猪ATF4基因真核超表达载体的构建及鉴定

摘 要：通过克隆猪ATF4基因CDS,构建pcDNA3.1(+)-ATF4真核超表达载体,为下一步在细胞水平和个体水平研究该基因功能奠定条件。提取RNA,运用RT-PCR技术和巢式PCR技术扩增猪ATF4全部编码序列,克隆转化到pMD18-T载体中,测序证实后,在克隆至真核超表达载体pcDNA3.1(+),构建了pcDNA3.1(+)-ATF4真核超表达载体,并进行酶切和测序鉴定。在细胞水平上进行转染鉴定。成功克隆了ATF4基因,并构建了pcDNA3.1(+)-ATF4真核超表达载体,瞬时转染C2C12细胞,超表达效果明显。为进一步研究该基因的功能奠定了基础。     

Transient stability simulation methods incorporating PMU measurements

Incorporating the PMU measurements information in transient stability simulation can increase computing speed and enhance accuracy in the time domain simulation. Based on the conventional alternating iteration technique, this paper presents two simulation methods combined with the PMU measurements information, in which the number of differential algebraic equations is reduced and so is the computational scale. The proposed methods have been tested and compared with the conventional method using the New England 10 machine 39 bus system and IEEE 50 machine 145 bus system. The cases with errors of the dynamic model parameter and PMU have been also simulated and discussed. The simulation results demonstrate the effectiveness of the presented methods.     

Fast algorithm for apparent resistivity calculation based on back propagation neural networks

To avoid the complex numerical calculation for the electromagnetic field and determine underground abnormality, a neural network based method is proposed. In consideration of turn off transmitter current, the effect of a linear ramp turn off current on transmitter is corrected. The characteristics of transient expression and the traditional calculation algorithm for apparent resistivity are analyzed, and a predigest structure of network is obtained based on the kernel expression. The three layer back propagation(BP) neural network is trained by using sample data in homogeneous half space, and its number in hidden layer was determined. The method proposed is compared with two traditional calculation methods with simulation experiments. The result demonstrates that BP neural network has a high speed of processing data and is useful in explanation of the transient electromagnetic method.     

PEI介导外源基因进入植物细胞的瞬时表达

 【目的】医学方面的大量研究证实,聚乙烯亚胺（polyethylenimine,PEI）作为一种新型阳离子多聚物基因载体可以吸附和浓缩DNA,通过与细胞膜亲和粘附和细胞吞噬作用运载外源基因进入细胞并实现表达。本实验研究旨在探索PEI作为非病毒基因载体介导外源基因进入植物细胞并获得瞬时表达的可能性。【方法】制备PEI/DNA复合物,利用凝胶阻滞分析PEI与DNA的结合情况,采用电子显微镜观察PEI/DNA复合物的形态。以绿色荧光蛋白基因为报告基因研究不同N/P比条件下PEI/DNA复合物对拟南芥原生质体细胞的转化效率,并与PEG转化方法进行比较分析。【结果】PEI与DNA的质量比为5﹕1～1﹕4,PEI可以与DNA稳定结合,形成粒径约100～200 nm的球形复合物。PEI/DNA复合物对拟南芥原生质体细胞的转化效率随其N/P比的增大而提高,N/P＝5时PEI的转化效率达到最高且明显高于PEG介导的转化效率,N/P＞5时转化效率反而下降,容易使细胞破碎和变形。【结论】PEI作为一种新型的外源基因运送载体对拟南芥原生质体细胞具有良好的介导基因转移效果。     

枝状管网水力瞬变工况的试验与数值模拟

采用试验并结合特征线法(MOC)对带有一个分支管管网阀门关闭时产生的水锤压力及波动规律进行了研究,试验采用压力传感器监测水锤压力波动、角位移传感器测定阀门的关闭时间及关闭规律,结果表明:MOC方法能较好地预测管网阀门关闭产生的最大水锤压力值,管网监测点计算与试验值相对误差小于13％,但MOC方法计算所得压力波的衰减比较缓慢,而试验得到的压力波衰减很快,是由于在计算中使用的恒定流摩阻所致;在此基础上对5种阀门关闭方案产生的瞬变流动进行了数值计算,表明管路末端2个阀门同时关闭时管网中产生的水锤压力最大,在实际工程中应避免阀门同时关闭的操作;管网最大水锤压力随关阀时间的延长而减小,当阀门关闭时间一定时,采用曲线关闭规律产生的最大水锤压力比直线关闭规律产生的要小,而采用两阶段直线关闭规律产生的最大水锤压力压力小于线性关闭的,对于实际工况中的两阶段关闭应通过延长第一阶段关闭时间以减小最大水锤压力．     

维管特异表达启动子BSP的克隆与活性检测

为了研究维管特异表达启动子BSP的活性和组织特异性,从杨树基因组中克隆得到维管特异表达启动子BSP,利用该启动子与绿色荧光蛋白（GFP）报告基因构建植物表达载体,采用基因枪法转化烟草叶片,并在高渗培养基上培养过夜,后转移到诱导培养基上(MS+6-BA 0.5 mg/L+NAA 0.05 mg/L+Kan 100 mg/L)培养生根并长到一定高度后,剪下已形成维管组织的叶脉和根,制成切片,然后在OLYPUS BX51/BX52型荧光显微镜下用蓝光激发,观察各个组织细胞中的绿色荧光,同时以未转化的烟草材料做为对照。结果表明：BSP启动子所驱动的GFP基因在烟草的维管组织细胞中得到了高水平的表达,而在烟草的其他组织及对照的任何组织中几乎不表达。通过GFP在烟草维管组织中的瞬时表达,表明了该启动子具有维管组织特异性表达活性特点。     

Transient Expression of Exogenous Gene into Plant Cell Mediated by PEI Nanovector

This study was carried out to investigate the transfection effect of exogenous gene into plant protoplast cell mediated by polyethylenimine (PEI) nanovector, based on PEI gene delivery system in the field of medical science. PEI/DNA complexes were prepared by using PEI polymer to bind the plant expression plasmid, pCMI205-GFPn. The ability of PEI combining and protecting DNA was investigated by agarose gel electrophoresis retardation assay. The surface characteristics of PEI/DNA complexes were observed with transmission electron microscope. The transfection efficiency of Arabidopsis thaliana protoplasts mediated by PEI/DNA complexes at different N/P ratios was analyzed based on observation of transient expression of green fluorescent protein with confocal laser scanning microscope. PEI could bind and condense DNA, and form stable 100-200 nm PEI/DNA complexes when the proportion of PEI and DNA is in the range of 5:1-1:4.Transfection efficiency of PEI/DNA complexes increased with N/P ratios in range of N/P＜5 and reached the highest at N/P=5, and began to decrease beyond N/P＞5 as higher toxicity to cells. The transfection efficiency of PEI/DNA complexes at N/P=5 was higher than PEG. This study confirmed that PEI nanovector could effectively mediate foreign gene entering into A. thaliana protoplast cell to obtain transient expression, which may be developed as a hopeful and novel transgenic method combined with plant protoplast regeneration.     

The application to simplify the complicated power grid through adjacent two-node equivalent method

Most real power systems are not radial networks and evaluation of the stability often needs to establish electromagnetic transient simulation models. So the network equivalent is required first and foremost. The adjacent two-node equivalent method, which considers the coupling impedance between equivalent nodes, is used to realize the simplification and modeling to complex power grid. The analysis and simulation on both the HVDC system from Deyang to Baoji and a real extra-high voltage AC transmission system in South China shows that the static performance of the simulated system by using the adjacent two-node equivalent method is completely equivalent to the original one, as well as the performance of dynamic equivalent is in a satisfied level. Thus the transient system model simplified can be well established to support research of power grid’s security and stability.     

Promoter identification and effect on activation of NF-κB of porcine ISG58

Interferon (IFN)-stimulated gene (ISG) 56 family (composed of ISG54, ISG56, ISG58, and ISG60) plays important roles in defense against viral infection in mammalian cells. Numerous studies have been conducted on ISG54, ISG56, and ISG60; however, little is known on ISG58. In the present study, the upstream sequence of porcine ISG58 gene was first characterized as functional promoter by luciferase reporter assay, and then two directly adjacent IFN-stimulated response elements (ISREs), one at ?206 to ?194 (ISRE-I) and a second one, directly upstream of this element at ?219 to ?207?bp (ISRE-II), were identified using the bioinformatics method. The subsequent site-directed deletion and transient transfection experiments showed the candidate ISREs are functional. ISRE-I works better than ISRE-II and synergistic cooperation exists between two ISREs. Additionally, the effect of porcine ISG58 on activation of NF-κB was analyzed using the dual-luciferase reporter assay. The results will contribute to revealing the role of ISG58 in immune response.     

瞬时表达比较2种不同的RNAi载体的干涉效果

 RNAi with natural defence mechanism of homologous RNA degradation is widely used in research of antiviral plant. It is important to construct a highly efficent RNAi vector for transgenic plants of virus resis-tance. In this study, part fragments of coat protein gene of Potato virus Y (PVY) (451-750 bp) were inserted into the two expression vectors. Vector pROKY300 without intron and pHelY300 with PDK and CAT introns on the hpRNA stem were constructed. The silence efficiency of virus resistance of the two vectors was investigated as 88% (22/25)for pROKY300 and 92% (23/25) for pHelY300 through transient expression mediated by agroinfiltration. The results showed that both vectors were highly antiviral and elucidated the validity of RNAi-medicates resistance to virus.