Drugs and pesticide residues in broiler feed can compromise the therapeutic and production benefits of antibiotic(ANT)application and affect gene expression.In this study,we analyzed the expression of 13 key pancreati...Drugs and pesticide residues in broiler feed can compromise the therapeutic and production benefits of antibiotic(ANT)application and affect gene expression.In this study,we analyzed the expression of 13 key pancreatic genes and blood physiology parameters after administering one maximum residue limit of herbicide glyphosate(GLY),two ANTs,and one anticoccidial drug(AD).A total of 260 Ross 308 broilers aged 1-40 d were divided into the following four groups of 65 birds each:control group,which was fed the main diet(MD),and three experimental groups,which were fed MD supplemented with GLY,GLY+ANTs(enrofloxacin and colistin methanesulfonate),and GLY+AD(ammonium maduramicin),respectively.The results showed that the addition of GLY,GLY+ANTs,and GLY+AD caused significant changes in the expression of several genes of physiological and economic importance.In particular,genes related to inflammation and apoptosis(interleukin 6(IL6),prostaglandin-endoperoxide synthase 2(PTGS2),and caspase 6(CASP6))were downregulated by up to 99.1%,and those related to antioxidant protection(catalase(CAT),superoxide dismutase 1(SOD1)and peroxiredoxin 6(PRDX6))by up to 98.6%,compared to controls.There was also a significant decline in the values of immunological characteristics in the blood serum observed in the experimental groups,and certain changes in gene expression were concordant with changes in the functioning of the pancreas and blood.The changes revealed in gene expression and blood indices in response to GLY,ANTs,and AD provide insights into the possible mechanisms of action of these agents at the molecular level.Specifically,these changes may be indicative of physiological mechanisms to overcome the negative effects of GLY,GLY+ANTs,and GLY+AD in broilers.展开更多
Different-sized hollow SiO2 spheres of 249–1348 nm in diameter were successfully prepared by using Na2SiO3 as the precursor and using polystyrene and polystyrene-methyl acrylic acid latexes as the templates. The diam...Different-sized hollow SiO2 spheres of 249–1348 nm in diameter were successfully prepared by using Na2SiO3 as the precursor and using polystyrene and polystyrene-methyl acrylic acid latexes as the templates. The diameter and shell thickness of the hollow SiO2 spheres increase with increasing the latex template diameter at a given mass ratio of SiO2 to latex template. The diameter and shell thickness of the hollow SiO2 spheres also increase with increasing the mass ratios of SiO2 to latex template. The presence of carboxylic acid groups on the surfaces of polystyrene-methyl acrylic acid latex templates favors the formation of dense and uniform SiO2 shells. The hollow SiO2 sphere is constructed by mesoporous shell with large specific surface area. When glyphosate is used as a release model chemical, glyphosate release rate is tuned by varying the shell thickness.展开更多
[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistan...[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistant soybean. [Method] First,a screening for glyphosate resistant varieties among sixty-seven wild soybean materials was done in a field trial; subsequently, physiological indexes of the screened resistant variety ZYD0685 and the sensitive variety ZYD0790 were studied. [Result]At the glyphosate dose of 1.23 kg a.i/hm2, glyphoaste resistance varied greatly among different wild soybean materials, with the highest survival rate of 87% and83% occurring in ZYD0685 and ZYD2405, respectively, and that of another seven accessions ranged from 2.7% to 38%, and all the remaining fifty-eight soybean materials died. After treatment with glyphoaste at different doses, there were no significant differences in chlorophyll content and shikimate content in the resistant ZYD0685, but there was an evident increase in the activity of gultathione-S-transferases（GSTs）; while in the sensitive ZYD0790, the content of shikimic acid increased significantly, and chlorophyll content decreased significantly, and GSTs activity revealed a slight change. [Conclusion] Therefore, lowering the amount of accumulated shikimic acid is the major physiological response to glyphosate in wild soybean.展开更多
Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSP...Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.展开更多
The use of glyphosate-resistant corn has facilitated a shift from a reliance on preemergence residual herbicides to postemergence (POST) herbicides, and in some cases exclusively glyphosate. Glyphosate is a non-select...The use of glyphosate-resistant corn has facilitated a shift from a reliance on preemergence residual herbicides to postemergence (POST) herbicides, and in some cases exclusively glyphosate. Glyphosate is a non-selective herbicide that is relatively slow-acting, which may allow weeds to continue to compete with corn after application and potentially decrease crop yield. The addition of several POST corn herbicides, with some residual control, to an early-season glyphosate application was examined to determine if the tankmix combination would improve the speed of weed control compared to glyphosate applied alone. Seven field trials were conducted over three years (2009, 2010 and 2011) near Ridgetown and Exeter, Ontario. The control of common ragweed was improved 3 days after application (DAA) with three POST glyphosate tankmixes compared to glyphosate alone. However control was still less than 55%. Depending on the weed species examined, at 28 DAA two of the glyphosate tankmix treatments tested provided better common ragweed, common lambsquarters, or green foxtail control than glyphosate alone. Treatments providing better weed control at 28 DAA also typically decreased weed density compared to glyphosate alone.展开更多
Horseweed(Conyza canadensis),an invasive alien weed,is one of the main weeds in orchards in China.Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in C...Horseweed(Conyza canadensis),an invasive alien weed,is one of the main weeds in orchards in China.Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in China,a case of glyphosate-resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries.Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history.Potted seedlings with 11-13-leaf growth stage were treated with glyphosate at 0.035,0.07,0.14,0.28,0.56,1.12,2.24,4.48,and 8.96 kg a.i.ha-1.The dosage dependence response curve of each population was constructed with Log-logistic dose response regression equations.The ED50 value of each population was calculated and compared with the susceptible population from China.Different populations had different relative glyphosate-resistant levels which increased with the number of years of glyphosate application.Two populations with the highest resistance levels,8.28 and 7.95 times,were found in Ningbo,Zhejiang Province,China,where glyphosate was used for weed control in orchards twice each year for 15 yr.The two resistant populations accumulated approximately two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application.展开更多
A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated....A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein CrylAb/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer （Ostnnia furnacalis）, cotton boll worm （Helicoverpa armigera）, and armyworm （Mythimna separata）. This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.展开更多
In this work,samples consisting of BiVO4 with exposed(040)facets coupled with Bi2S3(Bi2S3/BiVO4)were prepared through a one-pot hydrothermal method,using ethylenediaminetetraacetic acid as directing agent and L-cystei...In this work,samples consisting of BiVO4 with exposed(040)facets coupled with Bi2S3(Bi2S3/BiVO4)were prepared through a one-pot hydrothermal method,using ethylenediaminetetraacetic acid as directing agent and L-cysteine as sulfur source and soft template.X-ray diffraction,field emission scanning electron microscopy,and high-resolution transmission electron microscopy measurements indicated that the Bi2S3 content had a significant influence on the growth of(040)and(121)facets as well as on the morphology of the Bi2S3/BiVO4 samples.When the Bi2S3 content reached 1 mmol,the Bi2S3/BiVO4 samples exhibited a peony-like morphology.The results of transient photocurrent tests and electrochemical impedance spectroscopy measurements confirmed that a more effective charge separation and a faster interfacial charge transfer occurred in Bi2S3/BiVO4 than BiVO4.The enhanced photocatalytic activity of the Bi2S3/BiVO4 samples could be attributed to the improved absorption capability in the visible light region and the enhanced electron-hole pair separation efficiency due to the formation of the Bi2S3/BiVO4 heterostructure.In addition,the Bi2S3/BiVO4 samples showed relative stability and reusability.The simple method presented in this work could be used to fabricate composite photocatalysts with high activity for different applications,such as photocatalytic degradation of organic pollutants,photocatalytic splitting of water,and photocatalytic reduction of carbon dioxide.展开更多
Glyphosate （N-phosphonomethyl glycine） is the most used herbicide worldwide. The degradation of ^14C-labeled glyphosate was studied under controlled laboratory conditions in three different agricultural soils： a si...Glyphosate （N-phosphonomethyl glycine） is the most used herbicide worldwide. The degradation of ^14C-labeled glyphosate was studied under controlled laboratory conditions in three different agricultural soils： a silt clay loam, a clay loam and a sandy loam soil. The kinetic and intensity of glyphosate degradation varied considerably over time within the same soil and among different types of soil. Our results demonstrated that the mineralization rate of glyphosate was high at the beginning of incubation and then decreased with time until the end of the experiment. The same kinetic was observed for the water extractable residues. The degradation of glyphosate was rapid in the soil with low adsorption capacity （clay loam soil） with a short half-life of 4 days. However, the persistence of glyphosate in high adsorption capacity soils increased, with half-live of 19 days for silt clay loam soil and 14.5 days for sandy loam soil. HPLC analyses showed that the main metabolite of glyphosate, arninomethylphosphonic acid （AMPA） was detected after three days of incubation in the extracts of all three soils. Our results suggested that the possibility of contamination of groundwater by glyphosate was high on a long-term period in soils with high adsorption capacity and low degrading activities and/or acid similar to sandy loam soil. This risk might be faster but less sustainable in soil with low adsorption capacity and high degrading activity like the clay loam soil. However, the release of non-extractable residues may increase the risk of contamination of groundwater regardless of the type of soil.展开更多
Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO 2- 3 and -COOH groups, on cadmium adsorpti...Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO 2- 3 and -COOH groups, on cadmium adsorption in montmorillonite was studied in detail. The results showed that the cadmium adsorption quantity in montmorillonite increased with increasing soil solution pH and cadmium concentration as usual, but decreased with glyphosate, which is due to the formation of a low affinity complex of Cd and glyphosate and decreasing solution pH induced by glyphosate addition. When the equilibrium solution pH was below 6.7, glyphosate has little effect on cadmium adsorption, but when the equilibrium solution pH was above 6.7, glyphosate significantly decreased cadmium adsorption quantity in montmorillonite. In addition, the adding order of Cd and glyphosate also influenced Cd adsorption quantity in montmorillonite.展开更多
Glyphosate （GPS） is a non-selective, post-mergence herbicide that is widely used throughout the world. Due to the similar molecular structures of glyphosate and phosphate, adsorption of glyphosate on soil is easily ...Glyphosate （GPS） is a non-selective, post-mergence herbicide that is widely used throughout the world. Due to the similar molecular structures of glyphosate and phosphate, adsorption of glyphosate on soil is easily affected by coexisting phosphate, especially when phosphate is applied at a significant rate in farmland. This paper studied the effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils including two variable charge soils and one permanent charge soil. The results indicated that Freundlich equations used to simulate glyphosate adsorption isotherms gave high correlation coefficients（0.990-0.998） with K values of 2751, 2451 and 166 for the zhuanhong soil（ZH soil, Laterite）, red soil（ RS, Udic Ferrisol） and Wushan paddy soil（WS soil, Anthrosol）, respectively. The more the soil iron and aluminum oxides and clay contained, the more glyphosate adsorbed. The presence of phosphate significantly decreased the adsorption of glyphosate to the soils by competing with glyphosate for adsorption sites of soils. Meanwhile, the effects of phosphate on adsorption of glyphosate on the two variable charge soils were more significant than that on the permanent charge soil. When phosphate and glyphosate were added in the soils in different orders, the adsorption quantities of glyphosate on the soils were different, which followed GPS-soil 〉 GPS-P-soil = GPS-Soil-P 〉 P-soil-GPS, meaning a complex interaction occurred among glyphosate, phosphate and the soils.展开更多
Glyphosate is an important organophosphonate herbicide used to eliminate grasses and herbaceous plants in many vegetation management situations.Its extensive use is causing environmental pollution,and consequently,the...Glyphosate is an important organophosphonate herbicide used to eliminate grasses and herbaceous plants in many vegetation management situations.Its extensive use is causing environmental pollution,and consequently,there is a need to remove it from the environment using an eco-friendly and cost-effective method.As a step to address this problem,a novel bacterial strain Comamonas odontotermitis F2,capable to utilize glyphosate as a carbon(C)and/or phosphorus(P)source,was isolated from a glyphostate-contaminated field soil in Australia and characterized.Response surface methodology(RSM)employing a 2^3 full factorial central composite design was used to optimize glyphosate degradation by C.odontotermitis P2 under various culture conditions.The strain C.odontotermitis P2 was proficient in degrading 1.5 g L^-1 glyphosate completely within 104 h.The optimal conditions for the degradation of glyphosate were found to be pH 7.4,29.9℃,and an inoculum density of 0.54 g L^-1,resulting in a maximum degradation of 90%.Sequencing of glyphosate oxidoreductase(GOX)and C-P lyase(phnJ)genes from C.odontotermitis P2 revealed 999c and 93%identities to already reported bacterial GOX and phnJ genes,respectively.The presence of these two genes in C.odontotermitis indicates its potential to degrade glyphosate through GOX and C-P lyase metabolic pathways.This study demonstrates the potential of C.odontotermitis P2 for efficient degradation of glyphosate,which can be exploited for remediation of glyphosate.展开更多
The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new str...The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.展开更多
Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested ...Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.展开更多
Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an...Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an Agrobacterium-mediated transformation method,a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS）was transformed into cotton cultivar K312.The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.The results from polymerase chain reaction（PCR）and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.Further,even cotton treated with 45.0 mmol L^–1 of glyphosate was able to slowly grow,bloom and seed.The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.展开更多
The rapid detection of glyphosate resistance in goosegrass（Eleusine indica） will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence（Fluo） and P700（reaction ce...The rapid detection of glyphosate resistance in goosegrass（Eleusine indica） will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence（Fluo） and P700（reaction center chlorophyll of photosystem I） absorbance were analyzed in one biotype of goosegrass that is resistant to glyphosate and in another that remains sensitive to the herbicide. Both biotypes were treated with a foliar spray of glyphosate. Differences in photosystem II maximum quantum yield（Fv/Fm）, effective photochemical quantum yield（Y（II））, and non-photochemical quenching（NPQ） between the biotypes increased over time. Values for Fv/Fm and Y（II） differed between the two biotypes 24 h after treatment（HAT）. Differentiated activities and energy dissipation processes of photosystem II（PSII） and energy dissipation processes of photosystem I（PSI） were manifested in the two biotypes 24 HAT with 20 mmol L–1 glyphosate. Differentiated energy dissipation processes of PSI were still apparent 24 HAT with 200 mmol L–1 glyphosate. These results indicate that the Fluo parameters related to PSII activity and energy dissipation and the P700 parameters related to energy dissipation are suitable indicators that enable rapid detection of glyphosate resistance in goosegrass.展开更多
Cotton plants are recalcitrant with regards to transformation and induced regeneration.In the present study,5-enolpyruvylshikimate-3-phosphate（EPSPS）,a glyphosate resistant gene from the bacterium Agrobacterium sp.s...Cotton plants are recalcitrant with regards to transformation and induced regeneration.In the present study,5-enolpyruvylshikimate-3-phosphate（EPSPS）,a glyphosate resistant gene from the bacterium Agrobacterium sp.strain CP4,was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacteriummediated transformation of shoot apex.Primary transformants were initially screened using a 0.26%glyphosate spray and subsequently by PCR analysis.Five out of 4 000 transformants from T_1 seeds were obtained resulting in an in planta transformation rate of 0.125%.Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance.Transgene insertion was analyzed by Southern blot analysis.Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR,Western blot,and ELISA methods.Transgenic T_3 plants were resistant to as much as 0.4% of glyphosate treatments in field trials.Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars.Since this method does not require sterile conditions,the use of specialized growth media or the application of plant hormones,it can be conducted under the greenhouse condition.展开更多
It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In...It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase（rbcS） small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize（Zea mays） immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.展开更多
Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to s...Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.展开更多
Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimat...Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.展开更多
基金supported by the Russian Science Foundation(No.22-16-00128),“Investigation of the Toxic Effect of Glyphosates on the Functional State of the Bird Intestinal Microbial Community,Their Growth and Development,and the Development of a Biological Product Based on the Glyphosate Degrading Strain”.
文摘Drugs and pesticide residues in broiler feed can compromise the therapeutic and production benefits of antibiotic(ANT)application and affect gene expression.In this study,we analyzed the expression of 13 key pancreatic genes and blood physiology parameters after administering one maximum residue limit of herbicide glyphosate(GLY),two ANTs,and one anticoccidial drug(AD).A total of 260 Ross 308 broilers aged 1-40 d were divided into the following four groups of 65 birds each:control group,which was fed the main diet(MD),and three experimental groups,which were fed MD supplemented with GLY,GLY+ANTs(enrofloxacin and colistin methanesulfonate),and GLY+AD(ammonium maduramicin),respectively.The results showed that the addition of GLY,GLY+ANTs,and GLY+AD caused significant changes in the expression of several genes of physiological and economic importance.In particular,genes related to inflammation and apoptosis(interleukin 6(IL6),prostaglandin-endoperoxide synthase 2(PTGS2),and caspase 6(CASP6))were downregulated by up to 99.1%,and those related to antioxidant protection(catalase(CAT),superoxide dismutase 1(SOD1)and peroxiredoxin 6(PRDX6))by up to 98.6%,compared to controls.There was also a significant decline in the values of immunological characteristics in the blood serum observed in the experimental groups,and certain changes in gene expression were concordant with changes in the functioning of the pancreas and blood.The changes revealed in gene expression and blood indices in response to GLY,ANTs,and AD provide insights into the possible mechanisms of action of these agents at the molecular level.Specifically,these changes may be indicative of physiological mechanisms to overcome the negative effects of GLY,GLY+ANTs,and GLY+AD in broilers.
基金Projects (11KJB530002, CX10B-259Z) supported by Research Funds from Jiangsu Provincial Department of Education, ChinaProject (10zxfk35) supported by Sichuan Province Nonmetallic Composites and Functional Materials Key Laboratory Project, China
文摘Different-sized hollow SiO2 spheres of 249–1348 nm in diameter were successfully prepared by using Na2SiO3 as the precursor and using polystyrene and polystyrene-methyl acrylic acid latexes as the templates. The diameter and shell thickness of the hollow SiO2 spheres increase with increasing the latex template diameter at a given mass ratio of SiO2 to latex template. The diameter and shell thickness of the hollow SiO2 spheres also increase with increasing the mass ratios of SiO2 to latex template. The presence of carboxylic acid groups on the surfaces of polystyrene-methyl acrylic acid latex templates favors the formation of dense and uniform SiO2 shells. The hollow SiO2 sphere is constructed by mesoporous shell with large specific surface area. When glyphosate is used as a release model chemical, glyphosate release rate is tuned by varying the shell thickness.
基金Supported by the National Natural Science Foundation of China(30971834)~~
文摘[Objective] This study aimed to preliminarily determine the physiological mechanism of glyphosate resistance produced in wild soybean（Glycine soja） and further provide a basis for the breeding of glyphosate-resistant soybean. [Method] First,a screening for glyphosate resistant varieties among sixty-seven wild soybean materials was done in a field trial; subsequently, physiological indexes of the screened resistant variety ZYD0685 and the sensitive variety ZYD0790 were studied. [Result]At the glyphosate dose of 1.23 kg a.i/hm2, glyphoaste resistance varied greatly among different wild soybean materials, with the highest survival rate of 87% and83% occurring in ZYD0685 and ZYD2405, respectively, and that of another seven accessions ranged from 2.7% to 38%, and all the remaining fifty-eight soybean materials died. After treatment with glyphoaste at different doses, there were no significant differences in chlorophyll content and shikimate content in the resistant ZYD0685, but there was an evident increase in the activity of gultathione-S-transferases（GSTs）; while in the sensitive ZYD0790, the content of shikimic acid increased significantly, and chlorophyll content decreased significantly, and GSTs activity revealed a slight change. [Conclusion] Therefore, lowering the amount of accumulated shikimic acid is the major physiological response to glyphosate in wild soybean.
基金financially supported by the National Transgenic Major Program, China (2016ZX08004001-04)
文摘Glyphosate is a highly efficient, broad-spectrum nonspecific herbicide that inhibits the 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)-mediated pathway of shikimic acid. The screening of glyphosate-resistant EPSPS gene is a major means for the development of new genetically modified glyphosate-resistant transgenic crop. Currently, the main commercialized glyphosate-resistant soybean contains glyphosate-resistant gene CP4-EPSPS. In this study, a G10-EPSPS gene was reported providing glyphosate resistance in Zhongdou 32. Here, G10-EPSPS gene was introduced into soybeans through Agrobacterium-mediated soybean cotyledon node. PCR, Southern blotting, semi-quantitative RT-PCR, qRT-PCR, and Western blotting were used, and the results revealed that G10-EPSPS had been integrated into the soybean genome and could be expressed steadily at both mRNA and protein levels. In addition, glyphosate resistance analysis showed that the growth of transgenic soybean had not been affected by concentrations of 900 and 2 700 g a.e. ha–1 of glyphosate. All the results indicated that G10-EPSPS could provide high glyphosate resistance in soybeans and be applied in production of glyphosate-resistant soybean.
文摘The use of glyphosate-resistant corn has facilitated a shift from a reliance on preemergence residual herbicides to postemergence (POST) herbicides, and in some cases exclusively glyphosate. Glyphosate is a non-selective herbicide that is relatively slow-acting, which may allow weeds to continue to compete with corn after application and potentially decrease crop yield. The addition of several POST corn herbicides, with some residual control, to an early-season glyphosate application was examined to determine if the tankmix combination would improve the speed of weed control compared to glyphosate applied alone. Seven field trials were conducted over three years (2009, 2010 and 2011) near Ridgetown and Exeter, Ontario. The control of common ragweed was improved 3 days after application (DAA) with three POST glyphosate tankmixes compared to glyphosate alone. However control was still less than 55%. Depending on the weed species examined, at 28 DAA two of the glyphosate tankmix treatments tested provided better common ragweed, common lambsquarters, or green foxtail control than glyphosate alone. Treatments providing better weed control at 28 DAA also typically decreased weed density compared to glyphosate alone.
基金supported by the National Basic Research and Development Program of China(2007CB109202)the National Natural Science Foundation of China(30400059)the National Specific Program on Environmental Protection for Public-welfare Industry of China(200709017)
文摘Horseweed(Conyza canadensis),an invasive alien weed,is one of the main weeds in orchards in China.Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in China,a case of glyphosate-resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries.Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history.Potted seedlings with 11-13-leaf growth stage were treated with glyphosate at 0.035,0.07,0.14,0.28,0.56,1.12,2.24,4.48,and 8.96 kg a.i.ha-1.The dosage dependence response curve of each population was constructed with Log-logistic dose response regression equations.The ED50 value of each population was calculated and compared with the susceptible population from China.Different populations had different relative glyphosate-resistant levels which increased with the number of years of glyphosate application.Two populations with the highest resistance levels,8.28 and 7.95 times,were found in Ningbo,Zhejiang Province,China,where glyphosate was used for weed control in orchards twice each year for 15 yr.The two resistant populations accumulated approximately two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application.
基金Project supported by the Fundamental Research Funds for the Central Universities(No.2017FZA6011)the National Key Transgenic Research Projects(No.2016ZX08010003)of China
文摘A transgenic maize event ZD12-6 expressing a Bacillus thuringiensis （Bt） fusion protein CrylAb/Cry2Aj and a modified 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） protein G10 was characterized and evaluated. Southern blot analysis indicated that ZD12-6 is a single copy integration event. The insert site was determined to be at chromosome 1 by border sequence analysis. Expression analyses of Bt fusion protein CrylAb/Cry2Aj and the EPSPS protein G10 suggested that they are both expressed stably in different generations. Insect bioassays demonstrated that the transgenic plants are highly resistant to Asian corn borer （Ostnnia furnacalis）, cotton boll worm （Helicoverpa armigera）, and armyworm （Mythimna separata）. This study suggested that ZD12-6 has the potential to be developed into a commercial transgenic line.
文摘In this work,samples consisting of BiVO4 with exposed(040)facets coupled with Bi2S3(Bi2S3/BiVO4)were prepared through a one-pot hydrothermal method,using ethylenediaminetetraacetic acid as directing agent and L-cysteine as sulfur source and soft template.X-ray diffraction,field emission scanning electron microscopy,and high-resolution transmission electron microscopy measurements indicated that the Bi2S3 content had a significant influence on the growth of(040)and(121)facets as well as on the morphology of the Bi2S3/BiVO4 samples.When the Bi2S3 content reached 1 mmol,the Bi2S3/BiVO4 samples exhibited a peony-like morphology.The results of transient photocurrent tests and electrochemical impedance spectroscopy measurements confirmed that a more effective charge separation and a faster interfacial charge transfer occurred in Bi2S3/BiVO4 than BiVO4.The enhanced photocatalytic activity of the Bi2S3/BiVO4 samples could be attributed to the improved absorption capability in the visible light region and the enhanced electron-hole pair separation efficiency due to the formation of the Bi2S3/BiVO4 heterostructure.In addition,the Bi2S3/BiVO4 samples showed relative stability and reusability.The simple method presented in this work could be used to fabricate composite photocatalysts with high activity for different applications,such as photocatalytic degradation of organic pollutants,photocatalytic splitting of water,and photocatalytic reduction of carbon dioxide.
基金DIREN, AERM and DRAF of Lorraine (France) for their financial support
文摘Glyphosate （N-phosphonomethyl glycine） is the most used herbicide worldwide. The degradation of ^14C-labeled glyphosate was studied under controlled laboratory conditions in three different agricultural soils： a silt clay loam, a clay loam and a sandy loam soil. The kinetic and intensity of glyphosate degradation varied considerably over time within the same soil and among different types of soil. Our results demonstrated that the mineralization rate of glyphosate was high at the beginning of incubation and then decreased with time until the end of the experiment. The same kinetic was observed for the water extractable residues. The degradation of glyphosate was rapid in the soil with low adsorption capacity （clay loam soil） with a short half-life of 4 days. However, the persistence of glyphosate in high adsorption capacity soils increased, with half-live of 19 days for silt clay loam soil and 14.5 days for sandy loam soil. HPLC analyses showed that the main metabolite of glyphosate, arninomethylphosphonic acid （AMPA） was detected after three days of incubation in the extracts of all three soils. Our results suggested that the possibility of contamination of groundwater by glyphosate was high on a long-term period in soils with high adsorption capacity and low degrading activities and/or acid similar to sandy loam soil. This risk might be faster but less sustainable in soil with low adsorption capacity and high degrading activity like the clay loam soil. However, the release of non-extractable residues may increase the risk of contamination of groundwater regardless of the type of soil.
文摘Behaviors of soil heavy metals are often affected by coexisting herbicides due to their physical and chemical interaction. Effect of glyphosate, an herbicide containing -PO 2- 3 and -COOH groups, on cadmium adsorption in montmorillonite was studied in detail. The results showed that the cadmium adsorption quantity in montmorillonite increased with increasing soil solution pH and cadmium concentration as usual, but decreased with glyphosate, which is due to the formation of a low affinity complex of Cd and glyphosate and decreasing solution pH induced by glyphosate addition. When the equilibrium solution pH was below 6.7, glyphosate has little effect on cadmium adsorption, but when the equilibrium solution pH was above 6.7, glyphosate significantly decreased cadmium adsorption quantity in montmorillonite. In addition, the adding order of Cd and glyphosate also influenced Cd adsorption quantity in montmorillonite.
基金The Basic Research and Development Programof China(No.2002CB410808) andthe Director Foundation of Institute of Soil Science ,Chinese Academyof Sciences
文摘Glyphosate （GPS） is a non-selective, post-mergence herbicide that is widely used throughout the world. Due to the similar molecular structures of glyphosate and phosphate, adsorption of glyphosate on soil is easily affected by coexisting phosphate, especially when phosphate is applied at a significant rate in farmland. This paper studied the effects of phosphate on the adsorption of glyphosate on three different types of Chinese soils including two variable charge soils and one permanent charge soil. The results indicated that Freundlich equations used to simulate glyphosate adsorption isotherms gave high correlation coefficients（0.990-0.998） with K values of 2751, 2451 and 166 for the zhuanhong soil（ZH soil, Laterite）, red soil（ RS, Udic Ferrisol） and Wushan paddy soil（WS soil, Anthrosol）, respectively. The more the soil iron and aluminum oxides and clay contained, the more glyphosate adsorbed. The presence of phosphate significantly decreased the adsorption of glyphosate to the soils by competing with glyphosate for adsorption sites of soils. Meanwhile, the effects of phosphate on adsorption of glyphosate on the two variable charge soils were more significant than that on the permanent charge soil. When phosphate and glyphosate were added in the soils in different orders, the adsorption quantities of glyphosate on the soils were different, which followed GPS-soil 〉 GPS-P-soil = GPS-Soil-P 〉 P-soil-GPS, meaning a complex interaction occurred among glyphosate, phosphate and the soils.
基金We acknowledge Higher Education Commission (HEC), Islamabad, Pakistan, for providing financial support through International Research Support Initiative Program (IRSIP) to conduct this research at University of Sydney, Australia.
文摘Glyphosate is an important organophosphonate herbicide used to eliminate grasses and herbaceous plants in many vegetation management situations.Its extensive use is causing environmental pollution,and consequently,there is a need to remove it from the environment using an eco-friendly and cost-effective method.As a step to address this problem,a novel bacterial strain Comamonas odontotermitis F2,capable to utilize glyphosate as a carbon(C)and/or phosphorus(P)source,was isolated from a glyphostate-contaminated field soil in Australia and characterized.Response surface methodology(RSM)employing a 2^3 full factorial central composite design was used to optimize glyphosate degradation by C.odontotermitis P2 under various culture conditions.The strain C.odontotermitis P2 was proficient in degrading 1.5 g L^-1 glyphosate completely within 104 h.The optimal conditions for the degradation of glyphosate were found to be pH 7.4,29.9℃,and an inoculum density of 0.54 g L^-1,resulting in a maximum degradation of 90%.Sequencing of glyphosate oxidoreductase(GOX)and C-P lyase(phnJ)genes from C.odontotermitis P2 revealed 999c and 93%identities to already reported bacterial GOX and phnJ genes,respectively.The presence of these two genes in C.odontotermitis indicates its potential to degrade glyphosate through GOX and C-P lyase metabolic pathways.This study demonstrates the potential of C.odontotermitis P2 for efficient degradation of glyphosate,which can be exploited for remediation of glyphosate.
基金supported by the National Basic Research Program of China(2007CB707805)the National High Technology Research and Development Program of China(2006AA020101)the National Natural Science Foundation of China(30470047and 30200007)
文摘The widely used herbicide glyphosate targets 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS).Glyphosate acetyltransferase(GAT)effectively detoxifies glyphosate by N-acetylation.With the aim of identifying a new strategy for development of glyphosate-tolerant crops,the plant expression vector pG2-GAT harboring gat and G2-aroA(encoding EPSPS)has been transformed into tobacco(Nicotiana tabacum)to develop novel plants with higher tolerance to glyphosate.Results from Southern and Western blotting analyses indicated that the target genes were integrated into tobacco chromosomes and expressed effectively at the protein level.Glyphosate tolerance was compared among transgenic tobacco plants containing gat,G2-aroA,or both genes.Plants containing both gat and G2-aroA genes were the most glyphosate-tolerant.This study has shown that a combination of different strategies may result in higher tolerance in transgenic crops,providing a new approach for development of glyphosate-tolerant crops.
基金supported by the National Key Project of transgenic varieties breeding(2009ZX08003-003B)the Light of West Talent Training Project of China(2010-2011)the Project of Sichuan Province Finance Genetic Engineering,China(2011JYGC01-002)
文摘Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.
基金supported by the Genetically Modified Major Projects, China (2012ZX08011-003 and 2014ZX08011-004B)
文摘Given that glyphosate weed control is an effective strategy to reduce costs and improve economic outcomes of agricultural production in China,the development of glyphosate-resistant cotton holds great promise.Using an Agrobacterium-mediated transformation method,a new G2-aroA gene that encodes 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS）was transformed into cotton cultivar K312.The transgenic cotton plants were regenerated from a callus tissue culture via kanamycin selection.Ten regenerated cotton plants were obtained and allowed to flower normally to produce fruit.The results from polymerase chain reaction（PCR）and Southern and Western blot analyses indicated that the target gene was integrated into the cotton chromosome and was expressed effectively at the protein level.The glyphosate tolerance analysis showed that the transgenic cotton had a high resistance to glyphosate.Further,even cotton treated with 45.0 mmol L^–1 of glyphosate was able to slowly grow,bloom and seed.The transgenic cotton may be used for cotton breeding research of glyphosate-tolerant cotton.
基金supported by the Agricultural Research Project in Guangdong Province,China (2012A020100009)
文摘The rapid detection of glyphosate resistance in goosegrass（Eleusine indica） will enhance our ability to respond to new resistant populations of this major weed. Chlorophyll fluorescence（Fluo） and P700（reaction center chlorophyll of photosystem I） absorbance were analyzed in one biotype of goosegrass that is resistant to glyphosate and in another that remains sensitive to the herbicide. Both biotypes were treated with a foliar spray of glyphosate. Differences in photosystem II maximum quantum yield（Fv/Fm）, effective photochemical quantum yield（Y（II））, and non-photochemical quenching（NPQ） between the biotypes increased over time. Values for Fv/Fm and Y（II） differed between the two biotypes 24 h after treatment（HAT）. Differentiated activities and energy dissipation processes of photosystem II（PSII） and energy dissipation processes of photosystem I（PSI） were manifested in the two biotypes 24 HAT with 20 mmol L–1 glyphosate. Differentiated energy dissipation processes of PSI were still apparent 24 HAT with 200 mmol L–1 glyphosate. These results indicate that the Fluo parameters related to PSII activity and energy dissipation and the P700 parameters related to energy dissipation are suitable indicators that enable rapid detection of glyphosate resistance in goosegrass.
基金supported by the National Biotechnology Development Plan, China (2016ZX08005-004)
文摘Cotton plants are recalcitrant with regards to transformation and induced regeneration.In the present study,5-enolpyruvylshikimate-3-phosphate（EPSPS）,a glyphosate resistant gene from the bacterium Agrobacterium sp.strain CP4,was introduced into an elite Bt transgenic cotton cultivar with a modified technique involving in planta Agrobacteriummediated transformation of shoot apex.Primary transformants were initially screened using a 0.26%glyphosate spray and subsequently by PCR analysis.Five out of 4 000 transformants from T_1 seeds were obtained resulting in an in planta transformation rate of 0.125%.Four homozygous lines were produced by continuous self-fertilization and both PCR-based selection and glyphosate resistance.Transgene insertion was analyzed by Southern blot analysis.Gene transcription and protein expression levels in the transgenic cotton lines were further investigated by RT-PCR,Western blot,and ELISA methods.Transgenic T_3 plants were resistant to as much as 0.4% of glyphosate treatments in field trials.Our results indicate that the cotton shoot apex transformation technique which is both tissue-culture and genotype-independent would enable the exploitation of transgene technology in different cotton cultivars.Since this method does not require sterile conditions,the use of specialized growth media or the application of plant hormones,it can be conducted under the greenhouse condition.
基金funded by the National Major Project for Transgenic Organism Breeding, China (2014ZX08010-004)
文摘It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase（EPSPS） encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops（Cao et al. 2012）. In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301 Ubi Sp AM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase（rbcS） small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize（Zea mays） immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.
基金supported by the National Natural Science Foundation of China(31601326)the National Transgenic Major Program of China(2016ZX08004001 and 2016ZX08011003)the China Postdoctoral Science Foundation(2016M591300)。
文摘Glyphosate-tolerant soybean is the most widely planted genetically modified crop worldwide. However, soybean remains recalcitrant to routine transformation because of the low infection efficiency of Agrobacterium to soybean and lack of useful selectable markers. In this study, several Agrobacterium strains and cell densities were compared by transient expression of the GUS gene. The results showed that Agrobacterium strain Ag10 at cell densities of OD_(600) of 0.6–0.9 yielded the highest infection efficiency in Agrobacterium-mediated soybean cotyledonary node transformation system. Meanwhile, a simple and rapid method was developed for identification of glyphosate tolerance in putative T_0 transgenic plants, consisting of spotting plantlets with 1 μL Roundup~?. The whole cycle of genetic transformation could be shortened to about 3 mon by highly efficient selection with glyphosate during the transformation process and application of the spot assay in putative T_0 transgenic plantlets. The transformation frequency ranged from 2.9 to 5.6%. This study provides an improved protocol for development and identification of glyphosate-tolerant transgenic soybeans.
基金support of the National Natural Science Foundation of China(30771383)the Genetically Modified Organisms Breeding Major Projects, China(2013ZX08003-001)the National Basic Research Program of China (2009CB118902)
文摘Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.
