GAPB基因原核表达载体的构建

以拟南芥cDNA为模板,用PCR扩增出GAPB的基因全长,然后将GAPB基因片段连接到PET28a(+)载体上,构建重组质粒并转化大肠杆菌DH5α,经菌落PCR和酶切鉴定筛选出阳性克隆,测序正确后,再将阳性克隆的质粒转化大肠杆菌BL21(DE3)。结果表明:成功构建了原核表达载体PET28a(+)-GAPB,为后续的GAPB融合蛋白的表达以及纯化奠定了基础。     

天祝白牦牛SRY基因克隆与原核表达

从天祝白牦牛的基因组DNA中扩增了SRY（Sex-determining Region on the Y Chromosome，SRY）基因编码区序列，将其克隆至pGEM-T easy载体并送至生物公司测序，天祝白牦牛SRY基因编码区长687 bp，编码229个氨基酸；对牦牛和奶牛的SRY基因编码区进行序列比对，发现存在2个碱基的变异，造成1个氨基酸的变异；将牦牛SRY基因编码区连接至pET-28a(+)载体，成功构建了表达载体pET-28a/SRY；把表达载体pET-28a/SRY转入大肠杆菌E.coli BL21（DE3）中，在合适的条件下诱导该大肠杆菌，SRY蛋白得到了大量表达；对表达产物进行了Western-blot检测，进一步确定牦牛SRY蛋白得到表达。     

Overlap PCR克隆黄牛脂肪特异性磷脂酶A2基因(AdPLA)及其原核表达

脂肪特异性磷脂酶A2基因(adipose-specific phospholipase A2,A dPLA)在脂肪组织甘油三酯水解过程中发挥着重要的调节作用,为了构建黄牛AdPLA基因原核表达系统,本研究通过Overlap PCR方法从黄牛(Bos taurus)基因组DNA中得到了AdPLA基因编码区全长,将其克隆到pGM-T载体上,阳性克隆经测序表明,与NCBI公布的序列(GenBank No.NM_001075280)完全一致.阳性质粒经BamH Ⅰ和HindⅢ双酶切后,将其定向重组到pET28a(+)原核表达载体上,转化感受态大肠杆菌(Escherichia coli) BL21 (DE3)中,用IPTG诱导融合蛋白表达.SDS-PAGE电泳表明,融合蛋白His-AdPLA在大肠杆菌中表达,证明成功构建了AdPLA基因原核表达系统,为进一步研究黄牛AdPLA基因的功能和AdPLA蛋白产品开发提供了依据.     

猪圆环病毒2型河南分离株进化分析及ORF2基因的表达

根据GenBank中猪2型圆环病毒的核苷酸序列,设计两对引物,对来源于河南省不同地市的4株PCV2细胞培养物进行鉴定,并扩增出ORF2基因,克隆到pMD18－T载体上,获得重组质粒pMD18-T-ORF2,并对其进行测序,结果表明所克隆的ORF2基因与其它PCV2 ORF2基因核苷酸同源性在92.4%～99.6%之间,氨基酸同源性在90.6％～97.9％之间。同时采用PCR从重组质粒pMD18-T-ORF2中扩增出587bp的ORF2基因,克隆到表达载体pET-32a,成功构建了重组质粒pET-32a-ORF2,经诱导表达了ORF2基因编码的结构蛋白,表达的重组蛋白为融合蛋白,分子量为40kD,经Western-Blot检测,重组蛋白可被PCV2阳性血清识别。     

天祝白牦牛Y染色体性别决定基因(SRY)编码区的克隆和原核表达

从天祝白牦牛(Bos grunneins)的基因组DNA中扩增了Y染色体性别决定基N(SRY)编码区序列,将其克隆至pGEM-T easy载体,天祝白牦牛SRY基因编码区长687 bp,编码229个氨基酸(GenBank:FJ373272);对牦牛和奶牛(B.grunneins)的SRY基因编码区进行序列比对,发现存在2个碱基的变异,造成1个氨基酸的变异;将牦牛SRY基因编码区连接至pET-28a(+)载体,成功地构建了表达载体pET-28a/SRY;把表达载体pET-28a/SRY转入大肠杆菌(Escherichia coli)BL21(DE3)中,用IPTG在30℃ 200 r/min条件下诱导,获得了SRY蛋白的大量表达;对表达产物进行Western blot检测,结果显示获得的蛋白能与抗-His单抗特异性结合,确定了牦牛SRY蛋白的表达.     

猪链球菌2型纤连蛋白/血纤蛋白原结合蛋白 全基因的克隆及融合表达

采用发表的引物对,以猪链球菌2型（Streptococcus suis type 2）江苏分离株HA9801的基因组DNA为模板,采用PCR方法扩增纤连蛋白/血纤蛋白原结合蛋白基因fbps（GenBank登录号为AY565303）克隆于pMD-T18载体构建成载体pMD-T-fbps.经内切酶酶切和测序鉴定后,将由pMD-T-fbps内切酶切下的片段定向克隆于表达载体pET-32a（＋）,得到重组质粒pfbps.将重组质粒pfbps转化大肠杆菌（Escherichia coli）BL21株,经IPTG诱导,可高水平地表达相对分子量为83 kD的融合蛋白.配体印迹结合试验表明,表达的融合蛋白可与人纤连蛋白结合.     

小反刍兽疫病毒N基因在大肠杆菌中的表达及鉴定

参照GenBank中小反刍兽疫病毒((Peste des petits ruminants virus,PPRV)N基因序列（Nigeria/75/1）(1 578 bp)设计了1对添加EcoRⅠ和Not Ⅰ酶切位点的引物,对从奥地利引进的pCI-neo-PPRN质粒进行PCR扩增,克隆至pGM-T载体,并转化大肠杆菌Escherichia. coli TOP10感受态细胞,获得重组质粒pGM-T-PPRN。将重组质粒经EcoRⅠ和NotⅠ双酶切的产物插入原核表达载体pET-32a（+）,构建原核表达质粒pET-PPRN,将其转化进BL21(DE3)感受态细胞中后用IPTG诱导表达, 收集菌液进行SDS-PAGE电泳和Western blot分析,结果表明小反刍兽疫病毒N基因在BL21(DE3) 成功表达。所表达融合蛋白的相对分子质量约为80 kD,并能被组氨酸单抗、PPRV标准阳性羊血清及PPRV疫苗免疫羊血清所识别。     

猪圆环病毒2型BF株ORF2基因在大肠杆菌中的表达

采用PCR从构建的猪圆环病毒2型／(PCV2)ORF2重组质粒(pGEM-T-ORF2）中扩增出大小为593bp的ORb2基因，克隆到表达载体pET-32a，经异丙基硫代半乳糖苷诱导，成功表达了ORF2基因编码的结构蛋白。表达的重组蛋白为融合蛋白，分子量40kD，表达量20％左右。经Western blot检测，重组蛋白可被PCV2阳性血清识别。     

Overlap-PCR克隆秦川牛HCRTR1基因及其在大肠杆菌中的表达分析

通过Overlap-PCR方法克隆了秦川牛的HCRTR1基因编码区全长,获得1278 bp编码区全长序列,将其克隆至pMD-18T载体上,经测序表明获得的cDNA序列与GenBank公布的序列同源性均为98%。提取阳性克隆的质粒经BamHI和HindⅢ双酶切后,回收到1278 bp的目的片段,定向克隆到pET32a（+）表达载体上,连接产物转化Bl21(DE3)感受态大肠杆菌,对长出的菌落进行菌落PCR和质粒酶切鉴定正确后,用IPTG诱导目的蛋白的表达。SDS-PAGE表明秦川牛HCRTR1基因在大肠杆菌中不表达,生物信息学分析表明,HCRTR1基因的表达产物为具有7个跨膜螺旋的G蛋白偶联受体,可能是此类蛋白在原核系统的表达将会影响细菌本身存活,因此原核表达系统也成为研究此类蛋白的瓶颈。     

重组马槟榔甜蛋白MabinlinⅡ在大肠杆菌中的表达

MabinlinⅡ是我国所特有且唯一的植物甜蛋白，在体外至今没有得到具有甜味的基因表达产物。本文根据己知马槟榔甜蛋白的序列设计引物克隆MabinlinⅡ基因，对基因进行剪切重组。将重组基因克隆至大肠杆菌表达载体pET-30a（＋）中，构建了3个重组表达载体，转化大肠杆菌BL21（DE3），得到三株表达重组马槟榔甜蛋白的大肠杆菌工程菌。经IPTG诱导，3个重组MabinlinⅡ基因可在大肠杆菌BL21（DE3）中高效表达。     

低氮和干旱胁迫对富士和秦冠生长及氮素利用的影响

【目的】以富士（Fuji）、 秦冠（Qinguan）嫁接在平邑甜茶（Malus hupehensis Rehd.）上的当年生盆栽苗为试验材料,采用砂培方法,研究了缺氮胁迫和干旱对富士和秦冠生长情况、 光合参数、 植株各部位氮磷钾含量及氮素利用效率的影响,分析比较了低氮干旱条件下富士和秦冠生长及氮素利用的差异,以期为果树生产高效肥水利用提供理论指导。【方法】试验共设四个处理: 正常氮正常水（ZZ）、 低氮正常水（DZ）、 正常氮干旱（ZG）、 低氮干旱（DG）。氮素和水分均设置两个水平,分别为正常氮（6 mmol/L NO-3-N）、 低氮（0.3 mmol/LNO-3-N）、 正常供水（保持盆中砂子相对含水量为饱和含水量的80%~85%）、 干旱处理（保持盆中砂子相对含水量为饱和含水量的60%~65%）。【结果】富士和秦冠的生物量（茎和叶）、 株高茎粗等生长指标以及光合速率、 气孔导度、 蒸腾速率均为正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）,并且相对应处理下秦冠的以上指标均高于富士;正常供水下,缺氮处理使富士、 秦冠的根冠比比正常氮处理均有所增加,富士提高了2.05%,秦冠提高了22.40%。富士和秦冠的氮、 磷、 钾含量均表现出正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）; 氮、 钾元素含量在植株各部位的分布顺序依次是叶＞根＞茎,磷元素则是根＞叶＞茎;光合氮素利用效率（PNUE）和氮素利用效率表现为秦冠处理之间差异极显著,富士处理之间差异不显著;秦冠的PNUE和NUE明显高于富士,在低氮正常水（DZ）处理下,秦冠氮肥利用率比富士高42.07%,在低氮干旱（DG）处理下高64.14%;低氮胁迫下富士和秦冠的NUE显著提高,并且秦冠提高的幅度高于富士。【结论】施用氮肥能够显著提高富士与秦冠的干物质量,同等水肥条件下,秦冠生长优于富士;水分亏缺会减少叶片对氮的吸收,干旱条件下适度增施氮肥,可提高果树的抗旱能力;低氮干旱胁迫下秦冠的生长指标、 光合指标及氮素利用效率指标均优于富士,表现出较强的抗低氮干旱胁迫的能力。     

Biodiversity and ecogeography of wild Lactuca spp. in some European countries

Staff members of the Department of Botany of Palacký University in Olomouc and Gene Bank Department – Workplace Olomouc, Research Institute of Crop Production in Prague, Czech Republic, conducted an expedition in seven European countries (Austria, Czech Republic, France, Germany, Italy, the Netherlands, Switzerland) in August/September 1999 to collect wild Lactuca spp. germplasm and study its geographic distribution, ecology and biodiversity. During the mission, more than 600 locations were visited resulting in the collection of 602 seed samples (accessions) of wild Lactuca species and 13 seed samples of related genera (Chondrilla and Mycelis). Lactuca serriola f. serriola, L. serriola f. integrifolia, L. saligna and L. viminea subsp. chondrilliflora were prevalent in southern Europe (Italy, France), however, only L. serriola was common in central and western Europe (Austria, Czech Republic, Germany, Netherlands, Switzerland). The greatest diversity of Lactuca species was found in France, where also the most seed samples (165) were collected. The most characteristic habitats with a high density of Lactuca spp. populations were observed along roads and highways, grassy ditches, ruderal communities, and dust-heaps. Natural infections by powdery mildew (Erysiphe cichoracearum) and downy mildew (Bremia lactucae) on some wild Lactuca spp. were observed. Recent observations concerning the geographic distribution, population structure, habitats, and natural occurrence of diseases of Lactuca spp. are discussed. This assemblage of genetic resources of Lactuca spp. can serve as the basis of future studies of species diversification, spatial population structure, plant microevolution, domestication processes, and genetic variability of host-parasite interactions.     

Pasture Vegetation Elemental Analysis by Laser-Induced Breakdown Spectroscopy

Laser-induced breakdown spectroscopy (LIBS) is a new technique for the analysis of plant material. This study investigates the application of LIBS to pasture-based plant samples. The LIBS measurements were obtained from pelletized pasture samples (100 samples) that had been also analyzed by inductively coupled plasma–optical emission spectroscopy (ICP-OES) following microwave digestion for calibration and comparison purposes. Comparisons for elements sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), boron (B), phosphorus (P), and sulfur (S) showed that LIBS could be used for almost all the standard profile total elements with concentrations down to low mg/kg levels (observed error of Na: 0.024 percent, K: 0.18 percent, Mg: 0.016 percent, Ca: 0.073 percent, P: 0.017 percent, Mn: 31 mg/kg, Fe: 150 mg/kg, Zn: 6.6 mg/kg, and B: 1.1 mg/kg). Elemental analysis at less than mg/kg levels was not possible using LIBS. The elements S and Cu were particularly difficult to analyze with reliability using LIBS at the concentration levels found in the plant samples. Replacing microwave digestion and subsequent ICP analysis with a direct analysis of dried plant samples using LIBS has the potential to improve the productivity and reduce the cost of testing.     

Properties of anaerobically digested and composted municipal solid waste assessed by linking soil mesofauna dynamics and nitrogen modelling

We have studied the effect of anaerobically digested (ADMSW) and composted municipal solid waste (CMSW) on mineralization and foodweb dynamics to verify the hypothesis that ADMSW would immobilize N right after addition to soil in contrast to addition with CMSW. Another hypothesis was that the mesofauna (enchytraeids and microarthropods) would stimulate N release from the decomposer community. We measured excretion of -N and urea-N from the mesofauna and hypothesized that enchytraeids would release urea. ADMSW and CMSW were amended to pots with sandy loam and barley. The pots were divided into treatments with or without mesofauna. Mesofauna, plant N and biomass, soil N and ergosterol (fungal biomass) were measured over a 113-day period of four equidistant samplings. Soil respiration, N mineralization and N release by the mesofauna were modelled from concurrent studies. ADMSW- and CMSW-treated soils initially (<20 days) immobilized N. The amendments did not increase plant growth substantially, and this was probably due to N-limitation in the early stages of plant growth. Enchytraeid abundance was about three times higher in ADMSW- than CMSW-treated soils, indicating that ADMSW contained more labile compounds, bacteria, and microfauna. The mesofauna did not affect N-content, but the cumulated -N excreted by the mesofauna was estimated to be substantial and about one-fifth of total plant N in ADMSW. An explanation for this discrepancy might be that in the absence of the mesofauna, other members of the detrivore and microbivore community performed the mesofauna’s function. Neither enchytraeids nor microarthropods excreted urea.     

Faculty positions: Center for Agricultural Resources Research,Chinese Academy of Sciences

正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,     

Faculty positions: Center for Agricultural Resources Research,Chinese Academy of Sciences

正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,     

Faculty positions:Center for Agricultural Resources Research,Chinese Academy of Sciences

正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,     

Assessing the effectiveness of conservation measures: Resolving the “wicked” problem of the Steller sea lion

“Wicked” problems are those that are complex and that change when solutions are applied. Many conflicts in conservation fall into this category. The study approached the problem of how to constrain the apparent wickedness of a problem in the conservation management of a species by using simple empirical indicators to carry out iterative assessment of the risk to a population and to document how this risk evolves in relation to the addition of new data and the implementation of management actions. Effects of high levels of uncertainty within data and also concerning population structure were examined through stochastic simulation and by exploration of scenarios. Historical trends in the example used, the Steller sea lion, showed rapid declines in abundance in some regions during the 1980s. The current total population is 130,000-150,000 Steller sea lions through Alaska and British Columbia and this number has been stable since about 1990 in spite of regional differences in population dynamics. Regional differences in the sequence of changes in the number of pups and non-pups, suggested that an internal re-distribution of juveniles could have happened between 1980 and 1990. Current productivity also appears close to the long-term mean. Stochastic population projection using various scenarios showed that, based upon this history, the risk of extinction for the population has declined and is below reasonable thresholds for considering the population to be endangered. The trends in risk suggest that management actions taken since 1990 have probably been effective. Consequently, the conservation management objectives for the Steller sea lion are probably being met. The approach provides a mechanism, based upon experience and scenario analysis, for exploring future policy options and may help to constrain the debate amongst stakeholders about the cost-benefit trade-offs associated with different options.     

Soil-profile distribution of primary and secondary plant-available nutrients under conventional and no tillage

Nutrient distributions under no tillage (NT) compared with conventional disk-and-bed tillage (CT) management in the warm, humid region of the southeastern USA need to be assessed so that future placement, quantity, and type of fertilizers can be altered, if necessary, to efficiently match crop demands. We determined soil-profile distributions of pH, N, P, S, K, Ca, Mg, Na, Zn, Fe, Mn, and Cu to a depth of 0.9 m at the end of 8.5 years of continuous CT and NT management on a Weswood silty clay loam (fine, mixed, thermic Fluventic Ustochrept) in southcentral Texas. Most dramatic changes occurred within the 0–0.05 m depth, where soil under NT had lower pH, Fe, and Cu than under CT, but greater P, K, Zn, and Mn. Greater P and K under NT than under CT also occurred below the till-zone (0.15–0.3 m). At a depth of 0–0.3 m, soil under NT contained greater amounts of extractable P, K, Zn, Fe, Mn, and Cu than under CT. Nitrogen fertilization had little effect on nutrient distributions, except resulting in greater extractable K at 0–0.05 m and greater nitrate at 0–0.15 m. Few changes in soil-profile distributions were observed for extractable S, Ca, Mg, and Na. Long-term continuous use of NT on this fine-textured, high-fertility (except for N) soil had no apparent adverse effects on nutrient distributions relative to CT, but enhanced conservation and availability of P, K, Zn, Fe, Mn, and Cu near the soil surface where crop roots proliferate.     

Macroinvertebrates in North American tallgrass prairie soils: effects of fire, mowing, and fertilization on density and biomass

The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.