转基因产品中PAT蛋白的酶联免疫检测

建立并优化了转基因油菜(Brassica campestris )中的PAT蛋白酶联免疫检测技术。首先通过Western杂交和酶活性分析鉴定了PAT蛋白的纯度和活性。然后以其为抗原免疫新西兰大白兔制备了PAT蛋白的多克隆抗体，并采用硫铵沉淀法和protein A-Sepharose 4B对其进行了纯化。所得抗体对PAT蛋白的检测限为2×10-5 mg/mL，且与植物源的几种结构功能相关蛋白均无交叉反应。然后对植物蛋白进行初步提取，应用酶联免疫检测技术对转基因油菜(MS1/RF1 and MS8/RF3)中的PAT蛋白进行了检测，结果表明ELISA能高效地鉴别转基因和非转基因油菜.     

白血病中C-myb蛋白的作用

据《科学日报》（ScienceDaily）2010年1月19日报道,美国费城宾夕法尼亚大学医学院的Alan Gewirtz和他的同事现在已经鉴定了一个对诱导MLL关联的白血病的形成起重要作用分子途径。     

The Detection of Catastrophic Discontinuities in the Acid-Phosphorus Relationship (A Complex Systems Model)

Recognising the continual emergence of sudden catastrophic environmental changes resulting from the complex interactions of apparently disparate phenomena, and utilising an existing spatio-temporal model of two such phenomena applied to the Rutland Water Catchment, this paper presents a complex systems approach to the modelling of such environmental catastrophes which may be described as Chemical Time Bomb (CTB) phenomena. This is achieved through the definition and spatial observation of a catastrophe indicator which indicates both the proximity and magnitude of catastrophe arising from the non-linear and discontinuous acid-phosphorus relationship within the soil domain. Definition of this indicator facilitates the assessment of the vulnerability of a river catchment to potential propagation of this CTB whereby the soil suddenly changes from being a sink to a source of phosphates; long-term accumulations of phosphate in the soil being released as a consequence of soil acidification in the short-term. This complex systems approach – hinged upon the concept of a catastrophe indicator – highlights the non-linearities and discontinuities between apparently disparate phenomena which are 'competing' for a common resource, with the heuristic value of the indicator arising from the ability to understand more clearly the dynamics of these inter-relationships through examination of the temporal changes in the dynamic spatial landscapes which emerge from simulations.     

Iron Promoted Decontamination Studies of Nitrophenylphosphate in Aqueous and Microemulsion Media: A Model for Phosphate Ester Decontamination in the Environment

Over 250 years of metal smelting in Swansea (South Wales) left metal-rich slag across the Nant-y-Fendrod valley floor and aerial metal pollution over the wider landscape. Reclamation since 1965 included: (1) partial removal of spoil, flattening the rest and capping it with topsoil, (2) culverting watercourses and creation of two flood-relief lakes and (3) revegetation of the aerially polluted landscape. This paper assesses downstream changes along the Nant-y-Fendrod stream–lake system in metal levels of (a) fine bed-sediment and (b) streamwater and seepages. In the upper, ‘aerial-pollution zone’ total Zn, Cu, Pb and Cd in surface bed-sediment increase marginally within the first lake to 892, 207, 212 and 7.2 mg/kg, respectively. Farther downstream, Cu and Pb rise sharply, and Zn and Cd progressively, to 12,853, 595, 871 and 155 mg/kg, respectively, as the stream traverses the ‘capped metal-rich waste zone.’ Zn and Cd reach 22,671 and 229 mg/kg, respectively, in the second lake, before falling 35–56% below its outlet. Streamwater metal levels rise (but seepage metal concentrations remain stable) in most storm events, though patterns (including whether levels are reduced downstream of the lake) vary with antecedent conditions. Possible interactions between seepages, bed sediment and streamwater metal dynamics are explored.     

转基因水稻中CP4-EPSPS蛋白质的检测及其表达特征研究

为了建立转基因水稻中CP4-EPSPS蛋白质的免疫学检测方法,并了解其在水稻生长过程中的表达特征,本研究在大肠杆菌中表达了CP4-EPSPS蛋白质,纯化后以其为免疫原免疫小鼠,经筛选获得了可识别CP4-EPSPS的单克隆抗体。建立了用免疫印记技术检测转基因水稻中CP4-EPSPS蛋白质的方法,该方法可以从单粒稻米的0.63%材料中检测到CP4-EPSPS信号。对水稻不同发育时期代表性组织的检测结果表明,CP4-EPSPS蛋白质呈组成型表达,在花药、穗轴、颖壳及不同时期的叶片中的表达量较高,但在分蘖期基部茎节、叶鞘及成熟期种子胚中的表达量稍低。本研究建立的免疫学方法在转基因水稻检测中具有潜在的应用价值。     

转nsLTPs类抗菌蛋白基因烟草对赤星病和青枯病的抗病性分析

以烟草叶盘为受体，通过根癌农杆菌(Agrobacterium tumefaciens)介导法将益母草 (Leonurus japonicus Houtt.) 非专一性脂转移蛋白(nsLTPs)类抗菌蛋白基因Lj-L和相应改造基因Lj-S分别导入烟草(Nicotiana tabacum cv. Xanthi.)，并进行抗病性分析。GUS染色和PCR分子验证表明外源基因已整合进烟草基因组中。RT-PCR分析表明，外源基因能够在烟草中稳定转录。T0代离体抗性实验表明，外源基因对烟草赤星病(Alternaria alternata)的抗性已达极显著水平; T1代活体抗性实验表明，Lj-L和Lj-S基因对青枯病(Pseudomanas solanacearum)的抗性也均达显著水平。抗性结果比较发现，Lj-L基因的抗病性效果优于Lj-S基因，且离体和活体抗病结果较一致，但并不与基因的转录水平成正比，似乎存在一个阈值。     

重组E2蛋白-乳胶凝集试验检测猪瘟病毒血清抗体

人工合成4条两两互补的DNA序列,经磷酸化和退火后与经BglⅡ酶切的pET-E2载体连接,得到pET-E2HA重组质粒.该质粒除表达E2蛋白A/D抗原区外,还表达3个串联的人流感病毒(Human influeoza virus)血凝素表位.重组质粒转化到大肠杆菌(Escherichia coli)BL21(DE3),用IPTG诱导表达.利用纯化后的表达产物与流感病毒血凝素单抗及乳胶建立了诊断猪瘟抗体水平的乳胶凝集试验.利用盐酸胍溶解包涵体并过His·Bind柱,获得纯化的重组蛋白.研究结果表明,乳胶凝集方法具有操作简便、快速、敏感性高、特异性强、价格低廉且可用于现场检测等优点,是一种适合基层兽医单位用于猪瘟病毒(Classical swinefever virus,CSFV)血清抗体检测的新方法.     

红颊草莓离体培养及其增殖过程中可溶性蛋白研究

本文以红颊草莓为试材,对红颊草莓茎段进行组织培养,筛选出离体快繁最适的培养基条件,并诱导再生植株,也初步探索了培养物增殖过程中可溶性蛋白的动态变化。结果表明：红颊草莓适宜的诱导分化培养基为MS＋6-BA0.5mg/mL＋IAA0.25mg/mL,不定芽分化率可达100%;继代增殖最适宜培养基为MS＋6-BA0.25mg/L＋KT0.25mg/L＋IAA0.05mg/L,增殖倍数达到6.44;最佳生根培养基为1/2MS＋IAA0.05～0.1mg/L,生根数为5～6条,平均根长2cm左右,且生长健壮;最佳移栽基质为河沙,移栽成活率达88.9%。对红颊草莓增殖过程中的可溶性蛋白研究结果表明：可溶性蛋白含量在第10天、第30天呈现明显的两个高峰,SDS-PAGE电泳结果显示16.7kD、15.6kD和14.4kD的蛋白是草莓生长所必须的,而分子量为26.2kD和23.7kD的蛋白则只在继代增殖旺盛时期出现,为增殖时的特异蛋白组分。本研究结果为红颊草莓快繁育苗提供了一条高效途径,也为进一步研究草莓离体培养过程中体内生理变化的分子机理提供了有意义的参考。     

甜瓜细菌性叶枯病菌胶体金ICA检测试纸条的研发与应用

甜瓜(Cucumis melo)作物上会存在甜瓜细菌性叶枯病菌(Pseudomonas syringae pv.lachrymas)和瓜类细菌性果斑病菌(Acidovorax citrulli)两种细菌病害,二者混合发生,症状极其相似,不易区分.为了快速、准确地在田间检测区分出两种病害,本研究利用甜瓜细菌性叶枯病菌单克隆抗体制备了免疫层析试纸条,并建立一种快速、简便地现场检测甜瓜细菌性叶枯病菌的胶体金免疫层析分析方法(immunochromatography assay, ICA).在定性检测中,本方法对甜瓜细菌性叶枯病菌的定性检测限为2.5×103cfu/mL,与细菌性果斑病菌不存在交叉反应,具有很强的特异性.在半定量检测中,甜瓜细菌性叶枯病菌在2.5× 103～1×105 cfu/mL浓度范围内,T线和C线读数的比值与甜瓜细菌性叶枯病菌浓度的对数值呈显著的线性关系,线性方程=0.392 2x-1.263 8,相关系数R2达到0.988 1.因此,本研究制备的快速、简便、经济的高特异性甜瓜细菌性叶枯病菌ICA是一种非常实用的检测方法,不仅可以在田间实时原地快速检测甜瓜细菌性叶枯病菌,同时还能与瓜类细菌性果斑病菌进行有效区分,为甜瓜等瓜类种植业的发展带来很大帮助.     

抗朊蛋白单抗用于牛海绵状脑病检测和细胞型朊蛋白在中枢神经组织中的定位和分布

朊病毒病（又称传染性海绵状脑病）的病原是致病型朊蛋（PrP^Sc），聚集在牛脑中能够引发牛海绵状脑病（BSE）。利用自制的单抗4C11和国外同类单抗进行免疫组织化学（IHC）的比较染色，结果显示单抗4C11和国际上常用的单抗在BSE阳性切片的IHC染色结果上没有显著差异；利用单抗4C11结合IHC方法检测中国山东省150例牛脑样品的实验表明，所有送检样品中IHC的检测结果均为阴性，而且IHC方法比较适合于我国BSE的检测；对中枢神经组织内细胞型PrP^c的IHC定位结果显示，PrP^c在牛脑组织不同部位的分布有差异。     

A Threshold Model for Heron Productivity

We demonstrate the potential of conditionally Gaussian state-space models in integrated population modeling, when certain model parameters may be functions of previous observations. The approach is applied to a heron census, and the data are best described by a model with three population-size thresholds which determine the population productivity. The model provides an explanation of how the population rebounds rapidly after major falls in size, which are characteristic of the data. By contrast, a simple logarithmic regression of productivity on population size was not significant. The results are of ecological interest, and suggest hypotheses for further investigation. Supplementary figures are available online.     

杨桃细菌性斑点病菌的ITS序列分析及PCR检测

由丁香假单胞菌(Pseudomonas syringae pv.averrhoi)引起的杨桃(Averrhoa carambola)细菌性斑点病是杨桃生产上的重要病害.本研究利用细菌16S～23S rDNA间的内源转录间隔区(internal transcribed spacer ITS)序列通用引物Ll(5'-AGTCGTAACAACGTAGCCGT-3')和L2(5’-GTGCCAAGGCATCCACC-3’)扩增参试菌株的基因组DNA;并对其PCR产物进行克隆测序,经多重比对分析后设计出杨桃细菌性斑点病菌的特异性引物PsaveF(5'-CTTATCGACGACTCAGCTGCG-3’)和PsaveR(5’-TCATGCGTTGATCGTCAGGATC-3').此引物可以从杨桃细菌性斑点病菌基因组DNA中扩增出373bp的特异性片段,而其余参试的细菌无扩增条带,该引物检测的灵敏度可达10 pg,且可从自然发病的杨桃组织中扩增出特异性条带.表明该方法可以应用于杨桃细菌性斑点病的快速、灵敏、可靠的检测.此外,本研究对多种病原细菌的ITS序列进行比对分析,发现该病菌与核果树细菌性溃疡病菌Pseudomonas syringae pv.morsprunorum是近源种.该检测技术对杨桃细菌性斑点病害的控制有一定的实用意义.     

Proteomic analysis of shade-avoidance response in tomato leaves

The aim of this project was to investigate the molecular mechanisms of shade-avoidance response in tomato ( Solanum lycopersicum) plants. Plants were grown in direct sunlight in ambient temperature and in an adjacent environment under shade cloth. Leaves were harvested, and protein expression differences were investigated using two-dimensional differential in-gel electrophoresis and nanoflow high-performance liquid chromatography-tandem mass spectrometry. Striking differences in plant physiology and protein expression were observed. Plants grown in the shade grew very tall but bore almost no fruit and displayed a dramatic reduction in the accumulation of Rubisco and a number of other metabolic enzymes. We have identified, quantified, and classified 59 protein features found to be up- or down-regulated as part of a shade-avoidance response in S. lycopersicum and correlated these with phenotypic data. A large group of proteins related to metabolism and respiration were greatly reduced in accumulation in shade-grown plants, and there was also evidence of significant proteolysis occurring. Four stress-related proteins appear to be constitutively expressed as a result of heat acclimation, while three distinct stress-related proteins appear to accumulate as part of the shade-avoidance response. The identification and functional classification of all 59 differentially accumulating proteins is presented and discussed.     

Proteomic identification of actin-derived oligopeptides in dry-cured ham

An intense proteolysis of muscle proteins, mainly due to the action of endogenous proteolytic enzymes, has been reported to occur during the processing of dry-cured ham. This gives rise to an important generation of free amino acids and peptides of small size that contribute directly or indirectly to flavor characteristics of the final product. The nature and properties of the free amino acids generated during postmortem proteolysis have been well established. However, little is known about the identity of peptides generated during the processing of dry-cured ham. In the present paper, we describe the isolation (by ethanol precipitation followed by size exclusion and reverse phase chromatographies) and identification (by matrix-assisted laser desorption/ionization time-of-flight MS and collision-induced dissociation MS/MS) in a Spanish dry-cured ham of the following four oligopeptides: (A) TKQEYDEAGPSIVHR, (B) ITKQEYDEAGPSIVHRK, (C) DSGDGVTHNVPIYE, and (D) DSGDGVTHNVPIYEG. This is the first time that these peptide fragments have been reported in dry-cured ham at the end of processing. Sequence homology analysis revealed that the four peptides originated from muscle actin, indicating an extensive hydrolysis of this protein during dry-curing. Some of the cleavage sites corresponding to these fragments in actin were reproduced by other authors through the incubation of this myofibrillar protein in the presence of cathepsin D (EC 3.4.23.5), thus supporting a relevant action of this enzyme during the processing of dry-cured ham.     

一种圆形颗粒检测的图像处理技术

为进行圆形农业颗粒物料的分析与识别,根据图像处理的基本方法,设计了高效检测圆形物体的算法,给出了算法的详细描述。与传统检测方法比较,该算法具有较高的计算效率和可靠性,计算结果描述直观。     

渝西地区森林虫害的气象预报模型

对永川区1950—2002年逐年森林虫害发生面积的周期分析表明,渝西地区森林虫害大发生存在5～7a周期,80年代呈准2a周期高位震荡;通过对森林虫害与气象要素的数理统计分析得出,春季、盛夏期间温度、湿度及初夏强降水是影响渝西地区森林虫害的主要气象要素。在相关分析的基础上,利用人工神经网络方法建立了当地森林虫害面积的气象预测模型,1985—2002年的模拟预报的平均误差为4．85％,回代检验效果较好。     

A Rapid Protein Digestibility Assay for Identifying Highly Digestible Sorghum Lines

Protein digestibility in sorghum (Sorghum bicolor (L.) Moench) lines was determined using two standard procedures (pepsin digestibility and pH‐stat) and compared with a newly developed, rapid electrophoresis‐based screening assay. The new assay was based on the rate of α‐kafirin disappearance after pepsin digestion. α‐Kafirin, the major sorghum storage protein, makes up ≈60–70% of the total protein in the grain. In the new assay, samples were first digested with pepsin for 1 hr, and undigested proteins were then analyzed by SDS‐PAGE. The intensitizes of the undigested α‐kafirin bands were measured. Higher band intensity indicated lower protein digestibility. The new assay was significantly correlated with the standard pepsin digestibility assay (r = −0.96, n = 16) after which it was patterned. The same was true of the pH‐stat procedure (r = −0.85, n = 16). This implies that the new assay is comparable to existing procedures and can be used for screening sorghum lines for protein digestibility. Two groups consisting of high‐protein digestibility and wild‐type sorghum lines were identified when the new assay was tested on 48 sorghum lines derived from crosses of wild‐type and mutant high protein digestibility lines, indicating that the new assay was efficient in differentiating between the two groups. Advantages of the new assay over the standard procedures include considerable reduction in analysis time and sample size required for the analysis. For example, analysis time was reduced by 20% and sample size by 10% when the new assay was used as compared with the pH‐stat procedure. We estimate that ≈60 sorghum lines can be screened in a day by a single operator using the new assay.     

叶蛋白专用打浆机的研究

从提取叶蛋白专用打浆机的特殊要求出发，提出叶蛋白专用打浆机的工作原理，研制了９ＤＹ－５０型叶蛋白专用打浆机，通过试验分析得出了生产率与功耗的计算公式     

在板栗疫病菌线粒体中检出低毒病毒编码蛋白p29

p29蛋白是低毒病毒基因组编码的一个木瓜蛋白酶样蛋白。前人研究发现,在宿主板栗疫病菌（Cryphonectria parasitica）体内表达p29,会引起真菌毒力降低,色素产生减少,丧失产生无性孢子的能力。除已知p29与源于高尔基体的膜结构共分离之外,p29在细胞内的其它分布形式未明。本研究在成功制备p29特异抗体和高效分离板栗疫病菌线粒体的基础上,尝试用p29抗体检测线粒体中是否存在p29蛋白。Western印迹结果表明,受CHV1-EP713感染的EP713菌株线粒体中存在与p29抗体特异作用的病毒蛋白。本研究结果暗示,低毒病毒蛋白p29可能参与调控宿主线粒体功能。