碱性成纤维细胞生长因子在苜蓿中的表达

为了降低bFGF(basic fibroblast growth factor)的生产成本,结合植物生物反应器的优点,就bFGF在转基因苜蓿中的表达进行了探索.将bFGF插入植物表达载体pBⅡ21中,获得了含有bFGF基因的植物表达pBIcbFGF,再将pBIcbFGF利用冻融法转到农杆菌中.利用农杆菌介导法将基因转化保定苜蓿,转基因苜蓿在TM-1培养基+20 mg/L卡那霉素(Kan)+200 mg/L特美汀(Tim)中诱导分化,在生根培养基中生根,获得再生植株.再生植株通过PCR检测、RT-PCR及Western blot证实外源基因已经在苜蓿中成功表达.获得含有目的蛋白的阳性植株.为苜蓿作为植物生物反应器生产bFGF奠定了理论及技术基础.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

过量铜、锌对外生菌根菌牛乳牛肝菌生物量、呼吸和糖酵解酶活性的影响

在过量铜或锌胁迫下,独立培养的外生菌根菌牛乳牛肝菌（Suillus     

融合基因umcel5N-CBM的构建、表达及融合酶性质分析

BM,并实现了融合基因在大肠杆菌BL21(DE3)pLysS中的表达.研究结果表明,融合酶Umcel5N-CBM与结晶纤维素(avicel)以及滤纸粉末的结合能力比原始酶Umcel5N提高了约一倍,但未显示出降解结晶纤维素的新活性,说明在结晶纤维素的降解过程中,纤维素酶的催化功能域起到关键作用.     

DNA methylation changes in photoperiod-thermo-sensitive male sterile rice PA64S under two different conditions

Epigenetic modification can occur at a high frequency in crop plants and might generate phenotypic variation without changes in DNA sequences. DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by regulating gene expression. Rice Photoperiod-Thermo-Sensitive Genic Male Sterile (PTGMS) lines can transform from sterility to fertility under lower temperatures and short-day (SD) conditions during anther development. So far, little is known about the DNA methylation variation of PTGMS throughout the genome in rice. In this study, we investigated DNA cytosine methylation alterations in the young panicles of PTGMS line PA64S under two different conditions using methylation sensitive amplified polymorphism (MSAP) method. Compared with the DNA methylation level of PA64S under lower temperatures and SD conditions (fertility), higher methylation was observed in PA64S (sterility). The sequences of 25 differentially amplified fragments were successfully obtained and annotated. Three methylated fragments, which are homologous to D2, NAD7 and psaA, were confirmed by bisulfite sequencing and their expression levels were also evaluated by qPCR. Real time quantitative PCR analysis revealed that five of the six selected methylated genes were downregulated in PA64S (sterility). These results suggested that DNA methylation may be involved in the sterility–fertility transition of PA64S under two different environmental conditions.     

Inhibition of IFN-gamma receptor signaling by hepatitis C virus non-structural protein NS4B]

The function of NS4B is incompletely understood. The aim of the study is to understand the influence of NS4B on anti-viral response. After cell line stably expressing NS4B established, the influence of IFN-alpha of different concentration on VSV was studied using plaque assay; cell expression profiling caused by NS4B was studied using DNA microarray, and the IFNGR1 fluorescence intensity was analyzed. Our data showed that HCV-NS4B could suppress immuno-associated gene expression, in particular, IFN-gamma receptor signal transduction-related genes. Taken together, NS4B could play some roles in HCV resistance to IFN therapy.     

基于酵母水平的高通量药物筛选

随着后基因组时代的到来,越来越多的药物靶标蛋白将会被发现,基于靶标蛋白设计出的化合物也将大量涌现,高通量药物筛选日趋重要。酵母基因组的易操作性及其简单稳定的培养条件,使得该真核微生物成为一种理想的药物筛选工程细胞。本文讨论了选择酵母系统进行细胞水平筛选的优缺点,并从基于靶点和表型两种筛选模式对酵母水平的高通量药物筛选做一总结。     

外源基因表达造成的群体感应细菌生存压力的建模分析

外源基因的表达及其对细菌种群的影响对于群体感应系统和合成生物学产业的研究具有重要意义。然而,人们对于表达外源蛋白的细菌本身的行为模式仍然知之甚少。为了研究菌落生长和外源基因表达的过程究竟受到哪些因素的影响,文中测量了受Lux类受体调控的外源基因在N-酰基高丝氨酸内酯 (N-acyl homoserine lactone,N-AHL) 信号分子诱导下的表达,并模拟了其对细菌种群动态的影响。文中建立了一个假设性的数学模型,对信号分子诱导表达下细菌种群生长受影响的现象进行了分析。先前的研究通常将细菌种群生长受群体感应系统影响的现象归咎于合成群体感应信号分子的消耗与N-AHL信号分子的毒性,文中提供了对于这种生存压力的另一种可能的解释。     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.