肺炎链球菌SP0306蛋白的表达纯化及结晶研究

SP0306蛋白是肺炎链球菌TIGR4菌株中的一种假想的转录因子,但其蛋白三维结构及生物学功能尚未明了,生物信息学分析提示其可能调控碳水化合物代谢相关基因的表达。成功构建了SP0306蛋白的全长表达载体PET28a-sp0306,利用大肠杆菌BL21(DE3)菌株进行原核表达,获得了以可溶形式表达的目的蛋白。经Ni-NTA柱亲和层析及DEAE阴性离子交换层析纯化后,获得了高纯度的目的蛋白。采用悬滴气相扩散法获得了质量较好的SP0306蛋白晶体,并初步进行了晶体X射线衍射,为其最终的三维结构解析及生物学功能研究奠定了基础。     

应用群分析研究薯蓣的分类

本文是应用群分析研究薯蓣属根状茎组中国分类群分类的一次尝试。分类特征取用了形态学、细胞学、花粉形态和生物化学等多方面的性状进行综合分析;方法和手段上使用了距离系数、相关系数等各种群分析运算方法,并且以综合系数进行评价。不仅取得了与传统分类基本一致的分类结果,为该组植物的系统分类提供富有启发性的参考,同时也为高等植物数量分类研究找到一种最优分类方法一相关系数UPGMA法。     

Characterization of antiproliferative and cytotoxic properties of the HSV-1 immediate-early ICPo protein

BACKGROUND: The identification of novel proteins displaying cytostatic and/or cytotoxic actions that could eventually be used for gene therapy is a major issue in cancer research. Data from the literature suggested that the immediate-early ICP0 protein from herpes simplex virus type 1 (HSV-1) could fulfill these properties as it had been observed that this protein is involved in arrest of cell growth at the G1/S and G2/M phases of the cell cycle and that deletion of ICP0 from HSV-1 or HSV-1-recombinant vectors significantly reduced their cytotoxicity. The molecular basis of its action is likely related to the ability of ICP0, which possesses E3-ubiquitin ligase activity, to target destruction of key cellular proteins, including centromeric proteins, resulting in abnormal chromosome segregation, unusual cytokinesis, and emergence of nuclear morphological aberrations. However, neither the gene therapy potential of ICP0 on its own nor its action on primary quiescent cells has been assessed to date. The aim of this work was therefore to evaluate the antiproliferative and cytotoxic properties of ICP0 on a human glioblastoma cell line and on quiescent primary cells, and to explore whether this protein has a potential for gene therapy of cancer. METHODS: HSV-1-based amplicon particles were generated following a recently described method that produces relatively high titers of vector stocks that are essentially free of helper virus. These vectors express either wild-type ICP0 or FXE, a RING finger mutated inactive form of ICP0, together with reporter green fluorescent protein (GFP). The vectors were used to infect Gli36 cells, which derive from a human glioblastome, and cultured rat primary cardiomyocytes and brain cells, two well-established models of non-dividing cells. Expression and localization of ICP0 and FXE, as well as their action on centromeres and nuclear morphology, were evaluated by Western blotting, indirect immune fluorescence, and confocal microscopy using specific antibodies and DAPI labeling. The impact of ICP0 on cell growth, toxicity, and viability was evaluated in the different cells using a variety of methods, including FACS analysis after propidium iodide and AnnexinV staining, crystal violet staining, clonogenic capability, caspase 3 activation, MTT tests, and release of lactate dehydrogenase, after infection with the different vectors. RESULTS: The three cell types under study showed high levels of transduction by amplicons and strong expression of GFP, ICP0, and FXE transgenic proteins. Wild-type ICP0, but not FXE, induced centromeric disruption, appearance of micronuclei, arrest of proliferation, and significant cell death in glioblastoma Gli36 cells. In contrast, neither micronuclei formation nor any other sign of cell toxicity could be observed in cultured primary cardiomyocytes or brain cells, as evaluated by MTT tests and crystal violet staining. Furthermore, in the case of cardiomyocytes, expression of ICP0 did not interfere with beating as cells continued to beat at the same frequency as non-infected cells for several days post-infection. Neither AnnexinV early staining nor caspase 3 activation was observed in dying infected Gli36 cells, suggesting that these cells were not entering apoptosis. In contrast, release of lactate dehydrogenase by infected Gli36 cells suggested a necrotic way of death. CONCLUSIONS: ICP0 induced a strong cytostatic action followed by significant cell death on the glioblastoma Gli36 cell line. In contrast, neither cell death nor any other evidence of ICP0-induced toxicity affecting major physiological parameters was observed in primary cultured cardiomyoctes and brain cells, two models of primary non-cycling cells. These results suggest that ICP0 has gene therapy potential and could represent the first member of a novel family of directly acting proteins that could be used to treat cancers.     

用改进的重叠PCR引入血管内皮生长因子基因突变

血管内皮生长因子（vascular endothelial growth factor, VEGF）的PCR产物克隆于T载体上,经转化JM109感受态菌株后,随机挑取8个白斑菌落,混合后制成混合模板.采用3条引物,做两轮重叠PCR反应,获得了VEGF的突变基因,经PCR鉴定,酶切鉴定和测序分析表明所得基因为目的产物.实践证明这种突变方法简单快速,为下一步实验大量引入突变奠定了实验基础.     

北方引种二乔木兰育苗技术及经济意义

二乔木兰(Magnolia soulangeana Soul.-Bod.)分布于我国广州、杭州、昆明等地。经引种,二乔木兰在辽宁西部(北纬41°12′～42°17′)这块高寒之地也能生长,且已开花结实并培育出引种后的子代苗木。1991年引入二乔木兰栽植成功。1995年开花,2000年该批树木结实。2005年试验播种、扦插等育苗,直到2015年才初步完成播种育苗关。9月下旬采种,种子需经低温储藏100～150天,种子6℃始萌动,催芽需20～25℃,播种25～30天出苗。二乔木兰是辽宁乃至东北少有的阔叶花卉树种,其栽培育苗成功具有重要经济价值。     

Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: β-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

不同作物两苗同穴互作育苗的生理生态效应

为了探明不同作物两苗同穴互作育苗提高目的作物幼苗素质的机理,本试验在塑料温棚20-30℃、自然光照条件下,采用532mm×280mm具有200方形孔的塑料育苗盘,用土壤作基质,分别以小麦、玉米、谷子、高粱和目的作物棉花、油菜、番茄、花生、牡丹、烟草同穴播种,研究了互作育苗对育苗土壤微生物、酶活性及根系分泌物的影响,以及对目的作物幼苗根系活力、叶片可溶性糖含量和ATP含量的影响,结果表明:随互作苗的加入,育苗土壤中细菌数量显著增加52.80％-102.76％、放线菌数量显著增加34.11％-76.48％、真菌数量显著降低44.33％-56.14％;所测土壤酶活性显著提高,其中脱氢酶活性显著提高30.57％-66.37％、中性磷酸酶活性显著提高38.17％-54.37％、转化酶活性显著提高23.74％-35.04％、脲酶酶活性显著提高60.25％-85.47％;所测根系分泌物积累量显著减少,其中2,4-二叔丁基苯酚显著减少32.80％-51.65％、2,6-二叔丁基苯酚显著减少36.60％-56.59％、邻苯二甲酸二丁酯显著减少10.42％-49.99％、9-16碳烯酸甲酯显著减少25.62％-55.59％;目的作物则表现为根系活力、叶片可溶性糖含量和ATP含量显著提高,增加了目的作物幼苗根重、苗重和侧根数,离床存活期延长,栽后缓苗期缩短,表现互作促进.在所有互作处理中,以棉花+小麦、棉花+谷子、油菜+谷子、番茄+小麦、番茄+谷子、花生+小麦、花生+谷子、牡丹+谷子、烟草+谷子处理中目的作物幼苗素质表现较好.不同作物两苗同穴互作育苗改善了育苗土壤微生物数量和结构,这可能是提高土壤酶活性和降低土壤有害根系分泌物积累的主要原因,进而提高了目的作物幼苗素质.