Transgenic cotton could safely be grown since CpTI toxin rapidly degrades in the rhizosphere soil

Abstract

Cultivation of transgenic plants is debated worldwide. Potential environmental risks have to be considered, before acceptance of expanding cultivation, despite the advantages of the use of fewer pesticides. Here, the potential effects on soil ecosystems of transgenic plants have been studied. As a model, genetically engineered cotton producing cowpea trypsin inhibitor (CpTI) has been used. The degradation of CpTI in the rhizosphere of the transgenic CpTI+Bt (Bacillus thuringiensis) cotton cultivar SGK321 was assessed. During plant development, concentrations of CpTI toxin in the rhizosphere were measured using an enzyme-linked immunosorbent assay (ELISA). As the plants developed, the residue of CpTI in the rhizosphere increased, and reached a peak at topping stage (100 days after planting). After this stage, the residue began to decrease, and was nil the following year (258 days after planting). The conclusion is that genetically engineered cotton can safely be cultivated since no accumulation of substances released from the transgenic plants was persistent in the soil.     

High sensitive detection of Cry1Ab protein using a quantum dot-based fluorescence-linked immunosorbent assay

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs.     

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。     

检测多杀性巴氏杆菌毒素抗体的单抗竞争ELISA方法的建立

本研究以纯化的多杀性巴氏杆菌毒素基因片段的原核表达产物作为抗原免疫小鼠制备单抗,并利用表达蛋白和多杀性巴氏杆菌毒素单抗酶结合物建立了竞争ELISA方法检测多杀性巴氏杆菌毒素抗体。经过研究确定抗原包被浓度为223ng／mL,待检血清最佳稀释度为1：2,酶标单抗工作浓度为1：3200,血清抑制率大于50%为阳性。应用单抗竞争ELISA和细胞毒性中和试验同时对82份血清进行猪多杀性巴氏杆菌毒素抗体检测,竞争ELISA的检出率为40.2％,细胞毒性中和试验检出率为36.6％,两者符合率达91.5％。试验结果表明,该ELISA方法特异性强,敏感性高,稳定性和重复性好,操作简便。本方法的建立在实验室诊断的标准化、猪群萎缩性鼻炎疫苗免疫效果的评价及流行病学调查方面具有应用价值。     

H5N1型禽流感病毒血凝素HA1蛋白在苏云金芽胞杆菌细胞表面的展示及其免疫原性

利用苏云金芽胞杆菌S-层蛋白CTC表面展示系统研究在细胞表面展示H5N1型禽流感病毒HA1蛋白的可行性及其免疫原性，为研制既安全有效又能常温长期保藏和运输的禽流感口服基因工程疫苗奠定基础。用部分ha1基因（ha1p）代替S-层蛋白ctc基因中部且位于表面锚定序列slh下游的片段，构建了融合基因ctc-ha1p和 csa-ctc-ha1p；利用电脉冲转化法将含融合基因的重组质粒转入苏云金芽胞杆菌无质粒突变株BMB171中，获得了重组菌株BCCH（含ctc-ha1p，并导入一个载有协助细胞表面展示的csaAB操纵子的质粒）和CH（含csa-ctc-ha1p）。通过血凝和血凝抑制试验以及小鼠免疫学实验证实，2个重组菌株均成功地在细胞表面展示了重组HA1蛋白并具有一定的特异性和免疫原性，其中，CH的效果强于BCCH，说明csa-ctc-ha1p这种融合基因的构建方式更胜一筹。研究结果表明，苏云金芽胞杆菌S-层蛋白CTC表面展示系统可用来研制禽流感口服疫苗。     

Molecular quantification of slug density in the soil using monoclonal antibodies

Molecular ecology techniques are increasingly used to study invertebrate foodwebs and trophic interactions in the field. However, the study of subterranean foodwebs is currently constrained by the difficult, laborious and often expensive methods that need to be employed to simply measure invertebrate population densities accurately. Here we describe and field-test a novel monoclonal antibody-based system for tracking slug populations. Proteins were extracted from soil blocks using sodium chloride and a new slug-specific monoclonal antibody was developed, capable of detecting the proteins liberated by the salt. Detection sensitivity and limits, using enzyme-linked immunosorbent assays (ELISA), were measured for a range of soils and the system proved to be effective, whether the soil was heavy clay or sandy. The sensitivity of the assay varied between soils and needed to be calibrated by ELISA. There was a linear relationship between slug biomass in any given soil and slug proteins detected by ELISA. A field experiment was performed comparing ELISA with the most accurate conventional approach. The latter involved taking blocks of soil from the field and flooding them gradually over 10 days to drive slugs to the surface, where they were collected and weighed. Parallel blocks of soil were taken 1 m away and subjected to the salt extraction/ELISA approach. Results using the two systems proved to be very similar, but ELISA produced results more rapidly. The many advantages of using ELISA to measure slug density are discussed.     

Nutrient uptake and growth of potato: Arbuscular mycorrhiza symbiosis interacts with quality and quantity of amended biochars

Aims : The aim of this study was to explore interactive effects between quality (types) and quantity (application rates) of biochar as well as of arbuscular mycorrhiza (AM) symbiosis on the growth of potato plants. Methods : A low P sandy loam soil was amended with 0%, 1.5%, or 2.5% (w/w) of either of 4 types of biochar, which were produced from wheat straw pellets (WSP) or miscanthus straw pellets (MSP) pyrolyzed at temperatures of either 550°C or 700°C. Potato plants grown in pots containing the soils or soil biochar mixture were inoculated with or without AM fungus (AMF), Rhizophagus irregularis. The experiment was carried out under fully irrigated semi‐field conditions and plants were harvested 101 days after planting. Results : Application of high temperature biochar decreased growth, biomass and tuber yield of potato plants, while the low temperature biochar had a similar effect on yield as plants grown without biochar amendment. Total biomass of potato plants were decreased with the increasing rate of biochar. Arbuscular mycorrhizal fungus inoculation stimulated the growth of potato plants in all organs, increased tuber biomass significantly in 1.5% MSP700 amended plants, and to a lesser degree for WSP700, MSP550, and WSP550. In addition, plant biomass gain was linearly related to N, P, and K uptake, the ratio of P to N in the leaf of plants indicated that all treatments were mainly P‐limited. A multiple linear regression using P uptake and biochar rate as independent variables explained 91% of the variation in total biomass. The single effect of AMF inoculation, type and rate of biochar affected plant N, P and K uptake similarly. While AMF inoculation significantly increased P uptake in potato plants grown in soil with WSP700 or MSP700 despite of the rate of biochar. In general, application of biochar significantly increased AMF root colonization of potato plants. Conclusions : The application of MSP550 at 1.5% combined with AMF stimulated growth of potato the most. Furthermore, the results indicated that the interactive effect of AMF inoculation, biochar type and application rate on potato growth to a large extent could be explained by effects on plant nutrient uptake.     

Multiplexed protein quantification in maize leaves by liquid chromatography coupled with tandem mass spectrometry: an alternative tool to immunoassays for target protein analysis in genetically engineered crops

A multiplexing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method to quantify three proteins in maize leaves was developed and validated. For each protein, a hybrid Q-TRAP mass spectrometer was operated in the information-dependent acquisition (IDA) mode to select optimal potential signature peptides. The respective signature peptides were then further optimized and quantified as protein surrogates by multiple reaction monitoring (MRM). Leaf crude extracts were subject to microwave-assisted trypsin digestion for 30 min and then injected directly onto a high-performance liquid chromatography (HPLC) column without further separation or enrichment. The minimum sample process enabled us to achieve high recovery and good reproducibility, with a throughput of 200 samples per day. Using recombinant proteins as standards, a linear dynamic quantitative range of 2 orders of magnitude was obtained (correlation coefficient > 0.997) with good accuracy (deviation from nominal concentration < 15%) for all three proteins. Our study demonstrates that LC-MS/MS can be used as an alternative to immunoassays to quantify multiple low abundant proteins in genetically engineered crops.     

Assessment of screening methods for the identification of genetically modified potatoes in raw materials and finished products

Qualitative polymerase chain reaction methods for the detection of genetically modified potatoes have been investigated that can be used for screening purposes and identification of insect-resistant and virus-resistant potatoes in food. The presence of the nos terminator from Agrobacterium tumefaciens and the antibiotic marker gene nptII (neomycin-phosphotransferase II) was demonstrated in three commercialized Bt-potato lines (Monsanto Co., St. Louis, MO, USA) and one noncommercial GM-potato product (high amylopectin starch, AVEBE, Veendam, The Netherlands) and allows for general screening in foods. For further identification, specific primers for the FMV promoter derived from the figwort mosaic virus, the CryIIIA gene (delta-endotoxin from Bacillus thuringiensis subsp. tenebrionis), potato leafroll virus replicase gene, and the potato virus Y coat protein gene, were designed. The methods described were successfully applied to processed potato raw materials (dehydrated potato powders and flakes), starch samples, and finished products.     

玉米赤霉烯酮的高灵敏时间分辨荧光免疫分析

采用时间分辨荧光技术建立高灵敏的玉米赤霉烯酮（ZEN）间接竞争免疫分析法（ZEN-TRFIA）。以玉米赤霉烯酮人工抗原（ZEN-BSA）包被96孔板为固相抗原，与ZEN标准或样品中的ZEN共同竞争有限的抗ZEN的单抗；用稀土离子Eu3+标记的羊抗鼠抗体进行示踪。该方法的灵敏度为0.01 μg/L（10ppt），测量范围为0.01-20 μg/L，批内和批间变异分别为7.2%和14.6%，平均回收率为94.4%，与玉米赤霉醇（ZER）的交叉反应率为15.16%。7条不同时间进行的ZEN-TRFIA的效应点值ED20、ED50、ED80分别为0.156±0.035 μg/L、0.634±0.091 μg/L和2.595±0.274 μg/L。试剂盒4℃保质期超过6个月。研究表明，ZEN-TRFIA是目前报导的ZEN检测中较灵敏的方法，该分析方法稳定性好，货架期符合要求，可测范围宽，具有很好的应用前景。     

Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression

Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.     

猪圆环病毒2型ORF2/猪白介素2嵌合基因真核表达载体的构建、表达及在小鼠体内的免疫效果研究

摘要：为了研究猪圆环病毒2型（PCV2）核酸疫苗,本研究采用重叠延伸PCR（splicing by overlap extension-PCR , SOE-PCR）方法通过一基因柔性接头（linker）(G4S)3将PCV2的ORF2全长基因和猪白介素2的成熟肽基因（PoIL-2）构建成PCV2-linker-PoIL-2嵌合基因并克隆入pGEM-T Easy载体,对阳性重组质粒进行双酶切后回收嵌合基因亚克隆入pcDNA3.1(+)真核表达载体中;筛选阳性重组表达质粒（rpcDNA3.1/PCV2-linker-PoIL-2）瞬时转染COS-7细胞,分别采用PCV2抗原间接免疫荧光试验和PoIL-2蛋白ELISA试验方法检测COS-7细胞中表达的重组融合蛋白(rCap-linker-PoIL-2 )生物学活性;提取rpcDNA3.1/PCV2-linker-PoIL-2免疫Balb/c小鼠,采用PCV2抗体ELISA检测试剂盒检测rpcDNA3.1/PCV2-linker-PoIL-2在小鼠体内的免疫效果,并与只含PCV2 ORF2基因的重组表达质粒pcDNA3.1/OFR2 (rpcDNA3.1/OFR2)进行免疫效果比较。结果表明：成功构建了PCV2-linker-PoIL-2嵌合基因及其pcDNA3.1(+)重组表达质粒;rpcDNA3.1/PCV2-linker-PoIL-2在COS-7细胞内进行了成功表达,表达的rCap-linker-PoIL-2蛋白存在于COS-7细胞的细胞浆内,可与抗PCV2和抗PoIL-2蛋白抗血清发生特异性免疫反应,表明rCap-linker-PoIL-2蛋白具有Cap蛋白和PoIL-2蛋白的双重生物学活性; rpcDNA3.1/PCV2-linker-PoIL-2质粒免疫小鼠后可诱导小鼠产生明显的抗PCV2抗体,且其诱导的抗体水平明显高于rpcDNA3.1/OFR2。本研究为PCV2的核酸疫苗研制奠定了基础。     

Development of a certified reference material for genetically modified potato with altered starch composition

The presence of genetically modified organisms (GMOs) in food and feed products is subject to regulation in the European Union (EU) and elsewhere. As part of the EU authorization procedure for GMOs intended for food and feed use, reference materials must be produced for the quality control of measurements to quantify the GMOs. Certified reference materials (CRMs) are available for a range of herbicide- and insect-resistant genetically modified crops such as corn, soybean, and cotton. Here the development of the first CRM for a GMO that differs from its non-GMO counterpart in a major compositional constituent, that is, starch, is described. It is shown that the modification of the starch composition of potato (Solanum tuberosum L.) tubers, together with other characteristics of the delivered materials, have important consequences for the certification strategy. Moreover, the processing and characterization of the EH92-527-1 potato material required both new and modified procedures, different from those used routinely for CRMs produced from genetically modified seeds.     

Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes

Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.     

赤羽病病毒囊膜糖蛋白G1基因的截短表达、纯化及间接ELISA诊断方法的建立

为获得赤羽病病毒（Akabane Virus, AKAV）囊膜糖蛋白重组抗原作诊断应用研究,通过软件分析AKAV OBE-1株的囊膜糖蛋白G1的氨基酸序列,筛选出抗原性较好的基因片段G1-2作为目的片段,经RT-PCR扩增后,插入pMD18-T载体。经测序鉴定正确后,将该片段定向亚克隆于pET-28a (+)表达载体中,转化至BL21 (DE3)感受态细胞中进行诱导表达。经SDS-PAGE和Western-blot分析,诱导表达产物以包涵体的形式存在,大小约为40ku,且具有免疫学活性。亲和纯化后的融合蛋白浓度为2mg/ml,纯度为92.6%。以该融合蛋白作为诊断抗原,建立了间接ELISA诊断方法。确定的抗原包被浓度为20g/ml,血清最佳稀释度为1:200。交叉试验表明该方法对牛常见的6种疾病阳性血清无交叉反应。应用该方法对病毒微量中和试验检测过的云南（77份）和内蒙（70份）牛血清样品进行了检测。以中和试验为参照,通过统计学处理,得出两地临界值分别为0.493和0.488,本方法的特异性为73%和86.9%,二者的符和率分别为80%和85.9%。     

Increase in lipid content in potato tubers modified by 14-3-3 gene overexpression

Recently, transgenic potato plants were created with overexpression of the 14-3-3 protein derived from Cucurbita pepo. Detailed analysis of those plants suggested that the function of the isolated 14-3-3 isoform is in the control of carbohydrate and lipid metabolism in the plants. 14-3-3 protein overexpression gave rise to an increase in soluble sugar and catecholamine contents in both leaves and tubers. It is proposed that 14-3-3 protein affects carbohydrate metabolism in potato plants via regulation of catecholamine synthesis. Furthermore, genetically modified potato tubers with 14-3-3 protein overexpression showed changes in lipid content and composition. The transgenic potato tubers contained 69% more total fat compared to the wild-type plant. Separation of tuber lipids into polar and nonpolar fractions revealed that the transgenic potato tubers contained almost 3 times more nonpolar lipids than the control. Analysis of fatty acid composition, conducted by the means of gas chromatography, showed that linoleic acid was the main fatty acid present in the tubers of both modified and control potato plants. In the nonpolar fraction of the fat of the transgenic tubers the unsaturated fatty acids exhibited a higher participation in the sum of all fatty acids.     

Transgenic Bt plants decompose less in soil than non-Bt plants

Bt plants are plants that have been genetically modified to express the insecticidal proteins (e.g. Cry1Ab, Cry1Ac, Cry3A) from subspecies of the bacterium, Bacillus thuringiensis (Bt), to kill lepidopteran pests that feed on corn, rice, tobacco, canola, and cotton and coleopteran pests that feed on potato. The biomass of these transgenic Bt plants (Bt+) was decomposed less in soil than the biomass of their near-isogenic non-Bt plant counterparts (Bt−). Soil was amended with 0.5, 1, or 2% (wt wt⁻¹) ground, dried (50 °C) leaves or stems of Bt corn plants; with 0.5% (wt wt⁻¹) ground, dried biomass of Bt rice, tobacco, canola, cotton, and potato plants; with biomass of the near-isogenic plants without the respective cry genes; or not amended. The gross metabolic activity of the soil was determined by CO₂ evolution. The amounts of C evolved as CO₂ were significantly lower from soil microcosms amended with biomass of Bt plants than of non-Bt plants. This difference occurred with stems and leaves from two hybrids of Bt corn, one of which had a higher C:N ratio than its near-isogenic non-Bt counterpart and the other which had essentially the same C:N ratio, even when glucose, nitrogen (NH₄NO₃), or glucose plus nitrogen were added with the biomass. The C:N ratios of the other Bt plants (including two other hybrids of Bt corn) and their near-isogenic non-Bt counterparts were also not related to their relative biodegradation. Bt corn had a significantly higher lignin content than near-isogenic non-Bt corn. However, the lignin content of the other Bt plants, which was significantly lower than that of both Bt and non-Bt corn, was generally not statistically significantly different, although 10-66% higher, from that of their respective non-Bt near-isolines. The numbers of culturable bacteria and fungi and the activity of representative enzymes involved in the degradation of plant biomass were not significantly different between soil amended with biomass of Bt or non-Bt corn. The degradation of the biomass of all Bt plants in the absence of soil but inoculated with a microbial suspension from the same soil was also significantly less than that of their respective inoculated non-Bt plants. The addition of streptomycin, cycloheximide, or both to the soil suspension did not alter the relative degradation of Bt+ and Bt− biomass, suggesting that differences in the soil microbiota were not responsible for the differential decomposition of Bt+ and Bt− biomass. All samples of soil amended with biomass of Bt plants were immunologically positive for the respective Cry proteins and toxic to the larvae of the tobacco hornworm (Manduca sexta), which was used as a representative lepidopteran in insect bioassays (no insecticidal assay was done for the Cry3A protein from potato). The ecological and environmental relevance of these findings is not clear.     

Structural characterization of potato protease inhibitor I (Cv. Bintje) after expression in Pichia pastoris

In the present study the structural properties of potato protease inhibitor 1 (PI-1) were studied as a function of temperature to elucidate its precipitation mechanism upon heating. A cDNA coding for PI-1 from cv. Bintje was cloned and expressed in Pichia pastoris. Using the recombinant PI-1 it was suggested that PI-1 behaves as a hexameric protein rather than as a pentamer, as previously proposed in the literature. The recombinant protein seems either to have a predominantly unordered structure or to belong to the beta-II proteins. Differential scanning calorimetry analysis of PI-1 revealed that its thermal unfolding occurs via one endothermic transition in which the hexameric PI-1 probably unfolds, having a dimer instead of a monomer as cooperative unit. The transition temperature for the recombinant PI-1 was 88 degrees C. Similar results were obtained for a partially purified pool of native PI-1 from cv. Bintje.     

微垄覆膜沟播对阴山丘陵地区旱作马铃薯土壤水分及产量的影响

阴山丘陵地区土壤水分状况是限制该地区马铃薯发展的主要因素,如何对天然降雨进行高效、永续利用,提高马铃薯对自然降水的利用率,是该地区马铃薯生产发展的战略考虑。本研究提出了微垄覆膜沟播的种植模式,结果表明:(1)微垄覆膜沟播种植模式明显提高土壤0~60 cm土层贮水量,在马铃薯需水关键时期,出苗后35 d和55 d,微垄覆膜沟播土壤贮水量分别比平作覆膜和露地平作提高25.68%、14.26%和28.92%、18.47%。(2)微垄覆膜沟播种植模式可明显提高马铃薯产量及对降雨的利用效率。(3)微垄覆膜沟播种植模式下的马铃薯每公顷吸收的水分数量比平作覆膜和露地平作分别高25.70%和33.52%。因此,微垄覆膜沟播种植模式可以明显提高马铃薯产量及对自然降水的利用效率,可作为发展内蒙古阴山丘陵地区旱作马铃薯的有效途径。