红豆杉内生真菌中巴卡亭Ⅲ产生菌的筛选及培养基的初步优化

目的:筛选出产巴卡亭Ⅲ的红豆杉内生真菌,为抗癌药物紫杉醇提供一个新的来源。方法:从云南红豆杉树皮中分离纯化出内生真菌,对其进行液体发酵培养,过滤,收集菌丝体并破碎,用二氯甲烷萃取,萃取液进行薄层层析,与巴卡亭Ⅲ标准品有相同比移值的样品再利用高效液型色谱与标样进行比照,进而筛选出产巴卡亭Ⅲ的内生真菌。同时以发酵培养基基础,对筛选出的真菌进行碳源和氮源的优化。并在此基础上探讨几种常见的紫杉烷类化合物代谢诱导剂乙酸钠、苯甲酸钠、苯丙氨酸和亮氨酸的浓度对巴卡亭Ⅲ积累的影响。结果:得到一株产巴卡亭Ⅲ的内生真菌NSZJ043,该菌株易培养,生长迅速,经鉴定属于曲霉属。NsZJ043产巴卡亭Ⅲ的培养条件的初步优化结果表明:最适碳源、氮源分别是蔗糖和蛋白胨;在含20g/L蔗糖、1．5g/L蛋白胨、0．1 mmol/L乙酸钠、0．01mmol/L苯甲酸钠的优化培养基中培养8d,巴卡亭Ⅲ含量为34．6μg/L。结论:筛选出一株产巴卡亭Ⅲ的内生真菌,其具有优良的发酵特性,巴卡亭Ⅲ前体物质苯甲酸钠和乙酸钠对巴卡亭Ⅲ的合成有促进作用;苯丙氨酸和亮氨酸对其含量影响不显著,优化后NSZJ043产巴卡亭Ⅲ含量有较大的提高。     

产紫杉醇内生真菌N-15的分离及鉴定

产紫杉醇内生真菌的研究是解决紫杉醇药源紧缺问题的重要途径之一。本研究主要报道了从南方红豆杉树皮的内表皮中筛选得到一株产紫杉醇的内生真菌N-15,高效液相色谱法(high performance liquid chromatography,HPLC)检测结果表明其提取物具有与紫杉醇及其前体巴卡亭Ⅲ、10-去乙酰巴卡亭Ⅲ标准品一致的色谱特征峰和保留时间,表明真菌N-15可以产紫杉醇及其两个前体物。质谱法(mass spectrometry,MS)检测结果表明,N-15的发酵液提取物具有和紫杉醇标准品相同质谱特征峰,进一步说明N-15可以产紫杉醇。通过结合形态特征观察和18S rRNA序列分析,将N-15初步确定为黑孢霉属(Nigrospora sp.)真菌。     

南方红豆杉枝叶中6种紫杉烷类化合物含量季节变化

紫杉醇(taxol)是主要来源于红豆杉属植物的一种天然抗肿瘤药物,紫杉烷(taxane)是紫杉醇的代谢前体或支路代谢产物,同样具有开发成为抗肿瘤新药的潜质。本文采用高效液相色谱法研究了紫杉醇(Taxol)、10-去乙酰巴卡亭Ⅲ(10-DAB)、7-木-10-去乙酰紫杉醇(7-xyl-10-DAT)、10-去乙酰紫杉醇(10-DAT)、三尖杉宁碱(CE)和7-表-10-去乙酰紫杉醇(7-epi-10-DAT)6种紫杉烷类化合物在南方红豆杉枝叶中含量的季节变化,结果显示Taxol和10-DAT在8、9月份含量最低,10-DAB和7-xyl-10-DAT在8、9月份含量相对最高,Taxol含量季节变化和10-DAB呈负相关,与CE呈显著正相关。7-xyl-10-DAT含量季节变化和10-DAB、10-DAT分别呈正相关。本文为研究南方红豆杉中紫杉醇及相关紫杉烷的代谢、积累规律提供了依据,不但有助于阐明紫杉醇的生物合成的关键步骤及调控的生理机制,而且对红豆杉资源的深度开发具有指导意义。     

产紫杉醇内生真菌MHZ-32的鉴定

从曼地亚红豆杉树皮内表皮分离获得一株内生真菌MHZ-32,通过高效液相色谱法检测发现,内生真菌MHZ-32的紫杉醇提取物中含有与紫杉醇标品 (15.02 min）、巴卡亭Ⅲ标品 (7.07 min)保留时间相近的色谱特征峰. 进一步通过质谱法检测发现,MHZ-32的紫杉醇提取物中具有与紫杉醇标品((M+Na)+=876)、巴卡亭Ⅲ标品((M+Na)+=609)相同的质谱特征峰,表明内生真菌MHZ-32可以产紫杉醇和巴卡亭Ⅲ. 其紫杉醇和巴卡亭III的产量分别约为0.6 μg/g和0.2 μg/g（紫杉醇或巴卡亭Ⅲ/菌丝干重）.并通过18S rRNA序列分析和形态学鉴定,将内生真菌MHZ-32初步鉴定为拟茎点霉属（Phomopsis sp.）真菌.     

南方红豆杉10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶基因的克隆与生物信息学分析

对中国重要药用植物南方红豆杉中的10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶(DBAT)基因进行分离、测序以及生物信息学分析。根据GenBank中已登录的10-去乙酰巴卡亭Ⅲ-10-乙酰转移酶(DBAT)基因cDNA序列设计引物,采用RT-PCR技术从南方红豆杉叶片中克隆了1个DBAT基因Tc-DBAT的全长cDNA。结果显示,Tc-DBAT cDNA含有1个1 323 bp的开放阅读框(open reading frame,ORF),编码440个氨基酸,对应基因组序列含有1个内含子。Tc-DBAT蛋白N端含有5个N-酰基化作用位点和2个保守的N-糖基化作用位点,具有1个保守的B ig-1结构域,多个重要的磷酸化位点以及1个类似钙结合蛋白重复结构。序列同源性比较、系统发生分析以及蛋白质高级结构预测均表明,Tc-DBAT属于转移酶超家族(transferase superfamily),是一个具有乙酰转移酶活性的功能蛋白,能够催化10-去乙酰巴卡亭Ⅲ(10-DAB)10位的乙酰化,从而生成抗癌新药紫杉醇生物合成途径中的最后一个双萜中间体——巴卡亭Ⅲ。有望通过成功克隆和鉴定紫杉醇生物合成途径中的关键酶基因达到提高其半合...     

一株能水解巴卡亭ⅢC-10位乙酰基的成团泛菌的筛选和鉴定

10-脱乙酰巴卡亭Ⅲ是合成紫杉醇和多烯紫杉醇的前体。以巴卡亭Ⅲ为底物,结合TLC、HPLC、HPLC-MS分析方法,通过设计专门的筛选方法筛选产酶菌株,得到一株巴卡亭ⅢC-10位脱乙酰基酶产生菌株Z1-56。以形态特征、生理生化特征、16S rDNA序列分析作为菌株的鉴定手段,Z1-56被鉴定为成团泛菌(Pantoea agglomerans),首次发现成团泛菌产生巴卡亭ⅢC-10-脱乙酰酶。     

一株产紫杉醇内生真菌YN6的分离及鉴定

从云南红豆杉（Taxus yunnanensis）树皮内表皮中分离得到75株内生真菌,采用基于紫杉醇合成关键酶10-去乙酰巴卡亭Ⅲ-10-O-乙酰基转移酶（10-deacetylbaccatin Ⅲ-10-O-acetyl transferase,DBAT）和C-13苯丙氨基侧链CoA乙酰基转移酶（C-13-phenylpropanoid side chain-CoA acyltransferase,BAPT）基因为标志分子的快速筛选方法获得一株可产紫杉醇的内生真菌YN6,并通过高效液相色谱法和质谱法对其紫杉醇进行分析.同时,通过对内生真菌YN6的形态特征分析以及18S rDNA序列分析将其初步鉴定为拟盘多毛孢属（Pestalotiopsis sp.）真菌. 拟盘多毛孢YN6的紫杉醇产量约为120~140 μg/L,是目前已报道的紫杉醇产量较高的野生菌株之一. 拟盘多毛孢YN6的发现为微生物发酵生产紫杉醇提供了潜在的优良种质资源.     

巴卡亭ⅢC-10位乙酰基水解酶产生菌的筛选和鉴定

10-脱乙酰巴卡亭Ⅲ是合成紫杉醇和多烯紫杉醇的前体.以巴卡亭Ⅲ为底物,结合TLC、HPLC、HPLC-MS分析方法,通过设计专门的筛选方法筛选产酶菌株,得到一株巴卡亭ⅢC-10位脱乙酰基酶产生菌株Z1-34.以形态特征、生理生化特征、16S rDNA序列分析作为菌株的鉴定手段,Z1-34被鉴定为稻草假单胞菌(Pseudomonas straminea),首次发现稻草假单胞菌产生巴卡亭ⅢC-10-脱乙酰酶.     

红豆杉低温半致死温度和低温胁迫下紫杉烷含量

通过测定低温胁迫下红豆杉电阻抗参数和紫杉烷含量变化,确定适合红豆杉低温半致死温度测定的电阻抗参数,探讨低温对红豆杉紫杉烷生物合成的影响,为扩大红豆杉引种范围及利用低温条件提高红豆杉中紫杉醇含量奠定了理论基础。以云南红豆杉、南方红豆杉(山西长治)、中国红豆杉、曼地亚红豆杉和东北红豆杉为材料,分别采用电导率法和电阻抗法测定上述树种低温半致死温度,并利用LC-MS法测定低温胁迫下红豆杉叶片中紫杉烷含量和脱落酸(abscisic acid, ABA)含量。结果表明,5种红豆杉中,中国红豆杉、南方红豆杉(山西长治)、东北红豆杉和曼地亚红豆杉均能耐受-14.668--9.106℃低温;云南红豆杉能耐受-8.802--3.521℃低温,高于其他4种红豆杉。电阻抗τ_m参数与电导法测定的5种红豆杉一年生枝低温半致死温度显著相关,相关系数为0.912。低温胁迫下,红豆杉一年生叶片中紫杉醇含量高于当年生叶片。随着处理温度的下降,云南红豆杉和东北红豆杉一年生叶片中紫杉醇含量显著增加,中国红豆杉当年生叶片中巴卡亭Ⅲ含量和10-去乙酰基紫杉醇含量显著增加。此外,红豆杉叶片中ABA含量随温度的...     

南方红豆杉产紫杉醇内生真菌的分离

对从广东乳源县的南方红豆杉(Taxus chinensis var. mairei)内生真菌中分离和筛选产紫杉醇的内生真菌进行了研究。从南方红豆杉的树皮、茎部、叶片及叶片研磨物中分离纯化了145株内生真菌,对其中的53株内生真菌采用摇瓶发酵培养的方法筛选产紫杉醇的内生真菌。发酵物和菌丝体经研磨、离心、乙酸乙酯萃取和浓缩,经硅胶薄层层析(TLC),高效液相色谱(HPLC)以及液相色谱-质谱(HPLC-MS)分析和检测,结果表明,从茎部分离的1株内生真菌能够产紫杉醇或其异构体, 产量达180 μg L^-1。通过对产紫杉醇内生真菌进行诱变、筛选以及优化培养条件等措施,大规模培养生产紫杉醇是具有可行性的。     

Coccystes undoxylophus

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UeberAquila pennata undminuta

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Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Ester production in E. coli and C. acetobutylicum

Genetic engineering has improved the product yield of a variety of compounds by overexpressing, inactivating, or introducing new genes in microbial systems. The production of flavor-enhancing ester compounds is an emerging area of heterologous gene expression for desired product yield in Escherichia coli. Isoamyl acetate, butyl acetate, ethyl acetate, and butyl butyrate are reported here to be produced by expressing Saccharomyces cerevisiae genes ATF1 or ATF2 and the strawberry gene SAAT in E. coli when the appropriate substrates are provided. Increasing the concentration of alcohol added to the reaction generally resulted in increased ester production. ATF1 expression was found to produce more isoamyl acetate and butyl acetate than ATF2 expression or SAAT expression in the strains and culture conditions examined. Additionally, SAAT expression resulted in greater isoamyl acetate and butyl acetate production than ATF2 expression. Butyl butyrate is produced by cell-free extracts of E. coli harboring SAAT but not ATF1 or ATF2.     

Aquila pennata undminuta

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Chemical composition, in situ ruminal degradability and post-ruminal disappearance of dry matter and crude protein from the halophytic plants Kochia scoparia, Atriplex dimorphostegia, Suaeda arcuata and Gamanthus gamacarpus

Samples of Kochia (K. scoparia), Atriplex (A. dimorphostegia), Suaeda (S. arcuata) and Gamanthus (G. gamacarpus) were collected and analyzed for chemical composition including crude protein (CP), ether extract (EE), ash, neutral detergent fiber (NDFom), acid detergent fiber (ADFom), non-protein N (NPN), Ca, P, Na, K, Cl, Mg, Fe, Cu and Se. In addition, in situ ruminal degradability and post-ruminal disappearance of dry matter (DM) and CP of the samples using a mobile bag technique were determined. Results indicate that the chemical composition of Kochia and Atriplex was notably different from those of Suaeda and Gamanthus. All of these halophytic plants had high concentrations of Na, K, Cl, Cu and Se, and low levels of Ca, P and Mg. The rapidly degradable fractions of DM and CP (g/g) of Kochia (0.31 and 0.35, respectively) and Atriplex (0.39 and 0.50, respectively) were lower than for Suaeda (0.53 and 0.55, respectively) and Gamanthus (0.56 and 0.66, respectively). Ruminal DM and CP disappearance of Kochia (444 and 517 g/kg, respectively) and Atriplex (472 and 529 g/kg, respectively) were lower (P<0.05) than those of Suaeda (553 and 577 g/kg, respectively) and Gamanthus (663 and 677 g/kg, respectively) (P<0.05) using the mobile bag technique. Suaeda had the lowest (P<0.05) NDFom and ADFom disappearance (214 and 232 g/kg, respectively) in the rumen. Kochia scoparia and Atriplex dimorphostegia have more beneficial chemical nutritive components and digestible values versus Suaeda arcuata and Gamanthus gamacarpus.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

The role of the genesnrf EFG andccmFH in cytochromec biosynthesis inEscherichia coli

It has been suggested that two groups ofEscherichia coli genes, theccm genes located in the 47-min region and thenrfEFG genes in the 92-min region of the chromosome, are involved in cytochromec biosynthesis during anaerobic growth. The involvement of the products of these genes in cytochromec synthesis, assembly and secretion has now been investigated. Despite their similarity to other bacterial cytochromec assembly proteins, NrfE, F and G were found not to be required for the biosynthesis of any of thec-type cytochromes inE. coli. Furthermore, these proteins were not required for the secretion of the periplasmic cytochromes, cytochromec ₅₅₀ and cytochromec ₅₅₂, or for the correct targeting of the NapC and NrfB cytochromes to the cytoplasmic membrane. NrfE and NrfG are required for formate-dependent nitrite reduction (the Nrf pathway), which involves at least twoc-type cytochromes, cytochromec ₅₅₂ and NrfB, but NrfF is not essential for this pathway. Genes similar tonrfE, nrfF andnrfG are present in theE. coli nap-ccm locus at minute 47. CcmF is similar to NrfE, the N-terminal region of CcmH is similar to NrfF and the C-terminal portion of CcmH is similar to NrfG. In contrast to NrfF, the N-terminal, NrfF-like portion of CcmH is essential for the synthesis of allc-type cytochromes. Conversely, the NrfG-like C-terminal region of CcmH is not essential for cytochromec biosynthesis. The data are consistent with proposals from this and other laboratories that CcmF and CcmH form part of a haem lyase complex required to attach haemc to C-X-X-C-H haem-binding domains. In contrast, NrfE and NrfG are proposed to fulfill a more specialised role in the assembly of the formate-dependent nitrite reductase.