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1.
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。 相似文献
2.
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. 相似文献
3.
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。 相似文献
4.
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. 相似文献
5.
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. 相似文献
6.
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。 相似文献
7.
向日葵种子特异性启动子Ha ds10G1的克隆及其功能验证 总被引：3，自引：0，他引：3
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。 相似文献
8.
9.
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。 相似文献
10.
作为人们广泛食用的叶用蔬菜,莴苣(Lactuca sativa L.)一直被科学家认为是一种理想的植物生物反应器平台。本研究首先以生产上常用的两个莴苣品种美国大速生(Grand Rapid)与香港玻璃生菜(Hong Kong Glass)的叶片为外植体,放置在添加不同激素成分的培养基中诱导再生小芽,在生根培养基上生根后即建立了组织培养再生体系;依据两个莴苣品种在不同培养基中的诱导再生率、出芽效率和生根率,确定表现较好的品种美国大速生作为后续叶绿体遗传转化外植体的供体。其次,根据莴苣叶绿体基因组16S-trnI-TrnA-23S区域的序列,设计特异引物,利用PCR扩增技术获得上述区域的部分片段,并作为同源重组序列插入T载体中;在同源重组序列中的Ecl136Ⅱ酶切位点处,插入带有绿色荧光蛋白基因(gfp)和aadA抗性筛选基因的化学合成表达框,构建适合莴苣叶绿体转化的特异表达载体PTLE,其中表达框内为gfp基因,壮观素酶抗性基因(aadA)具有壮观霉素和链霉素抗性。最后,利用基因枪轰击法将PTLE载体转化到莴苣叶绿体基因组中,在含有30 mg/L壮观霉素的培养基中进行两轮抗性筛选后,PCR分子检测和绿色荧光观察证明:获得的莴苣叶绿体转化植株仍处于杂合状态,还未达到完全同质化的程度。上述研究结果表明,已经初步建立了莴苣叶绿体的遗传转化体系,为进一步获得同质化纯合体和后续的植物生物反应器研究与开发提供了基础资料。 相似文献
11.
M. Bernard P. Dudoignon Y. Pons C. Chevallier & L. Boulay 《European Journal of Soil Science》2007,58(5):1115-1126
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. 相似文献
12.
Determination of ochratoxin a with a DNA aptamer 总被引：2，自引：0，他引：2
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. 相似文献
13.
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. 相似文献
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15.
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. 相似文献
16.
Kazuhira Yokoyama Takahiro Tateishi Masanori Saito Takuya Marumoto 《Soil Science and Plant Nutrition》2005,51(1):125-128
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. 相似文献
17.
《Agricultural and Forest Meteorology》1987,39(1):67-80
The photosynthetic light curves of eight Scots pine (Pinussylvestris L.) shoots, each with a different structure, were measured in a direct radiation field at different positions relative to the radiation beam. Using a model of shoot geometry, the irradiance distributions on the needle surface area of the same shoots were simulated in each position. Using measurements of shoot photosynthesis, the photosynthetic light curve for a needle surface area element (a small area on the needle surface) was estimated indirectly for each shoot. Shoot photosynthesis was then calculated from the estimated photosynthetic light response curve of a needle surface area element and the irradiance distribution on the surface area of needles. Calculated rates for shoot photosynthesis agreed well with the measured rates. The mean irradiance on the needle surface area was the major component causing variation in the light response of a shoot irradiated from different directions, but the shape of the irradiance distribution on the needle surface area had a considerably smaller effect. 相似文献
18.
K. Hayano 《Biology and Fertility of Soils》1987,3(3):159-164
Summary A bis-(p-nitrophenyl) phosphatase (BPN-Pase) was extracted from a forest soil and fractionated by DEAE-cellulose column chromatography into seven fractions (1, 2, 3, 4, 5, 6 and 7). The main fraction (fraction 5) was further fractionated into 3 subfraction (fractions 1, 2 and 3) by affinity chromatography for nuclease. The properties of the BPNPase in subfraction 3 were characterized and the results are reported in this article. Subfraction 3, which had a peak at about 278 run in the UV absorption spectrum, hydrolyzed 2,3-cyclic-nucleotides more readily than 3,5-cyclicnucleotides, adenylyl-(3 5)uridine, uridylyl-(3 5)adenosine, thymidine 3-p-nitrophenyl phosphate, thymidine 5-p-nitrophenyl phosphate, p-nitrophenyl phosphate and BPNP. Subfraction 3 hydrolyzed BPNP into 2 mol p-nitrophenyl and 1 mol inorganic phosphate during incubation. Apparent molecular weight of the BPNPase was estimated to be about 58 000 by gel filtration. The BPNPase activity had a pH optimum at 5.0 and was inhibited by Hg2+ and slightly inhibited by F– and PO
4
3–
.These observations suggest that the BPNPase is subfraction 3 has been constituted mainly with 2,3cyclic-nucleotide 2-phosphodiesterase [EC 3.1.4.16] or 2,3-cyclic-nucleotide 3-phosphodiesterase [EC 3.1.4.37]. 相似文献
19.
Joke Van De Steene Hannelore Van Vooren Hubert Verplancke 《Water, air, and soil pollution》2007,183(1-4):403-413
A quasi steady state respiration test based on Fick’s law with a correction term for advective flux, for estimating petroleum hydrocarbon degradation rates, was evaluated in a full-scale (3,000 m3) biopile study. A contaminated clayey sand soil with an average TPH content of 1,421?±?260 mg kg?1 soil was treated in a biopile with a fixed venting and heating system. Temperature in the biopile ranged from 12.1 to 36.6°C and soil water content from 15.2 to 35.8 m3 H2O m?3 soil. Oxygen concentrations in the biopile showed a rapid decrease with depth, before venting and reached constant atmospheric concentration during venting. Measured oxygen consumption in the biopile ranged from ?0.04 to ?0.68 mol O2 m?3 soil day?1. Average oxygen consumption rates calculated with the quasi-steady-state method were significantly (P?<?0.05) lower then the oxygen consumption rates calculated with the transient method. It was suggested that the oxygen diffusion was underestimated by the diffusivity models used and that further research is needed to determine relative effective diffusion coefficients in biopiles. Although both respiration testing and petroleum hydrocarbon concentration showed a decrease of oxygen consumption in time, the estimated degradation rate was low compared to the actual decrease in petroleum hydrocarbon concentration. Additional work will have to be done to acquire a more precise knowledge of the relationship between respirometrically determined degradation rates and the actual change in petroleum hydrocarbon concentration in the soil. 相似文献
20.
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. 相似文献