阿维链霉菌NRRL8165中bldAa的克隆及其对形态分化与阿维菌素合成的影响

bldA编码天蓝色链霉菌中唯一有效识别UUA亮氨酸密码的tRNA(Leu)UUA。通过构建阿维链霉菌NRRL8165基因组亚文库,筛选得到含有阿维链霉菌bldAa及其侧翼序列的克隆。利用λRED介导的PCRtargeting技术构建了bldAa的基因置换质粒pHL358,将其跨属接合转移进入阿维链霉菌NRRL8165,筛选得到bldAa基因置换菌株TW10。TW10表现为光秃表型,表明bldAa调控阿维链霉菌的形态分化。摇瓶发酵TW10菌株并对发酵产物进行HPLC分析,发现TW10菌株均不合成阿维菌素组分,提示阿维菌素的合成受bldAa调控;考察阿维菌素生物合成基因簇,其中aveA3和aveR含有TTA密码,它们的翻译可能受bldAa调控,与实验结果一致。     

阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

以野生型阿维链霉茵NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643.将pilL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷.通过Southern杂交分析,证明whiEa基因簇被置换.通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象.     

链霉菌调控因子bldA的作用机制研究进展

链霉菌是一类具有特殊的形态分化和次生代谢的放线菌。bldA编码链霉菌中唯一能有效识别亮氨酸密码子UUA的tRNA~(Leu),是链霉菌次生代谢途径的必需基因,目前对于其全局调控功能已有详细报道。bldA突变或缺失会导致链霉菌不能完成气生菌丝分化和抗生素合成,这种调控主要是在翻译水平上完成。UUA密码子缺少有效的tRNA翻译,导致mRNA翻译受影响。bldA自身也受到其下游靶基因的负反馈调控。此外,bldA还有很多尚不明确的调控机制,回答这些问题能帮助我们更好地理解抗生素合成途径,为构建有应用前景的菌株奠定理论基础。     

利用Tn5型转座突变系统筛选高产阿维菌素菌株*

目的: 转座突变技术是发现新功能基因和获得高产天然产物菌株的一种有效策略。通过理性设计和构建Tn5型转座突变系统,并将其应用于阿维链霉菌,筛选高产阿维菌素的工程菌株。方法: 在转座突变载体pUCTN转座插入片段的上游和下游分别引入链霉菌常用的强启动子kasOp*和P21,强化插入位置上游和下游基因的转录表达;在插入片段两端分别添加双向转录终止子T1和T2,有效终止插入序列两端靶基因的转录,引入强启动子和终止子的目的在于增强对转座突变株生理代谢活动的扰动。结果: 通过优化供体菌和受体菌的比例,转座效率显著提高。随机选择500株转座突变株进行发酵和阿维菌素产量测试,筛选到3株突变株的阿维菌素产量明显高于出发菌株产量的50%以上。结论: Tn5转座突变系统为研究阿维链霉菌的基因功能和生理代谢提供了有效的分子遗传工具。     

卡西霉素生物合成基因簇中调控基因calR1的功能

【背景】卡西霉素(calcimycin)是重要的离子载体抗生素,其生物合成基因簇已从教酒链霉菌NRRL3882的基因组DNA中成功克隆,但基因簇内的部分生物合成基因及调控基因的功能有待研究。【目的】研究卡西霉素产生菌教酒链霉菌NRRL3882中编码TylR家族同源转录调控蛋白的calR1基因的功能。【方法】通过PCR-targeting的方法,构建calR1基因敲除突变株及回补菌株,对突变菌株及回补菌株进行发酵,通过HPLC分析其代谢产物。利用荧光定量PCR检测ΔcalR1突变菌株和野生菌株的生物合成基因转录水平。【结果】calR1基因敲除突变株丧失产生卡西霉素的能力,但仍有中间产物噻唑霉素的积累,回补菌株中卡西霉素的产量有一定程度的恢复。RT-qPCR结果表明,卡西霉素合成相关的一些重要基因calC、calG、calU3等基因的表达量明显改变。【结论】TylR家族转录调控基因calR1是卡西霉素生物合成的调控基因。     

阿维莲霉菌中aveD基因缺失对阿维菌素合成的影响

利用aveD基因的缺失载体pCZ8(pKC1139::△aveD)对阿维菌素（Avermectin）的产生菌阿维链霉菌（Streptomyces avermitilis)76-9的aveD基因进行缺失获得aveD缺失突变株。经摇瓶发酵和HPLC检测,发现该突变株只产生阿链菌素B组分。说明将阿维链霉菌的aveD基因缺失,并不影响下游aveF的表达。缺失突变株的阿维菌素的总产量与出发菌株的总产量基本相同,突变株中B1的产量略有提高,阿维菌素B2的含量显著提高。     

ECF-σ^5因子参与阿维链霉菌中阿维菌素合成和环境胁迫的研究北大核心CSCD

【目的】研究阿维链霉菌中ECF-σ^5子对阿维菌素合成、形态分化和环境胁迫的调控,为揭示阿维菌素生物合成的调控机制和ECF-σ因子的调控网络提供依据。【方法】构建sig5基因缺失、回补和过表达菌株,通过形态观察和摇瓶发酵实验初步确定σ^5形态分化、菌体生长和阿维菌素合成的影响。进一步通过RT-q PCR、EMSA和Ch IP实验寻找确定σ^5靶基因,再通过胁迫实验揭示σ^5能参与的胁迫反应。【结果】对sig5相关突变株的摇瓶发酵和形态观察结果表明,σ^5阿维菌素合成具有抑制作用,但不影响菌体生长和形态分化。sig5基因缺失导致阿维菌素生物合成途径特异性正调控基因ave R和结构基因ave A1的转录水平提高,但σ^5不与ave R和ave A1的启动子区结合。σ^5结合在自身基因及附近基因SAV612、SAV615、SAV618的启动子区,正调控这些基因及所在操纵子的表达。胁迫实验暗示σ^5能参与渗透压引起的胁迫反应。【结论】ECF-σ^5子在转录水平间接负调控阿维菌素的合成。     

阿维链霉菌中aveD基因缺失对阿维菌素合成的影响

利用aveD基因的缺失载体pCZ8(pKC1139∷△aveD)对阿维菌素(Avermectin)产生菌阿维链霉菌(Streptomyces avermitilis)76\|9的aveD基因进行缺失获得aveD缺失突变株。经摇瓶发酵和HPLC检测,发现该突变株只产生阿维菌素B组分。说明将阿维链霉菌的aveD基因缺失,并不影响下游aveF的表达。缺失突变株的阿维菌素的总产量与出发菌株的总产量基本相同,突变株中B1的产量略有提高,阿维菌素B2的含量显著提高。     

金色链霉菌J13基因敲除体系的构建

目的:建立金色链霉菌基因敲除体系,敲除金色链霉菌J13中的ctcF基因,研究工程菌的代谢变化.方法:采用基因置换和框内缺失技术,对ctcF基因进行敲除.结果:利用接合转移的方法,将质粒pFD109导入到金色链霉菌J13中,经抗性筛选及PCR验证获得ctcF基因置换菌株金色链霉菌A1 - 20;将质粒pFD111经接合转移导入到A1 -20中,获得ctcF基因框内缺失菌株金色链霉菌K2-46;以上工程菌的发酵组分经HPLC分析,发现金霉素发酵单位显著降低.结论:获得的接合子发生双交换的概率可达18％以上,建立及验证了金色链霉菌基因敲除体系的实用性和可操作性,并初步推测ctcF为金霉素生物合成中的调控基因.     

阿维链霉菌bkdAB的基因中断对阿维菌素合成的影响

以阿维菌B组分菌株Streptomyces avermitilis Bjbm0006为出发菌株,用PCR的方法构建bkdAB基因簇(Ｂranched_chain α-keto acid dehydrogenase gene AB) 的基因置换质粒pHJ5821 (pHZ1358∷bkdAB&erm),并对其进行基因中断,得到重组菌株Bjbm5821。Bjbm5821的发酵产物经HPLC检测发现,除了产生B1a和B2a外,还产生一种原菌株没有的新组分,3个组分的总含量只有出发菌株Bjbm0006的25%。结果表明bkdAB的中断不仅部分阻断了阿维菌素的合成,还阻断了阿维菌素b组分的合成,可以推测bkdAB的产物在阿维菌素合成途径中主要承担了α-酮基异戊酸脱氢酶 (α-ketoisovaleric acid dehydrogenase) 角色。     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

Comparison of parameters estimated from A/Ci and A/Cc curve analysis

The parameters estimated from traditional A/C _i curve analysis are dependent upon some underlying assumptions that substomatal CO₂ concentration (C _i) equals the chloroplast CO₂ concentration (C _c) and the C _i value at which the A/C _i curve switches between Rubisco- and electron transport-limited portions of the curve (C _i-t) is set to a constant. However, the assumptions reduced the accuracy of parameter estimation significantly without taking the influence of C _i-t value and mesophyll conductance (g _m) on parameters into account. Based on the analysis of Larix gmelinii’s A/C _i curves, it showed the C _i-t value varied significantly, ranging from 24 Pa to 72 Pa and averaging 38 Pa. t-test demonstrated there were significant differences in parameters respectively estimated from A/C _i and A/C _c curve analysis (p<0.01). Compared with the maximum ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation rate (V_cmax), the maximum electron transport rate (J_max) and J_max/V_cmax estimated from A/C _c curve analysis which considers the effects of g _m limit and simultaneously fits parameters with the whole A/C _c curve, mean V_cmax estimated from A/C _i curve analysis (V_cmax-C _i) was underestimated by 37.49%; mean Jmax estimated from A/C _i curve analysis (J_max-C _i) was overestimated by 17.8% and (J_max-C _i)/(V_cmax-C _i) was overestimated by 24.2%. However, there was a significant linear relationship between V_cmax estimated from A/C _i curve analysis and V_cmax estimated from A/C _c curve analysis, so was it J_max (p<0.05).     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Ribonucieic Acid and Protein Metabolism of t12/t12 Embryos and T/t12 Spermatozoa

RNA precursor uptake and incorporation, amino acid uptake and incorporation, and the characterization of newly synthesized RNA and protein in pools of normal morulae and pools containing one-third t ¹²/ t ¹² morulae were compared. Maturing spermatoza of +/+ and T / t ¹² animals were analyzed for RNA and protein content, and the RNA characterized. No differences in these parameters could be ascribed to the t ¹² gene in homozygous embryos or haploid sperm.     

C-Glycosylflavones and xanthones in leaves of Rigidella,sessilanthera and Fosteria

Four New World genera are included in the tribe tigrideae of the Iridaceae. These genera, which have been the subject of recent systematic treatments [1–4], share many characteristics; e.g. a haploid complement of two large and 12 small chromosomes and bisulcate pollen grains. However, the taxonomic and phyletic relationships within and between these genera have been based strictly on floral morphology and may simply reflect the primary pollinators of these genera [4,5].The flavonoid chemistry of eight species in three of the four genera, Rigidella, Sessilanthera and Fosteria, was investigated as part of a systematic study of Tigrideae. Our results are summarized in Table 1 nad Fig. 1. Although a recurrent pattern of $\text{C-}\text{glycosylflavones}$ and xanthones characterize the leaves of these taxa, each species exhibits a distinctive array of compounds. The same classes of compounds occur in other tribes of the Iridaceae: $\text{C-}\text{glycosylflavones}$ occur in the Irideae [6,7], Cipureae, and Mariceae and xanthones in Irideae [6] and Sisyrinchieae [6].     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

cis- and trans-phytoene in Verticillium agaricinum

cis-Phytoene and trans-phytofluene were identified in illuminated cultures of Verticillium agaricinum in addition to the other carotenoids     

Alkylation of isolated chromatin with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

When isolated chromatin is incubated with the carcinogens N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU), DNA and chromosomal proteins become alkylated to increasingly greater extents as the carcinogen concentrations increase. With either MeNU or EtNU, the core and linker DNA of chromatin are alkylated to essentially identical extents. Alkylation of chromatin DNA as well as free DNA is drastically reduced at physiological ionic strengths (e.g. 0.15 M NaCl). The presence of 0.15 M NaCl, on the other hand, enhances alkylation of chromosomal proteins. While EtNU is much less reactive to DNA than MeNU, alkylation of chromosomal proteins relative to that of chromatin DNA has been found to be markedly greater with EtNU than with MeNU. Such a difference in their relative reactivities toward DNA and proteins may be related to the known difference of carcinogenic potency between these N-nitroso compounds.     

Nδ-acetylornithine and S-methylcysteine in blood plasma

N^δ-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 ± 0.4 μmol/l (range 0.8–0.2 μmol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 μmol/day). Human blood plasma contains 3.9 ± 1.9 μmol/l (range 1.4–6.5 μmol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.