Expression Pattern of Ａ Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

以本实验室构建的重组南瓜(Cucurbita moschata)韧皮部特异启动子dENP构建了植物表达载体pBdENP。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化马铃薯(Solanum tuberosum)品种Favorita，经过抗生素筛选，共获得转pBdENP和对照pBI121的抗卡那霉素马铃薯再生植株106株。通过PCR初步筛查，筛选出65株为转基因阳性植株。通过Southern blot对部分植株进一步分析,确证外源gus基因已经插入到转基因马铃薯植株的基因组中，插入拷贝数在1个或2个以上。对这些转基因马铃薯植株进行GUS染色结果表明, dENP和CaMV35S启动子一样均能驱动gus基因的表达，前者仅在马铃薯的韧皮部内特异表达，而CaMV35S启动子驱动的gus基因为组成型性表达。GUS酶活力测定结果进一步表明dENP和CaMV35S启动子驱动gus基因表达水平没有明显区别。以上结果证明dENP启动子驱动的外源基因在马铃薯中也具有韧皮部特异而高效表达的特征，从而可用于马铃薯抗病、抗蚜虫转基因研究。     

低温诱导的位点特异性重组植物表达载体构建及小麦转化

选择标记基因的剔除是转基因植物商品化种植的重要基础.为建立小麦(Triticum aestivum)低温诱导的位点特异性标记基因删除体系,本研究以小麦西农928基因组DNA为模板,通过PCR扩增获得小麦低温诱导表达wcs120启动子,序列分析表明,该序列与GenBank中序列AF031235相比有一段21 bp插入,与AY493570.1序列完全一致.以含有CinH/RS2单向位点特异性重组系统的植物表达载体pXL5513为框架,成功构建了包含有抗旱相关性基因PYL5(pyrabactin-resistance like gene 5)表达框的低温诱导的位点特异性重组植物表达载体pXL5513-fwcs-RDA;并对小麦绵阳19(M19)幼胚愈伤组织进行基因枪转化,1 745块愈伤组织经筛选分化共获得9株转基因植株,经PYL5基因特异引物PCR检测,6株为阳性植株,低温春化处理后经删除引物PCR检测,6株阳性植株初步证实成功删除了选择标记基因.本研究为利用低温诱导的位点特异性重组系统进行小麦无标记基因转化奠定了基础.     

一个新的马铃薯patatin classⅠ基因的cDNA在烟草中的表达及其酶活性

摘要：将马铃薯(Solanum tuberosum )块茎特异蛋白patatin classⅠ基因cDNA与CaMV 35S启动子融合，构建了植物表达载体pBSSP。用电激法将表达载体导入根癌农杆菌（Agrobacterium tumefaciens）LBA4404，然后转化烟草(Nicotiana tabacum )叶片获得再生植株。经PCR、PCR-Southern、Northern杂交和蛋白质检测，证明patatin classⅠ基因cDNA已整合到烟草基因组中并在转基因植株中转录和表达。酯酰水解酶（lipid acylhydrolase, LAH）活性分析显示，转基因烟草植株的叶片中LAH活性明显提高。     

一个新的马铃薯patatin classⅠ基因的cDNA在烟草中的表达及其酶活性

将马铃薯(Solanum tuberosum)块茎特异蛋白patatin class Ⅰ基因cDNA与CaMV 35S启动子融合,构建了植物表达载体pBSSP.用电激法将表达载体导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404,然后转化烟草(Nicotiana tabacum)叶片获得再生植株.经PCR、PCR-Southem、Northern杂交和蛋白质检测,证明patatin class Ⅰ基因cDNA已整合到烟草基因组中并在转基因植株中转录和表达.酯酰水解酶(lipid acylhydrolase,LAH)活性分析显示,转基因烟草植株的叶片中LAH活性明显提高.     

油菜毛状体发育相关基因GLABRA2启动子的克隆与功能验证

GLABRA2(GL2)基因在拟南芥毛状体发育中具有重要作用.实验利用基因组步移巢式PCR的方法,通过两次步移克隆得到一个油菜(Brassica napus)GL2基因启动子序列,序列分析发现它与拟南芥GL2启动子同源性较差,但有一些共同的顺式作用元件如MYB结合位点、G box等,也有油菜GL2基因所特有的应答元件如E-box.将其中具有启动子的基本特征的序列GP1与GUS报告基因融合构建重组载体pBI121-GP1,转化拟南芥(Arabidopsis).用卡那霉素筛选得到10株阳性苗.在T2代转基因株系中有6个株系的绿苗与黄花苗的比例都接近3:1,推定T-DNA是以单拷贝的形式插入拟南芥基因组DNA.GUS组织化学染色表明,GP1调控下报告基因主要在子叶、真叶的表皮毛以及根部的幼嫩组织中表达,与拟南芥GL2基因启动子表达模式基本一致,但也有明显不同:油菜GL2启动子GP1只在表皮毛发育早期强烈表达,而拟南芥GL2启动子调控表皮毛发育的整个过程.     

油菜叶绿体多顺反子定点整合表达载体的构建

根据拟南芥叶绿体基因组序列设计PCR引物,从我国甘蓝型油菜栽培品种F4叶绿体基因组中克隆了长度为1.5kb的trnI和trnA2个基因序列,核酸序列分析表明它们与拟南芥基因的同源性高达94%和99%,这两个克隆的油菜叶绿体基因组序列trnI和trnA被用于构建叶绿体定点整合表达载体。利用烟草叶绿体基因强启动子Prrn和终止子TpsbA,以及筛选标记基因aadA和目的基因HSA,构建了多顺反子表达盒Prrn-aadA-HSA-TpsbA,将表达盒置于油菜叶绿体trnI和trnA序列之间,最后构建成油菜叶绿体多顺反子定点整合表达载体pIPaHTA。酶切鉴定及序列分析证实,构建的表达载体具有预期的调控元件及结构,这为后期油菜叶绿体转化体系的建立奠定了基础。     

向日葵种子特异性启动子Ha ds10G1的克隆及其功能验证

利用PCR技术从向日葵基因组DNA中克隆了Lea蛋白基因家族中Ha ds10 G1基因上游1414bp的调控序列，序列分析表明-1—1414bp与报道序列同源性为100%，将其与GUS基因融合构建植物表达载体后，通过农杆菌介导法转化烟草NC89，PCR扩增初步证明目的片段已整合到烟草基因组中，转基因植株的GUS活性检测表明，在茎、叶中无GUS活性，GUS活性只存在于种子中，因此，Ha ds10 G1启动子具有种子特异性的功能。     

拟南芥AtHOS10基因的克隆及其表达载体的构建

AtHOS10基因对植物冷调节起到重要作用,可以通过控制ABA合成来改变植物的脱水胁迫耐性。本文利用PCR方法从Columbia生态型的拟南芥基因组中扩增出冷调节基因AtHOS10,并插入克隆载体。序列分析表明,克隆片段长3453bp,与目前发表的序列完全一致。将pUC-AtHOS10和pBI-HEM进行酶切重组构建表达载体pBI-AtHOS10,然后将表达载体转化导入农杆菌中,为侵染烟草作准备。     

含粘质沙雷氏菌几丁质酶SchiA基因的植物转化质粒pBG1112构建和水稻遗传转化

摘要:将一个2.8 kb的CaMV35S启动子/SchiA编码区/Nos终止区融合基因插入到植物转化载体pCAMBIA1301的多克隆酶切位点，得到1个14.6 kb的新植物转化质粒pBG1112,用花器介导法转化水稻（Oryza sativa ），经PCR检测，初步证实已将目的基因整合到受体植物的基因组中。一部分转基因T3代潮霉素抗性阳性植株对水稻纹枯病(Rhizoctonia solani )和稻瘟病（Pyricularia oryzae）的抗性较非转化对照增强。RT-PCR表明抗病性植株为阳性，而不抗病的植株为阴性。将RT-PCR产物测序后，用BLAST软件分析序列可知，该序列为细菌几丁质酶基因核苷酸序列而不是植物几丁质酶基因的核苷酸序列。T4代转基因水稻的几丁质酶活力高于对照未转基因植株，表明转入的外源几丁质酶基因能正常表达。     

玉米花青素调节基因Lc对转基因烟草和矮牵牛花色的影响

利用PCR方法从油菜(Brassica napus)基因组中扩增得到花瓣特异性启动子XY355,构建该启动子与玉米(Zea mays)花青素调节基因Lc的植物表达载体pXY60。通过根癌农杆菌(Agrobacterium tumefaciens)介导,将pXY60分别转化烟草(Nicotiana tabacum)和矮牵牛(Petunia hybrida)。对再生植株进行PCR检测,结果表明,外源基因已经整合到转基因烟草和矮牵牛的基因组中。观察发现,转基因植株的表型发生了明显变化:转基因烟草的花色由浅红变成了红色,转基因矮牵牛的花色由白色变成了浅紫色。     

Structural characteristics of clay-dominated soils of a marsh and a palaeosol in a crossed diagram

Although the shrink‐swell phenomenon of clays has been thoroughly studied, the in situ relation of the shrinkage curve to the structure profile is rarely presented from the shrinkage limit to the liquid limit. We studied the consolidated structure of clay‐dominated (<2 μm) soils formed on ‘pseudo‐liquid’ marsh sediments in the ‘Marais de l’Ouest’ (France). The profiles were studied in a grassland field and in a sunflower field from unsaturated surface soils down to deeper, saturated, levels characterized by a very large water content (100% by weight). The consolidation states were quantified recording cone resistance (Qd) profiles using a dynamic penetrometer in successive seasons. These Qd profiles were compared with the associated wet density and gravimetric water‐content profiles. Two consolidation depths were evident, the surface soil and a 130‐cm deep palaeosol. The seasonal Qd profiles demonstrate the partial irreversibility of the consolidation peaks associated with the surface soil and with the palaeosol. The shrinkage properties were established through drying curves of undisturbed test samples. In the void ratio (e)‐water content (W) and water content (W)‐saturation index (Sr) diagrams, the profiles as a whole exhibit only one clay soil behaviour from their pseudo‐liquid to plastic to solid states. Each Qd profile is represented by a hyperbolic curve in the e/Qd diagram. Represented in a (e – W – Qd —Sr) crossed diagram, the vertical evolution of the successive profiles shows the soil structure behaviour from the initial pseudo‐liquid sediment to the consolidated soil. A simple cone resistance recording associated with gravimetric water‐content profiles, characterizes the evolution of structural layers of soils for the seasonal drying‐wetting cycles, for the over‐consolidation associated with the palaeosol, and also for the effect of ploughing.     

Determination of ochratoxin a with a DNA aptamer

This work describes the identification of an aptamer that binds with high affinity and specificity to ochratoxin A (OTA), a mycotoxin that occurs in wheat and other foodstuffs, and a quantitative detection method for OTA based on the use of this aptamer. Aptamers are single-stranded oligonucleotides selected in vitro to bind to molecular targets. The aptamer selected in this work exhibited a dissociation constant in the nanomolar range and did not bind compounds with structures similar to OTA such as N-acetylphenylalanine or warfarin. The aptamer bound with a 100-fold less affinity to ochratoxin B. The selected aptamers could be used for the determination of ppb quantities of OTA in naturally contaminated wheat samples. Further work is ongoing to broaden the application demonstrated here with the development of sensors, affinity columns, and other analytical systems for field and laboratory determination of this toxin in food and agricultural products.     

Use of a 6-steps microcosm for studying a wastewater discharge in a freshwater ecosystem: a multidisciplinary study

An experimental microcosm has been designed for simulating and studying impacts of a wastewater treatment plant (WTP) discharge on a freshwater/sediment ecosystem. The study was focused on the changes in biodiversity of benthic populations, especially bacteria and oligochaetes. Effluents were discharged in the Saône river, near Lyon (France) from a small treatment plant which treated domestic raw water by an activated sludge process. Freshwater and sediments were sampled in the Saône river upstream of the discharge point and placed in microcosms. Following the WTP discharge, physicochemical parameters of the overlying water column and sediments exhibited only a slight change, as compared to a reference. Characterization of the sediment bacterial populations was conducted with the Biolog and API systems. Strain identification and interpretation of data was difficult using thesetwo systems. Bacterial taxa in the sediments increased slightly below the WTP discharge. Gram negative strains dominated in the effluents, but G+ and G- bacteria were balanced in the sediments. Pseudomonas sp. and Bacillus sp., were the dominant strains. Invertebrate populations indicated an effect of the WTP discharge, with increasing of pollution resistant strains (Tubificidae) and disappearance of pollution intolerant strains such as Limnodrilus udekemianus and Quistadrilus multicoetosus. Taken as a whole, biological parameters indicated an environmental changes despite only slight changes in the physicochemistry of water. This experimental microcosm has proven to be a useful tool for studying impact of wastewater discharge on benthic populations.     

Impact of a ski basin on a mountain watershed

The input of heavy metals by automobile exhaust pollution associated with the ski basin activities is the primary concern of this paper. Stream, snowpack and lichen samples were collected and analyzed for Pb, Zn, and Cu. Some lichen samples were also analyzed for Ca, Mg, Na, K, Fe, and Mn, as well as Pb, Zn, and Cu. Snowpack samples from roadside areas demonstrated increased levels of Pb and Cu in comparison to areas up to 180 m from the road, but heavy metal levels at more remote areas were comparable to roadside levels. In all snow samples the heavy metals associated with the particulate matter was the major form of all inputs. Heavy metals in streams were also highly correlated with the particulate matter content. Heavy metal inputs to the watershed were greater than stream export, indicating an accumulation of heavy metals by the system. The forest canopy appears to be the major factor in the accumulation of heavy metals, probably due to better interception and impaction of the particulate matter by the canopy.     

Application of a Molecular Method for the Identification of a Gigaspora margarita Isolate Released in a Field

A molecular technique for the identification of the Gigaspora margarita isolate CK based on the detection of a DNA sequence of 235 bp as its diagnostic marker was evaluated to investigate the survival and establishment of introduced arbuscular mycorrizal fungi (AMF) in a field ecosystem. In March 2001, roots and rhizosphere soil of Eragrostis curvula and Miscanthus sinensis were collected from the Mizunashi River at Mt. Fugendake (Nagasaki Prefecture, Japan), where plant seeds and AMF including G. margarita CK had been introduced for reforestation after the occurrence of repeated pyroclastic flows. We detected the marker sequence from DNA preparations of E. curvula roots and Gigaspora spores in the rhizosphere. This clearly showed that the isolate occurred at both hyphal and sporal stages. It was shown that the isolate survived and developed a life cycle in the revegetation area for 4 years. It was confirmed that the method was effective for tracing the isolate in samples collected from field ecosystems.     

Remediation of a long-term copper-contaminated soil using a polyacrylate polymer

Abstract. We investigated whether a polyacrylate polymer could be used to remediate a soil which had been contaminated with copper for many years. Perennial ryegrass was grown in a loamy sand containing 230 mg ammonium-acetate-EDTA extractable Cu kg⁻¹ and amended with 0, 0.1 and 0.2% of polymer. Growth of perennial ryegrass was stimulated in the polymer-amended soil, especially in the soil with 0.1% of polymer. After plant growth for 177 days, the amount of water extractable copper present in the unamended soil was 17 times that of the original soil. In the soil amended with 0.1% of polymer the level of copper after plant growth was only 0.11 times the amount present in the unamended soil. When the soil was incubated with polymer in the absence of plants, the level of water extractable copper was not reduced. The polymer seems to compete with plants for copper, and to prevent the increase of copper in soil solution brought about by root exudates.     

Cyanides in a soil of a former coking plant site

Soils of former coking plant sites are frequently contaminated with cyanide, which mainly occurs as the pigment Berlin blue, Fe^III4 [Fe^IICN)₆]₃, and soluble iron cyanide complexes, Fe(CN)₆^3-/4?. Berlin blue is only slightly soluble under acidic conditions. The cyanide mobility in a strongly acid soil (pH about 3) of such a site was studied by assessing the distribution of cyanides in the soil and sediment and by conducting batch experiments at different pH levels. The soil is based on a disposal layer (0–32 cm) overlying sandy loess (32–145 cm) overlying glaciofluvial sand (145–250 cm) overlying marlstone (250–500 cm). Highest cyanide concentrations were found in the disposal layer (62–2865 mg CN^? kg^?1), medium concentrations in the sandy loess (16–29 mg CN^? kg^?1), concentrations of about 100 mg CN^? kg^?1 in the glaciofluvial sand and lowest concentrations in the marlstone (0.22–0.49 mg CN^?1 kg^?1). The surfaces of macropores, which occur in the sandy loess and finish in the glaciofluvial sand, are dark-blue dyed and have much higher cyanide concentrations than the surrounding bulk soil. Thus, the accumulation of cyanides in the sand layer may partly be the result of macropore flow. Batch experiments show a strong pH dependence of the solubility of cyanide in the soil as well as of Berlin blue which was found by Mössbauer spectroscopy to be the dominating or sole iron cyanide. The time necessary to transport the cyanides solely as hexacyanoferrate into the undisturbed horizons is estimated to 1000 yr. However, because Berlin blue is known to form colloids, we discuss the possibility of cyanide transport as colloid not requiring dissolution and reprecipitation. We postulate that colloidal Berlin blue transported by macropore flow is responsible for the high mobility of cyanides in this acid soil.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Dynamics of a black bear population within a desert metapopulation

Understanding metapopulation dynamics in large carnivores with naturally fragmented populations is difficult because of the large temporal and spatial context of such dynamics. We coupled a long-term database of visitor sighting records with an intensive 3-year telemetry study to describe population dynamics of recolonization by black bears (Ursus americanus) of Big Bend National Park in Texas during 1988-2002. This population, which occurs within a metapopulation in western Texas and northern Mexico, increased from a single pair of known breeding-age animals in 1988 to 29 bears (including 6 females of breeding age) in March 2000 (λ = 1.25/year). A migration and dispersal event in August-December 2000 reduced the population to 2 adult females and as few as 5-7 individuals. One-way movement distances from the study area during this event averaged 76 km for females (n = 7) and 92 km for males (n = 4), and 3 animals conducted migrations of at least 154, 178, and 214 km, respectively. Our observations exemplify the importance of stochastic events on demographics of small populations and highlight the potential scale of bear movement among montane islands of southwestern North America. They also provide insight into the use of dispersal data in parameterizing metapopulation models for large carnivores.