内蒙古荒漠草原植物遗传多样性对模拟增温处理的响应

为探究全球变暖对温带荒漠草原地上种群的遗传影响,对已经接受模拟增温处理6年的短花针茅草原4种不同生活型植物,即半灌木、多年生禾草、多年生杂类草和一年生植物,应用AFLP分子标记方法研究了其遗传多样性和遗传结构。结果显示,对照处理与增温处理下的木地肤、短花针茅、细叶葱、猪毛菜4种植物的多态位点百分率(PPB)分别为11.32%,11.32%;40.83%,39.91%;14.29%,13.10%;19.85%,19.12%。Nei's基因多样性指数(He)分别为0.0274,0.0259;0.0812,0.0899;0.0131,0.0084;0.0506,0.0456。Shannon's信息指数值(I)分别为0.0447,0.0430;0.1354,0.1466;0.0267,0.0182;0.0811,0.0733。分子方差分析(AMOVA)显示4种植物的变异主要来源于实验处理内部,木地肤为85.03%,短花针茅为66.35%,细叶葱为70.00%,猪毛菜为66.52%;增温处理间的变异分别占-2.81%,-5.47%,-3.60%,2.53%(P0.05)。4种植物增温处理与变异程度之间在统计学上并无相关性。研究表明虽然短时间的模拟增温并不足以使4种生活型植物种群遗传多样性和遗传结构发生显著变化,但相对于3种多年生植物,一年生植物猪毛菜更容易受到增温影响。多年生和一年生植物对增温具有不同的遗传响应。     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

寡肽转运蛋白PepT2原核表达质粒的构建及其表达初探

寡肤转运蛋白(PepT2,peptide transporter,SLC15A2)是哺乳动物体内能够转运二肤、三肽的蛋白.研究表明,一些类肽的小分子药物也是PepT2的底物,但PepT2的结构与生物学功能尚待研究.建立稳定表达PepT2的表达体系是研究PepT2的重要环节.根据GenBank中人PepT2基因序列,借助Primer5.0设计了1对寡核苷酸引物,经PCR合成长达2 190bp的目的序列,通过重组构建pET30a(+)/PepT2表达质柱,测序分析确认目的基因中的3个碱基发生突变.初步研究了pET30a(+)/PepT2在大肠杆菌BL21(DE3)pLysS中的表达,为PepT2原核表达的进一步科研和实际应用奠定了基础.     

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10^?7, 1.4×10^?7, and 1.3×10^?8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.     

Erythropoietin Prevents Blood Brain Barrier Damage Induced by Focal Cerebral Ischemia in Mice

Recombinant human erythropoietin (rhEPO), a neurovascular protective agent, therapeutically supports angiogenesis after stroke by enhancing endogenous up-regulation of vascular endothelial growth factor (VEGF). Increased VEGF expression has been characterized to negatively impact the integrity of the blood brain barrier (BBB), causing brain edema and secondary injury. The present study investigated the rhEPO-induced BBB protection after stroke and how it might be achieved by affecting VEGF pathway. rhEPO treatment (5,000 U/kg, i.p., 30 min before stroke and once a day for three days after stroke) reduced Evans blue leakage and brain edema after ischemia. The expression of the BBB integrity markers, occludin, α-catenin and β-catenin, in the brain was preserved in animals received rhEPO. rhEPO up-regulated VEGF expression; however, the expression of VEGF receptor-2 (fetal liver kinase receptor, Flk-1) was significantly reduced in rhEPO-treated animals three days after stroke. We propose that, disregarding increased VEGF levels, rhEPO protects against ischemia-induced BBB damage at least partly by down-regulating Flk-1 expression and the response to VEGF signaling in the acute phase after stroke.     

Colonization of endophyte Acremonium sp. D212 in Panax notoginseng and rice mediated by auxin and jasmonic acid

Endophytic fungi can be beneficial to plant growth. However, the molecular mechanisms underlying colonization of Acremonium spp. remain unclear.In this study, a novel endophytic Acremonium strain was isolated from the buds of Panax notoginseng and named Acremonium sp. D212. The Acremonium sp. D212 could colonize the roots of P. notoginseng,enhance the resistance of P. notoginseng to root rot disease, and promote root growth and saponin biosynthesis in P. notoginseng. Acremonium sp. D212 could secrete indole-3-acetic acid(IAA) and jasmonic acid(JA), and inoculation with the fungus increased the endogenous levels of IAA and JA in P. notoginseng. Colonization of the Acremonium sp. D212 in the roots of the rice line Nipponbare was dependent on the concentration of methyl jasmonate(Me JA)(2–15 μmol/L) and 1-naphthalenacetic acid(NAA)(10–20 μmol/L). Moreover, the roots of the JA signaling-defective coi1-18 mutant were colonized by Acremonium sp. D212 to a lesser degree than those of the wild-type Nipponbare and mi R393 boverexpressing lines, and the colonization was rescued by Me JA but not by NAA. It suggests that the cross-talk between JA signaling and the auxin biosynthetic pathway plays a crucial role in the colonization of Acremonium sp. D212 in host plants.     

Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.     

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO₂ in C₄ and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C₃-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.     

斑点叉尾和杂交鲟幼鱼昼夜摄食节律和胃肠排空时间的研究

采用一次饱食投喂(将一昼夜分为8个时间段,每个时间段作为一个处理组,每天每个处理组饱食投喂一次)和分段式连续投喂(将一昼夜分为8个时间段,每天每个实验缸连续投喂8次)两种方法研究斑点叉尾和杂交鲟幼鱼的昼夜摄食节律,同时研究它们在摄食后24h内胃和全肠的排空时间。结果显示,在两种投喂方式下,斑点叉尾均表现出24h 一周期的摄食节律,两个日摄食率高峰值均出现在06：00和18：00(P<0.05)。杂交鲟在一次饱食投喂下表现出24h一周期的摄食节律,高峰值分别出现在11：00、17：00和05：00,在分段式连续投喂时表现出48h 一周期的摄食节律,高峰值分别出现在11：00、17：00和36：00。在摄食后1-9h 内,斑点叉尾的胃内含物比率急剧降低(P<0.05),并在24h 时出现极低值(P<0.05),而1-9h 内全肠内含物比率迅速升高(P<0.05),在9h时达到最大(P<0.05),在24h出现极低值(P<0.05)。在摄食后1-7h内,杂交鲟的胃内含物比率迅速下降(P<0.05),在24h 时出现极低值(P<0.05),1-7h 内肠内含物比率迅速升高(P<0.05),24h 时呈现极低值(P<0.05)。结果表明,两种实验鱼表现出不同的昼夜摄食节律,该节律受各自胃肠排空时间的影响,也受投喂时间的影响。研究建议,在斑点叉尾和杂交鲟幼鱼的养殖中宜在光线较弱的清晨(05：00-06：00)和黄昏(17：00-18：00)进行投喂。