联合固氮菌Enteroabcter gergoviae 57-7氮代谢相关基因的克隆和泌氨工程菌的构建

从构建具有稳定泌氨能力的联合固氮工程菌的目的出发，首先构建Enterobacter gergoviae57-7 (E57-7)基因文库并与所筛选的泌氨突变株进行遗传互补实验，得到可互补 泌氨特性的克隆，经Southern杂交后推测其中包含与glnA、amtB基因无关的另一类与泌 氨相关的基因。同时根据铵载体基因amtB的已知序列设计两对简并性引物，经PCR从 E57-7 DNA中扩增得到约340bp的片段，序列分析和Blast序列同源性比较后确定为amtB 基因片段，申报并获得序列号AJ132232，最终从基因库中筛选到两个包含E57-7 amtB 基因的克隆。 用K. pneumoniae的glnA基因片段为探针，通过Southern杂交从E57-7基因库中筛选到包含有glnAntrBC基因的克隆，经亚克隆后对包含有这个操纵元的4316bp片段进行了全序列分析，申报GenBank获得序列号AF072440。在体外实验中构建了Km-cassette 插入glnA的重组质粒pA，将此质粒转入E57-7野生型菌株后经筛选同源重组子获得glnA 突变的具有稳定泌氨能力的菌株15、I9。并进行了盆栽玉米接种实验，确定在灭菌上壤实验体系下I5对玉米幼苗有显著促生效应。 利用绿色荧光蛋白(GFP-S65T，V68L，S72A)基因建立分子生物学研究手段，构建了新 型克隆载体pGreenLD，建立了绿白斑筛选重组质粒的的技术。构建组成型表达gfp的质粒载体研究了E57-7在玉米根际的定殖模式;构建nifH-gfp表达载体，确定在与植物联合生活时其固氮酶结构基因nifHDK的表达与碳源物质供应密切相关。利用不同抗性基因和gfp基因片段构建出在E57-7中组成型表达抗性和GFP的质粒载体，建立了监测接种菌在土壤中释放的双标记系统。 最后克隆了E57-7 glg cluster并测定部分glgCA和glgP基因序列，申报后获得序列号AJ132233和AJ132234，这是首例从联合固氮菌中克隆得到glg cluster的报道。

Seasonal variation in metabolism of cultured Pacific oyster, Crassostrea gigas, in Sanggou Bay, China

Rates of respiration and excretion of the Pacific oyster, Crassostrea gigas, were measured seasonally from June 2002 to July 2003 under ambient conditions of food, water temperature, pH, and salinity in Sanggou Bay, an important mariculture coast in north China. The aim of this study is to obtain fundamental data for further establishing an energy budget model and assessing the carrying capacity for cultivation of C. gigas in north China. Oysters were collected monthly or bimonthly from the integrated culture areas of bivalve and kelp in the bay. Oxygen consumption and ammonium and phosphorus excretion rates were measured, and ratios of O/N and NIP were calculated. One-way ANOVA was applied to determine differences among these parameters that act as a function of seasonal variation. All the physiological parameters yielded highly significant variations with season (P<0.01) The rate of respiration varied seasonally, with the highest oxygen consumption rate in July and the lowest rate in January, ranging from 0.07 to 2.13 mg O-2 h(-1) g(-1) dry tissue weight (DW). Maximum and minimum ammonium excretion rates were recorded in August and January, respectively, ranging from 0.51 to 5.40 mu mol NH4-N h(-1) g(-1) DW. Rates of phosphorus excretion varied from 0.11 (in January) to 0.64 (in July) mu mol PO4-P h(-1) g(-1) DW. The O/N and N/P ratios changed from 9.2 (in January) to 59.8 (in July) and from 4.6 (in January) to 10.9 (in August), respectively. For each season, the allometric relationship between the physiological response (e.g., rate of oxygen consumption, ammonium and phosphorus excretion) and DW of the animal was estimated using the formula: Y=a x DWb. (C) 2005 Elsevier B.V. All rights reserved.

Changes of urinary steroid conjugates and gonadotropin excretion in the menstrual cycle and pregnancy in the Yunnan snub-nosed monkey (Rhinopithecus bieti)

The Yunnan snub-nosed monkey (Rhinopithecus bieti) is one of the most endangered species in the world, and it is endemic to China. According to our knowledge, there was no information on reproduction for this species. The present study was designed to und

Effects of ammonium on growth, nitrogen and carbohydrate metabolism of Potamogeton maackianus A. Benn

Growth, nitrogen and carbohydrate metabolism in relation to eutrophication were studied for a submerged plant Potamogeton maackianus, a species common in East Asian shallow lakes. The plants were grown in six NH4+-N concentrations (0.05, 0.50, 1.00, 3.50, 5.00 and 10.00 mg/L) for six days. NH4+-N levels in excess of 0.50 mg/L inhibited the plant growth. The relationships between external NH4+-N availability and total nitrogen (TN), protein-N, free amino acid-N (FAA-N) and NH4+-N in plant tissues, respectively, conformed to a logarithmic model suggesting that a feedback inhibition mechanism may exist for ammonium uptake. The response of starch to NH4+-N was fitted with a negative, logarithmic curve. Detailed analysis revealed that the influx NH4+-N had been efficiently incorporated into organic-N and eventually stored as protein at the expense of starch accumulation. These data suggest that this species may be able to tolerate high levels of ammonium when dissolved oxygen is sufficient.

Acute biochemical responses of a submersed macrophyte, Potamogeton crispus L., to high ammonium in an aquarium experiment

Acute biochemical responses of Potamogeton crispus L. to high external ammonium were investigated in an aquarium experiment. Shoots of P. crispus were incubated in aquaria for 24 h or 48 h at five treatments of ammonium-0, 1, 5, 10 and 20 mg/L NH4-N. Soluble sugar content of the shoots declined markedly with increasing ammonium levels, whereas soluble amino acid content increased dramatically. Responses of two antioxidant enzymes as well as soluble protein content fit a lognormal distribution with increasing ammonium levels. High ammonium levels (NH4-N greater than or equal to 5 mg/L) caused significant acute biochemical changes in P. crispus, which potentially could lead to significant biochemical damage.

An improvement on Si-etching tetramethyl ammonium hydroxide solution

An optimal concentration of the etching solution for deep etching of silicon, including 3% tetramethyl ammonium hydroxide and 0.3% (NH4)(2)S2O8, was achieved in this paper. For this etching solution, the etching rates of silicon and silicon dioxide were about 1.1 mu m(.)min(-1) and 0.5 nm(.)min(-1), respectively. The etching ratio between (100) and (111) planes was about 34:1, and the etched surface was very smooth.

Clathrate Dissociation Conditions for Methane + Tetra-n-butyl Ammonium Bromide (TBAB) + Water

Hydrate equilibrium data of the CH4 + tetra-n-butyl ammonium bromide (TBAB) + water have been measured by using the isothermal pressure search method for four components of TBAB aqueous solutions. The three-phase equilibrium lines obtained in the present study are shifted to the low-temperature or high-pressure side from that of the stoichiometric TBAB solution. Moreover, methane uptake into semi-clathrates is confirmed by a shift in the clathrate regions when methane is present. The experiments are carried out in the pressure range of (0.5 to 11) MPa and in the temperature range of (281.15 to 295.15) K.