Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells

Background  

The investigation of protein-protein interactions is important for characterizing protein function. Bimolecular fluorescence complementation (BiFC) has recently gained interest as a relatively easy and inexpensive method to visualize protein-protein interactions in living cells. BiFC uses "split YFP" tags on proteins to detect interactions: If the tagged proteins interact, they may bring the two split fluorophore components together such that they can fold and reconstitute fluorescence. The sites of interaction can be monitored using epifluorescence or confocal microscopy. However, "conventional" BiFC can investigate interactions only between two proteins at a time. There are instances when one may wish to offer a particular "bait" protein to several "prey" proteins simultaneously. Preferential interaction of the bait protein with one of the prey proteins, or different sites of interaction between the bait protein and multiple prey proteins, may thus be observed.     

Non-native plants and illegitimate interactions are highly relevant for supporting hummingbird pollinators in the urban environment

Urbanization leads to the restructuring of plant-animal interactions due to environmental changes and introduction of non-native species that become part of local ecological processes. The conservation of pollinators in urban areas has received increasing attention. However, detailed quantification of available floral resources and their use by pollinators are rarely conducted. Here, we characterized the interaction network between hummingbirds and native and non-native plants, the behavior of resource use by pollinators and the temporal availability of nectar resources in an extensive urban green area from Brazil. We found that more than half of the interactions between hummingbirds and plants were illegitimate, which do not constitute potential pollination. The interaction network was generalized, indicating low level of niche partitioning, which is usual for urban environments. Inclusion of non-pollination interactions increased specialization. Although native and non-native plants provide a similar amount of nectar when considering each plant species for a given month, the latter contributed most to the total nectar availability owed to their higher abundance and longer flowering phenologies. Importantly, non-native plants provided resources when native flowers were scarce. Our results show how non-native plants may sometimes have relevant contributions for maintenance of pollinators in cities throughout the year, supporting them during periods of resource shortage. At the same time, urban pollinator-plant communities may be characterized by high prevalence of illegitimate interactions, which highlights the opportunistic use of resources by animals. In conclusion, our study suggests that biodiversity-friendly urban planning should consider the relevant role played by non-native species and that pollinator support may require different types of interactions with flowers.     

芥菜开花信号整合子的两个核心转录因子FLC和SVP相互作用的体外检测

 为了深入研究调控芥菜开花信号整合子的两个核心转录因子Flowering Locus C（FLC）和SHOTR VEGETATIVE PHASE（SVP）的作用机理和进行人工调控,以芥菜‘QJ’为材料,采用RT-PCR技术分别扩增FLC和SVP的编码序列,构建原核表达质粒pET43.1a-FLC、pET43.1a-SVP,转化宿主菌大肠杆菌BL21,通过SDS-PAGE检测该蛋白的表达。利用免疫共沉淀原理及pET43.1a-FLC、pET43.1a-SVP融合蛋白序列中的6 × His标签能与Ni+结合的特点,对芥菜FLC与SVP的相互作用进行SDS-PAGE检测。结果表明：FLC与SVP能在体外相互作用并形成复合体,为深入分析FLC与SVP间的作用机理以及探讨其与下游开花信号整合子元件的相互作用提供了理论和技术基础。     

Protein and isozyme electrophoresis and isoelectric focusing for the characterization of apple clones

To identify apple (Malus domestica Borkh.) clones, electrophoretic and isoelectric focusing of protein and isozyme pattern differences were investigated. Existing electrophoretic methods were adapted to obtain satisfactory separation and reproducibility of patterns. Several protein extraction buffers and procedures were evaluated to optimize protein recovery and enzyme activity from shoot bark tissue and other organs and to counteract the interference of other cell components. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) did not reveal useful differences among cultivars, nor did total native protein patterns (non-denaturing PAGE). Peroxidase, esterase, acid phosphatase and indoleacetic acid oxidase extracted from shoot bark tissue did, however, show isozymic differences useful for identification. These enzymes expressed constant isozyme patterns at different times of the year and from trees growing under different conditions, and patterns were independent of wood age and rootstock influence.     

山葡萄花序蛋白质双向电泳技术体系的建立

【目的】为开展山葡萄花序蛋白质组学研究。【方法】以山葡萄(Vitis amurensis Rupr)花序为试材,通过对蛋白质提取方法、裂解液成分、胶条pH梯度的改进,建立了适于山葡萄花序蛋白质组学研究的最佳双向电泳技术体系。【结果】采用酚提取法提取花序蛋白质,用含有硫脲和DTT的裂解液Ⅱ裂解,选用17 cm pH 4～7的IPG胶条在适宜的双向电泳条件下分离,可以获得分辨率高、背景清晰且重复性好的双向电泳图谱,可以检测出1 088个清晰的蛋白点。【结论】酚提取法适于山葡萄花序蛋白质的提取,硫脲和DTT可增强花序蛋白质的溶解性。     

桃果实蛋白质双向电泳影响因素的研究

以‘大久保’桃（^{Prunus persica} L.'Okubao'）果实为试材,通过比较不同上样量、不同上样缓冲液、不同平衡缓冲液、不同染色方式等条件下的双向电泳图谱,探讨以上各因素对桃果实双向电泳图谱效果的影响。结果表明考马斯亮蓝染色后的电泳图谱虽然蛋白质点的数量少于银染色后的图谱,但背景较为清晰,适用于蛋白质组分析。采用固相pH梯度17 cm胶条,100 μg蛋白质上样量,样品干粉溶于375 μL含有硫脲、磺基三甲基胺乙内脂3~10的上样缓冲液中,等电聚焦后用含有磷酸三丁酯的平衡缓冲液还原,进行SDS-PAGE,银染色得出的双向电泳图有蛋白点1 209个,纹理较少,适用于桃果实蛋白质组学分析且兼容于下游技术的蛋白质质谱鉴定。     

葡萄叶片蛋白质双向电泳技术体系的建立

现对葡萄叶片中总蛋白质的双向电泳技术体系进行了初步探讨,比较了不同等电聚集程序、不同pH胶条以及不同SDS-PAGE凝胶浓度的双向电泳效果。结果表明:葡萄叶片蛋白质使用等电聚焦程序B、pH范围为4~7的胶条和12%的SDS-PAGE凝胶可以得到较为理想的双向电泳分析结果;采用7 cm胶条最大可以分离出143个有效蛋白质点。     

Expression of recombinant myocardin and its effect on human bone marrow mesenchymal stem cell reprogramming into vascular smooth muscle cells

AIM: To optimize the expression and purification of recombinant myocardin, and to explore the effect of recombinant myocardin on the transdifferentiation of human bone marrow mesenchymal stem cells (hBMSCs) into vascular smooth muscle cells (vSMCs). METHODS: The prokaryotic expression vector for myocardin was constructed. The best ways to induce the expression of myocardin recombinant protein and to optimize the isolation and purification methods were explored. Nano-transducers were applied to encapsulate myocardin to form nano-protein complexes. Nano-myocardin was transducted into hBMSCs and the protein transduction efficiency was measured. After transduction of the protein was successful, smooth muscle myosin heavy chain, a specific marker of vSMCs, was detected, and the transformation of hBMSCs into vSMCs was also observed. RESULTS: Compared with the traditional methods, the expression of myocardin recombinant protein was higher by high-concentration induction method, and the new elution method was able to obtain more myocardin recombinant protein. The nano-myocardin complexes were successfully transduced into the hBMSCs and induced the transformation of hBMSCs into vSMCs. CONCLUSION: The recombinant myocardin was efficiently transduced into the cells and reprogrammed hBMSCs into vSMCs.     

我国榛属植物种质资源的研究、利用与创新

榛子为榛科榛属植物,全世界有16种,我国原产8个种和2个变种;我国现有榛林的95%是野生平榛,选育的平欧杂种榛和引进的欧洲榛是目前主要的栽培种。从平榛资源调查和选优、平欧杂种榛种质创新和品种选育、我国其他榛属资源的利用和国外榛属资源的引种等方面对我国榛子的育种现状进行了介绍;对我国及国外榛属植物种质资源的研究进展进行了总结,对比了我国与世界先进水平的差距;并从现有平欧杂种榛在科研和生产中亟待解决的问题、第1代平欧杂种榛的目标性状改良及今后榛属植物的育种策略等方面,对榛属育种与资源利用提出了一些见解,以期与国内同行专家进行交流。     

枇杷叶片和果实总蛋白质提取及双向电泳的优化方法

以枇杷叶片和果实为材料,探索适于枇杷总蛋白质分析的双向电泳方法.比较2种不同蛋白质提取方法对双向电泳结果的影响,并对双向电泳蛋白质上样量、等电聚焦条件等进行了优化,探索出一种适于枇杷叶片和果实总蛋白质分析的高分辨率、高重复性双向电泳方法.采用酚抽法提取枇杷叶片和果实蛋白质,以24 cm、pH 4~7的IPG胶条,按15...     

不同方法提取青藏高原蕨麻总DNA的比较研究

以改良CTAB法、改良SDS法、试剂盒法提取青藏高原蕨麻总DNA;用紫外分光光度法测定提取的青藏高原蕨麻的总DNA浓度和质量;用琼脂糖凝胶电泳法、分光光度法测定提取的青藏高原蕨麻的总DNA质量,以期筛选出青藏高原蕨麻总DNA提取的最佳方法。结果表明:用改良SDS法提取的蛋白质含量最高,OD260/OD2801.61左右,用改良CTAB法提取的DNA的OD260/OD2801.76左右;用试剂盒法提取的DNA的OD260/OD2801.70左右;用改良CTAB法提取DNA的浓度在100μg/mL。     

The use of native turf mixtures to approach sustainable lawn in urban landscapes

Native grasses are excellent candidate species for manipulation to produce dwarf and turf type varieties as well as for producing cultivars with higher attractiveness and requiring less maintenance to be used as a turf grass in arid and semi-arid regions of the world. This investigation was conducted to explore visual qualities of native grasses and their mixtures compared to commercial turf. The field experiment was set out in a split-plot in time based on a randomized experimental design with three replications. We used two native monoculture accessions, perennial ryegrass (Lolium perenne L. ‘Yarand’) and (Lolium perenne L. ‘Shadegan’), Native low-variety Mixture (NM1): consisting of 50% Lolium multiflorum ‘Shadegan’? 50% Festuca spp. ‘Shadegan’, Native high-variety Mixture (NM2): consisting of 55% Lolium perenne L. ‘Yarand’? 35% Lolium perenne L. ‘Shadegan’? 5% L. multiflorum ‘Shadegan’ and 5% Festuca spp. ‘Shadegan’ and compared this with one commercial turf mixture that is commonly used in landscaping. Results indicated the effects of seasons and turf grass types and their interaction had significant effects on most variables including quality, season color, leaf texture, density, quality after clipping (p < 0.01). The visual quality measurements indicated the superiority of L. perenne ‘Shadegan’ over other native monoculture and polyculture and its ablility to compete with the commercial turf. The native turf mixture of NM2 showed several good characteristics. L. perenne ‘Yarand’ had statistically the lowest score for visual appeal as compared with the other turf types. This research suggests that the use of native grass species of L. perenne ‘Shadegan’ is worth investigating for better performance of the native landscape.     

Gene expression of spleen lymphocytes induced by gp96-peptide complexes and its anti-tumor effect in vitro

AIM: To study the expressing variation of TNF-α and IFN-γ mRNA in mouse splenocytes induced by H22 tumor cells derived heat shock protein gp96-peptide complexes in vitro,and to observe the morphologic change of H22 tumor cells which treated with the culture supernatant.METHODS: H22 tumor cells derived HSP gp96 was obtained by the techniques for protein extraction and purification and was identified by Western blotting method.The expression values of TNF-α and IFN-γ mRNA in spleen lymphocytes were detected by semi-quantitative RT-PCR.The morphologic changes of H22 tumor cells induced by the culture supernatant were observed by laser scanning confocal microscopy (LSCM) and transmission electron microscope (TEM).RESULTS: Purified heat shock protein gp96 was identified by Western blotting.The expression value of TNF-α and IFN-γ mRNA in activated spleen lymphocytes induced by gp96-peptide complexes was higher than that in control groups (P<0.05).The morphologic change of apoptosis of H22 tumor cells,which treated by the culture supernatant of experimental group was observed with LSCM and TEM.CONCLUSION: Heat shock protein gp96-peptide complexes increase the expression value of TNF-α and IFN-γ mRNA in spleen lymphocytes of mouse in vitro.Besides,apoptosis of H22 cells is induced by immunologic active material secreted by activated splenocytes.     

Native plant enthusiasm reaches new heights: Perceptions,evidence, and the future of green roofs

The use of native plants on green roofs has attracted considerable attention in recent years. With this comes the implicit assumptions that native plants are better adapted, provide greater environmental benefit, and are more aesthetically pleasing than non-native plants. We examined papers published in scholarly journals and papers presented at the annual North American green roof conference to identify who is promoting the use of native plants on green roofs, their rationale for doing so, and the scientific evidence to support the assertion that natives are better adapted. Architects, landscape architects, and biologists were the most likely to promote native plants and engineers were the least likely. Many of the reasons for using native plants on green roofs originate from ground-level landscaping and have simply been transplanted to the roof, without regard for the fact that the rooftop is a fundamentally different environment than the ground. Nearly half of all pro-native papers used the term “native” without definition. This review highlights the need for greater rigor and transparency when promoting the use of native plants and further demonstrates how misconceptions can result in sub-optimal green roof design and performance.     

Expression of proteins in mouse thymocytes induced by low dose X-rays and their biological activity

AIM:To observe the expression of proteins induced by whole-body irradiation in mice with low dose X-rays and their biological activity.METHODS:To analyse the expression of protein induced by whole-body irradiation with 75mGy X-rays and sham-irradiation, the method of gel filtration with Sephadex G-100 and HPLC were used.The biological activity of protein expressed in thymocytes was observed by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes.RESULTS:HPLC analysis showed that there was a marked decrease in expression of 24.5 kD protein molecule in the fractions of thymocyte extract in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocytes of the irradiated mice at the concentration of 6.25 mg/L showed both inhibitory effect on normal T cell proliferation and protective effect on the chromosome damage induced by high dose radiation. CONCLUSION:Down regulation of 24.5 kD protein molecule expression may have implications in the mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation.     

天津木本蔬菜资源调查研究

通过野外调查、走访、市场调研以及查阅大量文献资料,对天津木本蔬菜资源进行了系统调查。结果表明:初步确定天津有木本蔬菜资源23科35属41种,并依据食用器官的不同将其分为4类。为天津木本蔬菜资源的保护和开发提供初步参考。     

竹荪多糖的分离纯化和鉴定

人工栽培的竹荪子实体干品经热水提取，乙醇沉淀，蛋白酶法和Ｓｅｖａｇ法相结合去蛋白，ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５和Ｓｅｐｈａｄｅｘ Ｇ－２００柱纯化，得到竹荪多糖，命名为ＤＩ。用聚丙烯酰胺凝胶电泳和葡聚糖凝胶柱层析，分析表明，该多糖为均一物质，竹荪多糖的分子量为１４４０００。经紫外扫描，无核酸和蛋白质的特征吸收峰，红外扫描表明有典型的多糖吸收峰。该多糖经纸层析和气相色谱分析表明：其单糖组成为     

生物技术在果树上的应用

现代生物技术有很大发展潜力,其成就备受关注.近年来,生物技术在果树上的研究进展很快,尤其在种苗的生产和育种上已取得了可喜进步.该文就离体繁殖、花药和花粉培养、胚培养、原生质体培养及融合、果树的遗传转化及与果树遗传育种相关的其它分子生物技术的优点、应用原理以及在果树上的研究进展方面作了简要概述,并提出了今后研究重点及方向.     

Bird populations in native forest patches in south-eastern Australia: the roles of patch width,matrix type (age) and matrix use

We surveyed birds in patches of native eucalypt forest and in surrounding exotic matrix (Radiata pine forests) in south-eastern Australia. Our objectives were: (1) to examine the influence of the width of native forest patches and the age of surrounding pine forests on bird occurrence in patches of native forest; and (2) to verify the relationship between the use of the surrounding pine matrix and bird species response to variation in width of patches of native forests. A total of 32 study sites (boundaries between eucalypt and pine forests) were surveyed. Birds were counted by the area search method within 0.5-ha quadrats. Data were analysed using generalised linear models. Wide patches of eucalypt forest supported higher species richness and greater numbers of birds, such as foliage searchers and nectarivores, than narrow patches. Matrix age also influenced the occurrence of some species in native patches. The abundance of species in wide and narrow patches of native forest was related to their use of the matrix. This was true for native forests surrounded by old but not by young pine forests. We suggest that management in wood production landscapes take into account both characteristics of native patches and the surrounding matrix. Negative impacts of fragmentation in managed landscapes might be reduced by promoting matrix types that are suitable for bird species.