MSPD-HPLC法分析云南红豆杉中的10-DAB Ⅲ和紫杉醇

为建立云南红豆杉(Taxus yunnanensis)中10-去乙酰基巴卡亭Ⅲ(10-DAB Ⅲ)和紫杉醇(Taxol)的分析方法,该研究采用基质固相萃取-高效液相色谱分析(MSPD-HPLC)对云南红豆杉中的10-DAB Ⅲ及Taxol进行定量分析,探究硅胶、佛罗里硅土、酸性氧化铝、中性氧化铝、碱性氧化铝、C₁₈、C₁₈-ME、C₁₈-G1、C₁₈-HC、Diol、Xamide、Xion 共12种样品预处理分散剂及各种分散剂的质量、洗脱剂的种类、洗脱剂的浓度、洗脱剂体积对两种成分分析的影响,并对优化后的条件进行方法学验证,同时与超声提取、热回流提取预处理方法进行比较分析证明其有效性。结果表明:(1)12种固相分散剂中,碱性氧化铝具有良好的目标化合物萃取物检出量,并且当碱性氧化铝与样品的质量比为3:1、6 mL甲醇为洗脱剂洗脱时,10-DAB Ⅲ和Taxol的萃取检出量较高。(2)建立的云南红豆杉枝叶中10-DAB Ⅲ和Taxol的分析方法具有良好的线性关系(r≥0.999 9),10-DAB Ⅲ和Taxol的检出限和定量限分别介于0.023 9～0.030 1 μg·mL^-1和0.142～0.178 μg·mL^-1之间; 各目标分析物平均加样回收率在93.6%～109.0%之间。(3)比较分析显示,3种方法对2种紫杉烷类化合物的萃取检出量基本无差别,但MSPD法溶剂消耗少、操作简单,分析时间短,净化效率高,可应用于云南红豆杉原料的快速分析。该研究以碱性氧化铝为分散剂应用于云南红豆杉紫杉烷类化合物的萃取,建立了快速、高效的云南红豆杉中10-DAB Ⅲ和紫杉醇的分析方法,为云南红豆杉中的紫杉烷类化合物的定量分析提供了依据。     

用Blue Sepharose CL-6B快速纯化天花粉蛋白

差光谱显示染料cibacron blue F3GA与天花粉蛋白(TCS)有特异性结合,复合物在可见光部分的最大吸收波长在690 nm,摩尔消光系数ε=2.6×10^-3(mol/L)^-1·cm^-1,解离常数K_d=1.8 μmol/L,0.5 mol/L NaCl可使复合物解离.根据这一特点,用Blue-Sepharose CL-6B凝胶从栝篓块茎中亲和纯化了TCS.此法快速、简便、高效,易于大量制备.     

Reaction of winter oilseed rape callus to different concentrations of elicitors: pectinase or chitosan

The aim of the presented methodical experiments was 1) the evaluation if callus of winter oilseed rape (Brassica napus var. oleifera L.) initiates a defence reaction to fungal elicitors: pectinase (polygalacturonase) or chitosan, and 2) the choice of the elicitor doses, which evoke the strongest tissue reaction. The results obtained will be used in the next experiments relating the studies of pathogenesis mechanisms proceeding in rape plants infected by necrotrophic fungi. The defence response was estimated on the basis of changes in electrolyte leakage from cells, metabolic efficiency, phenolic content and catalase activity. In the experiment pectinase was used at concentration of 3, 8, 16, 133 and 166 μl per 1 cm³ of culture medium while chitosan at: 25, 50, 75 and 100 μg·cm⁻³. Both elicitors increased cell membrane permeability: pectinase at the doses equal or greater to 16 μl·cm⁻³ while chitosan of 25 μg·cm⁻³. The greatest metabolic inefficiency was observed in calli elicited with 16 μl·cm⁻³ pectinase and with chitosan of 100 μg·cm⁻³. The decrease in phenolic content was noted under influence of most doses of both elicitors. The highest catalase activity was evoked by pectinase of 8 μl·cm⁻³ and chitosan of 75 and 100 μg·cm⁻³. The results indicated that 8–16 μl·cm⁻³ of pectinase and 100 μg·cm⁻³ of chitosan caused the strongest defence reaction of oilseed rape tissue.     

没食子酸对水稻细菌性条斑病菌细胞结构的影响

没食子酸(gallic acid, GA)是一种植物酚类化合物,具有多种生物活性。前期实验发现,GA对水稻细菌性条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)具有较强的抑制作用。为了解该物质对Xoc的细胞结构和细胞膜的影响,该研究用电子显微镜观察GA对Xoc的形态结构的影响,通过测定GA处理后的Xoc培养液的电导率、紫外吸收物含量(260 nm的吸光值)、乳酸脱氢酶的活性以及菌体的二乙酸荧光素(fluorescein diacetate, FDA)的强度等,探讨了GA对Xoc细胞膜的完整性和通透性的影响。结果表明:经浓度为200 μg·mL^-1的 GA处理后,Xoc的菌体形态结构发生改变,表面有明显的凹陷或不规则囊泡状突起,表明GA对Xoc细胞壁有损伤作用。200 μg·mL^-1的GA处理24 h后,病菌培养液的电导率为135.48 μS·cm^-1(对照处理为127.85 μS·cm^-1)。GA处理2 h后,Xoc细胞荧光强度下降58.10%,说明病菌细胞内电解质外渗和细胞溶质发生渗漏; 同时,乳酸脱氢酶的活性增加,表明菌体的细胞膜受到破坏。此外,GA处理24 h后,Xoc培养液在260 nm下的吸光值为1.004(对照处理为0.018),表明病菌细胞膜的完整性受到破坏。这表明GA不仅破坏Xoc的细胞膜通透性,而且还影响膜的完整性。     

模拟氮沉降对石栎和苦槠幼苗土壤呼吸的影响

用LI-8100开路式土壤碳通量测量系统测定模拟氮沉降4种不同处理水平(0、60、120\,240 kg · hm^-2 · a^-1)下石栎(Lithocarpus glabra)和苦槠(Castanopsis sclerophylla)幼苗的土壤呼吸速率及土壤温度、含水量对其土壤呼吸的影响。结果表明,氮沉降对土壤呼吸的影响根据施氮水平和幼苗的种类不同而异。低氮(60 kg · hm^-2 · a^-1)处理下石栎和苦槠的土壤呼吸速率平均值分别为(4.014±0.812)μmol · m^-2 · s^-1和(5.170±0.689)μmol · m^-2 · s^-1,比对照组(0 kg · hm^-2 · a^-1)土壤呼吸速率平均值(3.802±0.948)μmol · m^-2 · s^-1和(3.557±0.906)μmol · m^-2 · s^-1分别高5%和45%;两树种在中、高氮处理下均出现对土壤呼吸明显的抑制。其中石栎中、高氮实验组的土壤呼吸速率分别为(2.653±0.681)μmol · m^-2 · s^-1、(2.592±0.736)μmol · m^-2 · s^-1, 比对照组低27%和29%。苦槠中、高氮实验组的土壤呼吸速率为(3.563±0.402)μmol · m^-2 · s^-1、(3.466±0.994)μmol · m^-2 · s^-1, 比对照组低7%和8%;石栎在高氮(240 kg · hm^-2 · a^-1)处理水平下,其土壤呼吸速率同10cm土壤温度之间呈现显著的指数关系(R²=0.811,P=0.001),而在低、中氮实验均未发现有明显指数关系。苦槠各处理水平下其土壤呼吸与土壤温度之间均未发现有明显的指数关系;在土壤呼吸与5cm土壤含水量的相关性方面,仅有苦槠高氮实验组表现出明显的二次方程关系(R²=0.722),而其低、中氮实验组及石栎各实验组均未有明显的相关性;与单因素(温度、含水量)拟合它们与土壤呼吸速率的方程相比,多元回归分析得到的土壤呼吸速率同土壤温度和含水量之间的拟合方程在P=0.05水平上能更好地解释土壤呼吸的变化情况。石栎和苦槠在氮沉降处理下的土壤呼吸温度系数Q₁₀值分别为2.29、1.95、1.59和1.46、1.41、1.76,同对照组2.64和1.78相比,均有明显降低,且两者Q₁₀值的变化分别呈递减和先减小后增大的趋势,表明氮沉降是影响石栎和苦槠土壤CO₂通量的一个重要因素。     

盐分胁迫下杠柳叶片光合特性的光响应研究

利用CID型便携式光合作用仪测定不同NaCl浓度下杠柳叶片净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(G_s)、水分利用效率（WUE）及光能利用率(LUE)生理参数的光响应过程,阐明盐分胁迫下其对光照响应的规律,探讨有利于杠柳正常生长的盐分浓度和光照条件。结果表明:（1）各盐分浓度下杠柳叶片光补偿点（LCP）在21.89～65.05 μmol·m^-2·s^-1之间变动,介于阴性植物与阳性植物之间;杠柳随土壤盐分的不同,其光合作用参数对光照强度表现出一定的适应性和可塑性;50 mmol·L^-1盐分浓度下杠柳光合同化能力最强,最有利于其干物质的积累,表现出一定的耐盐性。（2）轻度的盐分胁迫（小于50 mmol·L^-1）可以提高杠柳叶片的P_n、G_s、WUE和LUE,而盐分胁迫对杠柳的T_r有抑制作用,并随着盐分浓度的增加其抑制作用愈强烈。（3）维持杠柳正常生长的土壤盐分浓度小于50 mmol·L^-1,最佳PAR为1 000～2 000 μmol·m^-2·s^-1;而保持杠柳最大WUE和LUE的光照强度分别为800和100 μmol·m^-2·s^-1。     

不同地理种源南方红豆杉中紫杉醇和10-DAB含量及影响因子

选择福建省梁野山、龙栖山、闽侯旗山、南靖书洋乡和南平大坪村南方红豆杉（Taxus chinensis var. marei）天然群体分布相对集中区,采集胸径相同的南方红豆杉的当年生针叶和树皮并取树下土壤样品。利用超高效液相色谱分析测定南方红豆杉当年生针叶及树皮中紫杉醇及10-去乙酰基巴卡亭Ⅲ（10-deacetylbaccatin Ⅲ, 10-DAB）含量,另测定土壤pH值、速效磷、速效氮、铁、钙、镁、锌含量。结果表明,不论是紫杉醇还是10-DAB含量,不同地理种源南方红豆杉间差异均较大,说明地理位置对于红豆杉中紫杉醇和10-DAB含量具有明显的影响;树皮中紫杉醇含量与土壤中铁含量显著相关（P＜0.01）;南方红豆杉当年生针叶中及树皮中紫杉醇含量与各种源地气象因子的相关性不显著（P＞0.05）。     

γ-氨基丁酸(GABA)种子引发缓解辣椒盐胁迫的效果及生理生化的变化

种子引发是提高作物生长期耐盐性的有效方法,而γ-氨基丁酸(GABA)种子引发对辣椒(Capsicum annuum)耐盐性的效果和作用机制尚不清楚。该研究以‘茂蔬360''朝天椒为材料,分析了不同浓度γ-氨基丁酸(0、1.0、2.0、4.0、6.0、8.0 μmol·L^-1)种子引发对4～6叶期100 mmol·L^-1 NaCl胁迫下的植株生物量、渗透调节物质、抗氧化能力、光合作用系统及钾钠离子吸收的影响。结果表明:(1)种子引发能显著增加盐胁迫下辣椒植株的生物量,以6.0 μmol·L^-1 GABA引发处理的效果最佳。(2)种子引发处理增加了盐胁迫下植株的可溶性糖、可溶性蛋白及脯氨酸的含量,同时,O₂^-和MDA的含量下降,抗氧化酶(SOD、POD、CAT和APX)活性增强,叶片叶绿素含量增加,叶片叶绿素荧光参数受胁迫影响程度低,叶绿素荧光指标包括F_v ''/F_m ''、qP_Lss、QY_Lss、NPQ_Lss和Rfd均有所上升。根、茎中的K⁺含量和K⁺/Na⁺比值下降。(3)灰色关联度分析表明,GABA种子引发主要通过提高POD、CAT的活性和渗透调节物质含量来缓解盐胁迫对辣椒植株的伤害。综上所述,6.0 μmol·L^-1 GABA种子引发可有效提高辣椒苗期的耐盐性,其作用机制可能是提高了盐胁迫下辣椒植株的抗氧化能力和渗透调节能力。     

Hyporheic biofilms — a potential food source for interstitial animals

Glass-beads (diam. = 250 μm) were buried 10 cm deep in the sediment of a stream. After an exposure of eight weeks, bacterial densities on the beads varied between 2.7 × 10⁵ and 2.4 × 10⁷/cm², and the length of the fungal mycelium between 0.2 and 5.3 mm/cm². Bacterial densities did not show any correlation with the DOC content of the water, but were positively correlated with respiration on the beads. Fungal mycelium was negatively correlated with water temperature. Acid hydrolysis of stream-exposed beads released sugars and amino acids, whose combined carbon content exceeded that of the microbial cells by a factor of at least 4. Gut extracts of Gammarus tigrinus and Tipula caloptera released amino acids and sugars from stream-exposed beads.     

云南红豆杉培养细胞系的建立

紫杉醇(Taxol)最初是从红豆杉属植物短叶红豆杉(Taxus brevifolia)树皮中分离出的一种二萜类化合物^[1]．对卵巢癌,转移性乳腺癌和恶性黑色寮瘤等患者疗效显著^[2],全世界红豆杉属植物有近11种,都含紫杉醇成分．但含量很低,加之现存数量很少,生长极为缓慢．造成了紫杉醇原料供应的危机^[3]。紫杉醇化学合成已经成功^[4-6],但繁杂的反应过程及前体化合物来源的限制使得它们无法实现商业化生产。最近从短叶红豆杉中分离出一种生产紫杉醇的内寄生真菌Tgromyer andreanae^[7]．由于紫杉醇含量仅为24～50ng/L．没有实用价值。植物细胞和组织培养可能是解决天然抗肿瘤药物长期供应的有效方法之一^[8]。自1991年Christen等人申请利用红豆杉细胞培养物生产紫杉醇专利以来^[9]．有关红豆杉细胞培养的研究已有不少报^[10-12]。但云南红豆杉(T.yunnanensis)仅见愈伤组织诱导的报道^[13]。本文报道云南红豆杉愈伤组织诱导和细胞培养的初步结果,并分析了细胞培养物中紫杉醇含量。     

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.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.