二性霉素B与β-escin混合穿孔电极液在人心房肌细胞SK2电流记录中的应用

目的:为研究钙离子对人心房肌细胞小电导钙激活钾通道电流(ISK2)的调节作用,建立用二性霉素B(amphotericin B)与β-escin穿孔的膜片钳(PPR)技术。方法:体外循环术中取得右心耳,应用急性酶分离获得单个人体心房肌细胞,用二性霉素B和/或β-escin作为穿孔电极液,进行穿孔膜片钳实验,在此模式下测试钙离子对人心房肌细胞SK2电流的调控作用,并用胞内钙测试系统验证穿孔前后胞内钙变化。结果:混合使用穿孔电极液6.88μg/mlβ-escin和150μg/ml二性霉素B与单独用150μg/ml二性霉素B或6.88μg/mlβ-escin相比,前一种方法细胞容易封接,能形成稳定的穿孔膜片钳记录模式,可观察到SK2电流有激活,且胞内钙测试系统检测时可观察到穿孔后电极液至细胞内的游离钙离子浓度的增加,F340/380增强。结论:适当浓度的二性霉素B与β-escin混合使用进行穿孔膜片钳实验是一种稳定的全细胞膜片的记录技术,可以用于胞内游离钙离子对SK2电流调控的研究。     

Quorum Sensing System Used as a Tool in Metabolic Engineering

Quorum sensing (QS) is a ubiquitous cell–cell communication mechanism in microbes that coordinates population‐level cell behaviors, such as biofilm production, virulence, swarming motility, and bacterial persistence. Efforts to engineer QS systems to take part in metabolic network regulation represent a promising strategy for synthetic biology and pathway engineering. Recently, design, construction, and implementation of QS circuits for programmed control of bacterial phenotypes and metabolic pathways have gained much attention, but have not been reviewed recently. In this article, the architectural organizations and genetic contributions of the naturally occurring QS components to understand the mechanisms are summarized. Then, the most recent progress in application of QS toolkits to develop synthetic networks for novel cell behaviors creation and metabolic pathway engineering is highlighted. The current challenges in large‐scale application of these QS circuits in synthetic biology and metabolic engineering fields are discussed and future perspectives for further engineering efforts are provided.     

Reagentless amperometric immunosensor for human chorionic gonadotrophin based on direct electrochemistry of horseradish peroxidase

A novel amperometric immunosensor for determination of human serum chorionic gonadotrophin (HCG) was constructed by immobilization of HCG with titania sol-gel on a glassy carbon electrode and the direct electrochemistry of horseradish peroxidase (HRP) labeled to HCG antibody (HRP-anti-HCG). The morphologies of the HCG membrane were characterized to be chemically clean, porous and homogeneous. HRP-anti-HCG was functionally conjugated with the immobilized HCG after incubation in phosphate buffer (PBS) containing HRP-anti-HCG. A direct electron transfer of HRP with a rate constant of 1.35+/-0.40 s(-1) was observed at the HRP-anti-HCG-HCG/titania sol-gel membrane modified electrode in 0.1 M PBS pH 7.0. With a competitive mechanism the differential pulse voltammetric peak current of the immobilized HRP decreased linearly with an increasing HCG concentration from 2.5 to 12.5 mIU/ml in the incubation solution. The HCG immunosensor showed a detection limit of 1.4 mIU/ml, a good accuracy and acceptable precision and reproducibility with an intra-assay CV of 4.7% at 5.0 mIU/ml and an inter-assay precision of 8.1% obtained at 10 mIU/ml. The biosensor displayed a good stability in a storage period of 30 days.     

鱼腥藻PCC7120藻蓝蛋白α亚基体内重组研究

为了研究藻蓝蛋白α亚基的生物合成途径,通过构建相容的3种重组质粒pETDuet-cpcA、pCOLADuet-cpcE-cpcF和pACYCDuet-ho1-pcyA,将裂合酶基因cpcE和cpcF、血红素氧化酶基因ho1、藻蓝胆素合成酶基因pcyA和脱辅基藻蓝蛋白α亚基基因cpcA共同转入大肠杆菌BL21(DE3)。通过色素蛋白锌电泳和光谱检测表明产生了生物活性的CpcA-PCB。成功实现了大肠杆菌内藻蓝蛋白α亚基84位半胱氨酸残基与PCB的连接。而在裂合酶基因cpcE和cpcF不转入大肠杆菌的情况下,大肠杆菌内只有0.2%的CpcA-PCB产生。以上研究为进一步在大肠杆菌内合成天然的藻蓝蛋白奠定了基础。     

喀斯特小流域栓皮栎林土壤螨类群落结构特征

为比较石漠化环境与喀斯特森林土壤螨类的群落结构差异,对贵州喀斯特地区朝营小流域栓皮栎林的土壤螨类群落结构本底进行了研究,经2014年各季节的4次调查,共发现土壤螨类3目54科83属.对螨类属数、个体数量、个体密度、Shannon多样性指数(H)、Margalef丰富度指数(SR)、Pielou均匀性指数(J)、捕食性螨类成熟度指数(MI)、甲螨MGP类群和甲螨营养结构等进行了分析.结果表明:土壤螨类在类群属数和个体数量上均以甲螨亚目的属占优势,夏季和秋季具有丰富的属数、较高的个体密度与多样性,春季和秋季具有丰富的个体数量,群落分布具有明显的表聚性.捕食性螨类夏季生态类群以K选择型为主,其他季节以r选择型为主;甲螨生态类群主要为P型和O型,缝甲螨属、异珠足甲螨属和合若甲螨属等属构成了栓皮栎林土壤螨类的营养功能集团.研究表明,该区山毛榉林与其他地区山毛榉林、其他不同类型森林的土壤螨类主要类群存在差异,其中含丰富属组成的派伦螨科、厉螨科、奥甲螨科和单翼甲螨科以及多奥甲螨属、派伦满属、菌甲螨属和单翼甲螨属等数量上占优势的类群属可作为山毛榉林土壤环境的生物指示.     

Valuing urban green spaces in mitigating climate change: A city‐wide estimate of aboveground carbon stored in urban green spaces of China's Capital

Urban green spaces provide manifold environmental benefits and promote human well‐being. Unfortunately, these services are largely undervalued, and the potential of urban areas themselves to mitigate future climate change has received little attention. In this study, we quantified and mapped city‐wide aboveground carbon storage of urban green spaces in China's capital, Beijing, using field survey data of diameter at breast height (DBH) and tree height from 326 field survey plots, combined with satellite‐derived vegetation index at a fine resolution of 6 m. We estimated the total amount of carbon stored in the urban green spaces to be 956.3 Gg (1 Gg = 10⁹ g) in 2014. There existed great spatial heterogeneity in vegetation carbon density varying from 0 to 68.1 Mg C ha^‐1, with an average density of 7.8 Mg C ha^?1. As expected, carbon density tended to decrease with urban development intensity (UDI). Likely being affected by vegetation cover proportion and configuration of green space patches, large differences were presented between the 95th and 5th quantile carbon density for each UDI bin, showing great potential for carbon sequestration. However, the interquartile range of carbon density narrowed drastically when UDI reached 60%, signifying a threshold for greatly reduced carbon sequestration potentials for higher UDI. These findings suggested that urban green spaces have great potential to make contribution to mitigating against future climate change if we plan and design urban green spaces following the trajectory of high carbon density, but we should be aware that such potential will be very limited when the urban development reaches certain intensity threshold.     

微卫星标记分析中国梨木虱种群的遗传多样性

中国梨木虱Cacopsylla chinensis (Yang et Li)是梨树主要害虫之一。为了从分子水平评估中国梨木虱种群内的遗传变异和种群间的遗传分化, 本文应用7对微卫星DNA引物对中国16个地区中国梨木虱种群进行遗传多样性分析。结果表明： 各位点有效等位基因为2.2927～10.0610, 多态信息含量(PIC)值在0.5073～0.8735之间。16个种群的平均期望杂合度为0.7876, 遗传距离在0.0951～1.0139之间, Nei氏期望杂合度为0.4771～0.7892, Shannon信息指数在0.8396～1.9989之间, 群体分化率F_ST为11.61%, 基因流平均值为2.2236。结果表明, 7个微卫星位点均具有较高的多态性, 各种群间的遗传分化水平较低, 基因交流程度较高, 遗传变异主要存在于种群内的个体间。研究结果为制定针对中国梨木虱的有效防治策略提供部分分子生物学的基础资料。     

三叶鬼针草与不同本地植物竞争对土壤 微生物和土壤养分的影响

入侵植物三叶鬼针草(Bidens pilosa)对我国农牧业生产造成了重大的损失。本文主要研究三叶鬼针草入侵与不同本地植物竞争对土壤微生物群落结构和土壤养分的影响。利用磷脂脂肪酸方法(phospholipid fatty acids, PLFAs)测定土壤微生物群落组成, 同时测定土壤养分和酶活性, 并利用Canoco4.5软件分析了土壤微生物、土壤养分和土壤酶活性的相关性。结果表明: (1)三叶鬼针草对革兰氏阳性菌、革兰氏阴性菌、丛枝菌根真菌等土壤微生物具有较强的聚集能力, 且其根际土壤聚集的微生物类群与本地植物种类密切相关。(2)三叶鬼针草入侵显著增加了入侵地土壤的有机碳含量, 降低了铵态氮的含量; 土壤中的速效钾、速效磷和硝态氮的含量则与本地植物种类密切相关。(3)相关性分析表明, 16:00和16:1 ω5c对铵态氮的含量影响较大, 而三叶鬼针草入侵地16:00和16:1 ω5c的含量显著高于裸土对照, 进而推测这一状况导致了铵态氮含量的降低。(4) 15:1 anteiso A和18:1 ω5c与速效钾的含量呈显著正相关, 而其含量在狗尾草(Setaria viridis)中显著高于其他处理, 三叶鬼针草与狗尾草混种处理中土壤中速效钾的含量高于其他处理。以上结果说明, 三叶鬼针草通过改变土壤微生物群落结构影响了土壤酶活性和土壤养分, 且这种改变与入侵地本地植物种类有关。     

超嗜热酯酶EST2在不同宿主中的异源高效表达研究

来源于超嗜热古菌Alicyclobacillus acidocaldarius的酯酶EST2是目前报道的活性最高的超嗜热酯酶,具有极大的工业应用价值。为促进EST2的生产应用,将其分别在大肠杆菌及毕赤酵母中进行异源表达,并就不同宿主对表达情况和重组酶酶学性质的影响进行了分析。在大肠杆菌和毕赤酵母中重组表达的EST2酶学性质基本一致:最适温度分别为75℃和77.5℃,最适pH均为8.0,比活力分别为4656.6 U/mg和4078.3 U/mg,70℃水浴保温4.5 h,残余活力均在70%以上。在摇瓶发酵的基础上,于5 L发酵罐中进行了重组大肠杆菌及毕赤酵母的高密度发酵。毕赤酵母高密度发酵120 h菌体干重达68 g/L,最大表达酶活力为959.6 U/ml。大肠杆菌高密度发酵25 h菌体干重达60.8 g/L,最大酶活力14825.6 U/ml,表达量是毕赤酵母的15.4倍,单位时间产量是酵母的74.2倍。结果表明大肠杆菌发酵周期短、表达量高,更适合进行嗜热酯酶EST2的高效生产,这为促进嗜热酯酶在工业生物技术产业的应用奠定了基础。     

TaqMan-MGB荧光定量RT-PCR技术快速检测H5亚型禽流感病毒

建立以TaqMan-MGB荧光探针为特点的荧光定量RT-PCR方法,用于检测H5亚型禽流感病毒.针对H5亚型禽流感病毒血凝素(HA)基因保守区域设计特异性引物与TaqMan-MGB荧光探针,筛选并优化荧光定量RT-PCR反应体系与反应条件,用以提高方法的特异性、敏感性与准确性;并通过体外克隆技术建立病毒基因拷贝数进行定量分析.结果表明引物与探针的优化浓度分别640nmol/L和480nmol/L,体系具有良好的保守性和特异性,与其他呼吸道病毒均无交叉反应.方法检测灵敏度为100拷贝/反应,标准曲线线性范围为107～102拷贝/反应,从病毒核酸提取至检测完成仅需3h左右,操作简便,重现性好.本研究建立的TaqMan-MGB荧光定量PCR方法特异、敏感、快速,适合于临床实验室进行H5亚型禽流感病毒的快速定量检测.