绿色荧光蛋白基因导入胡萝卜愈伤组织并获得表达

目的:将绿色荧光蛋白基因(green fluorescent protein,GFP)重组到胡萝卜愈伤组织细胞中,使其获得表达,为今后利用GFP基因作为植物报告基因提供条件。方法:通过冻融法将含有GFP基因的重组表达载体PBI1121转入到根癌农杆菌EHA105中,再利用根癌农杆菌介导的方法将GFP基因导入到胡萝卜愈伤组织细胞中,经过除菌和抗性筛选后观测转化结果。结果:荧光显微镜观测到被转化的愈伤组织在受蓝光激发后发出绿色荧光,利用PCR法扩增出约740bp的目的基因片断。结论:GFP基因在胡萝卜愈伤组织细胞中获得了表达。     

城市公园绿地水、热与CO2通量观测与分析

水、热和CO2通量是评价城市绿地生态效益的重要指标。在北京海淀公园中部和边缘架设涡度相关系统,连续观测和定量研究城市公园绿地影响下的通量变化特征。结果表明,公园绿地所获得的净辐射在植被生长季节大部分用于植被的蒸散作用,潜热大于显热;而在植被非生长季节大部分用于显热,潜热数值非常小。晴天显热与潜热的比值在春季随着植被枝叶的生长逐渐减少,到夏季达到最小值约1/3,在秋季随着植被叶片枯黄逐渐变大,冬季达到数倍。公园绿地能量平衡率在52%～83%,普遍存在能量不平衡。公园中部的潜热观测值大于公园边缘,而显热小于公园边缘;公园中部CO2通量日均值为负,公园绿地是CO2汇。公园绿地在植被生长季节具有明显的降温增湿、吸收CO2等生态效应。     

Dnd1的蛋白亚细胞定位及其对HeLa细胞增殖的抑制作用

小鼠睾丸生殖细胞瘤易感基因Dnd1编码的蛋白是一个在进化中保守的RNA结合蛋白.为探讨小鼠Dnd1的蛋白亚细胞定位和对细胞增殖的影响及其机制, 利用生物信息学技术, 采用组合的亚细胞定位分析软件对Dnd1进行真核生物亚细胞定位预测; 利用融合绿色荧光蛋白(green fluorescent protein, GFP)定位的方法, 通过构建pEGFP-Dnd1重组质粒, 将重组质粒pEGFP-Dnd1转染HeLa细胞和GC-1细胞, 在荧光显微镜下观察Dnd1的蛋白亚细胞定位; 用MTT法和流式细胞技术测定Dnd1过表达对HeLa细胞的增殖能力的影响和细胞周期的改变; 在HeLa细胞系中检测Dnd1对AP-1转录活性的影响. 结果表明: ① 生物信息学预测Dnd1主要在细胞核表达, 在细胞质中也有少量表达; 荧光显微镜下观察发现,Dnd1蛋白主要定位在细胞核, 在细胞质中也有少量分布; ② Dnd1基因在HeLa细胞系中的过表达抑制细胞增殖和诱导细胞周期G1期阻滞;③ Dnd1抑制AP-1的转录活性,从而抑制AP-1介导的转录是Dnd1抑制细胞增殖的可能机制.本研究初步明确了Dnd1的蛋白亚细胞定位及其对HeLa细胞的生长抑制作用, 这为进一步研究Dnd1基因的功能建立基础.     

Monitoring the transport of recombinant Candida rugosa lipase by a green fluorescent protein-lipase fusion

In a batch cultivation of Pichia pastoris expressing Candida rugosa lipase 1 (CRL1), secretion of 200 microg lipase ml(-1) of culture was achieved in sorbitol-based medium. However, a large amount of recombinant protein was retained intracellularly throughout the fermentation, pointing to the transport step as a major bottleneck. Therefore a translational fusion with the green fluorescent protein (GFP) was constructed that was expressed and transported similarly to the native lipase and retained catalytic activity. This analytical tool enables a rapid monitoring of product localization and amount, based on GFP-associated fluorescence.     

酵母表面展示脂肪酶合成己二酸二异辛酯

展示酶的酵母细胞既具有固定化酶的优点,又有制备简单、成本较低的特点.采用表面展示南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)的毕赤酵母细胞催化合成己二酸二异辛酯(Diisooctyl adipate,DIOA),对该反应体系进行优化,并实现了初步工艺放大制备.经条件优化后,在10mL反应体系中,DIOA的产率可达85.0％.该工艺放大到200mL反应体系时,DIOA产率可达97.8％.经减压蒸馏,DIOA纯度可达到98.2％.该酵母表面展示脂肪酶在合成绿色润滑油己二酸二异辛酯中具有良好应用前景.     

Viral Membrane Protein Topology Is Dictated by Multiple Determinants in Its Sequence

The targeting, insertion, and topology of membrane proteins have been extensively studied in both prokaryotes and eukaryotes. However, the mechanisms used by viral membrane proteins to generate the correct topology within cellular membranes are less well understood. Here, the effect of flanking charges and the hydrophobicity of the N-terminal hydrophobic segment on viral membrane protein topogenesis are examined systematically. Experimental data reveal that the classical topological determinants have only a minor effect on the overall topology of p9, a plant viral movement protein. Since only a few individual sequence alterations cause an inversion of p9 topology, its topological stability is robust. This result further indicates that the protein has multiple, and perhaps redundant, structural features that ensure that it always adopts the same topology. These critical topogenic sequences appear to be recognized and acted upon from the initial stages of protein biosynthesis, even before the ribosome ends protein translation.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

基于农业生态服务价值的农业绿色GDP核算——以安塞县为例

将传统GDP绿色化,也就是“绿色GDP”是国民经济核算研究和探索的热点。从微观层面对农业生产进行绿色GDP核算,认为农业绿色GDP不仅包含传统的产品价值,还应包括农业对人类社会经济系统的生态服务价值,并以基于生态系统服务价值的“农业绿色GDP=农业常规GDP＋农业生态GDP”的公式核算了安塞县的绿色GDP。结果表明：（1）安塞县2000年的农业生产总值为29187万元,减去中间消耗为18972万元;（2）若考虑农业生态环境代价,则农业生产产值仅为6272．30万元,是农业常规GDP的33．06％,农业常规GDP背后至少隐含着66．94％的农业生态环境代价;（3）应用市场价值法、机会成本法、替代工程法、影子价格法等核算农业生态系统服务价值结果为504218．37万元,把农业生态系统服务价值作为农业系统的间接产出,则该县2000年的农业绿色GDP为510490．67万元。安塞县农业经济的获得背后至少有66．94％生态环境代价是依靠其农业生态系统的服务来提供和补偿的。     

贵州玉米田害虫绿色灯光防控技术研究

为探明贵州玉米田昆虫群落结构和杀虫灯对害虫的防治效果,选取贵州玉米田3个具有代表性的点分别安装诱虫灯,进行诱集调查,并对近灯区（距离灯20 m以内）、远灯区（距离灯20~150 m）和对照区的昆虫种群消长情况进行田间调查。结果表明,贵州玉米田昆虫共有11目66科132种,主要为同翅目、鞘翅目、鳞翅目、膜翅目、直翅目、双翅目和半翅目类昆虫;灯诱共获得89个种类,其中害虫占64.77%,益虫占15.91%,中性昆虫占19.32%。田间调查结果显示,近灯区物种丰富度最高,对照区次之,远灯区最低;近灯区玉米螟Ostrinia furnacalis、黏虫Mythimna seperata (Walker)、灰飞虱Laodelphax striatellus和条赤须盲蝽Trigonotylus coelestialium (Kirkaldy)等趋光性昆虫的种群数量长期高于其他区域。综上所述,风吸式诱虫灯对玉米田害虫有较好的防治效果,为玉米田害虫的绿色防控提供了参考。     

灰盖拟鬼伞核定位蛋白重组表达系统的构建

为构建灰盖拟鬼伞Coprinopsis cinerea的核定位蛋白重组表达系统,本研究通过蛋白序列比对和信息学分析,预测了灰盖拟鬼伞组蛋白H2B的核定位序列,构建了融合组蛋白H2B核定位序列的绿色荧光蛋白(green fluorescent protein,GFP)重组表达载体,将该载体转入灰盖拟鬼伞AmutBmut菌...