慈姑属4种同域分布植物的人工种间杂交

泽泻科（Alismataceae）是现存最原始的单子叶植物类群之一,慈姑属（Sagittaria）是泽泻科相对进化的分类群,地域分布广泛,其物种大多集中分布于北温带,且有种间同域分布现象。为了探究同域分布物种之间的杂交可能性,我们通过选取同域分布且在系统进化树上具有代表性的冠果草（S.guayanensis subsp. lappula）、小慈姑（S. potamogetonifolia）、利川慈姑（S. lichuanensis）和野慈姑（S. trifolia）为研究对象,调研中国数字植物标本馆（CVH）的标本信息和已有文献,分析这4种慈姑的地理分布格局,并分别以这4种慈姑为母本进行人工授粉实验以检验种间杂交的可能性。基于对1 172份4种慈姑属腊叶标本的分析,表明这4种慈姑在福建省武夷山地区存在同域分布情况。通过对4种慈姑之间的人工授粉实验结果分析表明,冠果草、小慈姑、利川慈姑和野慈姑异种授粉后均出现心皮膨大现象,所有异种授粉产生的种子均不可育,败育胚种子比例各不相同,且该比例与父本和母本在系统进化树上的位置有关。     

Expression and purification of homogenous proteins in Saccharomyces cerevisiae based on ubiquitin-FLAG fusion

The construction of an expression vector for increased expression of cytoplasmic proteins in Saccharomyces cerevisiae is described. To enhance the yield of expressed proteins, fusion of ubiquitin to an octapeptide (a FLAG tag) upstream of the respective model genes was applied. During protein maturation ubiquitin is efficiently removed by yeast autologous hydrolases, generating the FLAG octapeptide at the N-terminus. Fusion proteins were recognized by the specific monoclonal antibody M1 directed against the FLAG tag. The FLAG-tagged proteins were purified to homogeneity by immunoaffinity chromatography using an anti-FLAG M1 agarose. Different model proteins, green fluorescent protein, green fluorescent protein-human lysozyme, green fluorescent protein elongation-initiahon factor 5a, green fluorescent protein-rapamycin-selective 25-kDa immunophilin, and green fluorescent protein-heat shock protein 90 beta have been selected to demonstrate the efficiency of the new vector construct.     

中国盾盘菌属分类研究概况

本文对中国已记载的盾盘菌属成员名称做了分类和命名方面的评述,报道该属六个中国新记录种,须孢盘菌广西变型描述为新变型。     

Septoria的新种和国内新记录种

作者在1987-1991年间从东北地区收集和采集Septoria真菌标本221份,鉴定出Septoria 84种,其中2个新种:Septoria dioscorical Lu Guo-zhong et Bai Jin-kai和Septoria saposhinikoviae Lu Guo-zhong et Bai Jin-kai,以及13个国内新记录种. Septoria属真菌的产孢方式为全壁芽生合轴式(holoblastic sympodial)。     

异源表达海苏特氏菌Argonaute蛋白基因提高酿酒酵母脂肪酸含量研究

Argonaute蛋白作为一类高度保守的蛋白,是小RNA调控代谢机制的重要参与者,是RNA干扰所必须的,然而此蛋白在酿酒酵母中却未被发现。为初步探究海苏特氏菌Argonaute蛋白基因对脂肪酸含量的影响,以海苏特氏菌全基因组为模板,克隆并表达了海苏特氏菌Argonaute蛋白基因的CDS区。生物信息学分析发现该基因具有基因沉默调节因子SIR2结构域。将海苏特氏菌Argonaute蛋白基因连接至载体p ZMG中获得重组载体p ZMG-Jm-Argonaute并转化至酿酒酵母BY4742中,GC-MS检测显示,重组菌株总脂肪酸含量相对于野生菌株显著提高了6. 4%,其中不饱和脂肪酸C161含量显著提高了8. 3%,结果表明Jm-Argonaute蛋白基因能够提高酿酒酵母BY4742不饱和脂肪酸C161的含量,从而为深入研究该基因在菌体内对脂肪酸代谢的调节机制提供参考。     

木糖醇脱氢酶基因高拷贝表达载体构建及在酿酒酵母中的表达

木糖醇脱氢酶（xylitol dehydrogenase, XDH）可以氧化木糖醇生成木酮糖,处于木糖代谢的节点位置。利用PCR方法克隆得到了休哈塔假丝酵母（Candida shehatae） 20335的木糖醇脱氢酶基因、质粒pKT0150的ADH1终止子序列和G418抗性基因（KanR）,以及酿酒酵母(Saccharomyces cerevisiae) W5特定的2.2 kb的rDNA片段。以酿酒酵母整合载体p406ADH1为骨架,利用基因工程手段构建一个多拷贝整合表达载体pLX-AGRX。将重组载体pLX-AGRX线性化转入到酿酒酵母W5后,通过高浓度G418筛选和PCR双重鉴定,证实重组载体pLX-AGRX已整合到酿酒酵母W5基因组上,测定木糖醇脱氢酶酶活可达65.957 4 U/mg。     

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

宿主遗传背景对重组酿酒酵母木糖共发酵影响的研究进展

木糖是纤维素原料水解液中最主要的五碳糖成分,由于野生的酿酒酵母缺乏有效的木糖利用途径,将外源木糖代谢途径整合至酿酒酵母中使其具有发酵木糖生产乙醇的能力是构建纤维素乙醇发酵菌株的关键。国内外学者的研究表明,同一木糖代谢途径导入不同酿酒酵母菌株中,所得到的重组菌发酵性能存在明显差异,表明宿主的遗传背景对菌株利用木糖能力和发酵性能具有重要的影响。就酿酒酵母宿主对重组菌株的木糖发酵性能的影响进行了综述,分析了产生宿主差异的内在机理,为进一步选育高效木糖共发酵菌种提供借鉴。     

Electron paramagnetic resonance studies and effects of vanadium in Saccharomyces cerevisiae

Vanadium uptake by whole cells and isolated cell walls of the yeast Saccharomyces cerevisiae was studied. When orthovanadate was added to wild-type S. cerevisiae cells growing in rich medium, growth was inhibited as a function of the VO₄ ^3- concentration and the growth was completely arrested at a concentration of 20 mM of VO₄ ^3- in YEPD. Electron paramagnetic resonance (EPR) spectroscopy was used to obtain structural and dynamic information about the cell-associated paramagnetic vanadyl ion. The presence of EPR signals indicated that vanadate was reduced by whole cells to the vanadyl ion. On the contrary, no EPR signals were detected after interaction of vanadate with isolated cell walls. A mobile and an immobile species associated in cells with small chelates and with macromolecular sites, respectively, were identified. The value of rotational correlation time _r indicated the relative motional freedom at the macromolecular site. A strongly immobilized vanadyl species bound to polar sites mainly through coulombic attractions was detected after interaction of VO²⁺ ions with isolated cell walls.     

Effect of pH,aeration and sucrose feeding on the invertase activity of intactS. cerevisiae cells grown in sugarcane blackstrap molasses

S. cerevisiae was grown in a blackstrap molasses containing medium in batch and fed-batch cultures. The following parameters were varied: pH (from 4.0 to 6.5), dissolved oxygen (DO) (from 0 to 5.0 mg O₂L^–1) and sucrose feeding rate. When glucose concentration (S) was higher than 0.5 g L^–1 a reduction in the specific invertase activity of intact cells (v) and an oscillatory behavior of v values during fermentation were observed. Both the invertase reduction and the oscillatory behavior of v values could be related to the glucose inhibitory effect on invertase biosynthesis. The best culture conditions for attainingS. cerevisiae cells suitable for invertase production were: temperature=30°C; pH=5.0; DO=3.3 mg O₂L^–1; (S)=0.5 g L^–1 and sucrose added into the fermenter according to the equations: (V–V_o)=t²/16 or (V–V_o)=(V_f–V_o)·(e^0.6t–1)/10.This work was supported by FAPESP     

The molecular genetic differentiation of cultured <Emphasis Type="Italic">Saccharomyces</Emphasis> strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that bakers yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae × S. bayanus var. uvarum hybrids. The molecular karyotyping of bakers, brewers, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)₅ can be used to study the populations of cultured S. cerevisiae strains.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 215–223.Original Russian Text Copyright © 2005 by Naumova, Zholudeva, Martynenko, Naumov.     

Antioxidant activity of extracts from leaves and roots of Salvia miltiorrhiza Bunge, S. przewalskii Maxim., and S. verticillata L

Three Salvia species have been studied for antioxidant activity in methanol extracts from roots and leaves. The presence of the polyphenols and tanshinones was screened by HPLC and spectrophotometric assays and related to the antioxidant potential. The antioxidant capacity of the studied species is high, but differences between species and organs have been also revealed. Salvia przewalskii leaf extract was the strongest one in all tests, followed by Salvia miltiorrhiza root and Salvia verticillata leaf. Among the roots, the most active was S. miltiorrhiza extract, followed by S. verticillata. The antioxidant activity correlates to the total polyphenol and, depending on the assay, to the hydroxycinnamic acids content. The high content of tanshinones in both S. miltiorrhiza and S. przewalskii roots is unlikely to contribute to the antioxidant activity.     

Pollen Aerobiological Monitoring with the personal volumetric air sampler (PVAS). Correlation with a fixed Hirst type sampling station

Summary Monitorings both with a fixed Hirst type volumetric sampler, placed on the balcony of the Medicine Division in Lanciano City Hospital and with a portable volumetric sampler PVAS type by Burkard, placed outside the dwelling-houses of the examined patients have been carried out simulaneously. This has given us further information about the quantity of pollen that single patients affected by pollinosis inhale. Preliminary controls on the capture efficiency of both samplers have been conducted. The results of this survey have shown that the symptom score cards of the eight patients enrolled for the study, have always been well correlated with the average daily pollen concentration of the fixed station (Pearson correlation), and the data obtained by the two samplers have pointed out remarkable differences in the pollen concentration. According to the PVAS, placed near patients, the concentrations were twofold to sixfold as compared to those of the fixed station. This was probably due to the different height of the samplers. However, owing to the short sampling time (30 min.), we aren't able to draw any conclusions. Nevertheless we do hope that more and more patients will undergo this test, so that with several daily surveys over longer periods, we will have more information on the real pollen exposure of single patients. We will verify the threshold of symptoms onset, which is only theoretical nowadays, in the monosensitized patients. Such a program will allow us to carry on useful research both in the respiratory physiopathological and in the allergological fields.