几种因素诱导鱼腥藻7120短藻丝体的形成

丝状蓝藻鱼腥藻7120（Anabaena sp.PCC7120）中可成功表达外源基因,但其转化和表达效率不高,改变细胞的生理状态可能会影响外源基因的转化和表达效率。将鱼腥藻7120藻丝体通过几种因素诱导形成短藻丝体（具有25个左右细胞）,并对其光合活性进行了测定。结果表明：红光和高温对鱼腥藻7120短藻丝体较为有效,且红光诱导在48h时,短藻丝体细胞数占总细胞数的比例达到85％;DCMU单独诱导效果不明显,适当浓度的DCMU＋红光诱导时诱导效率略有增加;高温以45℃诱导12h比例最高,约达87％;高温45℃诱导时,对数生长后期的鱼腥藻7120较易形成短藻丝体。光合活性测定结果显示,诱导形成的短藻丝体光合放氧速率比正常营养藻丝体的低,这种具有光合放氧能力的短藻丝体显示出作为表达外源基因受体的可能性。     

T7 RNA聚合酶基因表达系统在鱼腥藻7120中构建及hG-CSF的表达

外源基因的表达效率低是蓝藻基因工程发展的瓶颈之一,T7 RNA聚合酶表达系统实现了大肠杆菌中外源基因的高效表达,蓝藻与大肠杆菌同为革兰氏阴性菌,具有较高的遗传同源性,在蓝藻中构建T7 RNA聚合酶表达系统有可能提高外源基因在蓝藻中的表达效率。为了在鱼腥藻7120中构建T7 RNA聚合酶表达系统,采用重叠延伸PCR技术和酶切连接等方法构建能够表达T7 RNA聚合酶的定点整合载体pEASY-T1-F1-TacT7RNAPCmR-F2以及由T7启动子驱动hG-CSF基因表达的穿梭表达载体pRL-T7-hG-CSF;采用电击转化法将定点整合载体导入野生型鱼腥藻中,通过三亲接合的方法将穿梭表达载体转入已定点整合T7 RNA聚合酶的转基因鱼腥藻中。利用PCR技术鉴定外源基因在蓝藻中的存在;RT-PCR方法检测外源基因在蓝藻中的转录情况;Western blotting实验检测外源基因在蓝藻中的蛋白表达情况。结果表明两种载体构建成功,T7 RNA聚合酶基因和hG-CSF基因被转入鱼腥藻中,两个基因均在藻中表达,T7 RNA聚合酶表达系统在鱼腥藻中构建成功,与传统蓝藻表达系统相比,文中在鱼腥藻中构建的T7表达系统使hG-CSF基因的表达量提高2倍。该表达系统将为蓝藻基因工程的应用提供更优的工具,将促进蓝藻作为底盘细胞在合成生物学等领域的发展。     

启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较

为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。     

应用生物膜干涉技术检测鱼腥藻HetR结合靶DNA的亲和力

正鱼腥藻PCC7120是一种固氮丝状蓝藻,当环境中化合态氮源充足时,其藻丝只有进行光合作用的营养细胞;在环境中缺乏可利用的氮源时,部分营养细胞会在藻丝上以一种半规律的格式分化成异形胞(异形胞间隔约10个营养细胞),从而进行固氮作用。早在1984年,Wolk实验室通过与大肠杆菌接合转移的方式,成功地将穿梭质粒转入鱼腥藻PCC7120,建立了遗传转移系统~([1])。在2001年,日本Kazusa研究中心完成了对鱼腥藻PCC7120全基因组测序~([2])。有规律的细胞分化格式,成熟的遗传转移系统以及完整可用的基因组序列使得鱼腥藻PCC7120     

鱼腥藻7120遗传转化的研究进展

鱼腥藻7120作为模式生物被广泛用于光合、固氮、进化、代谢等基本生命现象的研究。近几年, 对其基因工程的研究使人们看到它在医药、环保、能源等方面的应用潜力, 但表达效率低是其发展的瓶颈。为了提高其表达效率, 研究者从鱼腥藻7120的载体(包括启动子、复制子、选择标记基因等)的改进、目的基因的优化(密码子和SD序列)、宿主的改善、转化方法的改变等方面进行了大量探索, 除了用于功能基因的研究, 已经有几十个外源基因在鱼腥藻7120中表达。除了研究载体, 诱变鱼腥藻7120形成有利于外源基因表达的突变体和摸索转基因蓝藻最佳生长条件和表达条件, 可能是新的发展方向。     

重组人粒细胞集落刺激因子(rhG-CSF)基因在鱼腥藻中的克隆

为了将rhG-CSF基因在鱼腥藻PCC 7120中克隆,用于制备口服制剂,利用DNA重组技术,在不改变阅读杠的前提下,将hG-CSF基因进行突变,并插入到pUC-19载体上,构建中间载体pUC=G-CSF;将pUC-G-CSF插入到pRL-489的启动子PpsbA的下游,构建穿梭表达载体pRL-G-CSF;通过三亲接合转移方法,将pRL-G-CSF转入丝状体蓝藻鱼腥藻PCC 7120内。本试验得到了有抗生素性的鱼腥藻,并用PCR技术检测到rhG-CSF基因在转基因鱼腥藻中存在。     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段,与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM,然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选,酶谱鉴定,斑点杂交,质粒的交叉转化以及内源glnA基因表达的GS活性分析,GS相关的胞外泌氨分析及所获藻株的形态学变化,证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。     

转基因鱼腥藻7120适宜生长条件的初步研究

以前的研究报导了将人肿瘤坏死因子基因(TNF-α)成功转入鱼腥藻7120中,这项研究在实验室小规模范围内探讨了其转TNF-α基因鱼腥藻7120的适宜生长条件。结果表明,转基因鱼腥藻7120最适生长温度在25～30℃,最适pH7.0～7.5。不同光照强度下的净光合放氧速率测定结果表明,转基因鱼腥藻7120与野生型具有一致的光饱和点和光补偿点,且适合在10～700μE.m-2.s-1下生长。外加有机碳源(4g/L葡萄糖)一定程度上可以增加转基因鱼腥藻7120生长速度。对不同藻龄转基因鱼腥藻7120中TNF-α的累积量的检测发现:生长8d左右时转基因鱼腥藻7120中TNF-α累积量达到最大值。这将为产业化生产、适时收获转基因蓝藻提供理论指导,同时讨论了葡萄糖对转基因鱼腥藻7120代谢的调节。     

人肿瘤坏死因子α基因穿梭表达载体的构建和在鱼腥藻7120中的表达

报道了人肿瘤坏死因子α(hTNFα)基因穿梭表达载体的构建及其在丝状体蓝藻鱼腥藻 712 0中的表达和鉴定结果 .将质粒pRL rhTNF上hTNFα的cDNA连于pRL 43 9质粒上的PpsbA启动子下游 ,得到中间载体pRL TC .进一步与穿梭质粒pDC 8重组 ,得到可在大肠杆菌和蓝藻中均可表达的穿梭表达载体pDC TNF .pRL rhTNF ,pRL TC和pDC TNF三者在大肠杆菌中的表达量分别为 11.8% ,16 9%和 15 .0 % .通过三亲接合转移将pDC TNF引入鱼腥藻 712 0中并获稳定遗传的转基因株 .从转基因的鱼腥藻 712 0中检测到pDC TNF质粒的存在 ,且在和TNFα的cDNA探针进行的Southern杂交中呈阳性反应 .抽提转基因藻的蛋白样品进行检测 ,在Western印迹中和TNFα单克隆抗体呈阳性反应 .采用TNF对L92 9细胞的细胞毒性方法 ,证明转基因藻粗提液中 ,TNF确有细胞毒活性 .     

苏云金芽胞杆菌杀蚊基因在鱼腥藻中克隆和表达的初步研究

将苏云金芽胞杆菌以色列亚种的杀蚊晶体蛋白基因cry11A亚克隆到大肠杆菌－蓝藻的穿梭质粒载体pRL25C,然后用三亲本杂交的方法将重组质粒转移到一种具有固氮能力且可被蚊幼虫吞食的鱼腥藻（Anabaena）PCC7120中。Southernblot及Westernblot分析表明cry11A基因在鱼腥藻PCC7120中得以克隆和表达,但生物测定未能检测到转基因鱼腥藻对库蚊（Culex）的毒性,可能是因为带有苏云金芽胞杆菌自身启动子的Cry11A基因在鱼腥藻PCC7120中表达量不够高的缘故。     

中华鳖（Trionyx sinensis）幼体能量转换的初步研究

在突验室内,对中华鳖幼体的能量转换进行了初步研究。结果表明：１）温度结中华鳖幼体对食物的同化效率无显著影响,但影响总转化效率和生长效率。二者在２２℃左右最高,２６—３２℃温度范围内各温度组之间元显著差异,３５℃则显著下降。２）体重与同化效率的关系为：ＡＥ＝９４．１８６＋２．７６０ｌｇＷ,而生长效率和总转化效率在各体重组间无显著变化;３）中华鳖幼体能量收支方程为：１００Ａ＝７４Ｒ＋２６Ｇ,并认力中华鳖将较多能量（７４％）投入代谢,而将较少能量（２６％）投入生长是适应其生态习性的缘故。     

日光温室采光面三效率计算模式及其优化选择研究

本文针对日光温室常见的3种采光面（圆弧面、椭圆和抛物面）,给出了各自采光效率、土地使用率和保温效率（简称三效率）的理论计算模式,并通过分析得出,各个高效采光面上的采光效率均随纬度增加而降低,随距冬至的日数而增加,随作物高度而降低,随高跨比增加而增加。在三种采光面中,无论时间、地点、高跨比和种植高度怎样变化,均以圆弧面的采光效率为最高,椭圆面次之,抛物面最低。另一方面,从土地使用效率来看,椭圆面最高,但它的保温效率最低。因此,采光面形状的选择应因使用对象而宜,本文给出了最佳选择建议。     

Dimensionless criteria for estimating oxygen transfer in aeration systems

The effect of power consumption on oxygen transfer efficiency has been evaluated for both surface and subsurface aeration systems. Two new dimensionless criteria (numbers) have been proposed for this purpose. They are termed the efficiency criterion or T(o) number and the power consumption effect criterion or Av number. The analysis presented in this article is based on data obtained on various full-scale aeration systems, and it shows that for each aerator there is an optimum power consumption at which maximum oxygen transfer efficiency results. The oxygen transfer efficiency has maximum values when the efficiency criterion (T(o) number) reaches an approximately constant value. The results have proven that when there are several aerators with identical diameters and the same rotational velocity but different geometrical shapes, the most efficient one will be (after having reached the maximum transfer efficiency) the one with the highest power consumption. For two aerators varying in diameter but with the same parameter as above (maximum transfer, equal rotational velocity), different efficiencies are obtained, the difference being a function of d(-0.5). The results of investigating subsurface aeration systems show that the efficiency of decreased.     

Revised algorithm of ecosystem water use efficiency for semi-arid steppe in the Loess Plateau of China

Aims We evaluated the applicability of different measures of water use efficiency through analyzing the coupled dynamics of GPP and evapotranspiration in the semi-arid steppe in the Loess Plateau of China. Our objective is to explore the applicability of two quantitative measures of ecosystem water use efficiency—inherent water use efficiency (IWUE) and underlying water use efficiency (uWUE) —for the semi-arid steppe and to endeavor necessary modifications.Methods The consistency and stability of three indices of water use efficiency formulations (i.e. WUE, IWUE, uWUE) were calculated and compared at hourly, daily and annual time scales before proposing an optimal water use efficiency (oWUE). These indices were additionally used to quantify their importances in modeling the diel change of gross primary production (GPP). The yielded-accuracy of the prediction was used for justifying their uses.Important findings IWUE and uWUE appeared suitable for examining the coupled water-carbon characteristics of vegetation at hourly and daily scales, whereas WUE was more plausible on the annual and interannual scales. The optimized water use efficiency index did not improve the prediction of the coupled water-carbon characteristics as compared with uWUE, but it improved the prediction of GPP and its dynamics. oWUE and uWUE improved the predictions of GPP in the peak growing period, while WUE predicted the GPP better at the early and late growing season. Interestingly, we found that IWUE was not suitable for predicting GPP and its dynamics. The results will be of great importance in modeling the effects of climate change on the carbon assimilation and water cycle for the future.     

Nutritional properties of four marine microalgae for albino rats

The nutritive value of the marine microalgaeTetraselmis suecica, Isochrysis galbana, Dunaliella tertiolecta andChlorella stigmatophora was studied in diets given to rats. Biological assays were carried out in order to determine the Protein Efficiency Ratio (PER) and the Food Conversion Efficiency (FCE). Each dried microalga was fed to weaning Wistar albino rats as the sole protein source at a protein level of 12%. Control rats were given diets containing 12 % casein. Food consumption was similar in all groups. PER values obtained were 1.14 withT. suecica diet, 1.13 withI. galbana diet, 2.07 withD. tertiolecta diet and 1.13 withC. stigmatophora diet (casein, 2.50). FCE values followed a similar pattern. The data showed that the marine microalgaD. tertiolecta is a source of protein of good quality. Its PER is quite high, compared to vegetable and cereal proteins, and compares favourably with other microbial protein sources, such as yeasts or different freshwater microalgae. Haematological tests showed no significant differences among the groups in haemoglobin levels, red and white blood cell counts, differential count and mean corpuscular volume. Different blood parameters were also determined and a significant decrease in triglycerides levels appeared with all the microalgal diets, whereas a significant decrease in cholesterol appeared inD. tertiolecta andC. stigmatophora diets.     

Cycling efficiency and pedalling frequency in road cyclists

The purpose of this study was to determine the influence of pedalling rate on cycling efficiency in road cyclists. Seven competitive road cyclists participated in the study. Four separate experimental sessions were used to determine oxygen uptake (VO(2)) and carbon dioxide output (VCO(2)) at six exercise intensities that elicited a VO(2) equivalent to 54, 63, 73, 80, 87 and 93% of maximum VO(2) (VO(2max)). Exercise intensities were administered in random order, separated by rest periods of 3-5 min; four pedalling frequencies (60, 80, 100 and 120 rpm) were randomly tested per intensity. The oxygen cost of cycling was always lower when the exercise was performed at 60 rpm. At each exercise intensity, VO(2) showed a parabolic dependence on pedalling rate (r = 0.99-1, all P < 0.01) with a curvature that flattened as intensity increased. Likewise, the relationship between power output and gross efficiency (GE) was also best fitted to a parabola (r = 0.94-1, all P < 0.05). Regardless of pedalling rate, GE improved with increasing exercise intensity (P < 0.001). Conversely, GE worsened with pedalling rate (P < 0.001). Interestingly, the effect of pedalling cadence on GE decreased as a linear function of power output (r = 0.98, n = 6, P < 0.001). Similar delta efficiency (DE) values were obtained regardless of pedalling rate [21.5 (0.8), 22.3 (1.2), 22.6 (0.6) and 23.9 (1.0)%, for the 60, 80, 100 and 120 rpm, mean (SEM) respectively]. However, in contrast to GE, DE increased as a linear function of pedalling rate (r = 0.98, P < 0.05). The rate at which pulmonary ventilation increased was accentuated for the highest pedalling rate (P < 0.05), even after accounting for differences in exercise intensity and VO(2) (P < 0.05). Pedalling rate per se did not have any influence on heart rate which, in turn, increased linearly with VO(2). These results may help us to understand why competitive cyclists often pedal at cadences of 90-105 rpm to sustain a high power output during prolonged exercise.     

Practical application of ligand efficiency metrics in lead optimisation

The use of composite metrics that normalise biological potency values in relation to markers of physicochemical properties, such as size or lipophilicity, has gained a significant amount of traction with many medicinal chemists in recent years. However, there is no consensus on best practice in the area and their application has attracted some criticism. Here we present our approach to their application in lead optimisation projects, provide an objective discussion of the principles we consider important and illustrate how our use of lipophilic ligand efficiency enabled the progression of a number of our successful drug discovery projects. We derive, from this and some recent literature highlights, a set of heuristic guidelines for lipophilicity based optimisation that we believe are generally applicable across chemical series and protein targets.