甘薯草甘膦抗性基因EPSPS克隆和序列分析

EPSPS基因编码5-烯醇式丙酮酰莽草酸-3-磷酸合成酶,该酶是芳香族氨基酸合成的关键酶,该基因在细菌、真菌、藻类和植物中被广泛克隆和研究。EPSPS酶是草甘膦除草剂的靶点酶,过量表达EPSPS基因可以提高作物的草甘膦抗性。该研究根据甘薯基因组数据库设计引物,以‘广薯87’为材料提取RNA,通过RT-PCR方法扩增甘薯IbEPSPS基因,测序后进行生物信息学分析和表达分析。结果表明:(1)成功克隆获得甘薯IbEPSPS基因,该基因全长CDS为1569 bp,编码522个氨基酸,其中在第98~113、173~183位氨基酸序列具有2个EPSPS的保守结构域。(2)系统进化树分析结果表明,甘薯IbEPSPS基因与三裂叶薯(Ipomoea triloba)、打碗花(Calystegia hederacea)、田旋花(Convolvulus arvensis)和牵牛(Ipomoea nil)聚在一类,其中与三裂叶薯的亲缘关系最近。(3)实时荧光定量PCR分析结果表明,甘薯IbEPSPS基因在茎、叶和茎尖表达量较高,同时受到草甘膦胁迫后IbEPSPS基因表达量提高。该研究结果为进一步探讨甘薯IbEPSPS基因的功能及甘薯对草甘膦的耐药性机制奠定了基础。     

Importance of Seed and Microsite Limitation: Native Wildflower Establishment in Non‐native Pasture

Barriers to establishing native plant communities on former pasture include dominance by a single planted species, hydrologic and edaphic alteration, and native species propagule limitation. Establishment may be dispersal‐limited (propagules do not arrive at the site), microsite‐limited (areas suitable for seedling emergence and survival do not exist), or both. Successful restoration strategies hinge on identifying and addressing critical limitations. We examined seed and microsite limitation to establishment of a native wildflower (Coreopsis lanceolata ) in a former pasture dominated by Paspalum notatum (bahiagrass). We determined the relative and interactive effects of microsite (irrigation and disturbance) and seed limitation on C. lanceolata establishment. We tested (1) irrigation (none, pre‐seeding, and pre‐ and post‐seeding), (2) disturbance (none, sethoxydim, glyphosate, and topsoil removal), and (3) C. lanceolata seeding rate (three seeding densities). Applying glyphosate before seeding increased C. lanceolata establishment compared to other disturbance treatments. Ultimately, C. lanceolata establishment was not affected by irrigation. Coreopsis lanceolata establishment was limited when seeded at 100 live seeds/m² but not at 600 or 1100 live seeds/m². Seed and microsite availability interactively affected C. lanceolata establishment, in that microsite limitation was biologically relevant only when a minimum number of seeds were present. In practice, both seed and microsite requirements must be met for successful establishment, and increasing the availability of seeds or microsites does not compensate for limitations of the other. Here, it is the relative importance of seed and microsite limitations that drives plant establishment; these limitations do not represent a simple dichotomy.     

Pre-harvest retting of flax with glyphosate: effects of novel methods of spray application on penetration of the canopy and uniformity of deposition

Four methods of spray application were compared in 1986 with the recommended method of glyphosate application for pre-harvest retting of flax (Linum usitatissimum). Sampling individual plants immediately after application of glyphosate and dye demonstrated that spray penetration through the crop canopy, measured as dye deposition on the lower stem as a percentage of total deposition per plant, was the same for all methods of application. None of the methods of application improved the uniformity of dye deposition.
The following year, effects of deseeding prior to spraying and of using an air-assisted sprayer were investigated. Deseeding increased dye deposition on the lower stem but did not improve the efficacy of glyphosate. Application with an air-assisted sprayer increased the proportion of spray deposited on the lower stem but not the absolute amount deposited or the efficacy of glyphosate. Dye deposition on individual plants varied greatly for all methods of application and, when glyphosate was applied using the same methods, undesiccated plants occurred in all plots.
Considerable improvements in spray penetration and uniformity of spray deposition appear necessary for successful pre-harvest retting of flax with glyphosate.     

Integrating perennial biomass crops into crop rotations: How to remove miscanthus and switchgrass without glyphosate

Perennial energy grasses have gained attention in recent years as a promising resource for the bioeconomy because of their benign environmental profile, high stress tolerance, high biomass yields and low input requirements. Currently, strong breeding efforts are being made to extend the range of commercially available miscanthus and switchgrass genotypes. In order to foster farmers' acceptance of these crops, and especially of novel hybrids, more information is required about how they can be efficiently integrated into cropping rotations, how they can be removed at the end of their productive lifespan, and what effect they have on subsequently grown crops. Farmers in Europe are meanwhile increasingly constrained in the methods available to them to remove these crops, and there is a risk that the herbicide glyphosate, which has been used in many studies to remove them, will be banned in coming years. This study looks at the removal of seven-year-old stands of miscanthus and switchgrass over 1 year at an experimental site in Southern-Germany. Three novel miscanthus genotypes were studied, alongside one variety of switchgrass, and the impact of each crop's removal on the yield of maize grown as a follow-on crop was examined. A combination of soil tillage and grass herbicides for maize cultivation was successful in controlling miscanthus regrowth, such that yields of maize grown after miscanthus did not differ significantly from yields of maize grown in monoculture rotation (18.1 t dry biomass ha⁻¹). Yields of maize grown after switchgrass (14.4 t dry biomass ha⁻¹) were significantly lower than maize in monoculture rotation caused by insufficient control of switchgrass regrowth by the applied maize herbicide. Although some regrowth of miscanthus and switchgrass was observed in the follow-on crop maize, complete eradication of both crops was achieved by subsequent winter wheat cultivation.     

Simultaneous LC-MS/MS Analysis of Glyphosate,Glufosinate, and Their Metabolic Products in Beer,Barley Tea,and Their Ingredients

Glyphosate and glufosinate are non-selective herbicides that have been extensively used worldwide. Their ionic and water-soluble characteristics often make it difficult to analyze them, especially in food components. A method was developed in this study for the simultaneous analysis of glyphosate, glufosinate, and three metabolic products in beer, barley tea, and their ingredients (malt and corn). The analytical samples were extracted with H₂O, purified with a strong anion-exchange solid-phase extraction (SPE) cartridge, and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) with an anion-exchange high-performance liquid chromatography (HPLC) column. This method enabled a rapid and sensitive analysis [limit of quantification (LOQ) = 10 µg/kg] of the herbicides to be achieved.     

A radiometric assay for 5-enolpyruvylshikimate-3-phosphate synthase

A new assay for 5-enolpyruvylshikimate-3-phosphate synthase is described. This enzyme of the shikimate pathway of aromatic amino acid biosynthesis generates 5-enolpyruvylshikimate 3-phosphate and orthophosphate from phosphoenolpyruvate and shikimate 3-phosphate. The shikimate pathway is present in bacteria and plants but not in mammals. The assay employs a paper-chromatographic separation of radiolabeled substrate from product. The method is specific, is sensitive to 50 pmol of product, and is suitable for use in crude extracts of bacteria. This enzyme appears to be the primary target site of the commercial herbicide glyphosate (N-phosphonomethyl glycine). A procedure for the enzymatic synthesis of [¹⁴C]shikimate 3-phosphate from the commercially available precursor [¹⁴C]shikimic acid is also described.     

Influence of conservation reserve program mid-contract management and landscape composition on northern bobwhite in tall fescue monocultures

The United States Department of Agriculture (USDA) authorized mid-contract management (MCM) in 2004 to restore and maintain plant species composition and structural diversity in aging Conservation Reserve Program (CRP) fields for the northern bobwhite (Colinus virginianus) and other grassland-dependent wildlife. We implemented 3 USDA-approved MCM regimes (i.e., strip disking, strip glyphosate spraying, and strip glyphosate spraying in combination with legume interseeding) in 60 tall fescue (Festuca arundinaceae) CRP monocultures in south-central Illinois, USA, during 2005–2008. We hypothesized that adult bobwhite relative densities and brood presence would increase following MCM that effectively restored early successional plant communities in otherwise monotypic stands of tall fescue. We estimated annual adult bobwhite relative densities and brood presence-absence in managed and unmanaged CRP. We modeled vegetation characteristics and landscape composition to identify factors influencing adult densities and brood presence. Adult relative densities were 2-fold greater in managed fields than in unmanaged fields, and were negatively correlated with greater percentages of grass cover. Adult densities were positively correlated with greater plant species diversity, and greater percentages of bare ground and legume cover. Logistic regression and odds ratio estimates indicated that fields managed with glyphosate-interseed and glyphosate treatments were 39.6% more likely to have broods than unmanaged CRP, whereas disked fields were 10.0% more likely than unmanaged CRP. These models indicated that the probability of brood presence was greater in fields with increased percentage of bare ground, greater plant species diversity, and decreased percentage of grass and litter cover. These findings suggest that a 3-year rotation of glyphosate or glyphosate-interseed treatments can enhance habitat conditions for adult bobwhites and broods in CRP tall fescue monocultures. © 2011 The Wildlife Society.     

除草剂对蜜蜂健康影响的研究进展

除草剂是农业生产中一种被广泛使用的农药,是当前种植业重要的一环,但其长期使用也导致了自然界中广泛存在除草剂残留和污染.蜜蜂作为自然界最主要的传粉者,具有重要的经济和生态价值.近年来的研究发现,除草剂对蜜蜂有着明显的负面作用,可能会导致蜜蜂个体健康受损、蜂群损失和其在蜂产品中残留等问题.本文综述了除草剂对蜜蜂生理、行为、...     

Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation

The lack of alternative selectable markers in crop transformation has been a substantial barrier for commercial application of agricultural biotechnology. We have developed an efficient selection system for wheat transformation using glyphosate-tolerant CP4 and GOX genes as a selectable marker. Immature embryos of the wheat cultivar Bobwhite were bombarded with two separate plasmids harboring the CP4/GOX and GUS genes. After a 1 week delay, the bombarded embryos were transferred to a selection medium containing 2 mM glyphosate. Embryo-derived calli were subcultured onto the same selection medium every 3 weeks consecutively for 9–12 weeks, and were then regenerated and rooted on selection media with lower glyphosate concentrations. Transgenic plants tolerant to glyphosate were recovered. ELISA assay confirmed expression of the CP4 and GOX genes in R₀ plants. Southern blot analysis demonstrated that the transgenes were integrated into the wheat genomes and transmitted to the following generation. The use of CP4 and GOX genes as a selectable marker provides an efficient, effective, and alternative transformation selection system for wheat.     

抗草甘膦转基因大豆PCR的检测

目的:建立快速、有效的鉴别转基因作物与非转基因作物及其产品的检测方法体系。方法:用抗草甘膦转基因大豆中的外源CaMV35S启动子和CP4-EPSPS基因引物,应用PCR方法,从中扩增出预期大小的DNA片段,将扩增产物回收后测序。结果:经同源性分析,扩增产物为CaMV35S启动子和CP4-EPSPS基因的一部分序列。结论:初步建立了转基因大豆的检测方法,同时讨论了PCR检测过程中假阴性和假阳性的原因。