Development of Transgenic Glyphosate-Resistant Rice with G6 Gene Encoding 5-Enolpyruvylshikimate-3-Phosphate Synthase

Glyphosate-resistant crops have been a huge economic success for genetic engineering. The creating of new glypbosateresistant plants would increase the available choices for planting and lower the price of genetically modified crop seeds. A novel G6 gene from Pseudomonas putida that encoded 5-enolpyruvylshikimate-3-phosphate synthase （EPSPS） was previously isolated. The G6 gene was transfected into rice via Agrobacterium-mediated transformation. The transgenic rice obtained was confirmed by PCR, Southern, and Western blots. The lab experiment and field trials further confirmed that the transgenic rice can survive glyphosate spraying at a dose of 8 g L^-1. In contrast, conventional rice was killed at a weed control glyphosate spray dose of 1 g L^-1. Altogether, the present study showed that the G6 gene works well in rice in vivo for glyphosate-resistance.

Overexpression of GmBIN2,a soybean glycogen synthase kinase 3 gene,enhances tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots

Glycogen synthase kinase 3 （GSK3） is a kind of sedne/threonine kinase widely found in eukaryotes. Many plant GSK3 kinases play important roles in regulating stress responses. This study investigated BRASSINOSTEROID-INSENSITIVE 2 （GmBIN2） gene, a member of the GSK3 protein kinase family in soybean and an orthologue of Arabidopsis BIN2/AtSK21. GmBIN2 expression was increased by salt and drought stresses, but was not significantly affected by the ABA treatment. To examine the function of GrnBIN2, transgenic Arabidopsis and transgenic soybean hairy roots were generated. Overexpression of GmBIN2 in Arabidopsis resulted in increased germination rate and root length compared with wild-type plants under salt and mannitol treatments. Overexpression of GmBIN2 increased cellular Ca2~ content and reduced Na~ content, enhancing salt tolerance in transgenic Arabidopsis plants. In the soybean hairy root assay, overexpression of GmBIN2 in transgenic roots also showed significantly higher relative root growth rate than the control when subjected to salt and mannitol treatments. Measurement of physiological indicators, including proline content, superoxide dismutase （SOD） activity, and relative electrical conductivity, supported this conclusion. Furthermore, we also found that GmBIN2 could up-regulate the expression of some stress-related genes in transgenic Arabidopsis and soybean hairy roots. Overall, these results indicated that GmBIN2 improved tolerance to salt and drought in transgenic Arabidopsis and soybean hairy roots.

See the color,see the seed:GmW1 as a visual reporter for transgene and genome editing in soybean

A fast and efficient recognition method of transgenic lines will greatly improve detection efficiency and reduce cost.In this study,we successfully identified the transgenic soybean plants by the color.We isolated a GmW1 gene encoding a flavonoid 3'5'-hydroxylase from a soybean cultivar ZH42(purple flower).We found that purple flowers occurred in the overexpression lines in the Jack and Williams 82 backgrounds(white flower).All plants with purple flowers were positive,and this trait seems stably inherited in the offspring.We have also obtained the editing plants,which were classified into three types according to the different flower colors appeared.We analyzed the phenotype and the homozygous types of the T_1mutants.We also found that a correspondence between flower color and stem color.This study provides a visible color reporter on soybean transformation.It can be quickly and early to identify the transgenic soybean plants by stem color of seedlings,which substantially reduces the amount of labor and cost.

Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x＋35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x＋35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

Development and identification of glyphosate-tolerant transgenic soybean via direct selection with glyphosate

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.

Sexual compatibility of transgenic soybean and different wild soybean populations

The introduction of genetically modified(GM) soybean into farming systems raises great concern that transgenes from GM soybean may flow to endemic wild soybean via pollen. This may increase the weediness of transgenic soybean by increasing the fitness of hybrids under certain conditions and threaten the genetic diversity of wild soybean populations. Although pollen-mediated gene flow between GM crops and wild relatives is dependent on many factors, the sexual compatibility(SC)determined by their genetic backgrounds is the conclusive factor. The considerable genetic variation among wild soybean populations may cause compatibility differences between different wild and cultivated soybeans. Thus, an evaluation of the SC between transgenic soybean and different wild soybeans is essential for assessing the environmental consequences of cultivated soybean–wild soybean transgene flow. The podding and seed sets were assessed after artificial hybridization using transgenic glyphosate-resistant soybean as the paternal parent and 18 wild soybean populations as the maternal parents. Then, the average number of filled seeds produced in 200 flowers(AFS) was calculated for each wild soybean under natural self-pollination as well as under artificial crossing with transgenic soybean. Finally, the index of cross-SC was calculated(ICSC) as the ratio of the AFS of wild soybean artificially crossed with transgenic soybean and the AFS of naturally self-pollinated wild soybean. The results demonstrated that after self-pollination and crossing with transgenic soybean, the average podding rates of 18 wild soybean populations ranged within 96.50–99.50% and 4.92–18.03%, and the average filled seed numbers per pod varied from 1.70 to 2.69 and 0.20 to 0.48, respectively. The results showed that approximately 89% of wild soybeans displayed either medium or higher than medium SC with transgenic soybean(ICSC>1.0%). This implied the high possibility of gene flow via pollen from transgenic soybean to wild soybean.

Agronomic Traits of Transgenic Soybean Overexpressing TaDREB3

Dongnong50 was overexpressing TaDREB3, a stress-tolerance gene, and transgenic progenies with TaDREB3 showing stable heredity characters were obtained in this study. The seeds of T4 generation were sowed on saline land of which salt concentration was 0.19% and pH was 8.43, and agronomic traits including growth period, plant height, numbers of main nodes, branching numbers, pods per plant, numbers of seeds per plant, and weight of 100-seed were collected and comparing to control plants Dongnong50 were made. The impacts of TaDREB3 gene on agronomic traits of soybeans under high concentration of salt were discussed. The results showed that the growth period was not altered in transgenic line with TaDREB3 under high concentration of saline and alkali, the extremely significant or significant increase in the numbers of main nodes, pods per plant and in branching numbers and other agronomic traits in transgenic lines in comparing with non-transgenic lines was an indicator of their resistance to saline-alkali stress

Advance of Multiplex PCR in Rapid Detecting Transgenic Soybean Oil

Transgenic food safety is a high-profile public health issue in worldwide, especially transgenic soybean (Glycine max L.) oil. To rapidly and effectively detect transgenic components of soybean oil, in the present study, we isolated DNA from transgenic soybean oil by modified method, and employed the multiplex PCR method to identify targeted genes, including CaMV35S promoter, Nos terminator, NPTII, CP4-EPSPS and endogenous gene Lectin. The research aims to build a method which is accurate, rapid and reliable for detection of genetically modified soybeans oil. The targeted gene including DNA was successfully established by the improved method, and then amplified by PCR. Five genes are simultaneously specifically detected. Commercial soybean, genetically modified soy bean and oil were detected with the Multiplex PCR. The improved method of DNA extraction was rapid and accurate to extract high quality total DNA which was amplified by PCR. The method could eliminate the PCR inhibitor. A way of detecting the genetically modified soybean and Oil was set up in this study.

Establishment of PCR-ELISA for Detecting Glyphosate Resistant Transgenic Soybean

A PCR-ELISA method for detecting the glyphosate resistant transgenic soybean was established and optimized. The results showed that the key parameters of PCR-ELISA were as follows： the concentration of digoxin tag probe was 0.5 μmol · L^-1, the time of hybridization reaction was 15 min and the chromogenic reaction should last for 30 min. The sensitivity and the repeatability of our PCR-ELISA method were evaluated, and the results showed that it could be detected when the concentration of DNA template from transgenic soybean samples was 0.01% or higher, and the coefficient of variation of this method was less than 5% in our research condition. These results suggested that PCR-ELISA method establishment in this study had good repeatability and high precision for detecting the transgenic soybean samples.

Influence of Herbicides on Competition Weed and Glyphosate-resistant Soybean

Competitions of light,nutrient and water between transgenic glyphosate resistant soybean and weed were evaluated with different herbicides(glyphosate,acetochlor,and the mixture of fomesafen and quizalofop-p-ethyl)in filed,which displayed that the application of the mixture of fomesafen and quizalofop-p-ethyl on soybeans had the best anti-herbicidal effect while glyphosate had the weakest effect.The half-life extended by herbicide mixture would affect the soil nutrients.Weed-soybean competitions affected the absorption of nutrients on soybean and yield.However,effective control of weeds could significantly reduce the weed density,improve the field through different layers of light transmittance and the absorption of nutrients on soybean,and enhance the comprehensive competitiveness of soybean.

Linuron Biologically Effective Dose for Glyphosate-Resistant Giant Ragweed (<i>Ambrosia trifida</i>L.) Control in Soybean (<i>Glycine max</i>L.)

Glyphosate-resistant (GR) giant ragweed (Ambrosia trifida L.) was first identified in Canada in 2008 and has since been found throughout southwestern Ontario. Six field trials were conducted over a two-year period (2012, 2013) on Ontario farms with GR giant ragweed to evaluate the efficacy of linuron applied pre-plant (PP) in soybean (Glycine max (L.) Merr.). The dose required for 50%, 80%, and 95% GR giant ragweed control was 1238, 2959, and 6018 g·ai·ha-1 four weeks after application (WAA), respectively. The linuron dose needed for 50%, 80%, and 95% reduction in density was 1554, 3181, and 5643 g·ai·ha-1 and 1204, 2496, and 4452 g·ai·ha-1 for dry weight, respectively. Application of 7874 g·ai·ha-1 linuron was needed to obtain soybean yields that were 90% of the weed-free control;approximately 3.5 times the maximum field recommended dose. To achieve 95% and 98% yields, greater than 8640 g·ai·ha-1 linuron was required. Application of linuron plus glyphosate PP in soybean will help to control GR giant ragweed as well as reduce GR selection pressure.

Regeneration Function Analysis of GmESR1 in Transgenic Soybean

ENHANCER OF SHOOT REGENERATION(ESR1)is an important regulator of plant regeneration in vitro,which promotes regeneration of plant.In this study,transgenic positive plants with normal expression of proteins were screened by molecular assay.Through the study of the transgenic plants and the control Dongnong 50,the difference between immature embryo-induced callus and induced shoot bud was observed.The increase in callus weight indicated that GmESR1 gene accelerated the formations of shoot buds.By measuring the changes of hormone in the process of induction callus of transgenic plants,it was found that the contents of indole-3-acetic acid(IAA)and zeatin(ZT)in transgenic lines were significantly increased.It could be concluded that GmESR1 gene promoted the accumulation of hormone and affected regeneration process.In addition,this study also verified the interaction between GmBIM1 gene and GmESR1 gene by bimolecular fluorescence complementation(BiFC).

Glyphosate-Resistant Giant Ragweed(Ambrosia trifida L.)in Ontario:Dose Response and Control with Postemergence Herbicides

Giant ragweed (Ambrosia trifida L.) is competitive with agronomic crops and can cause significant yield losses. Rapid adoption of glyphosate-resistant (GR) crops and a concomitant increase in the reliance on glyphosate for weed management has led to the evolution of GR giant ragweed in Ontario, Canada. Field studies were conducted to evaluate the level of resistance in giant ragweed biotypes from Ontario, and to evaluate the effectiveness of various postemer-gence (POST) herbicides in soybean (Glycine max L.). The effective dose (ED) to provide 50%, 80% and 95% giant ragweed control was up to 1658, 9991 and >43200 g?a.e.?ha–1 4 weeks after application (WAA), respectively. For effective control, growers would need to apply glyphosate 18 times greater than the recommended field application dose. Glyphosate applied at the recommended field dose of 900 g?a.e.?ha–1 provided up to 57% control and resulted in soybean yield equivalent to the weedy check. Cloransulam-methyl applied POST provided up to 99% control, reduced giant ragweed density 98%, reduced giant ragweed shoot dry weight 99% and resulted in soybean yield equivalent to the weedfree check. Chlorimuron-ethyl, fomesafen, imazethapyr and imazethapyr plus bentazon applied alone or with glyphosate did not provide adequate control of GR giant ragweed. Based on these results, some GR giant ragweed biotypes from Ontario have evolved a high level of resistance to glyphosate. Cloransulam-methyl applied POST was the only herbicide that provided adequate control and suggests that additional weed management tactics will need to be implemented in order to effectively manage GR giant ragweed.

Effects of Transgenic DREB Toybean Dongnong50 on Diversity of Soil Nitrogen-fixing Bacteria

Drought is a bottleneck for worldwide soybean production which is getting more serious as the climate continues to worsen. Dehydration responsive element binding（DREB） is a kind of transcription factor that regulates the expression of stress tolerance-related genes in response to drought, high salinity and cold stress in plant. Soybean with DREB gene possesses the drought resisting capability which is helpful to increase the yield. However, the potential risk of genetically modified plants（GMPs） on soil microbial community is still in debate. In order to understand the effects of transgenic DREB soybean on the nitrogen-fixing bacteria, the diversity of nif H gene in pot experiments planted transgenic soybean and near-isogenic nontransgenic soybean under normal water condition and drought stress condition was analyzed by PCR-DGGE and sequence analysis. The results showed that transgenic soybean under normal water condition decrease the diversity of the nitrogen-fixing bacteria in the seeding stage and flowering stage, but had no notable effect in other stages. Under drought stress, transgenic soybean reduced the diversity of the nitrogen-fixing bacteria in the flowering stage, but had no notable effects on other stages. Phylogenic analysis revealed that g7, g13, g15 and g19 had a close relationship with Alphaproteobacteria, g12 had a close relationship with Azonexus, others were related to Betaproteobacteria and Burkholderia.

Identification of transgene insertions in two genetically modified soybeans using high throughput next generation sequencing

Genetically modified(GM) organisms are widely adopted. However, their safety assessments and control are still of special concern to the public. Identifying and localizing transgene insertion is an essentially prerequisite step. In this study, 2 independent transgene soybean lines were selected(LB4-AtDCGS-1-20-5-2 and CGS-ZG11) as typical cases. Both lines contained expression cassette of At-DCGS that encoding a feedback-insensitive cystathionine gamma-synthase to produce higher level methionine(Met). LB4-AtDCGS-1-20-5-2 was whole genome sequenced with one paired-end 500 bp library and two mate-paired 1 kb and 2 kb libraries using Illumina HiSeq sequencing platform. CGS-ZG11 was sequenced with only one paired-end 500 bp library. Both genomes were assembled,and 2 scaffold sequences(1 for each line) were screened out by aligning with transgene.Then the transgene insertion and its flanking regions in soybean genome were further identified and confirmed by PCR cloning and Sanger sequencing. Results showed that these 2 transgene lines had single copy of inserted transgene. Their transgene insertion contents were identified, which facilitates further safety assessment. These results indicated that genome assembly using high throughput sequencing is a powerful tool for identifying transgene insertions, even with limited knowledge.

耐盐转基因大豆事件AtARA6-A001外源插入片段侧翼序列及其应用

转基因大豆AtARA6-A001事件采用农杆菌介导大豆遗传转化法获得,其受体为沈农9号,具有耐盐特性,目前已经进入环境释放阶段。为明确该转基因大豆材料的分子特征及其转基因检测方法,推进该转基因事件生物安全评价工作。本研究以转基因大豆AtARA6-A001为研究对象,利用Southern杂交方法及基因组重测序技术鉴定了外源基因的拷贝数及插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转AtARA6基因耐盐大豆A001事件的特异性定性PCR检测方法。此方法能特异性检测转基因大豆植株AtARA6-A001根、茎、叶、花和种子样品,并且能够特异性识别转基因大豆事件的身份,这为后续转基因大豆及其产品的检测和监管提供技术支持。

Overexpression of G10-EPSPS in soybean provides high glyphosate tolerance

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.

Specific Expression of a Novel Nodulin GmN479 Gene in the Infected Cells of Soybean (Glycine max) Nodules

A novel nodulin gene, GmN479 genomic clone composing of 3 630 nucleotides was isolated from mature soybean nodules using GmN479 cDNA as a probe by subtractive hybridization procedure. GmN479 encodes 170 amino acids with 2.09 kb nucleotides promoter region, and contains two important upstream promoter elements, one is a conserved cis-acting sequence motif 5′-AAAGAT-3′ controlling nodulin gene expression, and the other is typical CAAT boxes. GmN479 gene has a single zinc-finger C2H2 domain YSCAFCQRGFSNAQALLGGHMNIH and a conserved motif, QALGGHMN in the zinc-finger with a short leucine repeat in the LDLELRLGL motif closed to C-terminal. These two conserved motifs share respectively higher identity with those in the floral regulator SUPERMAN gene, indicating that GmN479 may function as a transcriptional regulator, and is a likely candidate for playing a role in nodule-morphogenesis. Blotting data showed that GmN479 is a single copy presenting in the genome of soybean nodule, and its expression profile is similar to that of Lb-a, but it is different from that of ENOD2. GUS staining showed that GmN479 promoter just functions in the infected cells of nodules, indicating that the GmN479 is one of the truly symbiotically induced host genes, and belongs to a late nodulin gene. The expression pattern of GmN479 gene seems to imply that it may be closely related to the development of the nodule. In a sense, it may be a useful marker for identifying the development of the infected cell system in the nodules of soybean.

Identification and Quantification of Corn, Soybean and Cotton Genetically Modified by Real Time PCR

In order to obtain a cheaper method for quantification of transgenic events in corn, soybeans and cotton, primers for real time PCR have been developed and optimized, with fluorescent BRYT Green system. The DNA was extracted from grains, with and without event, by CTAB method. The following events have been studied for corn: MON810, Bt11, MON89034, GA21, TC1507, NK603, MIR162, PRO3;Soybean: GTS-40-3-2, MON87701;MON89788;for cotton: MON1445, MON531, LLCotton25, 281-24-236;3006-210-23, GHB614, T304-40;GHB119, MON15985, MON88913, besides the respective primers for the endogenous genes of corn, soybean and cotton. The sensitivity was 0.057%, the coefficient of linearity R2 ranged from 0.98 to 0.99 and the efficiency of PCR 0.9 to 1.1. The quantification of events ranged from 92 to 115, with a relative error (RE) from 2 to 18%, and a variance of 0.33 to 3.0. The precision acceptance criterion was observed for all analyses, as well the repeatability and reproducibility. As it was found that the measurement of accuracy and reproducibility were within the international acceptance criterion, it may infer the robustness of the methodology. Therefore, the results from replicates with two different technicians, and validation of results by comparison with those obtained by Eurofins Brazil, showed the possibility of specific and quantitative analysis of transgenic events with a cheaper method with sensitivity, repeatability and robustness.

Resistance identification of bivalent fungi-resistant genes transformed soybean to Phytophthora sojae

Soybean is one of the most important sources of edible oil and proteins in the world. However, it suffers from many kinds of fungal diseases which is a major limiting factor in soybean production. The fungal disease can be effectively controlled by breeding plant cultivars with genetic transformation. In this study, the resistance to Phytophthora sojae of five bivalent transgenic soybean lines was identified using the hypocotyls inoculation technique. The lines were the T2 of the transgenic soybean which were transformed with kidney bean chitinase gene and barley ribosome inactivating protein gene, and were positive by Southern Blot analysis. The resistance difference was studied through comparing the death percentage of transgenic soybean with the control. The results showed that four lines were more resistant to P sojae, whereas other one had no significant difference in comparison with the control. These transgenic soybean lines with enhanced resistance to P sojae will be useful in soybean resistance breeding.