镍钴转运酶NiCoT基因的克隆表达及基因工程菌对镍离子的富集

利用PCR技术从Staphylococcus aureus/ ATCC6538基因组中扩增出大小为1?053 bp的镍钴转运酶基因NiCoT gene,将其连接到pET-3c载体上构建重组质粒,并转化至E.coli BL21.筛选阳性菌并经酶切分析和PCR扩增双重鉴定.核苷酸序列测定及分析结果与GenBank中报道的同类基因相似性高达97%以上,表明其具有正确的NiCoT基因核苷酸序列.重组菌的SDS-PAGE结果图谱中,在相对分子量为39?000附近有特异性蛋白条带,大小符合预测值,表明NiCoT基因在E.coli BL21中成功表达.基因工程菌在IPTG用量为1.00 mmol·L^-1,诱导时间为4 h的条件下培养对镍离子的富集能力最高.在不同镍离子浓度时,基因工程菌对溶液中Ni²⁺的平衡富集量为11.33 mg·g^-1,与原始宿主菌相比提高了3倍.对基因工程菌吸附镍和钴的实验表明,Staphylococcus aureus ATCC6538的NiCoT对镍具有较高的特异性和富集容量,属于第Ⅲ类镍钴转运酶.     

Evaluation of the infectivity, gene and antigenicity persistence of rotaviruses by free chlorine disinfection

The effects of free chlorine disinfection of tap water and wastewater effluents on the infectivity, gene integrity and surface antigens of rotaviruses were evaluated by a bench-scale chlorine disinfection experiments. Plaque assays, integrated cell culture-quantitative RT-PCR (ICC-RT-qPCR), RT-qPCR, and enzyme-linked immunosorbent assays (ELISA), respectively, were used to assess the influence of the disinfectant on virus infectivity as well as genetic and antigenic integrity of simian rotavirus SA11 as a surrogate for human rotaviruses. The ICC-RT-qPCR was able to detect rotaviruses survival from chlorine disinfection at chlorine dose up to 20 mg/L (60 min contact), which suggested a required chlorine dose of 5 folds (from 1 to 5 mg/L) higher than that indicated by the plaque assay to achieve 1.8 log₁₀ reductions in tap water with 60 min exposing. The VP7 gene was more resistant than the infectivity and existed at chlorine dose up to 20 mg/L (60 min contact), while the antigencity was undetectable with chlorine dose more than 5 mg/L (60 min contact). The water quality also impacted the inactivation efficiencies, and rotaviruses have a relatively higher resistant in secondary effluents than in the tap water under the same chlorine disinfection treatments. This study indicated that rotaviruses have a higher infectivity, gene and antigencity resistance to chlorine than that previously indicated by plaque assay only, which seemed to underestimate the resistance of rotaviruses to chlorine and the risk of rotaviruses in environments. Present results also suggested that re-evaluation of resistance of other waterborne viruses after disinfections by more sensitive infectivity detection method (such as ICC-RT-qPCR) may be necessary, to determine the adequate disinfectant doses required for the inactivation of waterborne viruses.     

Biological treatment of chicken feather waste for improved biogas production

A two-stage system was developed which combines the biological degradation of keratin-rich waste with the production of biogas. Chicken feather waste was treated biologically with a recombinant Bacillus megaterium strain showing keratinase activity prior to biogas production. Chopped, autoclaved chicken feathers (4%, W/V) were completely degraded, resulting in a yellowish fermentation broth with a level of 0.51 mg/mL soluble proteins after 8 days of cultivation of the recombinant strain. During the subsequent anaerobic batch digestion experiments, methane production of 0.35 Nm³/kg dry feathers (i.e., 0.4 Nm³/kg volatile solids of feathers), corresponding to 80% of the theoretical value on proteins, was achieved from the feather hydrolyzates, independently of the pre-hydrolysis time period of 1, 2 or 8 days. Cultivation with a native keratinase producing strain, Bacillus licheniformis resulted in only 0.25 mg/mL soluble proteins in the feather hydrolyzate, which then was digested achieving a maximum accumulated methane production of 0.31 Nm³/kg dry feathers. Feather hydrolyzates treated with the wild type B. megaterium produced 0.21 Nm³ CH₄/kg dry feathers as maximum yield.     

石家庄市土壤中喹诺酮类抗生素空间分布特征及其与微生物群落相关性

微生物群落作为土壤生态系统重要组分,长期低含量抗生素干扰会影响土壤中微生物群落结构及其功能.选取石家庄市为研究区,在2020年9月采集12个样点的表层土壤（0~25 cm）,并根据空间方位将其划分为4个区（S1、S2、S3和S4）;运用超高效液相色谱-质谱联用（HPLC-MS/MS）方法测定土壤中典型抗生素——喹诺酮类（quinolones,QNs）含量,明晰QNs在土壤中的空间分布特征,同时利用16S rRNA高通量测序技术对土壤中微生物群落结构及功能进行研究,识别其主要环境影响因子.结果表明：①4个区域的QNs总含量平均值由大到小依次为：S3（313.5 μg·kg^-1）>S4（65.54 μg·kg^-1）>S1（46.19 μg·kg^-1）>S2（12.63 μg·kg^-1）.其中诺氟沙星（NOR）含量最高（平均值为91.99 μg·kg^-1）,而喹草酸（OXO）含量最低（平均值为0.4486 μg·kg^-1）;②土壤颗粒以粉粒（2~50 μm）占比最高（66.7%~93.2%）,而黏粒（小于2 μm）占比最低（2.50%~9.10%）;土壤中总磷（TP）和氨氮（NH₄⁺-N）无显著空间差异,而硝氮（NO₃^--N）、亚硝氮（NO₂^--N）和土壤粒径呈现显著空间差异;③6种,优势菌属有5种,其中放线菌门（18.3%~34.6%）和变形菌门（13.6%~34.1%）为主要优势菌门,Arthrobacter（3.24%~8.61%）和Nitrososphaeraceae（2.93%~9.46%）为主要优势菌属;α多样性分析结果表明,Shannon值在S2区最高（6.48）,而在S3区最低（5.89）;④相关性结果表明,QNs和土壤理化参数均会显著改变微生物群落的结构组成,OXO、NO₃^--N和土壤粒径会影响微生物群落多样性,而FLU、NH₄⁺-N和NO₂^--N和土壤粒径会对微生物群落的功能产生影响.因此,需进一步加强石家庄市土壤环境中抗生素的风险管控.     

Cloning,expression, and functional identification of CaSWEET7 and CaSWEET15 genes in Canarium album北大核心CSCD

糖外排转运蛋白(sugars will eventually be exported transporters,SWEET)介导植物光合同化产物蔗糖的跨膜运输.以橄榄(Canarium album(Lour.)Raeusch.)果实为材料,通过气相色谱串联质谱(GC-MS)检测橄榄果实中糖组分及含量变化,利用RT-PCR技术克隆得到CaSWEET7和CaSWEET15基因开放阅读框(ORF)序列.结果表明:CaSWEET7和CaSWEET15基因ORF全长分别为774 bp和951 bp,分别编码长度为257个和316个氨基酸残基,具有2个MtN3_slv结构域;进化树分析表明,CaSWEET7属于CladeⅡ,CaSWEET15属于CladeⅢ.实时荧光定量PCR(qRT-PCR)技术检测其在不同发育时期的果实中相对表达量变化,结果表明,随着果实发育,CaSWEET7和CaSWEET15表达量逐渐递增,且与果实发育蔗糖含量变化呈显著正相关(相关性系数分别为0.931和0.904,P<0.05);利用酵母功能互补证明CaSWEET7和CaSWEET15基因编码的蛋白具有转运蔗糖能力.本研究表明CaSWEET7和CaSWEET15基因可能在橄榄果实蔗糖积累中发挥作用.(图9表2参44)     

Adsorption of herring sperm DNA onto pine sawdust biochar: Thermodynamics and site energy distribution

● Adsorption of environmental deoxyribonucleic acid on biochar was studied. ● π−π interaction and electrostatic repulsion worked in the adsorption. ● Thermodynamics indicated the adsorption was spontaneous and endothermic. Environmental deoxyribonucleic acid (eDNA), which includes antibiotic resistance genes, is ubiquitous in the environment. The interactions between eDNA and biochar, a promising material widely used in soil amendment and water treatment, greatly affect the environmental behavior of eDNA. Hitherto few experimental evidences are available yet, especially on the information of thermodynamics and energy distribution to explains the interactions between biochar and eDNA. This study investigated the adsorption of herring sperm DNA (hsDNA) on pine sawdust biochar, with a specific emphasis on the adsorption thermodynamics and site energy distribution. The adsorption of hsDNA on biochar was enhanced by an increase in the pyrolysis and adsorption temperatures. The higher surface area, stronger π−π interaction, and weaker electrostatic repulsion between hsDNA and biochars prepared at high pyrolysis temperatures facilitated the adsorption of hsDNA. The thermodynamics indicated that the adsorption of hsDNA on biochar was spontaneous and endothermic. Therefore, higher temperature was beneficial for the adsorption of hsDNA on biochar; this was well explained by the increase in E^* and F(E^*) with the adsorption temperature. These results are useful for evaluating the migration and transformation of eDNA in the presence of biochar.     

Identification and expression of the COI1 gene family in Camellia sinensis and its expression in oolong tea processing北大核心CSCD

茉莉酸受体蛋白COI1(coronatine insensitive 1)是茉莉酸信号转导途径的重要组成部分,为鉴定分析茶树茉莉酸受体COI1基因家族,预测其潜在的分子功能,了解茉莉酸受体COI1基因在乌龙茶加工中应答胁迫的分子机制,利用生物信息学方法对茶树茉莉酸受体COI1进行家族成员鉴定,氨基酸序列、结构域、基因结构、进化分析以及启动子顺式元件分析,结合实时荧光定量分析CsCOI1基因在乌龙茶加工中的表达.结果显示,茶树茉莉酸受体CsCOI1家族有两个成员,均含有F-box和富亮氨酸重复序列(LRRs)两个结构域;单子叶、双子叶的COI1蛋白各聚一支,且与蜜柑进化关系较近;茶树COI1基因家族两个成员均含有3个内含子,启动子顺势元件主要有胁迫响应元件、激素响应元件以及光响应元件;转录组数据说明茶树CsCOI1基因具有较强的组织表达差异性.实时荧光定量分析表明,CsCOI1a基因在室内萎凋后表达显著上调,且15 min、30 min日光萎凋后CsCOI1b基因的表达水平显著上调,同时茉莉酸含量发生显著变化.本研究推测CsCOI1基因可能通过茉莉酸信号转导途径参与乌龙茶加工中萎凋的胁迫响应过程,该结果可为乌龙茶加工品质调控奠定基础.(图8表2参30)     

竹林河岸带土壤反硝化脱氮效果模拟及机理研究

以太湖流域上游南苕溪流域农业区竹林河岸带土壤为研究对象,采用乙炔抑制法研究不同外源硝态氮（NO₃^--N）添加量(模拟添加NO₃^--N浓度分别为0、5和10 mg·L^-1的污水,分别用N0、N5和N10处理表示)对不同离水距离（0～3、3～6、6～12 m）、不同深度（0～10、10～20、20～40 cm）土壤反硝化速率的影响.结果表明,N0、N5和N10处理的土壤72 h内的反硝化速率均值分别为59.65、163.56和295.81μg·kg^-1·h^-1,不同处理间差异显著.N5和N10处理的土壤对外源NO₃^--N的脱氮率均值分别为0.45和0.51,且N5处理显著低于N10处理;土壤反硝化速率和对外源NO₃^--N脱氮率均随NO₃^--N添加量的增大而增加.土壤反硝化速率随离水距离的增加而降低,表层土壤的...     

Using historical spy satellite photographs and recent remote sensing data to identify high-conservation-value forests

High-conservation-value forests (HCVFs) are critically important for biodiversity and ecosystem service provisioning, but they face many threats. Where systematic HCVF inventories are missing, such as in parts of Eastern Europe, these forests remain largely unacknowledged and therefore often unprotected. We devised a novel, transferable approach for detecting HCVFs based on integrating historical spy satellite images, contemporary remote sensing data (Landsat), and information on current potential anthropogenic pressures (e.g., road infrastructure, population density, demand for fire wood, terrain). We applied the method to the Romanian Carpathians, for which we mapped forest continuity (1955–2019), canopy structural complexity, and anthropogenic pressures. We identified 738,000 ha of HCVF. More than half of this area was identified as susceptible to current anthropogenic pressures and lacked formal protection. By providing a framework for broad-scale HCVF monitoring, our approach facilitates integration of HCVF into forest conservation and management. This is urgently needed to achieve the goals of the European Union's Biodiversity Strategy to maintain valuable forest ecosystems.     

Effects of different concentrations of tetracycline and oxytetracycline on the growth and ecotoxicity in lettuce北大核心CSCD

四环素类抗生素(TCs)是目前我国应用广泛、用量最大的一类抗生素,畜禽粪便和土壤中存在TCs残留,影响蔬菜作物的生长发育. TCs因水溶性较高更容易被植物转运和积累,植物对TCs耐受性机理研究仍不足.为更全面探究土壤TCs对蔬菜的毒性作用,研究不同浓度四环素(TC)和土霉素(OTC)分别对生菜的抗生素残留、生长特征及抗氧化酶系统的影响.结果显示,在0(对照)、2、10、50、250 mg/kg 5个施用水平下,生菜叶片抗生素含量逐渐增加,且土霉素含量始终大于四环素含量.与对照相比,抗生素浓度在50 mg/kg以上时对生菜生长具有显著抑制作用,其中,株高、根长、地上部和地下部鲜重与叶片TC残留量具显著负相关.生菜叶片的脯氨酸含量随浓度增加呈先增加后降低的趋势,在10 mg/kg时达到最大.并且低浓度(2 mg/kg)促进抗氧化酶基因SOD、POD21和CAT的表达,高浓度抗生素(50、250 mg/kg)对其产生抑制作用,10 mg/kg的抗生素处理对SOD、POD21和CAT基因表达的影响在抗生素种类上存在差异.本研究表明抗生素浓度超过50 mg/kg对生菜生长产生抑制作用,脯氨酸和抗氧化酶SOD、POD、CAT的转录水平及其酶活性能快速响应抗生素胁迫,可作为生菜对抗生素抗性的辅助评价指标.(图8表3参19)