染料：马钱子的根茎可用于提取纺织染料，这种染料色泽鲜艳、牢度高，可用于染色棉、麻、丝等天然纤维和合成纤维。
中国的枝顶孢属_英文_王有智
菌物系统21（2）：192~195, 2002MycosystemaSURVEY OF ACREMONIUM SPECIES FROM CHINA WITHTHREE NEW RECORDS∗WANG You-Zhi GUO Fang ZHOU Yu-Guang(Institute of Microbiology, Chinese Academy of Sciences)ABSTRACT: Thirteen species of Acremonium from China are investigated, including three new records,A. alternatum, A. egyptiacum and A. potronii. A key is also provided in the paper.KEY WORDS: Hyphomycetes, Acremonium,new record speciesCephalosporium-like species are valuable antibiotic-producing fungi. Cepahlosporin has earned a good reputation in biosynthetic antibiotics. Recently, a novel antibiotic produced by an Acremonium species was reported (Nakai, 2000).In the monograph of Cephalosporium-like Hyphomycetes (Gams, 1971), the older generic name Acremonium Link replaced Cephalosporium Corda and A. alternatum Link per S.F. Gray was selected as the lectotype of the genus. Under the generic level, Section Acremonium (= Sect. Simplex), Section Gliomastix, Section Nectrioidea, Section Albo-lanosa, Section Chaetomioides and Section Lichenoidea were erected (Gams, 1971, 1975; Morgan-Jones & Gams, 1982; Lowen, 1995). The diagnostic feature of Acremonium is 1-celled (rarely 2-celled) phialospores in chains or heads producing on thin-walled orthophialides or on basitonously branched conidiophores (Gams, 1971). There are about 130 worldwide species accepted in Acremonium. Fourty of them were described after 1971 when the monograph of Acremonium has been carried out (Gams, 1971). Molecular phylogenetic study shows that Acremonium is a polyphyletic taxon with affiliations to at least three ascomycetous families: Hypocreaceae, Claviciptaceae and Chaetomiaceae (Glenn et al., 1996). Acremonium species have a worldwide distribution and occur as saprobic, plant parasitic or lichenized.Surveying on the Chinese species of Acremonium, seven species of Cephalosporium were recorded in Sylloge Fungurum Sinicum (Tai, 1979). However, according to Gams (1971), four of them were transferred to Acremonium and accepted as A. persicinum (Nicot) W. Gams, A. roseo-griseum (S.B. Saksena) W. Gams, A. rutilum W. Gams and A. strictum W. Gams. Species of A. kiliense Grütz and A. strictum were found in Hong Kong (Lu et al., 2000). In Taiwan three correct species of Acremonium were reported. They are A. charticola (J. Lindau) W. Gams, A. fusigerum (Berk. & Broome) W. Gams and A. zonatum (Sawada) W. Gams (Wang et al., 1999). Most recently, A. terricola (Miller et al.) W. Gams and A. tubakii W. Gams were reported in Anhui and Guangdong (Lu & Lu, 1999; Fan et al., 1999).∗ Supported by The Research Fund for Returned Overseas Chinese Scholars of CAS (no.20010712112454) and The Director Fellowship of IMCAS(2001)Received:2001-07-25, accepted: 2001-11-082期 王有智等: 中国的枝顶孢属 193When rechecking the mitosporic fungi cultures in CGMCC (China General Microbiological Culture Collection Centre), we found three species new to China,Acremonium alternatum Link, Mag. Ges. naturf. Fr. Berlin 3: 15, 1809; Gams,Cephalosporium-artige Schimmelpilze (Hyphomycetes) 62, 1971, Fig. 1.Colonies reaching 14 mm diam after 10 days at 26ºC on 2% malt agar, whitish, powdery, reverse brownish. Sporulation phalacrogenous to plectonematogenous. Vegetative hyphae thin-walled, 0.5~1.5 µm wide. Phialides simple, arising from aerial hyphae, awl-shaped, 15~25 µm long, tapering imperceptible from1.5~3.0 µm to 0.5~1 µm wide. Conidia in short chains at the beginning and later in heads, ellipsoidal, long obovate, with faintly apiculate base and round upper end, hyaline, smooth-walled, 4.2~5.0 × 1.5~2.0 µm. Chlamydospores absent.Material examined: China, CGMCC, 1 November 1969, Chen Qin-Tao (AS 3.3759, as Spicaria presina ).Acremonium egyptiacum (van Beyma) W. Gams, Cephalosporium -artige Schimmelpilze(Hyphomycetes), 64, 1971. Fig. 2.≡ Oospora egyptiaca van Beyma, Zentbl.Bakt. ParasitKde(Abt. II) 89: 242,1933.Colonies reaching 18 mm diam. after10 days at 26ºC on 2% malt agar. Aerialmycelium white, later cream, powdery,reverse luteous. Sporulation phalacro-genous to plectonematogenous. Vegetativehyphae thin-walled, 1.0~1.8 µm wide.Phialides simple, 25~40 µm long,gradually tapering from 1.5~2.0 µm to0.5~1 µm wide. Conidia cohering in longchains or forming loose heads, ellipsoidal,hyaline, smooth-walled, 4.4~5.2 × 1.4~2.0µm. Chlamydospores absent.Material examined: China, CGMCC,1 November 1969, Chen Qin-Tao (AS3.3796, as Cephalosporium sp.).Acremonium potronii Vuill., Soc. Sci. Nancy, 19., 1910 and Encyclop. Mycol. 2: 66, 1931; Gams,Cephalosporium- artige Schimmelpilze (Hyphomycetes), 59, 1971. Fig.3.≡ Cephalosporium potronii(Vuill.) Oomen in CBS list of Cultures, 1957 (invalid publication).194 菌物系统21卷Colonies reaching 6 mm diam. after 10 days 26ºC on 2% malt agar, powdery, white to pinkish, reverse yellow. Sporulation phalacrogenous. Vegetative hyphae 1.0~2.0 µm wide. Phialides simple, 10~25 µm long, tapering from 1.0~2.0 µm to 0.5~1.0 µm. Conidia in slimy heads, obovate, with an apiculate base, hyaline, smooth-walled, 2.5~4.0 × 1.4~2.5 µm. Chlamydospores absent.Material examined: China, CGMCC, 1 November 1969, Chen Qin-Tao (AS 4.4008, as Cephalosporium acremonium).KEY TO THE THIRTEEN SPECIES OF ACREMONIUM FROM CHINA1. Vegetative hyphae with refrative wall thickening, conidia incrusted dark or hyaline (2)1. V egetative hyphae lackinging pronounced wall thicking, conidia always hyaline (4)2. Conidia spindle-shaped with truncate ends, very dark, 17.5~19.0×7.0~8.5µm………………………………………………………………………...A.f u s i g e r u m2. Conidia subglobose, obovate, hyaline or pigmented (3)3. Conidia hyaline, 3.2~4.8 ×1.2~3.0 µm .………………………………A.persicinum3. Conidia pigmented,4.8~6.4 ×2.6~4.0 µm .………………………..A. roseo-griseum4. Conidiophores often repeatedly branched (5)4. Conidiophores usually consist of slender, unbranched phialides (6)5. Colonies intensely pink, conidiophores slightly branched, Conidia 3.0~6.0× 2.0~3.4µm…………………………………………………………………………….A.r u t i l u m5. Colonies whitish to pale pink, conidiophores several times branched, Conidia 5.0~6.8× 2.0~2.8µm ……………………………………………………….A.z o n a t u m6. Conidia at least partly in dry chains (7)6.Conidia always in heads (9)7. Conidia with sharply pointed ends, 3.8~5.4×1~1.5 µm ………………...A. terricola7. Conidia with rounded upper ends (8)8. Conidia sometimes in heads, 4.2~5.0 × 1.5~2.0µm …………………..A. alternatum8. Conidia always in chians, 4.4~5.2 × 1.4~2.0 µm ……………………..A. egyptiacum9. Conidia obovate, 2.5~4.0 × 1.4~2.5µm ………………………………….A. potronii9. Conidia ellipsoidal or cylindrical, length : width>2 (10)10. Conidia ellipsoidal, 3.2~4.4 ×1.4~2.0 µm ……………………………A. charticola10. Conidia typically cylindrical (11)11. Chlamydospores absent, conidia 3.2~5.4 ×1.0~1.8µm ………………...A. strictum11. Chlamydospores present (12)12. Chlamydospores usually more numerous than phialospores, conidia 3.5~5.7 × 1.5~2.3 µm……………………………………………………………………………..A.t u b a k i i12. Chlamydospores less numerous than phialospores, conidia 3.1~5.8 ×1.0~1.6µm…………………………………………………………………………...A.k i l i e n s e Notes: Acremonium chrysogenum (Thirum. & Sukap.) W. Gams has been used for producing2期王有智等: 中国的枝顶孢属195 Cephalosporin C, N in China for years, but the isolates are from abroad. No report on isolation of A. chrysogenum in China was traced. Therefore, this species is not included here.[REFERENCES]Fan M-Z, Huang B, Li C-R, Li Z-Z, 1999. A new record species of the genus A cremonium. Mycosystema18 (4): 449 (in Chinese). Gams W, 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes). Stuttgart: Gustav Fischer Verlag. 1~262.Gams W, 1975. Cephalosporium-like Hyphomycetes: some tropical species. Transactions of the British Mycological Society64 (3): 389~404.Glenn A E, Bacon C W, Price R, Hanlin R T, 1996. Molecular phylogeny of Acremonium and its taxonomic implications.Mycologia 88 (3): 369~383.Lowen R, 1995. Acremonium Section Lichenoidea Section nov. and Pronectria oligospora species nov. Mycotaxon 53: 81~95. Lu B-S, Hyde K D, Ho W-H et al., 2000. Checklist of Hong Kong Fungi. Hong Kong: Fungal Diversity Press, 1~207.Lu Y-J, Lu D-J, 1999. Rhizospheric fungal diversity of vegetables in suburbs of Guangzhou. Ecological Science18 (1): 23~25 (in Chinese).Morgan-Jones G, Gams W, 1982. Notes on Hyphomycetes. XLI. An endophyte of Festuca arundinacea and the anamorph of Epichloë typhina, new taxa in one of two sections of Acremonium. Mycotaxon15: 311~318.Nakai R, Ogawa H, Asai A, et al., 2000. UCS1025A, a novel antibiotic produced by A cremonium sp. The Journal of Antibiotic53 (1): 294.Tai F-L, 1979. Sylloge Fungurum Sinicum. Beijing: Science Press. 1~1572 (in Chinese).Wang Y-C, Wu S-H, Chou W-N et al., 1999. List of the fungi in Taiwan. Taiwan, 1~289.中国的枝顶孢属王有智郭芳周宇光（中国科学院微生物研究所）摘要：本文对中国的枝顶孢属丝孢菌进行了调查，其中包括3个新记录种，并给出了全部13个种的检索表。
affinity chromatography
ReviewStructural analysis and classification of native proteins from E.coli commonly co-purified by immobilised metal affinity chromatographyVictor Martin Bolanos-Garcia ⁎,Owen Richard DaviesDepartment of Biochemistry,University of Cambridge,80Tennis Court Road,Cambridge,CB21GA,EnglandReceived 25January 2006;received in revised form 23March 2006;accepted 24March 2006Available online 26April 2006AbstractImmobilised metal affinity chromatography (IMAC)is the most widely used technique for single-step purification of recombinant proteins.However,despite its use in the purification of heterologue proteins in the eubacteria Escherichia coli for decades,the presence of native E.coli proteins that exhibit a high affinity for divalent cations such as nickel,cobalt or copper has remained problematic.This is of particular relevance when recombinant molecules are not expressed at high levels or when their overexpression induces that of native bacterial proteins due to pleiotropism and/or in response to stress conditions.Identification of such contaminating proteins is clearly relevant to those involved in the purification of histidine-tagged proteins either at small/medium scale or in high-throughput processes.The work presented here reviews the native proteins from E.coli most commonly co-purified by IMAC,including Fur,Crp,ArgE,SlyD,GlmS,GlgA,ODO1,ODO2,YadF and Yf bG.The binding of these proteins to metal-chelating resins can mostly be explained by their native metal-binding functions or their possession of surface clusters of histidine residues.However,some proteins fall outside these categories,implying that a further class of interactions may account for their ability to co-purify with histidine-tagged proteins.We propose a classification of these E.coli native proteins based on their physicochemical,structural and functional properties.©2006Elsevier B.V .All rights reserved.Keywords:Affinity chromatography;E.coli contaminant protein;Metal-binding classification;Histidine-tagged protein;Protein purification1.IntroductionThe use of immobilised metal affinity chromatography (IMAC)has revolutionised protein biochemistry by allowing the production of a pure protein sample through a single puri-fication step.However,the concomitant expression of native bacterial proteins that exhibit a relatively high affinity for di-valent cations during the expression of heterologue protein domains,full-length proteins or macromolecular complexes in E.coli frequently results in their co-purification during IMAC [1].Most of these metal binding proteins are present in E.coli strains of different genetic backgrounds,such as BL21,BL21(DE3),BL21(DE3)pLysS,C41,C43,Rosetta (DE3)and (DE3)pLysS as well as Origami (DE3)and (DE3)pLysS.These strains contain a lamba-lysogen DE3bacteriophage that encodes T7RNA polymerase under the control of the lac UV5operator;theexpression of T7promoter and lac UV5operator controlled genes on pET-based vectors is thus permitted upon induction with isopropyl-2-D -thio-galactopyranoside (IPTG)[2,3].Since the 1970s,IMAC has remained the most important technique for single-step protein purification [4].The expression of a recombinant protein containing a histidine-tag (usually six consecutive histidine residues)allows it to be specifically bound by chelated divalent metal ions,and then eluted through com-petition by the addition of imidazole,or through the protonation of histidine residues by a reduction in pH.This often has dramatic results in the purification of target proteins to near homogeneity from bacterial cell lysate [5,6].Further advantages of IMAC include ligand stability,high protein loading capacity,mild or denaturing elution conditions,column regeneration,low cost and scalability [7,8].This has meant that it is now in widespread use in both low and high throughput environments [9,10].There are many different metal-chelator systems for IMAC,although the most common are the tridentate ligand IDA,Ni 2+bound to tetradentate ligand NTA (Ni-NTA;Qiagen Ltd.)and Co2+Biochimica et Biophysica Acta 1760(2006)1304–1313⁎Corresponding author.Tel.:+441223766029;fax:+441223766002.E-mail address:victor@ (V .M.Bolanos-Garcia).0304-4165/$-see front matter ©2006Elsevier B.V .All rights reserved.doi:10.1016/j.bbagen.2006.03.027bound to tetradentate ligand CM-Asp (TALON ™;BD Biosciences Clontech).The interaction between Ni-NTA resin and a histidine-tagged protein is illustrated in Fig.1.NTA and TALON ™have higher affinities for metal ions than IDA,but they exhibit lower protein binding due to the loss of one coordination site.Depending on the proximity,orientation and spatial accessibility of histidine residues as well as the density of the chelating groups and metal ions,multipoint binding of different histidine residues can be ually,one histidine is enough for weak binding to IDA-Cu 2+,while more proximal histidine residues are needed for efficient binding to Zn 2+and Co 2+[8].The reported capacities of these commercial sorbents are usually in the range of 5–10mg/ml or even higher.These values commonly refer to isolated pure proteins or synthetic mixtures,so the capacities for isolation of recombinant proteins from complex sources are often lower.In successful cases,over 80%of histidine-tagged proteins can be recovered from E.coli homogenates [11].However,the use of IMAC for some recombinant proteins may be limited by their low binding affinity to metal-chelating sorbents despite optimisation and/or the use of fresh resin.This situation is often due to the histidine-tag being partially hidden from the protein surface,which can in turn be due to inter or intra-molecular interactions.Although it is occasionally possible to fully expose the histidine tag by adding detergents,glycerol,polyethylene glycol of low molecular weight and/or chaotropic agents at low concentration,in many cases,a relatively low affinity for the sorbent persists in the presence of these and other additives.The length and position of the histidine tag can also affect other fundamental properties of a recombinant protein such as its expression level,stability,oligomerisation state,and ability to constitute suitable samples (for example,protein sam-ples that allow the formation of single crystals for X-ray crystal-lography)[7].In addition to histidine-tagged recombinant proteins,some native proteins also show affinity for metal chelating resins com-monly used in IMAC.Binding of native proteins is determined by many factors,including the accessibility of surface histidine residues to the metal ions present in chelating resins,the micro-environment of binding residues,cooperation between neighbour amino acid side groups,and local conformations.Interestingly,the intrinsic metal-binding properties of several non-histidine tagged proteins have been exploited for simple single-step puri-fication by Ni-NTA-sepharose.Examples are untagged HIV-1integrase expressed in E.coli [12],the alpha subunit of the human transcription factor A (TFIIA)[13]and a proteomic-wide analysis of copper-binding proteins in plants [14].The co-purification of contaminant proteins that bind to metal-chelating sorbents is particularly problematic when one or more E.coli native proteins are expressed at high levels and/or when they exhibit a size similar to that of the recombinant protein [15].This situation might ultimately result in the purification of an undesired bacterial protein rather than the recombinant protein of interest.One example of medical relevance is the purification of TNF-α,which allowed efficient host-cell endotoxin and DNA removal but resulted in the simultaneous recovery of some re-sidual E.coli proteins during the final elution step [8].Several innovations have been described in the literature to improve the yield and purity of recombinant histidine-tagged proteins purified by single-step chromatography,such as the use of isopropanol during washing steps for the removal of contaminants,including bacterial endotoxins [16].However,this treatment may result in protein unfolding,thus making the purification of properly folded recombinant proteins a difficult task.Additionally,there are se-veral other aspects of IMAC that must be further improved,in-cluding low dynamic capacity and efficiency of cleaning pro-cedures for eliminating contaminants.The co-purification of contaminant proteins exhibiting affinity for divalent cations has been observed not only with single-tagged proteins but also with double-tagged molecules,including the combination of histidine and thioredoxin (TRX),Nut,glutathi-one-S-transferase (GST),maltose binding protein (MBP)and GroEL tags.Although the use of double tags seems to be adequate in removing most of the native bacterial contaminants,this strat-egy is not exempt of other problems such as excessive sample manipulation and the occasional partial proteolysis and/or aggre-gation and/or insolubility of the recombinant protein after tag cleavage and removal.Moreover,the use of double tags is gen-erally undesirable in high-throughput processes.Although it is well known that certain E.coli native proteins are problematic contaminants during IMAC [1,15],to ourknowledge,Fig.1.The mechanism of binding between a histidine-tag and Ni-NTA resin (Qiagen Ltd.).Nickel ions are immobilised on a nitrilotriacetic acid (NTA)sepharose resin through co-ordination sites with three oxygen atoms and one nitrogen atom.This leaves two co-ordination sites,which may be taken up by nitrogen atoms of two adjacent histidine residues of a histidine-tagged recombinant protein,thus offering a mechanism for affinity chromatography.The interaction between the nickel ion and the histidine-tagged protein may be disrupted and protein eluted through competition by the addition of imidazole or through the protonation of histidine residues by lowering the pH.1305V .M.Bolanos-Garcia,O.R.Davies /Biochimica et Biophysica Acta 1760(2006)1304–1313a formal account of the specific proteins and their mechanisms of binding to metal chelating resins has not yet been reported.In recognition of this,we reviewed the physicochemical,functional and structural properties of native proteins from E.coli that in our experience are the most commonly co-purified by IMAC and proposed a classification based on their relative affinity for metal chelating resins.Our analysis shows that most of the contaminants are stress-response proteins that tightly bind to metal chelating sorbents through surface clusters of histidine residues or other metal-binding residues that are physiologically important.We conclude that the identification of contaminant proteins aids the design of a purification strategy for recombinant proteins expressed in intact E.coli cells as well as in E.coli lysates used in cell-free translation systems.1.1.The majority of E.coli contaminants are stress-responsive proteinsThe response of E.coli to stress conditions such as nutrient starvation,heat shock and oxidative damage results in a trans-criptional shutdown of protein synthesis and in the induction of genes encoding diverse stress proteins.According to our expe-rience,which is based on the purification of more than80different histidine-tagged proteins,discussions in open forums such as CCP4()as well as with colleagues of this and other research centres,we conclude that these stress responsive proteins are the main native proteins from E.coli that co-purify with recombinant proteins during IMAC under conventional purification conditions.Very importantly,our experience shows that the relative level of expression of a particular contaminant protein from E.coli is quite variable and appears to depend upon numerous factors,including culture conditions,media composi-tion and the genetic background of the expression strain.From our experimental observations,these stress responsive proteins have been classified into three groups on the basis of the concentration of imidazole that is required for their elution from IMAC columns,thus providing an indication of the strength of binding to metal-chelating resins.Class I proteins require≥80mM imidazole for elution(Fur,Crp,SlyD,ArgE,Cu/Zn-SODM and YodA),Class II proteins require55to80mM of imidazole(GlmS, ODO2,YadF,CA T,GlgA,Yf bG and G6-PD)and Class III to proteins that bind weakly,requiring only30to50mM(Hsp60and ODO1).A constant volume of fresh Ni-NTA sorbent,identical chro-matography protocols and buffer solutions of similar composition were used to assess meaningful comparisons.All proteins described in this work were identified by mass spectrometry(MALDI-TOF) and N-terminal sequence analysis according to Edman's degrada-tion at the Protein and Nucleic Acid Chemistry(PNAC)facility (Department of Biochemistry,University of Cambridge,UK).Full details of the identified proteins,their metal-chelating binding strengths and physicochemical properties are given in Tables1and2.Upon analysis of these contaminant proteins,two distinct mechanisms of binding to metal-chelating resins can be recognised:(1)the possession of native metal-binding sites that can bind to Ni2+or Co2+metal ions and(2)the presence of surface clusters of histidine residues that bind to the chelated metal in the same way as the tandem residues of a histidine-tag.As discussedbelow,different binding mechanism(s)may account for the resinbinding capacity of the other contaminants that do not satisfythese two conditions.Our analysis also shows that native E.coli proteins that are co-purified by IMAC exhibit a wide diversity of folds,oligomerisationstates and hierarchical organisation.A high percentage of thesecontaminant proteins correspond to those with more than onedomain.In terms of frequency,most of these proteins belong to the α+βclass,closely followed by the all-βclass.Interestingly,only a marginal number of contaminant proteins correspond to the all-αclass.With the possible exception of ODO2and Hfq,there seems tobe no correlation between the in vitro protein oligomerisation stateon the relative affinity for metal chelating sorbents.1.2.Native metal-binding proteinsThe presence of physiologically important metal-bindingsites is the most significant mechanism by which native E.coliproteins co-purify with the heterologue protein during IMAC.Common contaminants that bind through metal-binding sitesinclude Fur,YodA,Cu/Zn-SODM and ArgE(Class I),YadF andGlgA(Class II).It is noteworthy that most of these contaminantsbelong to the class I,and so such proteins are most likely to befound as problematic contaminants during IMAC purification ofheterologue histidine-tagged proteins.1.2.1.Ferric uptake regulator(Fur)The DNA-binding protein Fur(ferric uptake regulator)tightlycontrols the quantity of intracellular iron in E.coli through re-pressing the transcription of iron-starvation genes upon binding toFe2+[17].The structure of P.aureaginose Fur is40%helical and18%β-sheet,encompassing an N-terminal DNA-binding domainand C-terminal dimerisation domain[18:pdb entry1MZB].Thisfold classifies the protein in the DNA-binding domain superfam-ily[SCOP,19].In addition to a functionally important regulatoryiron-binding site in the dimerisation domain,Fur also contains astructural zinc-binding site that is crucial for its function in vivocalcium-regulatory functions of Fur meaninvolved in several cellular processes,in-cluding chemotaxis,protection against oxidative damage andacid-shock response[20].Thus,Fur may be overexpressed in E.coli upon the induction of foreign genes through pleiotropism andin response to acid-shock stress.The presence of two physiolog-ical metal-binding sites confers Fur a high affinity for metal-chelating resins and explains why it is commonly co-purifiedduring IMAC.1.2.2.Metal-binding lipocalin(YodA)The E.coli protein YodA plays an important role in thebacterial response to cadmium.Cadmium is readily taken up bybacteria but is highly toxic;its high redox potential allows it toblock the functions of metalloproteins and zinc finger proteins,thus leading to oxidative stress[21].YodAwas the first naturallyoccurring metal-binding lipocalin described;sequence similar-ities with putative proteins from other bacterial species havesuggested a family of proteins that are expressed in response to1306V.M.Bolanos-Garcia,O.R.Davies/Biochimica et Biophysica Acta1760(2006)1304–1313cadmium stress [22].The transcription of yodA is also dependent on fur and soxS ,both of which mediate the protection against reactive oxygen species.These findings and more recent geno-mic studies,where an increase in YodA levels in E.coli after acid-induction in the culture medium from pH 7to 5.8was noticed [23],indicate that YodA is over-expressed in response to oxidative and acid stress.Structural studies have revealed that YodA is composed of two domains:a major calyx domain (lipoclin/calycin-like domain)and a helical domain [22].Crystal structures of YodA bound to both cadmium and zinc (pdb accessions 1OEE and 1OEK,respectively)show a common metal-binding site made up of histidine residues that lie along the side of the calyx domain in a manner that resembles the metal binding sites of several proteases and oxidoreductases.The same authors also reported the structure of YodA crystallised in the absence of metal ions (pdb accession 1OEJ,shown in Fig.2B).Interestingly,they found that nickel ions had bound to the central metal-binding site during purification byNi-NTA,thus highlighting the high affinity of YodA for metal ions,and illustrating the mechanism by which metal-binding proteins can bind to metal-chelating resins.A similar three-histidine motif is found in several metalloproteins,lyases and oxygenases,raising the possibility that E.coli proteins from these classes may also exhibit high affinity for metal-chelating sorbents.Indeed,as shown in this work,some of the common contaminants we have identified belong to these classes.1.2.3.Cu/Zn-superoxide dismutase (Cu/Zn-SODM)Superoxide dismutases (EC 1.15.1.1)are metalloenzymes that protect against oxygen toxicity by catalysing the dismutation of superoxide into molecular oxygen and hydrogen peroxide [24].They are classified into three groups on the basis of their Fe 2+,Mn 2+,Cu 2+/Zn 2+catalytic centres.Unlike other superoxide dismutases,the Cu 2+/Zn 2+-superoxide dismutase (Cu/Zn-SODM)is a monomeric protein of 17kDa [25].The E.coli protein consists of the Greek-key β-barrel topology,formed by eight antiparallel β-strands [pdb entry 1ESO:26].Similar to some of the proteins previously described,Cu/Zn-SODM is expressed under stress conditions and likely binds metal-chelating resins through its Cu 2+and Zn 2+binding sites,which consist of conserved histidine residues (shown in Fig.2C).1.2.4.Acetylornithinase (ArgE)Prokaryotic arginine synthesis usually involves the transfer of an acetyl group to glutamate by ornithine acetyltransferase in order to form ornithine.However,in E.coli acetylornithine deacetylase (acetylornithinase,ArgE)(EC 3.5.1.16)catalyses the deacylation of N 2-acetyl-L -ornithine to yield ornithine and acetate [27].Phylogenetic evidence suggests that the clustering of the arg genes in one continuous sequence pattern arose in an ancestor common to Enterobacteriaceae and Vibrionaceae,where orni-thine acetyltransferase was lost and replaced by a deacylase.Table 1Native proteins from E.coli commonly co-purified during IMAC Protein SwissProt access code Molecular Mass (kDa)%Histidine residues Isoelectric point (pI)Metal requirement Fur P0697516.78.1 5.6Fe 2+,Zn 2+(a)YodAP7634422.3 5.2 5.6Cd 2+,Zn 2+(a)Cu-Zn-SODM AAC7471817.6 4.0 5.9Cu 2+(a),Zn 2+(a)ArgE P2390842.3 4.4 5.5Fe 2+,Ni 2+YadF P3685725.0 5.5 6.1Zn 2+,Hg 2+(a,b)GlgA P0832351.7 3.4 6.0Mg 2+(a,c)GlmS P1716966.8 3.9 5.5CAT AAA5708025.5 5.5 5.9Co 2+Crp P0302023.6 2.98.3Hfq P2552111.1 4.9 6.9SlyD P3085620.810.2 4.8Zn 2+,Ni 2+S15P0237110.2 5.610.4Yf bG P7739874.2 4.1 6.3Hsp60AAC7710357.00.2 4.8ODO1P0701510.5 3.6 6.0ODO2P0701644.0 1.7 5.5G6PDP2299255.71.25.5The table summarises the physicochemical properties of these proteins.aMetal ions reported to be present in the crystallization solution.bAs seen in the structure of its human counterpart (PDB:1CRM).cObserved in its human counterpart (PDB 1PYX).Table 2Relative resin affinity of native proteins from E.coli co-purified during IMAC Relative affinity for the metal chelating sorbent Class I aClass II b Class III c Fur YadF Hsp60YodAGlgA ODO1Cu-Zn-SODM GlmS ArgE CAT Crp YfbG Hfq ODO2SlyD G6-PDS15The table summarises the basis of the protein classification used in this work.The relative affinity is estimated as the millimolar concentration of imidazole required for their elution from a Ni-NTA sepharose column.a =N 80;b =55–80;c =30–50.1307V .M.Bolanos-Garcia,O.R.Davies /Biochimica et Biophysica Acta 1760(2006)1304–1313The383amino acid ArgE protein is made up of two domains: an N-terminal Zn2+-dependent exopeptidase domain and a C-terminal exopeptidase dimerisation domain.It forms a homo-dimer in solution and requires cobalt and glutathione as cofactors. ArgE contains the Co2+/Zn2+binding motifs,and shows a high degree of sequence and structural identity with other metalloen-zymes,explaining its high affinity for metal-chelating resins. 1.2.5.Carbonic dehydratase(YadF)Carbonic anhydrases(carbonic dehydratases)(EC4.2.1.1)are enzymes that catalyse the interconversion of carbon dioxide and bicarbonate,utilising Zn2+as a cofactor.This reaction is crucial to cellular growth;the low atmospheric CO2concentration and its rapid diffusion from cells means that spontaneously produced bicarbonate is insufficient to meet the metabolic requirements of growing cells.YadF from E.coli is essential for growth in the absence of another carbonic anhydrase,CynT[28].Although the transcription of yadf is not regulated by CO2nor subjected to self-regulation,YadF expression is dependent upon bacterial growth rate:its expression is maximal in slow-growing cultures,at high cellular densities and during starvation or heat stress conditions [29].1.2.6.Glycogen synthase(GlgA)The glgA gene encodes glycogen synthase(E.C.2.4.1.21),an enzyme of477amino acid residues in length.This enzyme participates in the biosynthesis of glycogen and contains the UDP-glycosyltransferase/glycogen phosphorylase domain[30].Gly-cogen is accumulated when there is a shortage of nutrients(such as nitrogen)even in the presence of excess carbon[31],so glycogen synthesis is induced when cells enter stationary phase, making E.coli glgA an example of an inducible bacterial gene [32].Even though a large number of bacterial genes are induced during transition into stationary phase,only a minority have been characterised to date.GlgA is also an example of a gene cluster containing the genes that encode for both catabolic andanabolic Fig.2.Three-dimensional structures of proteins that show native metal-binding properties.(A)The crystal structure of Fur with zinc ions bound at sites1and2;in the native state these metal-binding sites bind to iron and zinc,respectively.(B)Cu/Zn SODM contains metal binding sites for both copper and zinc ions,offering two sites for binding to metal chelating resins.Structural analysis and images were produced using Pymol[56].(C)YodA crystallised with nickel ions bound to the metal-binding site;nickel ions had bound to the structure during purification by IMAC,demonstrating the strength of binding to Ni-NTA by native metal-binding proteins. 1308V.M.Bolanos-Garcia,O.R.Davies/Biochimica et Biophysica Acta1760(2006)1304–1313proteins[33],which ensures the tight in vivo regulation of thesemetabolic pathways.Our analysis indicates that YadF and GlgA are the only naturalmetal binding proteins that correspond to Class II;they do nothave strong interactions with metal-chelating sorbents and can beeluted by imidazole concentrations in the range55–80mM.Indeed,GlgA is known to bind Mg+2rather than Ni+2.Therelative affinity of YadF by metal-chelating sorbents is lower thanexpected,taking into account the content of histidine residues(5%).This behaviour can be explained considering that thebiologically relevant metal binding residues and most of thehistidine residues are not surface exposed,as shown in the crystalstructure of its human counterpart(PDB1CRM).1.3.Surface histidine clustersIn considering E.coli proteins that bind to metal-chelatingresins along with histidine-tagged proteins,one of the mostobvious mechanisms of binding is through the possession of anative histidine tag or surface cluster of histidine residues thatcan bind to the resin in the same way as the recombinant protein.This is a relatively common scenario,as judged by the numberof contaminating E.coli proteins that bind to metal chelatingsorbents via surface expose histidine residues,including CRP,SlyD,Hfq and S15(Class I),GlmS and CAT(Class II).1.3.1.Glucosamine-6-phosphate synthase(GlmS)GlmS is an enzyme(E.C.2.6.1.16)that catalyses the formationof D-glucosamine6-phosphate from D-fructose6-phosphate using L-glutamine as the ammonia source.N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cellwall chitin.Thus,GlmS is a potential target for antibacterial andantifungal agents.In fact,potent carbohydrate-based inhibitors ofGlmS have already been reported,including2-amino-2-deoxy-D-glucitol6-phosphate,an analogue of the putative cis-enolamineintermediate that is formed during catalysis[34].GlmS from E.coli is a67-kDa protein,organised in twodomains:the N-terminal glutamine amidohydrolase domain isresponsible for the hydrolysis of L-glutamine,and the C-terminalglucosamine-6-phosphate synthase domain catalyses the ketoseto aldose isomerisation of fructose6-phosphate[34,35].Theisomerase domain comprises two topologically identical sub-domains,each of which is dominated by a nucleotide-bindingmotif of a flavodoxin type.The isomerase catalytic site of GlmSis assembled by the association of two monomers,implying thatthis protein has evolved by gene duplication and subsequentdimerisation[36].The crystal structures of both domains havebeen reported[37]and show the presence of four surface clustersof at least three histidine residues in close proximity,explainingits high affinity for metal-chelating resins.One particularlyinteresting histidine cluster occurs at the dimerisation interface atwhich three histidine residues from each protomer come togetherto form a six residues cluster(Fig.3A).1.3.2.Chloramphenicol-O-acetyl transferase(CAT)This is a25-kDa enzyme that belongs to the superfamily ofCoA-dependent acyltransferases(E.C.2.3.1.28).It catalyses the formation of chloramphenicol3-acetate from acetyl-CoA and chloramphenicol.The c at gene has been used in molecular biology for decades to confer chloramphenicol-resistance to chloramphenicol-sensitive bacterial strains,allowing the posi-tive selection of recombinant clones.C at has also been used in the transcriptional mapping of extrachromosomal elements [38].The crystal structure of this enzyme(pdb entry4CLA)shows that CAT is aα/βprotein of a two-layer sandwich architecture. Although cobalt ions are present in the crystal structure,these likely stabilise the crystal lattice rather than representing physiological important metal binding sites.Binding to metal-chelating resins is more likely to occur through a surface cluster of three histidine residues in close proximity.The co-purification of this protein during IMAC can clearly only be a problem if the gene of interest has been cloned in a bacterial expression vector containing this selection marker or if the recombinant gene is expressed in Rosetta™competent cells, which contain a chloramphenicol-resistant plasmid that encodes for“rare”codon tRNAs.1.3.3.cAMP-regulatory protein(CRP)Many cellular signalling pathways operate through the production of cAMP,which can regulate DNA transcription by binding to cAMP-regulatory protein(CRP;also known as catabolite gene activator protein,CAP).Homodimeric CRP forms a complex with cAMP,which is able to bind DNA at specific sites near the promoter region.This binding induces a dramatic conformational change in the DNA molecule,thus controlling the transcription of catabolite-sensitive operons [39].In addition to this role,CRP also regulates gene expression in response to osmotic changes[40].The crystal structure of CRP from E.coli shows that it is40%helical and29%β-sheet, constituting an N-terminal cAMP-binding domain and a C-terminal“winged helix”DNA-binding domain.Although this protein has low histidine content(2.9%)and no metal-binding sites,the flexible N-terminal chain of each protomer contains three surface-exposed histidine residues that may sequester metal ions.Thus,these residues may exhibit a cooperative effect on the binding of the dimeric protein to metal chelating resins.1.3.4.Host factor-I protein(Hfq)Hfq is a RNA-binding protein that is required for phage QβRNA genome replication.It also binds tightly to poly(A)RNA, oxyS RNA and the untranslated RNA dsrA,targeting several mRNAs for degradation possibly by increasing polyadenylation or by interfering with ribosome binding[41–43].Novel pro-teomic tools have allowed the identification of new mRNA targets of Hfq,including Fur and SodB,thus demonstrating roles in controlling iron uptake and scavenging[44].Hfq has also been implicated in negative post-transcriptional regulation by affecting the stability of the E.coli mutS,miaA,hfq[41]and ompA mRNAs[42].The functional hexamer of Hfq presents a central canal lined by six surface histidine residues(Fig.3C), and each monomer also contains a C-terminal run of histidine residues,thus offering multiple sites for interactions with metal-chelating resins.1309V.M.Bolanos-Garcia,O.R.Davies/Biochimica et Biophysica Acta1760(2006)1304–1313。
中国的枝顶孢属_英文_王有智
菌物系统21（2）：192~195, 2002MycosystemaSURVEY OF ACREMONIUM SPECIES FROM CHINA WITHTHREE NEW RECORDS∗WANG You-Zhi GUO Fang ZHOU Yu-Guang(Institute of Microbiology, Chinese Academy of Sciences)ABSTRACT: Thirteen species of Acremonium from China are investigated, including three new records,A. alternatum, A. egyptiacum and A. potronii. A key is also provided in the paper.KEY WORDS: Hyphomycetes, Acremonium,new record speciesCephalosporium-like species are valuable antibiotic-producing fungi. Cepahlosporin has earned a good reputation in biosynthetic antibiotics. Recently, a novel antibiotic produced by an Acremonium species was reported (Nakai, 2000).In the monograph of Cephalosporium-like Hyphomycetes (Gams, 1971), the older generic name Acremonium Link replaced Cephalosporium Corda and A. alternatum Link per S.F. Gray was selected as the lectotype of the genus. Under the generic level, Section Acremonium (= Sect. Simplex), Section Gliomastix, Section Nectrioidea, Section Albo-lanosa, Section Chaetomioides and Section Lichenoidea were erected (Gams, 1971, 1975; Morgan-Jones & Gams, 1982; Lowen, 1995). The diagnostic feature of Acremonium is 1-celled (rarely 2-celled) phialospores in chains or heads producing on thin-walled orthophialides or on basitonously branched conidiophores (Gams, 1971). There are about 130 worldwide species accepted in Acremonium. Fourty of them were described after 1971 when the monograph of Acremonium has been carried out (Gams, 1971). Molecular phylogenetic study shows that Acremonium is a polyphyletic taxon with affiliations to at least three ascomycetous families: Hypocreaceae, Claviciptaceae and Chaetomiaceae (Glenn et al., 1996). Acremonium species have a worldwide distribution and occur as saprobic, plant parasitic or lichenized.Surveying on the Chinese species of Acremonium, seven species of Cephalosporium were recorded in Sylloge Fungurum Sinicum (Tai, 1979). However, according to Gams (1971), four of them were transferred to Acremonium and accepted as A. persicinum (Nicot) W. Gams, A. roseo-griseum (S.B. Saksena) W. Gams, A. rutilum W. Gams and A. strictum W. Gams. Species of A. kiliense Grütz and A. strictum were found in Hong Kong (Lu et al., 2000). In Taiwan three correct species of Acremonium were reported. They are A. charticola (J. Lindau) W. Gams, A. fusigerum (Berk. & Broome) W. Gams and A. zonatum (Sawada) W. Gams (Wang et al., 1999). Most recently, A. terricola (Miller et al.) W. Gams and A. tubakii W. Gams were reported in Anhui and Guangdong (Lu & Lu, 1999; Fan et al., 1999).∗ Supported by The Research Fund for Returned Overseas Chinese Scholars of CAS (no.20010712112454) and The Director Fellowship of IMCAS(2001)Received:2001-07-25, accepted: 2001-11-082期 王有智等: 中国的枝顶孢属 193When rechecking the mitosporic fungi cultures in CGMCC (China General Microbiological Culture Collection Centre), we found three species new to China,Acremonium alternatum Link, Mag. Ges. naturf. Fr. Berlin 3: 15, 1809; Gams,Cephalosporium-artige Schimmelpilze (Hyphomycetes) 62, 1971, Fig. 1.Colonies reaching 14 mm diam after 10 days at 26ºC on 2% malt agar, whitish, powdery, reverse brownish. Sporulation phalacrogenous to plectonematogenous. Vegetative hyphae thin-walled, 0.5~1.5 µm wide. Phialides simple, arising from aerial hyphae, awl-shaped, 15~25 µm long, tapering imperceptible from1.5~3.0 µm to 0.5~1 µm wide. Conidia in short chains at the beginning and later in heads, ellipsoidal, long obovate, with faintly apiculate base and round upper end, hyaline, smooth-walled, 4.2~5.0 × 1.5~2.0 µm. Chlamydospores absent.Material examined: China, CGMCC, 1 November 1969, Chen Qin-Tao (AS 3.3759, as Spicaria presina ).Acremonium egyptiacum (van Beyma) W. Gams, Cephalosporium -artige Schimmelpilze(Hyphomycetes), 64, 1971. Fig. 2.≡ Oospora egyptiaca van Beyma, Zentbl.Bakt. ParasitKde(Abt. II) 89: 242,1933.Colonies reaching 18 mm diam. after10 days at 26ºC on 2% malt agar. Aerialmycelium white, later cream, powdery,reverse luteous. Sporulation phalacro-genous to plectonematogenous. Vegetativehyphae thin-walled, 1.0~1.8 µm wide.Phialides simple, 25~40 µm long,gradually tapering from 1.5~2.0 µm to0.5~1 µm wide. Conidia cohering in longchains or forming loose heads, ellipsoidal,hyaline, smooth-walled, 4.4~5.2 × 1.4~2.0µm. Chlamydospores absent.Material examined: China, CGMCC,1 November 1969, Chen Qin-Tao (AS3.3796, as Cephalosporium sp.).Acremonium potronii Vuill., Soc. Sci. Nancy, 19., 1910 and Encyclop. Mycol. 2: 66, 1931; Gams,Cephalosporium- artige Schimmelpilze (Hyphomycetes), 59, 1971. Fig.3.≡ Cephalosporium potronii(Vuill.) Oomen in CBS list of Cultures, 1957 (invalid publication).194 菌物系统21卷Colonies reaching 6 mm diam. after 10 days 26ºC on 2% malt agar, powdery, white to pinkish, reverse yellow. Sporulation phalacrogenous. Vegetative hyphae 1.0~2.0 µm wide. Phialides simple, 10~25 µm long, tapering from 1.0~2.0 µm to 0.5~1.0 µm. Conidia in slimy heads, obovate, with an apiculate base, hyaline, smooth-walled, 2.5~4.0 × 1.4~2.5 µm. Chlamydospores absent.Material examined: China, CGMCC, 1 November 1969, Chen Qin-Tao (AS 4.4008, as Cephalosporium acremonium).KEY TO THE THIRTEEN SPECIES OF ACREMONIUM FROM CHINA1. Vegetative hyphae with refrative wall thickening, conidia incrusted dark or hyaline (2)1. V egetative hyphae lackinging pronounced wall thicking, conidia always hyaline (4)2. Conidia spindle-shaped with truncate ends, very dark, 17.5~19.0×7.0~8.5µm………………………………………………………………………...A.f u s i g e r u m2. Conidia subglobose, obovate, hyaline or pigmented (3)3. Conidia hyaline, 3.2~4.8 ×1.2~3.0 µm .………………………………A.persicinum3. Conidia pigmented,4.8~6.4 ×2.6~4.0 µm .………………………..A. roseo-griseum4. Conidiophores often repeatedly branched (5)4. Conidiophores usually consist of slender, unbranched phialides (6)5. Colonies intensely pink, conidiophores slightly branched, Conidia 3.0~6.0× 2.0~3.4µm…………………………………………………………………………….A.r u t i l u m5. Colonies whitish to pale pink, conidiophores several times branched, Conidia 5.0~6.8× 2.0~2.8µm ……………………………………………………….A.z o n a t u m6. Conidia at least partly in dry chains (7)6.Conidia always in heads (9)7. Conidia with sharply pointed ends, 3.8~5.4×1~1.5 µm ………………...A. terricola7. Conidia with rounded upper ends (8)8. Conidia sometimes in heads, 4.2~5.0 × 1.5~2.0µm …………………..A. alternatum8. Conidia always in chians, 4.4~5.2 × 1.4~2.0 µm ……………………..A. egyptiacum9. Conidia obovate, 2.5~4.0 × 1.4~2.5µm ………………………………….A. potronii9. Conidia ellipsoidal or cylindrical, length : width>2 (10)10. Conidia ellipsoidal, 3.2~4.4 ×1.4~2.0 µm ……………………………A. charticola10. Conidia typically cylindrical (11)11. Chlamydospores absent, conidia 3.2~5.4 ×1.0~1.8µm ………………...A. strictum11. Chlamydospores present (12)12. Chlamydospores usually more numerous than phialospores, conidia 3.5~5.7 × 1.5~2.3 µm……………………………………………………………………………..A.t u b a k i i12. Chlamydospores less numerous than phialospores, conidia 3.1~5.8 ×1.0~1.6µm…………………………………………………………………………...A.k i l i e n s e Notes: Acremonium chrysogenum (Thirum. & Sukap.) W. Gams has been used for producing2期王有智等: 中国的枝顶孢属195 Cephalosporin C, N in China for years, but the isolates are from abroad. No report on isolation of A. chrysogenum in China was traced. Therefore, this species is not included here.[REFERENCES]Fan M-Z, Huang B, Li C-R, Li Z-Z, 1999. A new record species of the genus A cremonium. Mycosystema18 (4): 449 (in Chinese). Gams W, 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes). Stuttgart: Gustav Fischer Verlag. 1~262.Gams W, 1975. Cephalosporium-like Hyphomycetes: some tropical species. Transactions of the British Mycological Society64 (3): 389~404.Glenn A E, Bacon C W, Price R, Hanlin R T, 1996. Molecular phylogeny of Acremonium and its taxonomic implications.Mycologia 88 (3): 369~383.Lowen R, 1995. Acremonium Section Lichenoidea Section nov. and Pronectria oligospora species nov. Mycotaxon 53: 81~95. Lu B-S, Hyde K D, Ho W-H et al., 2000. Checklist of Hong Kong Fungi. Hong Kong: Fungal Diversity Press, 1~207.Lu Y-J, Lu D-J, 1999. Rhizospheric fungal diversity of vegetables in suburbs of Guangzhou. Ecological Science18 (1): 23~25 (in Chinese).Morgan-Jones G, Gams W, 1982. Notes on Hyphomycetes. XLI. An endophyte of Festuca arundinacea and the anamorph of Epichloë typhina, new taxa in one of two sections of Acremonium. Mycotaxon15: 311~318.Nakai R, Ogawa H, Asai A, et al., 2000. UCS1025A, a novel antibiotic produced by A cremonium sp. The Journal of Antibiotic53 (1): 294.Tai F-L, 1979. Sylloge Fungurum Sinicum. Beijing: Science Press. 1~1572 (in Chinese).Wang Y-C, Wu S-H, Chou W-N et al., 1999. List of the fungi in Taiwan. Taiwan, 1~289.中国的枝顶孢属王有智郭芳周宇光（中国科学院微生物研究所）摘要：本文对中国的枝顶孢属丝孢菌进行了调查，其中包括3个新记录种，并给出了全部13个种的检索表。
单词
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神经生长因子osteoporosis骨质疏松，骨关节炎(Osteoarthritis,OAphosphoric acid,orthophosphoric acid , diphosphate 二磷酸盐，，osteoclast破骨细胞，OB 成骨细胞。
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反义技术与成骨细胞干细胞stem cell BMP骨形成蛋白骨成型蛋白质（Bone Morphogenetic Proteinmorbidity 发病率，modulator调制器，Bisphosphonates硝酸甘油(nitroglycerin，NTG 茶多酚(tea polyphenols，TP)和茶黄素(tea flavanols，TF) 动脉粥样硬化(atherosclerosis ,As)平滑肌细胞(vascular smooth muscle cells ,VSMC)OPG：骨保护素；护骨素；人骨保护素Osteoporotegerin OPG：骨保护素rat OPG：大鼠骨桥素；大鼠骨保护素RANKL：受体活化因子配体；破骨细胞分化因子；人核因子活化因子受体配体The receptor activator of NF-kappaB (RANKL)RANK 受体活化因子二膦酸盐(bisphosphonates,BPsetidronate依替磷酸盐，clodronate氯替磷酸盐，pamidronate帕米磷酸盐，alendrnate阿仑磷酸盐，ibandrnate伊本磷酸盐APJ受体(apelinangiotensinrecepor．1ike，APJ，血管紧张素受体样受体白发性高血压大鼠(spontanouslyhypertensiverat，sHR)细胞外信号调节激酶1／2(extracellularregulatedkinase1／2，ERK1／2)颌骨中心性巨细胞病变(giant cell lesion, GCL)单核基质细胞(spindle stroma cel,l SSC)apoptosis[英汉医学名词3][细胞]凋亡apoptotic 细胞凋亡的mab [=man and biosphere programme]人和生物圈计划[=monoclonal antibody]单克隆抗体Lymphocytes淋巴细胞cytochrome 细胞色素thymocyte 胸腺细胞caspase 半胱天冬酶[=cysteine-containing aspartate-specific proteases]天冬氨酸特异性半胱氨酸蛋白酶半胱天冬酶peripheraladj. 次要的;无关紧要的a.①周围的；边缘的；表面的;a ~ war【军】边缘战争.②【解】(神经)末梢区域的;the ~ nervous system末梢神经系统.③较不重要的；非中心的;annexin膜联蛋白propidium普罗匹定cytochrome细胞色素camptothecin喜树碱enzymen. 酵素,酶apoptosis[英汉医学名词3][细胞]凋亡viable有活力的,有生机的purified纯化的purified精制的,纯净的FITC[=fluorescein isothiocyanate]异硫氰酸荧光素recombinant[英汉微生物学名词]重组体bind包扎,结合,困境或进退两难assay含量测定leukemian. 白血球过多症leukemian.白血病tum[=tuberactinomycin]结核放线菌素[=tumour-derived macrophages]肿瘤衍生的巨噬细胞视网膜母细胞瘤基因（Retinoblastoma Gene，Rbcyclin细胞周期调节蛋白,细胞周期蛋白程序性细胞死亡（programmed cell death,PCD凋亡与坏死apoptosis vs. necrosisfas[=fatty acid synthesis]脂肪酸合成[=fatty acid synthetase]脂肪酸合成酶[=fetal alcohol syndrome]胎儿酒精综合征[=fibrin adhesion system]纤维蛋白粘连系统FasFas蛋白bcl[=b-cell leukemia-lymphoma]b细胞白血病-淋巴瘤[=basal cell layer]基底细胞层[=basic cycle length]基础循环时间[=bekesy comfortable loundness test]贝凯西适宜响度试验prodomain前结构域IAP[=inhibitor of apoptosis protein]细胞凋亡蛋白的抑制物molecule ['m?likjul] n. 分子；微小颗粒，微粒mechanism ['mek?niz?m] n. 机制；原理，途径；进程；机械装置；技巧monocyte ['m?n?usait] n. [基医] 单核细胞；[基医] 单核白细胞macrophage ['m?kr?ufeid?] n. [组织] 巨噬细胞endocrinology [,end?ukri'n?l?d?i] 英汉翻译n. 内分泌学；内分泌科calvarial [k?l'vε?ri?l] 基本释义adj. 颅盖的；颅顶的thiazolidinediones网络释义噻唑烷二酮类药理学专业词汇英汉对照...thiazides 噻嗪类thiazolidinediones 噻唑烷二酮类thiophosphoramide 塞替派...基于6个网页-相关网页噻唑烷二酮...(Sulfonylurea)和双胍类(Biguanide)发展到目前的α-葡糖苷酶抑制剂(α-glucosidase inhibitor)、噻唑烷二酮(Thiazolidinediones)和非磺酰类胰岛素促分泌剂等,并相继出现了第二代、第三代产品.…基于4个网页- 相关网页短语thiazolidinediones TZDs噻唑烷二酮类Thiazolidinediones,TZDs噻唑烷二酮adverse ['?dv?:s, ?d'v?:s] 基本释义词组短语同近义词adj. 不利的；相反的；敌对的implications基本释义词组短语n. 蕴涵式；卷入counsel ['kauns?l] 基本释义词组短语同近义词n. 法律顾问；忠告；商议；讨论；决策vt. 建议；劝告vi. 商讨；提出忠告critique [kri'ti:k] 基本释义同近义词n. 批评；评论文章vt. 批判；评论optimal ['?ptim?l] 基本释义词组短语同近义词adj. 最佳的；最理想的pharmacological基本翻译adj. 药理学的facilitate基本翻译vt. 促进；帮助；使容易indications基本翻译n. 适应症，显示；标志hypercalcemia 高血钙division基本翻译n. 除法；部门；分割；师（军队）；[体]赛区endocrinology基本翻译n. 内分泌学；内分泌科metabolism基本翻译n. 新陈代谢diabetes基本翻译n. 糖尿病；多尿症cardiovasculara.心脏血管的cytokines细胞因子类cytokines[英汉药学名词]细胞活素类osteoclastogenesis骨诱裂发生critically基本翻译adv. 危急地；精密地；批评性地；用钻研眼光地sinusoid血窦,窦状隙interleukin白[细胞]介素,白介素,白细胞介素monocytic单核细胞的,单核细胞系的exogenous外生的,外生性,外源,外源的,外源性exogenousa.外长的, 外生的paraffin石蜡n. [化]石蜡；[化]链烷烃；[化]硬石蜡vt. 用石蜡处理；涂石蜡于…neutralize中和,成为无效vt.①使…中立;②【化】中和;bevacizumab(VEGF单克隆抗体)、sunitinib（VEGF的多靶点酪氨酸激酶抑制剂）calvarial颅盖的osteoarthritisn.骨关节炎prosthesisn.修复术，弥补术；假体，修补物;dental ~假牙dental ~假牙. 牙科synovial滑液的diethylpyrocarbonate (DEPC)焦碳酸二乙酯endocrinologyn.内分泌学vacuolar [vækju'əulə(r)]adj. 空泡的；有液泡的TIEG TNF-B inducibe early gene 早期反应基因agonist激动药[剂]stoichiometrical化学计算的collagenase胶原酶lysate裂解物,裂解液,溶胞产物assay含量测定Quantitative reversetranscriptase-polymerase chain reactionPCRPhenotype表型secrete分泌sialoprotein涎蛋白,唾液蛋白collagenolytic溶胶原的periodontal牙周病,牙槽浓溢,牙周症ligament韧带,系带endogenous内生的,内源的exogenous外生的,外生性,外源,外源的,外源性alternativelyadv. 非此即彼；二者择一地；作为一种选择dose dependent剂量依赖性osteonectin骨结合素osteopontin骨桥蛋白osteocalcin骨钙素diminish减少,减小accretion堆积,生长,添加生长,停滞堆积,外着生长,粘连molecule ['mɔlikjul]n. 分子；微小颗粒，微粒matrixn. 模子,矩阵simultaneousadj. 同时的；联立的；同时发生的n. 同时译员elaborationn. 详细说明,细致的工作extracellular胞外的,细胞外的。
园艺英语词汇
Amphiploid 双倍体Amphidiploid 双二倍体Autotetraploid 同源四倍体Allotetraploid 异源四倍体Allohexaploid 异源六倍体Allopolyploid 异源多倍体Autopolyploid 同源多倍体Natural selection 自然选择Euploid 整倍体Aneuploid 非整倍体Wild relatives 野生亲缘种Plant introduction 引种Genetic integrity 遗传完整性Genetic erosion 遗传冲刷Heritability 遗传力Genetic drift 遗传漂变Transgressive inheritance 超亲遗传Quantitative inheritance 数量遗传Qualitative inheritance 质量遗传(Fertility) Restoring gene 育性基因Additive effect 加性效应Additive genetic variance 加性遗传方差Dose effect 剂量效应Dominant effect 显性效应Male sterile gene 雄性不育基因Gene transfer 转基因Genic sterility 基因性不育Mutation 突变mutant 突变体Mutational site 突变位点Hot spot (in mutation) 突变热点Biochemical mutation 生化诱变karyotype 核型Cytoplasmic mutation 细胞质突变bud mutation 芽变Mutation frequency 突变频率Addition line 附加系Substitution line 代换系Translocation line 易位系Somatic hybridization 体细胞杂交Homogeneity 同质性Heterogeneity 异质性Xenia 异粉性Metaxenia 后生异粉性Male parent 父本Female parent 母本Progeny 后代Filial generation 子代，杂交后代Cross 杂交Hybrid 杂种，杂合物Cross breeding (Hybridization) 杂交育种Hybrid combination 杂交组合Pure line breeding (Pure line selection)纯系育种Single cross 单交Heterosis (hybrid vigor) 杂交优势Heterobeltiosis 超亲杂交优势Test cross 测交Double cross 双杂交Top cross 顶交Back cross 回交Recurrent parent 轮回亲本Non-recurrent parent 非回交亲本Reciprocal cross 正反交，互交Selfing 自交Inbreeding (Close breeding) 近交Outbreeding 远交，杂交繁殖Outcross 异型杂交，远交Intercrossing (intermating) 杂交，互交Intergenetic cross 属间杂交Interspecfic cross 种间杂交Intervarietal cross 品种间杂交Compatibility 自交亲和self-compatibility 自交亲合性Incompatibility 自交不亲和cross compatibility 杂交亲和性Self compatible line 自交亲和系Foundation seed 原种Crossability 杂交性Cross fertile 杂交可育Hybrid sterility 杂交不育Self-fertility 自交可育Self-sterility 自交不育Male sterile 雄性不育Female sterile 雌性不育Partial sterility 部分不育Male sterile line 雄性不育系Restoring line 恢复系Maintainer line 保持系Inbred line 近交系Reproductive isolation 生殖隔离Parthenogenesis 孤雌生殖，单性生殖Progeny test 后代测试Directional selection 定向选择Successive selection 持续选择General combining ability 普通配合力Specific combining ability 特殊配合力Selection differential 选择差数Breeding nursery 育种圃Emasculation 去雄Pollination 授粉Open pollination 自由授粉Cross pollination 异花授粉Self-pollination 自花授粉Self-fertilization 自花受精Artificial pollination 人工授粉Mixed pollination 混合授粉haploid breeding 单倍体育种Distant hybrid 远缘杂交Strain (Line) 系Breeding for disease resistance抗病育种Apomixis 无融合生殖Cultivar (Variety) 栽培品种Variety certification 品种鉴定Landrace Variety registration本地品种记录Improved variety 改良品种Commercial variety 商业品种Introduced variety 引进品种Kingdom 界Phylum 门Class 纲Subclass 亚纲Family 科Genus 属Species 种Germplasm 种质Fruit atlas 果树图集Germplasm resources 种质资源Germplasm of fruit tree 果树种质Germplasm resources of fruits果树种质资源Germplasm resources file种质资源档案Exploration of germplasm resources种质资源勘查Wild species野生种Cultivar 栽培品种Variety 变种Maintenance of germplasm resourcesspecies 种质资源保存Kindred wild National fruit germplasmrepository 国家果树种质库Passport data 基本数据Strain 株系、品系Classification of fruit plants 果树分类Characterization 鉴定Fruit cultivar inventory果树栽培品种目录Monograph of fruit trees 果树专著Degeneration of cultivars 品种退化Origin of cultivated fruits果树栽培品种的起源Cultivated species 栽培品种Palynology characterization孢粉学特征鉴定Science of germplasm resources种质资源学Biodiversity 生物多样性Science of germplasm resources of fruit果树种质资源学Variety test 品种试验Identification of characters 性状鉴定Early stage evaluation 早期鉴定Carbon (C)Hydrogen (H)Oxygen (O)Nitrogen (N)Phosphorous (P)Potassium (K)Calcium (Ca)Zinc (Zn)Magnesium (Mg)Sulphur (S)Sodium (Na)Iron (Fe)Manganese (Mn)Copper (Cu)Boron(B)Chloride (Cl)Molybdenum(Mo)Fertilizer 肥料Diagnosis 诊断Fertigation 施肥灌溉Nutrient balance 营养平衡Element 元素Starch 淀粉The balanced fertilization 平衡施肥Beneficial element 有益元素Essential element 必需元素Carbohydrate 碳水化合物Assimilate 吸收，同化Photosynthate 光合产物Ion 离子Deficiency 缺乏Mineral nutrition 矿质营养Toxicity 毒性Physiological disorder 生理失调Manure 施肥Green manure 绿色肥料Organic manure 有机肥Inorganic 无机肥Cover crops 覆盖作物cash crop 商品作物Pomology 果树学Deciduous fruit tree 落叶果树Annual vegetable crop 一年生蔬菜Biennial vegetable crop 两年生蔬菜Perennial vegetable crop 多年生蔬菜Aquatic 水生的Vegetables 蔬菜Ornamental horticulture 观赏园艺学Flower arrangement 花卉装饰Flower decoration 花卉装饰landscape design 景观设计Landscape gardening 传统园林学The standard leaf sample标准叶样Rhizosphere 根际Stress 胁迫Tolerance 耐受性The standard values of leaf mineral elements 标准叶矿质元素值mineral elements 矿质元素Photosynthesis 光合作用Chlorophyll 叶绿素Spray喷洒Nutrient 养分Content 含量，内容物Concentration 浓度Solution 溶液Soil application 土壤施肥Foliar application 叶面施肥Solution culture 液体培养abortion败育agar 琼脂anther花粉apical顶端的auxin生长素axillary bud 腋芽callus，calli愈伤组织cellular totipotency 细胞全能性cellulase 纤维素酶cellulose 纤维素centrifuge 离心chloroplast 叶绿体chromosome doubling 染色体加倍colony 细胞团，菌落cybrid(cytoplasmic hybrid)胞质杂种cytokinin 细胞分裂素cytoplasm 细胞质degeneration 败育dedifferentiation 脱分化redifferentiation 再分化dihaploid 二单倍体diploid 二倍体dissect 剥离dormancy 休眠eliminate 除去embryo 胚胎embryoid 胚状体embryogenesis 胚胎发生方式epidermis 表皮，上表皮excise 切除explants 外植体filter paper 滤纸agarose 琼脂糖genetype 基因型germplasm 种质global embryo 球型胚hormone 激素interspecific 种间的intraspecific 种内的in vitro 体外in vivo 活体kinetin 激动素medium 培养基membrane 膜meristem 分生组织meristem culture 茎尖培养micropropagation 微繁microspore 小孢子monocotyledon/monocots 单子叶植物nod culture 茎段培养organelle 细胞器organogenesis 器官发生方式osmotic 渗透的plantlet 小植株，苗pollen culture 花粉培养pollinate 授粉protoplast fusion 原生质体融合rapid propagation 快繁regeneration 再生self-incompatibility 自交不亲和shoot tip 茎尖somatic embryo 体细胞胚somatic hybridization 体细胞杂交somatic hybrid 体细胞杂种stem 茎sterile distilled water 蒸馏水sterilization 消毒stock plant 母株subculture 继代sucrose 蔗糖terminal bud 顶芽transfer 转移virus eradication 脱毒Gene 基因Intron 内含子Exon 外显子Operon 操纵子Replicon 复制子Promoter 启动子Ribonucleic acid RNANucleotide 核酸Amino acid 氨基酸Protein 蛋白质Mutant 突变体Mutation 突变Splicing 剪接Ligase 连接酶Kinase 激酶Transcriptional factor 转录因子Genome 基因组Action spectrum 光谱Photoautotroph 光合自养生物Photoheterotroph 光合异养生物Absorption 吸收Bioassay 生物测定Phytotron 人工气候室Photoreduction 光还原Photooxidation 光氧化Thermal death point(TDP) 热[致]死点Lignification 木质化Optimum temperature 最适温度Photochemical induction 光化学感应After-effect 后效Transmembrane potential 跨膜电位Reaction center 反应中心Gross photosynthesis 总光合作用率Net (Apparent) photosynthesis 净光合Law of limiting factor 限制因素定律Photosystem 光系统Quantum efficiency 量子效率Photosynthetically active radiation (PAR)光合成有效辐射Assimilation 同化作用Dissimilation 异化作用Compensation point 补偿点Light reaction 光反应Dark reaction 暗反应Photolysis 光解作用Photophosphorylation 光合磷酸化Light saturation point 光饱和点Respiration 呼吸作用Aerobic respiration 有氧呼吸Anaerobic respiration 厌氧呼吸Photorespiration 光呼吸Dark respiration 暗呼吸Cyanide-resistant respiration 抗氰呼吸Climacteric 呼吸跃变Metabolism 新陈代谢Catabolism 分解代谢（异化作用）Anabolism 合成代谢Secondary metabolism 次级代谢Electron transport 电子传递Electron carrier 电子载体Terminal oxidase 末端氧化酶Terminal electron acceptor末端电子受体Antitranspirant 抗蒸腾，抗蒸腾剂Plasmolysis 质壁分离Physiological drought 生理干旱Soil permeability 土壤渗透性Soil density 土壤密度Bulk density of soil 土壤Specific gravity of soil 土壤比重Soil solution 土壤溶液Soil moisture土壤湿度Soil water potential 土壤水势Soil drainage 土壤排水Soil aeration 土壤通气Soil hygroscopic water 土壤吸附水Soil gravitational water 地下重力水Water cycle 水循环Water regime 水分状况Water deficit 水分不足Water management 水分管理Water balance 水分平衡water compensation 水分补偿Water conservation 水分保持water retention capacity 保水能力Wilting point 萎蔫点water-storage capacity 储水能力Water consumption 水分消耗Seepage 渗漏Water requirement 水分需求Osmotic pressure 渗透压diffusion coefficient 扩散系数Percolation (leakage) 渗透Guttation 伤流现象Bleeding 伤流wilting coefficient 萎蔫系数Turgor pressure 膨胀压water potential 水势Permanent wilting 永久萎蔫Wilting 萎蔫Hypertonic solution 高渗溶液Hypotonic solution 低渗溶液Isotonic solution 等渗溶液Temporary wilting 临时萎蔫Osmotic pressure 渗透压Nitrogen fixation 固氮作用Permeability 渗透性Permeable membrane 渗透膜Physiological acidity 生理酸度Physiological alkalinity 生理碱度Acropetal translocation 顶向运输Bidirectional translocation 基向运输Ring gridling 环割Partitioning 分配Root pressure 根压Symplast 共质体Apoplast 质外体Aging 老化Tropism 向性Apical dominance 顶端优势Critical period 临界期Taxis 趋避性Nasty 感性Geotropism 向地性Phototropism 向光性Threshold value 临界值Stress 胁迫Lodging 倒伏Earliness 早熟Lateness 迟误Physiological maturity 成熟度。
胡萝卜素的分离和鉴定实验流程
胡萝卜素的分离和鉴定实验流程英文回答：Carotenoids are a group of pigments that areresponsible for the vibrant colors in fruits and vegetables, such as carrots. They are also known for their antioxidant properties and potential health benefits. In order to separate and identify carotenoids, several steps can be taken.Firstly, the extraction of carotenoids from the plant material is necessary. This can be done using a solvent, such as acetone or hexane, which can dissolve the pigments. The plant material, in this case, can be carrots. Thesolvent is added to the carrots and mixed thoroughly to ensure the extraction of carotenoids.After the extraction, the next step is to separate the carotenoids from other compounds present in the extract. This can be achieved through techniques such aschromatography. One commonly used method is thin-layer chromatography (TLC), where the extract is spotted onto a thin layer of adsorbent material, such as silica gel, and then developed using a suitable solvent system. The different carotenoids will move at different rates on the TLC plate, allowing for their separation.Once the carotenoids are separated, they can be identified using spectroscopic techniques. UV-Vis spectroscopy can be used to determine the absorption spectra of the carotenoids, which can provide information about their structure and functional groups. Additionally, other spectroscopic techniques, such as infrared spectroscopy or mass spectrometry, can be employed for further characterization.In conclusion, the process of separating andidentifying carotenoids involves the extraction of pigments from plant material, followed by their separation using chromatography, and finally their identification using spectroscopic techniques. This experimental flow allows for the isolation and characterization of carotenoids, whichcan contribute to our understanding of their properties and potential applications.中文回答：胡萝卜素是一类色素，负责水果和蔬菜（如胡萝卜）的鲜艳颜色。
普通野生稻绿色组织特异表达启动子的克隆与鉴定
普通野生稻绿色组织特异表达启动子的克隆与鉴定赵志强;薛满德;黄珂;张静文;龙艳;裴新梧;袁潜华【摘要】Green tissue-specific expression promoter,which makes the exogenous gene express efficiently in green tissues of receptor crop. The green tissue-specific promoter OrGSP was cloned from Oryza rufipogon,and the fusion vector of OrGSP and GUS gene was constructed and transferred into Arabidopsis thaliana in order to identify the functionof the promoter. Bioinformatics analysis shows that the length of 825 bp of OrGSP,contains the basic transcription initiation elements TATA-box and CAAT-box,and light responsive elements TCCC-motif,Sp1,G-box,I-box,GA-motif and as-2-box,etc. The GUS histochemical staining of transgenic Arabidopsis thaliana shows that OrGSP regulates the expression of GUS gene only in green tissue,and the GUS activity in leaf and stem is significantly higher than in roots. The promoter of Oryzarufipogon,OrGSP,is a green tissue-specific promoter and the results can provide new regulatory elements for crop molecular breeding.%绿色组织特异表达启动子可调控外源基因只在受体作物的绿色组织中定点、高效地表达.以普通野生稻为实验材料,克隆了绿色组织特异表达启动子OrGSP,构建OrGSP和GUS 基因融合的表达载体,转入拟南芥中鉴定功能.启动子OrGSP长度为825 bp,含有基本的转录起始元件TATA-box和CAAT-box,以及光响应元件TCCC-motif、Sp1、G-box、I-box、GA-motif和as-2-box等.转基因拟南芥GUS组织化学染色结果表明,启动子OrGSP调控GUS基因只在绿色组织中特异表达.GUS活性测定结果显示,叶和茎中的GUS活性比根中明显提高.普通野生稻中克隆的启动子OrGSP为绿色组织特异表达启动子,可为作物分子育种提供新的调控元件.【期刊名称】《生物技术通报》【年(卷),期】2017(033)008【总页数】7页(P51-57)【关键词】启动子;绿色组织特异表达;普通野生稻【作者】赵志强;薛满德;黄珂;张静文;龙艳;裴新梧;袁潜华【作者单位】海南大学热带农林学院,海口 570228;中国农业科学院生物技术研究所,北京 100081;海南大学热带农林学院,海口 570228;中国农业科学院生物技术研究所,北京 100081;中国农业科学院生物技术研究所,北京 100081;中国农业科学院生物技术研究所,北京 100081;海南大学热带农林学院,海口 570228【正文语种】中文启动子是基因的重要组成部分，它就像“开关”，调节着下游基因的活性。
辽宁西瓜和甜瓜细菌性果斑病的病原鉴定
收稿日期:2018-04-10基金项目:沈阳市重点科技研发计划项目(17-146-3-00);国家重点研发项目(2017YFD0201104)第一作者:于海博(1993-),女,硕士研究生,从事植物病害研究,E-mail :1440862240@通信作者:夏子豪(1988-),男,博士,讲师,从事植物病理学研究;E-mail :zihao8337@ ;吴元华(1963-),男,博士,教授,博士生导师,从事植物病理学和生物农药研究,E-mail :wuyh09@辽宁西瓜和甜瓜细菌性果斑病的病原鉴定于海博,毕馨月,夏博,安梦楠,夏子豪,吴元华(沈阳农业大学植物保护学院,沈阳110161)摘要:辽宁省锦州市凌海市西瓜、甜瓜果实上发生疑似细菌性果斑病,从发病西瓜果实上分离获得了菌株JZ17,从发病甜瓜果实上分离纯化获得了菌株JZX2和JZT8。
柯赫氏法则测定表明,接种的西瓜和甜瓜上均表现出与田间病害相同的症状,且从接种的发病植株上又重新分离到相同的菌株。
采用西瓜噬酸菌检测试剂盒进行DAS-ELISA 鉴定,表明3个菌株均呈阳性反应。
设计特异性引物YH1/YH2,对3个菌株的16S-23S rDNA ITS 序列进行扩增,3个菌株的ITS 序列与西瓜噬酸菌各菌株ITS 序列的同源性均高达99%以上。
综上可知,菌株JZ17,JZX2和JZT8可鉴定为西瓜噬酸菌(Acidovorax citrulli )。
利用特异性引物PL1/PL2对3个菌株进行亚群鉴定,结果表明菌株JZ17和对照菌株AAC00-1(亚群II)均可扩增出大小为332bp 的片段,而菌株JZX2、JZT8和对照菌株pslbtw8(亚群I)未扩增出此条带,因此可将菌株JZ17鉴定为西瓜噬酸菌亚群II 型菌株,菌株JZX2和JZT8鉴定为I 型菌株。
为比较3个菌株的亲缘关系,采用通用引物27F/1492R 对3个菌株的16S rDNA 序列进行扩增,获得了目的片段,在GenBank 中进行同源性比对的结果表明,菌株JZ17,JZX2,JZT8与A.citrulli 各菌株16S rDNA 的相似性均达99%以上。
黄海绿潮藻的形态学观察及分子鉴定英文
黄海绿潮藻的形态学观察及分子鉴定英文The concept of "None" is a fascinating one, as it encompasses both the presence and absence of something. It is a word that can be both definitive and ambiguous, depending on the context in which it is used. In this essay, we will explore the various facets of "None" and its significance in our lives.One of the most intriguing aspects of "None" is its ability to represent the absence of something. When we say that there is "None" of a particular item, we are conveying that there is a complete lack of that thing. This can be applied to a wide range of situations, from the absence of people in a room to the lack of a specific resource. In this sense, "None" serves as a powerful tool for communicating the void or emptiness that exists in a particular context.However, "None" can also be used to represent the presence of something. When we say that "None" of a certain condition or characteristic applies to a particular situation, we are actually affirming that the opposite is true. For example, if we say that "None of the students in the class failed the exam," we are implying that all the students passed. In this case, "None" is used to emphasize theuniversality of a positive outcome, rather than the absence of a negative one.The ambiguity of "None" can also be seen in its grammatical usage. In some cases, "None" is treated as a singular noun, while in others, it is considered a plural. This flexibility in its application can lead to confusion or debate, as individuals may interpret the word differently based on their own understanding of grammar and context.Furthermore, the concept of "None" can be deeply philosophical, as it raises questions about the nature of existence and the boundaries of our understanding. When we contemplate the idea of "Nothing," we are confronted with the paradox of trying to conceptualize the absence of all things. This exploration of the void or the unknown can lead to profound insights about the nature of reality and our place within it.In the realm of science, "None" can also play a significant role. In scientific experiments and observations, the absence of a particular phenomenon or result can be just as informative as its presence. The identification of "None" can lead to the refinement of theories, the rejection of hypotheses, and the discovery of new frontiers of knowledge.Additionally, "None" can have profound implications in the realm ofethics and decision-making. When faced with a situation where there are no clear-cut solutions or alternatives, the acknowledgment of "None" can be a powerful tool for navigating difficult choices. It can force us to think critically about our values, priorities, and the potential consequences of our actions.One example of the ethical implications of "None" can be seen in the context of medical decision-making. When a patient is faced with a terminal illness and there are no viable treatment options, the acknowledgment of "None" can be a difficult but necessary step. It can allow the patient and their loved ones to shift their focus towards palliative care, end-of-life planning, and finding meaning and purpose in the time that remains. In this situation, "None" is not a negative or hopeless statement, but rather a recognition of the reality of the situation and a call to embrace the present moment with compassion and dignity.Another example of the significance of "None" can be found in the realm of environmental conservation. When we are confronted with the depletion of natural resources, the loss of biodiversity, or the irreversible damage to ecosystems, the acknowledgment of "None" can be a powerful catalyst for action. It can compel us to rethink our relationship with the natural world, to prioritize sustainability over short-term gains, and to take collective responsibility for the preservation of our planet.In the context of social justice, "None" can also play a crucial role. When individuals or communities face systemic discrimination, oppression, or a lack of access to basic rights and opportunities, the acknowledgment of "None" can be a powerful statement of solidarity and a call for change. It can inspire us to challenge the status quo, to advocate for the marginalized, and to work towards a more equitable and inclusive society.In the realm of personal growth and self-discovery, "None" can also be a valuable concept. When we are faced with the limitations of our own knowledge, skills, or experiences, the acknowledgment of "None" can be a humbling and empowering experience. It can motivate us to seek out new perspectives, to embrace our vulnerabilities, and to continuously expand the boundaries of our understanding.In conclusion, the concept of "None" is a multifaceted and intriguing one, with far-reaching implications in various aspects of our lives. Whether it represents the absence of something or the presence of its opposite, "None" is a word that invites us to explore the boundaries of our understanding and to confront the complexities of the world around us. By embracing the nuances and ambiguities of "None," we can gain valuable insights and perspectives that canenrich our personal and intellectual growth, and inspire us to make a positive impact on the world.。
天然药物化学实验教学过程中科研反哺教学的实践——以冬凌草甲素的提取、分离和结构鉴定为例
C U R R I C U L U M T E A C H I N G“天然药物化学”实验教学过程中科研反哺教学的实践—以冬凌草甲素的提取、分离和结构鉴定为例赵杰吕洁丽张涛刘兆敏(新乡医学院河南•新乡453003)摘要“天然药物化学”是药学专业中理论和实践并重的专业基础课。
针对实验课程教学现状和不足，提出在教学过程中实施科研反哺教学，将科研精神用于教学，将科研成果引入课堂及开展实验、科研课题等方式，以改进“天然药物化学”课程教学质量，培养学生综合素质。
本文以冬凌草甲素的提取、分离和结构鉴定为例，进行科研反哺教学的探索和实践。
关键词天然药物化学科研反哺教学冬凌萆甲素中图分类号:G424 文献标识码:A DOI:10.16400/ki.kjdkz.2021.02.040Practice of Research Feedback Teaching in Experimental Teachingof Natural Medicinal Chemistry-------Take the extraction, isolation and structure identification o f oridonin as an exam ple ZHAO Jie, LV Jieli, ZHANG Tao, LIU Zhaomin(Sanquan Medical College, Xinxiang, Henan 453003)Abstract "Natural medicinal chemistry"is a basic course which emphasizes both theory and practice in pharmacy.In view of the current situation and shortcomings of experimental teaching,this paper proposes to implement scientific research feedback teaching in the teaching process,apply scientific research spirit to teaching,introduce scientific research achievements into the classroom and carry out experiments and scientific research projects,so as to improve the teaching quality of natural medicinal chemistry and cultivate students'comprehensive quality.Taking the extraction, separation and structure identification of oridonin as an example,this paper explored and practiced the teaching of scientific research feedback.Keywords natural medicinal chemistry;scientific research nurtures teaching;oridonin天然药物化学就是运用现代科学理论与方法研究天然 药物中化学成分的一门学科。
酸菜汁中白地霉菌的分离纯化与鉴定
分析检测酸菜汁中白地霉菌的分离纯化与鉴定张明玉1，卢志锋2（1.葫芦岛市检验检测中心，辽宁葫芦岛 125000；2.齐齐哈尔大学，黑龙江齐齐哈尔 161000）摘 要：本实验将自然发酵的酸菜样品中分离得到的霉菌进行种属鉴定，先对菌株进行形态学观察鉴定，再应用26S rDNA序列同源性分析的方法对所分离菌株进行基因扩增与测序。
将GenBank中该属内菌株的26S rDNA基因序列与测序结果进行同源性比较分析。
26S rDNA序列同源性分析结果表明，分离株G.wang 结合真菌鉴定手册得知为半知菌亚门-丝孢纲-丝孢目-从梗孢科-地霉属-白地霉种。
关键词：白地霉；分离鉴定；聚合酶链式反应Isolation, Purification and Identification of Geotrichumcandidum from Pickled Cabbage JuiceZHANG Mingyu1, LU Zhifeng2(1.Huludao Inspection and Testing Center, Huludao 125000, China; 2.Qiqihar Medical College, Qiqihar 161000,China)Abstract: In the present study, the species identification of mycete which was isolated from naturally fermented sauerkraut was conducted. The morphology identification was conducted. And 26S rDNA sequence analysis were applied for gene amplification. The 26S rDNA gene sequence of the strains within the genus in GenBank was compared with the sequencing results. The results of the 26S rDNA sequence homology analysis test showed that the isolate G.wang, combined with the fungal identification manual, was sub-phylum of Hemimycetes, Hyriospora, Hyriospora, Hyriosporaceae, Geotrichum and Geotrichum species.Keywords:Geotrichum candidum; isolation and identification; polymerase chain reaction白地霉是真核微生物，形态特征介于酵母菌和霉菌之间，具有生态适应性强、生长速度快的属性，在土壤、泡菜中分布广泛[1-2]。
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Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and Its Inhibitory Effect on Hela CellsWeihua JIN ，Junwei CAO *，Yi TU ，Yi TANG ，Ｒan GE ，Jingwen CHEN ，Fu XU ，Qijie XIONGDepartment of Life Science and Chemistry ，Wuhan Donghu University ，Wuhan 430212，ChinaAbstract ［Objectives ］The purpose of this study is to dissociate endophytic fungus producing diterpenoids from Torreya fargesii tissue and examines its inhibiting effect on tumor cells．［Methods ］Plant endophytes were isolated and purified to study their resistance to Gram-positive （G +）and Gram-negative bacteria （G －）．High performance liquid chromatography （HPLC ）was used for analysis of the retention time ，rela-tive peak area and percentage content of its metabolite．By liquid chromatography-mass spectrometry （HPLC-MS ），the material characteristic of the ion pair information of the metabolites was measured．The bacterial strain was also classified．［Ｒesults ］The results showed that the sec-ondary metabolites produced by the strain BP6T3possessed double resistance to G +and G －bacteria．The strain was identified as Penicilliumsp by preliminary classification．Through HPLC analysis ，the retention time of fermentation extracts was 12．8min with almost the same as the standard of taxol．According to the chromatograph ，the relative peak area was 12887．11，the average relative percentage was about 15．8%，and the content of taxol analogs in fermentation broth reached 16．59mg /L．The material characteristic of the formation of ion fragments of tax-ane analogues in metabolic extracts was identical to that of the taxol standard determined by HPLC-MS．It can be initially determined that strainBP6T3can produce taxane compounds．Taxol substance produced by this strain had obvious inhibitory effect on Hela cells with the concentra-tion increasing．Different precursors had a significant effect on the production of paclitaxel metabolites in this strain．L-phenylalanine was usedas the precursor and the yield increased most ，with an increase rate of 425．7%．［Conclusions ］The strain is expected to be used for mass production in antitumor drug taxol．Key words Endophytic fungus ，Penicillium sp ，Taxol ，Hela cells ，Inhibitory effectＲeceived ：January 15，2019Accepted ：June 10，2019Supported by Provincial College Students ’Innovation and Entrepreneurship Training Program of Colleges and Universities in Hubei Province in 2017（2013）；"Strategic Emerging （Pillar ）Industrial Talent Training Program"of Colleges and Universities in Hubei Province ［Hubei Provincial Department of Education EJiaoGao （201711798030）No．11］；Pilot Funded Project of "Comprehensive Professional Ｒeform"of Provincial Department of Education and Provincial Department of Finance ［EJiaoGaoBan （2014）No．6］；Hubei Educational Science "Twelfth Five-Year Plan"Project （2014B272）；SchoolYouth Natural Science Foundation （2013dhzk003）．*Corresponding author．E-mail ：215351231@qq．com1IntroductionTaxol was first isolated from Taxus ，but the content of taxol in Taxus is very low ，only 0．003－0．069%（V /V ）［1－4］．The guar-antee of taxol raw materials has become a key factor in the success of the drug to the market．Isolation of endophytic fungus producing taxol or analogues is a new way to effectively solve the problem oftaxol resources in recent years ［5］．The hosts of taxol-producing fungi are mostly found in the genera Taxus and Austrotaxus ［4－7］ofthe gymnosperms family Taxaceae．At present ，the endophytic fungus isolated are mainly from these genera ，but the content of taxol or analogues is generally low ，which does not meet the re-quirements of industrial production ［8］．A large number of studies have shown that the addition of precursors has a certain effect on increasing the content of endophytic taxol．Zamir et al．［9］reported that the tetracyclic diterpene skeleton of taxol came from hydroxy-methylvaleric acid ，the acyl group at position C-10came from ace-tic acid ，and the acyl side chain at position C-13came from phen-ylalanine．Therefore ，when endophytic fungus were cultured ，adding these precursors to the medium could effectively increase the content of taxol ［15］．In this study ，endophytic fungi were isolated from the tissues of Torreya fargesii collected from Shennongjia National Geological Forest Park．Then the strains producing taxol or analogues were screened by high performance liquid chromatography （HPLC ），liquid chromatography-mass spectrometry （LC-MS ）and antitumoractivity analysis．In order to increase the yield of taxol or ana-logues ，precursor substances and other methods were used to in-crease the yield of taxol or analogues．2Materials and methods2．1Materials The plant tissues of Torreya fargesii ，such as roots ，stems ，leaves and bark ，were collected from Shennongjia National Geological Forest Park ，Hubei Province （providing plant identification experts ）．taxol control （Beijing Solarbio Technology Co．，Ltd．，lot No．170922），methanol of chromatographicpurity ，other reagents of analytical purity．potato dextrose agar （PDA ）medium （g /L ）：potato ，200；dextrose ，20；KH 2PO 4，1．0；MgSO 4·7H 2O ，0．5；agar ，15－20；natural pH．Without agar ，it acted as a PDA seed liquid medium．Czapek medium （g /L ）：NaNO 3，2．0；K 2HPO 4，1．0；KCl ，0．5；MgSO 4·7H 2O ，0．5g ；FeSO 4·7H 2O ，0．01；sucrose ，30；agar ，15－20；natural pH．LBmedium （g /L ）：yeast powder ，5；NaCl ，10；peptone ，10；pH 7．4．15－20g of agar powder was added to the solid．Liquid chromatograph （HPLC ）model is Nexera LC-30AUPLC ；mass spectrometer （HPLC-MS ）model is AB scie Tri-ple Qaud 4500（triple quadrupole mass spectrometer ）．Medicinal Plant 2019，10（3）：45－48DOI ：10．19600/j．cnki．issn2152－3924．2019．03．0112．2Methods2．2．1Pretreatment of Torreya fargesii materials．The roots，stems，leaves and bark of Torreya fargesii were washed with asep-tic water and dried with aseptic absorbent paper．And they were sterilized with75%alcohol for5min and2%hypochloric acid for 8min，then rinsed with aseptic water for3times．The collected roots，stems，leaves and bark were then cut into small segments of roughly the same size with an aseptic knife．2．2．2Isolation of endophytic fungi．The plant materials treated by the above methods were placed in PDA solid medium plate and cultured at28ħuntil mycelium grew．2．2．3Purification of endophytic fungi．When the fine mycelium grew at the edge of the section of the inoculum in the plate，the mycelium was selected by inoculation needle and inserted into the newly prepared PDA solid plate．Then it was purified several times on PDA inclined medium until the pure culture was obtained．2．2．4Identification of endophytic fungus．The characteristics of endophytic fungus in plate culture were observed，and Shen Ping’s method［10］was used to observe the morphology of mycelium and conidia under microscope．2．2．5Extraction of taxol analogues from endophytic fungus．The purified endophytic fungi were activated in PDA liquid medium and inoculated in PDA liquid medium at28ħfor oscillating cul-ture．The filtrate was filtered and collected，and the filtrate was extracted with ethyl acetate to collect the upper organic phase．The organic solvent was evaporated in a rotary evaporator．2．2．6Detection by high performance liquid chromatography （HPLC）．（i）Chromatographic conditions were as follows：chro-matographic column，ODS（C18）column4．6mmˑ250mm，5μm；column temperature，room temperature；mobile phase，methanol/water=65/35（V/V）；flow rate，1．0mL/min；sample injection volume，20μL；UV detection wavelength，227nm．（ii）Sample treatment：the ethyl acetate extract was dissolved in the mobile phase，the supernatant was absorbed by centrifugation at 13200r/min for2min，and filtered by0．22μm pore size filtera-ble membrane．（iii）Quantitative analysis：the peak area was cal-culated according to the chromatogram．2．2．7Determination of fat-soluble metabolites by high perform-ance liquid chromatography-mass spectrometry（HPLC-MS）．（i）Chromatographic conditions were as follows：chromatographic col-umn，ODS（C18）column2．1mmˑ50mm，1．7μm；column temperature，room temperature，mobile phase，acetonitrile/water =65/35（V/V）；flow rate：0．2mL/min；sample injection vol-ume，1μL．（ii）Sample and mobile phase treatment：the extrac-ted product of ethyl acetate was dissolved in the mobile phase，the supernatant was absorbed by13200r/min centrifugation for 2min，and filtered by0．22μm pore size filterable membrane．（iii）MS parameters：（a）MS ion source：curtain gas（CUＲ），30．00；collision gas（CAD）：8．00；electrospray voltage（IS），5500．00；ion source temperature（TEM），550．00；nebulizer （GS1），40．00；auxiliary gas（GS2），45．00；inlet voltage （EP），10．00；collider outlet voltage（CXP），13．00．（b）Scan mode MＲM．2．2．8Inhibitory effect of extract on Hela tumor cells．Hela cells in logarithmic growth phase were inoculated in96-well plate at100μL/well and cultured in5%CO2incubator at37ħfor24h．Then different concentrations of taxol analogues（10μL）wereplaced into the sample wells．After being cultured in5%CO2in-cubator at37ħfor24h，the culture plate was taken out，the su-pernatant was absorbed，and90μL of fresh culture medium and 10μL of CCK-8were added to each well．After4h of culture un-der the same conditions，the A450value was read at450nm by mi-croplate reader（Elisa），and the cell growth inhibition rate was calculated．Cell growth inhibition rate was calculated as follows：Hela cell inhibition rate（IＲ）=（Control well A value－Sample well A value）/Control well A valueˑ100%．2．2．9Effects of precursors on the production of taxol metabolites by endophytic fungus．The endophytic fungi were inoculated in PDA liquid medium at28ħfor180r/min and the endophytic fungi were cultured for5d in oscillatory culture．Different precur-sors were added to the fermentation medium and sucrose solution was added at the same time to continue the culture．At the end of culture，taxol analogues of endophytic fungus were extracted．High performance liquid chromatography（HPLC）was used to de-termine taxol analogues in metabolic extracts and the content of taxol analogues was determined by HPLC．3Ｒesults3．1Isolation and purification of endophytic fungus from Torreya fargesii The material of Torreya fargesii was properly treated．After7d of culture，18strains of endophytic fungus were isolated from the tissues of Torreya fargesii．7plants were isolated from leaves，9from stems and2from bark．At the same time，no colony grew in the control group（uncut tissue），which confirmed that the isolated strains were endophytic fungus．3．2Identification of endophytic fungus BP6T3．The active BP6T3in18strains of endophytic fungus was classified and identi-fied．After observation of the characteristics of plate culture，it was found that the colony color of the strain was green and the col-ony texture was velvety（Fig．1）．Under the microscope，the hyphae were multicellular branches，the hyphae had transverse septum，and conidial pedicels also had transverse septum．There were no podocytes at the base and no enlarged apical sac at the tip，and its conidial pedicel was branched many times，resulting in two rounds of asymmetric pedicels，shaped like brushes．Conidia were spherical or oval，smooth or rough，and most of them were blue-green when they grew．The hyphae had erect multicellu-lar conidium pedicels on which each had a string of gray-green conidia（Fig．2），which could be preliminarily identified as Peni-cillium sp［11］．64Medicinal Plant2019Fig．1Bacterial lawn of strainBP6T3Fig．2Microscopic picture of strain BP6T33．3Analysis of taxol analogues in endophytic fungus BP6T3After the strain BP6T3was activated in PDA liquid medium ，the strain was cultured in PDA liquid medium at 28ħand 180r /minfor 7－10d．The filtrate was filtered and collected ，one third vol-ume ethyl acetate was added for countercurrent extraction ，the up-per organic phase was collected ，and the organic solvent was re-moved at 35ħin the rotary evaporator．The fermentation crude extract of strain BP6T3was detected by HPLC chromatography and determined under the same condi-tions of taxol control．The retention time （Ｒt ）of taxol control was 11－14min （Fig．3），and the Ｒt of strain BP6T3was 12．8min （Fig．4）．Therefore ，it was preliminarily determined that the me-tabolites of strain BP6T3contained taxol substances．The relativecontent of metabolites was 15．8%，and the content of taxol ana-logues in fermentation broth was 16．59mg /L （Fig．4）．Fig．3HPLC chromatogram of taxol referencesubstanceFig．4HPLC chromatogram of metabolic extract of strain BP6T33．4Determination of the characteristics of ion fragments formed by taxol analogues by HPLC-MS The characteristicsof taxol analogues forming ion fragments in the metabolic extractsof Penicillium BP6T3were determined by HPLC-MS ，and the re-sults showed that the ion fragment information of taxol analogues was completely consistent with that of taxol standard substance （Table 1）．The taxol analogues produced by Penicillium BP6T3can be preliminarily characterized as taxol．Table 1The ion pair information of the taxol standard and taxol analogues produced by strain BP6T3by the mass spectrometric analysisMass-to-charge ratio of parent ion ∥m /z Mass-to-charge ratio of daughter ion ∥m /zScanning time ∥msec Declustering potential （DP ）Collision energy （CE ）876．3307．95015036876．3591．25015036876．3531．250150403．5Inhibitory effect of extract on Hela tumor cells Helacell is a cell line isolated from human cervical cancer tissue ，which can be subcultured continuously ，the cell line will not die of ag-ing ，and can divide indefinitely．Compared with other cancer cells ，it has rapid proliferation and other characteristics．It has been widely used in tumor research ，biological experiments or cell culture ，and has become a very important tool in medical research．The inhibitory effect of taxol analogues on Hela cells was also used as the basis for judging the antitumor effect of taxol analogues．The inhibitory effect of strain BP6T3fermentation broth ex-tract on Hela tumor cells was tested by CCK-8reagent method ，and the inhibition rate on cell growth was calculated （Table 2）．Table 2Inhibitory effect of taxane from strain BP6T3on Hela cellsConcentration of sample ∥μg /mL OD 450value Ｒate of inhibition ∥%CK 1．100－0．10570．78324．141．0570．66735．4710．570．44756．6874Weihua JIN et al．Identification of the Diterpenoids Produced by Endophytic Fungus of Torreya fargesii and Its Inhibitory Effect on Hela CellsThe OD value calculation showed that taxol or taxol analogues produced by the strain had obvious inhibitory effect on Hela cells，and the inhibition rate increased with the increase of concentration．3．6Effects of precursors on the production of taxol metabo-lites by endophytic fungi Three precursor solutions，sodium benzoate，sodium acetate and L-phenylalanine，were prepared．Penicillium BP6T3was inoculated in PDA liquid medium at28ħfor180r/min．On the5th day of fermentation，the three precur-sors were added to the fermentation medium．The final concentra-tions of L-phenylalanine solution，sodium acetate solution and so-dium benzoate solution were3．0mg/L，3．5g/L and32．0mg/L，respectively．Sucrose solution was added to each fermentation me-dium，and the final concentration was5．0g/L．On the10th day，the fat-soluble metabolites of Penicillium BP6T3were extracted．High performance liquid chromatography （HPLC）was used to detect taxol in metabolic extracts qualitative-ly and quantitatively，and the growth rate of taxol was calculated （Table3）．It can be seen that with L-phenylalanine as precursor，the yield of taxol increased most，with a growth rate of425．17%．Table3Effects of different precursors on taxol produced by strain BP6T3Precursor materials Peak area Growth rate∥%CK12887．11－L-phenylalanine67678．78425．17Sodium acetate35735．06177．29Sodium benzoate35774．63177．604DiscussionsIn this study，endophytic fungi were isolated from the tissues of Torreya fargesii．Endophytic fungi with the ability to produce taxol were isolated from plants other than Taxus，which greatly expanded the range of resources to produce taxol or analogues．This is con-ducive to the wider use of microbial resources for the production of antitumor drugs［18－20］．Some studies have shown that the content of taxol in fermentation broth can reach more than9mg/L，and the cost recovery can be guaranteed in production［9－10］．At present，the main reason why endophytic fungus cannot be used in produc-tion is still the too low yield，generally only a few hundred micro-grams per liter［14－15］．The strain BP6T3obtained in this study could produce taxol analogues very stably．The content of taxol an-alogues in the fermentation broth was as high as16．59mg/L．This was higher than the previously reported taxol production from endo-phytic fungus［9－14］．The taxol produced had obvious inhibitory effect on Hela cells and increased with the increase of concentra-tion．Moreover，different precursors had obvious effects on the pro-duction of taxol metabolites by this strain，and the yield increased most when L-phenylalanine was used as the precursor．Therefore，the fermentation of taxol or analogues by the plant endophyte Peni-cillium BP6T3obtained in this study can be carried out in a short time and on a large scale without the limitation of resources，envi-ronment，conditions，equipment and so on．Ｒeferences［1］ＲEN YY，WANG P，ZAN N，et al．A survey of studies on activities and toxicities of diterpenoid alkaloids［J］．Chinese Journal of Experimental Traditional Medical Formulae，2010，16（11）：210－213．［2］MA XC，WU LJ，JIA JM．Development in the research on toxoids［J］．Journal of Shenyang Pharmaceutical University，2002，19（2）：147－156．［3］STIEＲLE A，STIEＲLE D，STＲOBEL G，et al．Endophytic fungi of Pa-cific yew（Taxus brevifolia），as a source of taxol，taxanes，and other pharmacophores［M］．ACS Symposium Series，1994，25（42）：64－77．［4］GUO BH，WANG YC，ZHOU XW，et al．An endophytic taxol-producing fungus BT2isolated from Taxus chinensis var．mairei［J］．African Journal of Biotechnology，2006，5（10）：875－877．［5］MA TY，DONG ZL．The study of isolating endophytic fungus synthesizing taxol from plant［J］．Journal of Northwest University（Natural Science Edition），1999，29（1）：47－49．［6］ZHOU DP，SUN JQ，YU HY，et al．Nodulisporium，a genus new to China［J］．Mycosystema，2001，20（2）：277－278．［7］ZHOU DP，PING WX，SUN JQ，et al．Isolation of taxol producing fungi ［J］．Journal of Microbiology，2001，21（1）：18－19．［8］QIAO W，LING F，YU L，et al．Enhancing taxol production in a novel endophytic fungus，Aspergillus aculeatinus Tax-6，isolated from Taxus chinensis var．mairei［J］．Fungal Biology，2017，121（12）：1037－1044．［9］ZAMIＲLO，NEDEA ME，GAＲALAU FX．Biosynthetic building blocks of Taxus cannadensis taxanes［J］．Tetrahedron Letters，1992，33（36）：5235－5237．［10］SHEN P，CHEN XD，et al．Experiment of microbiology（version5）［M］．Beijing：Higher Education Press，2007：241－242．［11］KONG HZ．Chinese fungi Penicillium and related sexual type（35）［M］．Beijing：Science Press，2007：5－10．［12］KASAEI A，MOBINIDEHKOＲDI M，MAHJOUBI F，et al．Isolation of taxol-producing endophytic fungi from Iranian yew through novel molecu-lar approach and their effects on human breast cancer cell line［J］．Cur-rent Microbiology，2017，74（6）：702－709．［13］JAYANTHI G．Isolation and characterization of anticancer compound，taxol from an endophytic fungus Phomopsis longicolla［J］．FEBS Letters，2017，316（2）：147－151．［14］PATEL VK，SAＲIM KM，PATEL AK，et al．Chapter14Synthetic mi-crobial ecology and nanotechnology for the production of taxol and its pre-cursors：A step towards sustainable production of cancer therapeutics ［J］．Design of Nanostructures for Theranostics Applications，2018：563－587．84Medicinal Plant2019。