农杆菌介导的红曲菌T-DNA插入突变库的构建及色素突变子的性质分析

以农杆菌EHA105为介导,将带有潮霉素抗性基因和gus基因的T-DNA片段转化到红色红曲菌(Monascusruber)中。通过转化条件的优化,成功构建了含有5132个转化子的T-DNA插入突变库。根据菌落颜色与色素分泌情况筛选到50株色素突变子,聚合酶链式反应(PCR)表明上述突变子均有T-DNA片段插入。经5代的继代培养后,94%的突变子具有稳定性(潮霉素抗性)。对突变子产色素和桔霉素能力的分析表明其分泌次生代谢产物的能力发生了较大变化。本方法的建立,为进一步深入研究红曲菌的代谢调节和基因功能奠定了基础。     

Purification and characterization of a chemotolerant alcohol dehydrogenase applicable to coupled redox reactions

The purification and characterization of an organic solvent tolerant, NADH-dependent medium-chain secondary alcohol dehydrogenase (denoted sec-ADH "A") from Rhodococcus ruber DSM 44541 is reported. The enzyme can withstand elevated concentrations of organic solvents, such as acetone (up to 50% v/v) and 2-propanol (up to 80% v/v). Thus, it is ideally suited for the preparative-scale enantioselective oxidation of sec-alcohol and the asymmetric reduction of ketones, using acetone and 2-propanol, respectively, as cosubstrates for cofactor-regeneration via a coupled-substrate approach. The homodimeric protein was found to bear tightly bound zinc and displayed a molecular mass of 38 kDa per subunit as determined by SDS gel electrophoresis. The optimal temperature ranged from 30-50 degrees C and the half-life at 50 degrees C was 35 h. In addition, excellent storage stability was found. The pH optimum for reduction is pH 6.5; pH 9.0 is preferred for oxidation. The enzyme followed a sequential reaction mechanism. The substrates are medium chain sec-alcohols or (omega-1)-ketones; primary alcohols or aldehydes are not accepted.     

氨基酸对于Monascus ruber生物合成Monacolin K影响的研究

在Monacolin K发酵中添加氨基酸后发现较高质量浓度的氨基酸高度抑制了Monacolin K的产量。0.1 g.L-1的D-甲硫氨酸在发酵第4 d添加可以提高产量30%以上,而L-甲硫氨酸则没有此功能,D,L-甲硫氨酸因D-甲硫氨酸的关系也有一定的增产效果。以甲硫氨酸代替蛋白胨作为主要氮源,则抑制了polyketide途径的前期步骤,因此严重抑制了色素及Monacolin K的生产。另外,发现1 g.L-1的L-苯丙氨酸的添加时间越早越有利于Monacolin K的生产,在起始时添加发酵单位可达135.9 mg.L-1,可能是因为L-苯丙氨酸经过脱氨后,可以进入polyketide途径从而促进了Monacolin K的生产。     

Genetic races associated with the genera and sections of host species in the holoparasitic plant Cytinus (Cytinaceae) in the Western Mediterranean basin

* Speciation via race formation is an important evolutionary process in parasites, producing changes that favour their development on particular host species. Here, the holoparasitic plant Cytinus, which has diverse host species in the family Cistaceae, has been used to study the occurrence of such races. * Amplified fragment length polymorphism (AFLP) analyses were performed on 174 individuals of 22 populations parasitizing 10 Cistaceae species in the Western Mediterranean basin. * Neighbour-joining, multivariate ordination analyses, and individual-based Bayesian analyses, clustered Cytinus populations into five well-characterized genetic races that, overall, agreed with the taxonomic sections of their hosts. In the AMOVA, among-races differences accounted for almost 50% of the genetic variation. The isolation-by-distance model was not supported by a Mantel test among Cytinus populations (r = 0.012; P = 0.456). All races showed low within-population genetic diversity, probably as a result of restricted pollen flow aggravated by flowering asynchrony, restricted seed dispersion, or stochastic processes. * The genetic differentiation among the five races of Cytinus is congruent with the view that these races are well-characterized lineages that have evolved independently as a result of selective pressures imposed by their hosts. This pattern, with genetically distinctive groups associated with the infrageneric sections of the host species, has not been reported previously for parasitic angiosperms.     

A photographic census of flamingos in the Rift Valley lakes of Tanzania

An aerial photographic census of flamingos ( Phoenicopterus minor and P. ruber) was conducted on 20 July 1994 on nine lakes in Tanzania: Lakes Natron, Manyara, Magadi (Ngorongoro), Burungi, Embagai, Eyasi, Lagarja, Masek, and Ambussel. This census, conducted concurrently with a survey in Kenya, documented the numbers and distribution of flamingos in the Rift Valley Lakes of East Africa. The total population size of flamingos in the Tanzanian lakes surveyed was |mF907,000. The largest concentrations in Tanzania were found in Lake Natron and Embagai Crater Lake. Although flamingos are known to breed in Lake Natron, no evidence of breeding was observed during the survey. The total estimate for the southern Kenya and northern Tanzania Rift Valley was |mF2·8 million. Trends in flamingo populations, survey methodology, and future monitoring needs are discussed.     

Biosynthesis of novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126 and related bacteria

Rhodococcus ruber and related Gram-positive bacteria synthesized and accumulated novel copolyesters containing 3-hydroxypivalic acid as constituent if the cells were cultivated in a mineral salts medium containing 3-hydroxypivalic acid and glucose as carbon sources. The copolyesters contributed 0.4–10% of the cellular dry mass, and they contained up to 78 mol% of 3-hydroxypivalic acid in addition to 3-hydroxybutyric acid and 3-hydroxyvaleric acid; a homopolyester of 3-hydroxypivalic acid was also synthesized under certain conditions. The presence of 3-hydroxypivalic acid in the accumulated copolyesters was confirmed by nuclear magnetic resonance spectrometry as well by coupled gas chromatography/mass spectrometry. This is the first time that the incorporation of 3-hydroxypivalic acid and therefore of a hydroxyalkanoic acid with two methyl group substituents at the α-carbon atom in a naturally occurring copolyester is reported. It indicates that 3-hydroxypivalic acid-coenzyme A is accepted by polyhydroxyalkanoic acid synthase as a substrate.     

红色亚栖热菌TPS/TPP海藻糖合成途径中相关基因的克隆、表达及功能鉴定

通过构建红色亚栖热菌(Meiothermus ruberCBS-01)的基因组DNA文库,克隆得到该嗜热菌海藻糖合成途径中的磷酸海藻糖合成酶(TPS)和磷酸海藻糖磷酸酯酶(TPP)基因。以pET21a为表达载体,将磷酸海藻糖合成酶和磷酸海藻糖磷酸酯酶在大肠杆菌中进行表达并纯化,利用薄层层析的方法验证了这两个酶的活性。同时,本研究检测了红色亚栖热菌在各种环境压力下细胞内含物成分的变化情况,发现在高渗环境压力的诱导下,该菌会在胞内积累大量的6-磷酸海藻糖,而并非海藻糖,这为进一步研究TPS/TPP和TreS途径在细胞体内的作用奠定了基础。     

根癌农杆菌介导的定点敲除技术在红色红曲菌中的应用

红曲菌(Monascus spp.)是一种重要的丝状真菌, 广泛应用于食品和医药领域中。目前, 由于对红曲菌遗传背景了解的很少, 因而对其重要功能基因的研究报道很少。本研究采用根癌农杆菌介导的转化技术对红色红曲菌(Monascus ruber)中推断的G-蛋白信号调节子mrfA基因的RGS功能域进行定点缺失研究, 探讨基于同源重组为基础的基因定点缺失技术在红曲菌基因功能鉴定中的可行性。构建的敲除载体pC805S左右同源臂长度分别为958 bp和824 bp, 将其转入受体菌M. ruber 中, 得到的138株转化子中, 有26株转化子发生了同源重组, 重组率达到18.8%。实验结果表明该技术作为红曲菌基因功能鉴定的方法是可行的。     

Temporal and spatial variation in dispersal in the greater flamingo (Phoenicopterus ruber roseus)

We studied movements of individually marked greater flamingos (Phoenicopterus ruber roseus) born in the Camargue, southern France, between their two most important breeding colonies in the western Mediterranean (Camargue and Fuente de Piedra, Spain) from 1986 to 1992. The two sites differ in the frequency with which they offer suitable conditions for breeding. Flamingos have bred each year in the Camargue since 1974, but in only 12 of the past 22 years at Fuente de Piedra. Higher colony fidelity is thus expected in the less variable environment (Camargue), but if dispersal occurs competition might be an important factor causing this dispersal. Following years during which breeding birds in the Camrgue were disturbed (1988 and 1990) a higher proportion of adults changed colonies between breeding attempts (= breeding dispersal, 12.4%), while only 0.4% of flamingos breeding in the Camargue dispersed in the other years. As expected, flamingos breeding at Fuente de Piedra showed a higher rate of breeding dispersal (8.14%). No differences were observed between males and females. The importance of breeding failure as a factor causing breeding dispersal in flamingos was also confirmed by the movements of individual birds. The proportion of young flamingos that moved from their natal colony to start breeding at Fuente de Piedra (= natal dispersal) was independent of sex and age, but increased when breeding access to the Camargue colony was more difficult. However, natal dispersal was also higher in 1988 and 1990 (40.5%) than in the remaining years (1.2%), as was breeding dispersal. We discuss possible ways in which the increased natal dispersal among inexperienced birds could be linked with the increased breeding dispersal of adults in the same year.