基因芯片技术在植物基因克隆中的应用研究进展

基因芯片是以预先设计的方式将大量的生物讯息密码(寡核苷酸、cDNA、基因组DNA等)固定在玻片、硅片等固相载体上组成的密集分子阵列.基因芯片技术本质是生物信号的平行分析,它利用核酸分子杂交原理,通过荧光标记技术检测杂交亲和与否,再经过计算机分析处理可迅速获得所需信息.由于其具有高通量、微型化、连续化、自动化、快速和准确等特点,已引起国际国内广泛的关注和重视,在许多领域得到了广泛的应用.本文简述了基因芯片的概念,技术特点及主要分类,着重对其在基因表达水平检测,基因突变和多态性的分析,基因组DNA分析,后基因组学研究以及转基因农作物检测等方面进行阐述,并说明其存在的问题及展望.     

Reproductive biology of the andromonoecious Cucumis melo subsp. agrestis (Cucurbitaceae)

Background and Aims

Cucumis melo subsp. agrestis (Cucurbitaceae) is cultivated in many African regions for its edible kernels used as a soup thickener. The plant, an annual, andromonoecious, trailing-vine species, is of high social, cultural and economic value for local communities. In order to improve the yield of this crop, the first step and our aim were to elucidate its breeding system.

Methods

Eight experimental pollination treatments were performed during three growing seasons to assess spontaneous selfing, self-compatibility and effects of pollen source (hermaphroditic vs. male flowers). Pollination success was determined by pollen tube growth and reproductive success was assessed by fruit, seed and seedling numbers and characteristics. The pollinator guild was surveyed and the pollination distance determined both by direct observations and by indirect fluorescent dye dispersal.

Key Results

The species is probably pollinated by several Hymenoptera, principally by Hypotrigona para. Pollinator flight distances varied from 25 to 69 cm. No evidence for apomixis or spontaneous self-pollination in the absence of insect visitors was found. The self-fertility index (SFI = 0) indicated a total dependence on pollinators for reproductive success. The effects of hand pollination on fruit set, seed number and seedling fitness differed among years. Pollen tube growth and reproductive success did not differ between self- and cross-pollinations. Accordingly, a high self-compatibility index for the fruit set (SCI = 1·00) and the seed number (SCI = 0·98) and a low inbreeding depression at all developmental stages (cumulative δ = 0·126) suggest a high selfing ability. Finally, pollen origin had no effect on fruit and seed sets.

Conclusions

This andromonoecious species has the potential for a mixed mating system with high dependence on insect-mediated pollination. The selfing rate through geitonogamy should be important.     

Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene

The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope ¹³C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO₂]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties.Isoprenoids represent the largest and most diverse group (over 50,000) of natural compounds and are essential in all living organisms (Gershenzon and Dudareva, 2007; Thulasiram et al., 2007). They are economically important for humans as flavor and fragrance, cosmetics, drugs, polymers for rubber, and precursors for the chemical industry (Chang and Keasling, 2006). The broad variety of isoprenoid products is formed from two building blocks, dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP). In plants, the plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Zeidler et al., 1997) produces physiologically and ecologically important volatile organic compounds (VOCs), the carotenoids (tetraterpenes; Giuliano et al., 2008; Cazzonelli and Pogson, 2010), diterpenes, the prenyl side-chains of chlorophylls (Chls) and plastoquinones, isoprenylated proteins, the phytohormones gibberellins, and side-chain of cytokinins (for review, see Dudareva et al., 2013; Moses et al., 2013). Industrially important prokaryotes (e.g. Escherichia coli) also use the MEP pathway for the biosynthesis of isoprenoids (Vranová et al., 2012), and there is an increasing interest in manipulating the MEP pathway of engineered microbes to increase production of economically relevant isoprenoids (Chang and Keasling, 2006). To achieve this, a mechanistic understanding of the regulation of the MEP pathway is needed (Vranová et al., 2012).Some plants, including poplars (Populus spp.), produce large amounts of the hemiterpene VOC isoprene. Worldwide isoprene emissions from plants are estimated to be 600 teragrams per year and to account for one-third of all hydrocarbons emitted to the atmosphere (Arneth et al., 2008; Guenther, 2013). Isoprene has strong effects on air chemistry and climate by participating in ozone formation reactions (Fuentes et al., 2000), by prolonging the lifespan of methane, a greenhouse gas (Poisson et al., 2000; Archibald et al., 2011), and by taking part in the formation of secondary organic aerosols (Kiendler-Scharr et al., 2012).Poplar leaves invest a significant amount of recently fixed carbon in isoprene biosynthesis (Delwiche and Sharkey, 1993; Schnitzler et al., 2010; Ghirardo et al., 2011) to cope with abiotic stresses (Sharkey, 1995; Velikova and Loreto, 2005; Behnke et al., 2007, 2010b, 2013; Vickers et al., 2009; Loreto and Schnitzler, 2010; Sun et al., 2013b), although there are indications that other protective mechanisms can partially compensate the lack of isoprene emission in genetically transformed poplars (Behnke et al., 2012; Way et al., 2013). It has been suggested that in isoprene-emitting (IE) species, most of the carbon that passes through the MEP pathway is used for isoprene biosynthesis (Sharkey and Yeh, 2001). However, a recent study using pulse-chase labeling with ¹⁴C has shown continuous synthesis and degradation of carotenes and Chl a in mature leaves of Arabidopsis (Arabidopsis thaliana; Beisel et al., 2010), and the amount of flux diverted to carotenoid and Chl synthesis compared with isoprene biosynthesis in poplar leaves is not known.Isoprene emission is temperature, light, and CO₂ dependent (Schnitzler et al., 2005; Rasulov et al., 2010; Way et al., 2011; Monson et al., 2012; Li and Sharkey, 2013a). It has been demonstrated that isoprene biosynthesis depends on the activities of IDP isomerase (EC 5.3.3.2), isoprene synthase (ISPS; EC 4.2.3.27), and the amount of ISPS substrate, DMADP (Brüggemann and Schnitzler, 2002a, 2002b; Schnitzler et al., 2005; Rasulov et al., 2009b). In turn, DMADP concentration has been hypothesized to act as a feedback regulator of the MEP pathway by inhibiting 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7), the first enzyme of the MEP pathway (Banerjee et al., 2013). Understanding the controlling mechanism of isoprene biosynthesis is not only of fundamental relevance, but also necessary for engineering the MEP pathway in various organisms and for accurate simulation of isoprene emissions by plants in predicting atmospheric reactivity (Niinemets and Monson, 2013).There is ample evidence that silencing the ISPS in poplar has a broad effect on the leaf metabolome (Behnke et al., 2009, 2010a, 2013; Way et al., 2011; Kaling et al., 2014). While some of those changes (e.g. ascorbate and α-tocopherol) are compensatory mechanisms to cope with abiotic stresses, others (e.g. shikimate pathway and phenolic compounds) might be related to the alteration of the MEP pathway (Way et al., 2013; Kaling et al., 2014). The perturbation of these metabolic pathways can be attributed to the removal of a major carbon sink of the MEP pathway and the resulting change in the energy balance within the plant cell (Niinemets et al., 1999; Ghirardo et al., 2011). In this work, we analyzed the carbon fluxes through the MEP pathway into the main plastidic isoprenoid products.We used the ¹³C-labeling technique as a tool to measure the carbon fluxes through the MEP pathway at different temperatures, light intensities, and CO₂ concentrations in mature leaves of IE and transgenic, isoprene-nonemitting (NE) gray poplar (Populus × canescens). Isoprene emission was drastically reduced in the transgenic trees through knockdown of PcISPS gene expression by RNA interference, resulting in plants with only 1% to 5% of isoprene emission potential compared with wild-type plants (Behnke et al., 2007).We measured the appearance of ¹³C in the isoprenoid precursors 2-C-methyl-d-erythritol-2,4-cyclodiphosphate (MEcDP) and DMADP as well as isoprene and the major downstream products of the MEP pathway, i.e. carotenoids and Chls. To reliably detect de novo synthesis of the pigments, which occur at very low rates (Beisel et al., 2010), we used isotope ratio mass spectrometry (IRMS).Here, (1) we quantify the effect of isoprene biosynthesis on the MEP pathway in poplar, and (2) we show that suppression of isoprene biosynthesis negatively affects the carbon flux through the MEP pathway by accumulating plastidic DMADP, which feeds back to inhibit PcDXS, leading to (3) a slight increase of carbon flux toward production of greater chain-length isoprenoids and (4) a strong decrease in the overall isoprenoid carbon fluxes to compensate for the much lower MEP pathway demand for carbon. This study strongly supports the hypothesis that an important regulatory mechanism of the MEP pathway is the feedback regulation of plastidic DMADP on DXS. The large carbon flux through the MEP pathway of IE poplar plastids demonstrates the potential of transgenically altered IE plant species to produce economically valuable isoprenoids at high rates in, for instance, industrial applications.     

Transgenic Mice Expressing Yeast CUP1 Exhibit Increased Copper Utilization from Feeds

Copper is required for structural and catalytic properties of a variety of enzymes participating in many vital biological processes for growth and development. Feeds provide most of the copper as an essential micronutrient consumed by animals, but inorganic copper could not be utilized effectively. In the present study, we aimed to develop transgenic mouse models to test if copper utilization will be increased by providing the animals with an exogenous gene for generation of copper chelatin in saliva. Considering that the S. cerevisiae CUP1 gene encodes a Cys-rich protein that can bind copper as specifically as copper chelatin in yeast, we therefore constructed a transgene plasmid containing the CUP1 gene regulated for specific expression in the salivary glands by a promoter of gene coding pig parotid secretory protein. Transgenic CUP1 was highly expressed in the parotid and submandibular salivary glands and secreted in saliva as a 9-kDa copper-chelating protein. Expression of salivary copper-chelating proteins reduced fecal copper contents by 21.61% and increased body-weight by 12.97%, suggesting that chelating proteins improve the utilization and absorbed efficacy of copper. No negative effects on the health of the transgenic mice were found by blood biochemistry and histology analysis. These results demonstrate that the introduction of the salivary CUP1 transgene into animals offers a possible approach to increase the utilization efficiency of copper and decrease the fecal copper contents.     

The Salicylic Acid Signaling Pathway Plays an Important Role in the Resistant Process of Brassica rapa L. ssp. pekinensis to Plasmodiophora brassicae Woronin

Journal of Plant Growth Regulation - Clubroot disease, caused by Plasmodiophora brassicae Woronin infection, leads to significant yield and economic losses in cruciferous vegetables. However, the...     

Conservation,propagation, and redistribution (CPR) of Hill’s thistle: paradigm for plant species at risk

Cryopreservation is a valuable tool that could potentially create an alternate plant preservation strategy for species at risk such as Hill’s thistle. The present study is focused on a successful paradigm involving conservation, propagation and redistribution (CPR), emaphasizing the usefulness of cryopreservation techniques for plant conservation using Hill’s thistle (Cirsium hillii. (Canby) Fernald). A cryopreservation protocol was established using the droplet-vitrification method for 5-week-old shoot tips of in vitro grown cultures. More than 90% of shoot tips showed regrowth and nearly all regenerated plants were able to survive in the greenhouse. The survival, growth, and development of plants from cryopreserved shoot buds and their performance in field conditions were all comparable or better than the plants from non-cryopreserved buds. Reintroduced plants flowered following overwintering and the magnitude of flowering was site dependent with ca. 80% flowering observed in one site. The site dependent flowering patterns were assessed using phytohormone profiling and compared to herbivory, a common biotic stressor of these plants. Lower tryptophan concentrations led to higher flowering except in alvars, where the limestone resisted root penetration resulting in poor flowering. The presence of tryptamine in the greenhouse acclimatized or alvar field leaves suggested the preparedness of the plants for herbivory/grazing. Serotonin and melatonin concentrations were lower in flowering plants and in sites where the biotic/abiotic stress was minimal. This study provides evidence of the effectiveness of the CPR model in species recovery programs for endangered species. Physiological characterization of plants developed from cryopreserved tissues can be useful for fundamental and applied research in stress adaptation and reproductive biology of plants.

     

金线蛙入蛰时几项生理常数间的关系研究

本文经统计学分析处理,表明金钱蛙（Ｒａｎａｐｌａｎｃｙｉ）入蛰时的体长与体重、胴体重和体重与肝重、脂肪体重、生殖腺重之间均存在非常显著的线性相关关系。     

中国株丙型肝炎病毒（HCV）结构区蛋白在昆虫细胞中的表达及加工

利用杆状病毒表达系统在昆虫细胞中表达了完整的中国河北株丙丙型肝炎病毒结构蛋白。免疫印迹实验结果显示,表达产物中有一系列分子量不同、可以与ＨＣＶ抗体阳性病人血清反应的蛋白,表明结构蛋白被宿主细胞蛋白酶切割与加工,相应分别为２０ｋＤ的核心蛋白、３２ｋＤ糖基化的Ｅ１蛋白４０ｋＤ的未糖化的Ｅ２蛋白和７０ｋＤ糖基化的Ｅ２蛋白,另有８０ｋＤ及１００ｋＤ的两组前体蛋白。利用表达产物检测慢性ＨＣＶ感染者血清,发现     

Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells

Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.     

Cytokine and Antibody Based Diagnostic Algorithms for Sputum Culture-Positive Pulmonary Tuberculosis

Background

Tuberculosis (TB) is one of the most serious infectious diseases globally and has high mortality rates. A variety of diagnostic tests are available, yet none are wholly reliable. Serum cytokines, although significantly and frequently induced by different diseases and thus good biomarkers for disease diagnosis and prognosis, are not sufficiently disease-specific. TB-specific antibody detection, on the other hand, has been reported to be highly specific but not sufficiently sensitive. In this study, our aim was to improve the sensitivity and specificity of TB diagnosis by combining detection of TB-related cytokines and TB-specific antibodies in peripheral blood samples.

Methods

TB-related serum cytokines were screened using a human cytokine array. TB-related cytokines and TB-specific antibodies were detected in parallel with microarray technology. The diagnostic performance of the new protocol for active TB was systematically compared with other traditional methods.

Results

Here, we show that cytokines I-309, IL-8 and MIG are capable of distinguishing patients with active TB from healthy controls, patients with latent TB infection, and those with a range of other pulmonary diseases, and that these cytokines, and their presence alongside antibodies for TB-specific antigens Ag14-16kDa, Ag32kDa, Ag38kDa and Ag85B, are specific markers for active TB. The diagnostic protocol for active TB developed here, which combines the detection of three TB-related cytokines and TB-specific antibodies, is highly sensitive (91.03%), specific (90.77%) and accurate (90.87%).

Conclusions

Our results show that combining detection of TB-related cytokines and TB-specific antibodies significantly enhances diagnostic accuracy for active TB, providing greater accuracy than conventional diagnostic methods such as interferon gamma release assays (IGRAs), TB antibody Colloidal Gold Assays and microbiological culture, and suggest that this diagnostic protocol has potential for clinical application.