利用长距离PCR扩增国内肠道病毒流行株基因组方法的建立和评价

目的:通过调查近年来我国肠道病毒EV-71型和柯萨奇病毒A16型流行株的全基因组序列,建立一种能够获得我国肠道病毒序列的通用扩增方法,为今后的手足口病流行病学分析、致病机理研究等打下基础。方法:收集我国近5年各地报道的肠道病毒流行株全基因组序列作为参考序列进行比对分析,在保守区设计通用引物,利用3'RACE、长距离PCR扩增及简并引物扩增肠道病毒全基因组序列,采用IonTorrentPGM二代测序仪对扩增产物进行深度测序,以对扩增方法进行验证和评价。结果:通过比对肠道病毒流行株序列设计了通用扩增引物,经二代测序实验获得了肠道病毒全基因组序列;以系列比例模拟混合病毒感染,该扩增方法能够同时获得2株肠道病毒的全基因组序列;能够完整地揭示肠道病毒重组情况。结论:建立了针对我国近年来肠道病毒流行株的通用全基因组扩增方法,在病毒培养液中肠道病毒的提取与扩增中显示了较高的灵敏度,能够反映混合病毒感染、重组病毒的情况。     

人H5N1流感病毒血凝素蛋白DNA疫苗的构建及其免疫原性研究

将我国分离的首株人H5N1亚型禽流感病毒A/Anhui/1/2005作为研究对象,扩增其HA和HA1基因片段并克隆至真核表达载体pStar,构建成真核表达质粒。通过Western blot和间接免疫荧光检测方法确认,构建的重组质粒在真核细胞中成功地表达了目的蛋白HA和HA1。将重组质粒免疫BALB/c小鼠,检测免疫后外周血中HA/HA1特异性抗体的效价,并比较HA和HA1的免疫原性。结果表明,重组质粒免疫后成功地诱导了体液免疫反应,且二者的血清抗体效价无显著性差异。     

甘蓝品种'争春'和'寒光2号'的DNA指纹图谱构建

用SDS法提取甘蓝（Brassfca oleraceavat．capitata）品种‘争春’、‘寒光2号’及其各自亲本的基因组DNA,通过SRAP、RAPD两种分子标记方法,构建其DNA指纹图谱,用于种子纯度鉴定。利用30对SRAP引物组合和200个RAPD随机引物,以各品种及其亲本的基因组DNA为模板组进行筛选,结果显示：多数SRAP引物组合对模板组的扩增带型一致,少数组合扩增出差异,但未能找到具有互补差异的引物组合;通过RAPD标记方法筛选出能鉴定2个品种纯度的引物分别为S42、S103、S193和S42、S89、S151,其中引物S42对2组材料均能扩增出特异的RAPD指纹图谱,并将RAPD指纹图谱转变为相应的数字指纹。     

生态效益评价内容和评价指标筛选

生态效益评价的重要性已经广为人知,但评价内容和指标不统一,大大影响了评价结果的可信度。基本上是各说各有理,很难相互比较。在分析国内外相关生态系统评价方法基础上,提出生态效益评价框架,强调生态效益与经济效益和社会效益共同构成了人类社会的价值判断标准和决策依据,是经济效益和社会效益的基础,应该包括生态系统整体贡献,考虑与生态系统的动态变化的关系;并探讨了生态效益评价指标筛选的原则(关联性、灵敏性、层级性、决策导向性、代表性、可行性、独立性及经济适用性和社会可接受性);提出了生态效益指标筛选的多准则综合法和生态效益评价指标检验的3个标准(可定量化、专一化和震撼性)。本研究将为生态效益指标体系的构建提供重要参考。     

黄土高原丘陵沟壑区柠条和沙棘树干液流的变化特征

近年来为防治黄土高原水土流失,我国政府开展了一系列的植被恢复工作。了解造林植被水分利用策略,对于在干旱半干旱黄土高原地区开展有效的植被恢复至关重要。以黄土高原甘肃省定西市安家沟小流域为研究区,选取黄土高原大规模植树造林灌木柠条(Caragana korshinskii)和沙棘(Hippophae rhamnoides)为研究对象,利用包裹式液流计于2020年6—9月对柠条和沙棘树干和枝条的液流进行观测,研究柠条和沙棘树干液流密度的日内与年内变化,以及与环境要素的关系。结果表明,柠条与沙棘的液流密度日内变化规律与光合有效辐射(Q₀)、饱和水汽压差(D_z)变化趋势一致。液流密度对环境要素的响应不同,在8月份,柠条和沙棘液流密度受D_z、Q₀和气温(T_a)的影响较大,其中,D_z占主导地位。在其他月份,液流密度主要受D_z、Q₀的影响较大。当柠条与沙棘经历了长期干旱无雨的条件下,土壤含水量对树干液流的影响较大,D_z...     

Response of terrestrial carbon uptake to climate interannual variability in China

The interest in national terrestrial ecosystem carbon budgets has been increasing because the Kyoto Protocol has included some terrestrial carbon sinks in a legally binding framework for controlling greenhouse gases emissions. Accurate quantification of the terrestrial carbon sink must account the interannual variations associated with climate variability and change. This study used a process‐based biogeochemical model and a remote sensing‐based production efficiency model to estimate the variations in net primary production (NPP), soil heterotrophic respiration (HR), and net ecosystem production (NEP) caused by climate variability and atmospheric CO₂ increases in China during the period 1981–2000. The results show that China's terrestrial NPP varied between 2.86 and 3.37 Gt C yr^?1 with a growth rate of 0.32% year^?1 and HR varied between 2.89 and 3.21 Gt C yr^?1 with a growth rate of 0.40% year^?1 in the period 1981–1998. Whereas the increases in HR were related mainly to warming, the increases in NPP were attributed to increases in precipitation and atmospheric CO₂. Net ecosystem production (NEP) varied between ?0.32 and 0.25 Gt C yr^?1 with a mean value of 0.07 Gt C yr^?1, leading to carbon accumulation of 0.79 Gt in vegetation and 0.43 Gt in soils during the period. To the interannual variations in NEP changes in NPP contributed more than HR in arid northern China but less in moist southern China. NEP had no a statistically significant trend, but the mean annual NEP for the 1990s was lower than for the 1980s as the increases in NEP in southern China were offset by the decreases in northern China. These estimates indicate that China's terrestrial ecosystems were taking up carbon but the capacity was undermined by the ongoing climate change. The estimated NEP related to climate variation and atmospheric CO₂ increases may account for from 40 to 80% to the total terrestrial carbon sink in China.     

研究报告: 基于核酸适配体的胶体金比色法检测鳗弧菌

目的 鳗弧菌（Vibrio anguillarum）可引起鲑鱼、鳗鲡、鲈鱼和牙鲆等多种水产养殖动物的疾病，是水产养殖中的一种重要病原菌，对其进行快速检测是确保水产养殖安全和食品安全所必需的。方法 本文利用鳗弧菌与其核酸适配体之间较强的亲和力，通过鳗弧菌夺取胶体金颗粒表面的核酸适配体，使胶体金溶液的吸光度发生变化，从而建立一种可定量检测鳗弧菌的方法。结果 该方法对鳗弧菌的吸光度值显著高于对溶藻弧菌、铜绿假单胞菌、变形假单胞菌、嗜水气单胞菌和迟钝爱德华氏菌等非目标菌的吸光度值（P<0.01），并在1~10⁵ CFU/ml的检测范围内呈现较好的线性关系。用该方法对不同盐度和鱼体组织样品进行加标回收检测，结果显示回收率和相对标准偏差等指标都符合相应检测标准。结论 该检测方法对鳗弧菌有较好的特异性，可用于水产品或食品中鳗弧菌的定量检测。     

Structural modeling and electrostatic properties of aspartate transcarbamylase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae the first two reactions of the pyrimidine pathway are catalyzed by a multifunctional protein which possesses carbamylphosphate synthetase and aspartate transcarbamylase activities. Genetic and proteolysis studies suggested that the ATCase activity is carried out by an independently folded domain. In order to provide structural information for ongoing mutagenesis studies, a model of the three-dimensional structure of this domain was generated on the basis of the known X-ray structure of the related catalytic subunit from E. coli ATCase. First, a model of the catalytic monomer was built and refined by energy minimization. In this structure, the conserved residues between the two proteins were found to constitute the hydrophobic core whereas almost all the mutated residues are located at the surface. Then, a trimeric structure was generated in order to build the active site as it lies at the interface between adjacent chains in the E. coli catalytic trimer. After docking a bisubstrate analog into the active site, the whole structure was energy minimized to regularize the interactions at the contact areas between subunits. The resulting model is very similar to that obtained for the E. coli catalytic trimer by X-ray crystallography, with a remarkable conservation of the structure of the active site and its vicinity. Most of the interdomain and intersubunit interactions that are essential for the stability of the E. coli catalytic trimer are maintained in the yeast enzyme even though there is only 42% identity between the two sequences. Free energy calculations indicate that the trimeric assembly is more stable than the monomeric form. Moreover an insertion of four amino acids is localized in a loop which, in E. coli ATCase, is at the surface of the protein. This insertion exposes hydrophobic residues to the solvent. Interestingly, such an insertion is present in all the eukaryotic ATCase genes sequenced so far, suggesting that this region is interacting with another domain of the multifunctional protein. © 1994 Wiley-Liss, Inc.     

In situPCR on Plant Material with Sub-cellular Resolution

In situPCR and reverse transcribedin situPCR have been testedon leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5%GA.In situPCR amplification of the gene coding for ribulose-1,5-bisphosphatecarboxylase/oxygenase (rbcL) was successfully performed followingall three fixation protocols. As expected the PCR product wasrestricted to the plastids of all cells. Reverse transcribedinsituPCR was performed onrbcL mRNA and in this case the PCR productwas restricted to the plastids of the bundle sheath cells. Thisis the first report ofin situPCR on plant material and onlythe second report ofin situPCR with sub-cellular resolution.InsituPCR onrbcL may prove to be a valuable positive control forfuturein situPCR studies on plant material.Copyright 1997 Annalsof Botany Company In situPCR; reversed transcribedin situPCR; rbcL; C₄-plants