• Journal of Microbiology and Biotechnology
• /
• v.22 no.2
• /
• pp.159-165
• /
• 2012

The rhizobacterial composition varies according to the soil properties. To test if the effect of herbicides on the rhizobacterial communities of genetically modified NK603 glyphosate-tolerant maize varies according to different soil locations, a comparison was made between the effects of glyphosate (Roundup Plus), a post-emergence applied herbicide, and a pre-emergence applied herbicide (GTZ) versus untreated soil. The potential effect was monitored by direct amplification, cloning, and sequencing of the soil DNA encoding 16S rRNA, and high-throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region. The results obtained using three different methods to analyze the herbicide effect on the rhizobacterial communities of genetically modified NK603 maize were comparable to those previously obtained when glyphosate-tolerant maize was grown in soil with different characteristics. Both herbicides decreased the bacterial diversity in the rhizosphere, with Actinobacteria being the taxonomic group most affected. The results suggest that both herbicides affected the structure of the maize rhizobacterial community, but glyphosate was environmentally less aggressive.

• Journal of Microbiology and Biotechnology
• /
• v.14 no.2
• /
• pp.390-394
• /
• 2004

The genetically modified glyphosate-tolerant soybean contains the following introduced DNA sequences: the EPSPS (5-enol-pyruvylshikimate-3-phosphate synthase) gene from Agrobacterium sp. strain CP4, the 35S promoter from the cauliflower mosaic virus, and the NOS terminator from Agrobacterium tumefaciens. In the present study, detection of these introduced DNAs was performed by amplification using the polymerase chain reaction (PCR). A multiplex PCR method was also applied to prevent false positive results. When primers for 35S promoter, nos3', CTP(chloroplast transit peptide), and CP4 EPSPS (EPSPS from Agrobacterium sp. CP4) were used, positive results were obtained in PCR reactions using DNA from genetically modified glyphosate-tolerant soybeans. There were no false positive results when using DNA from non-genetically modified soybeans. The CP4 EPSPS gene was detected when less than 125 pg glyphosate-tolerant soybean DNA was amplified. Lectin Lel and psb A were amplified from both non-genetically modified and genetically modified glyphosate-tolerant soybean DNA. Multiplex PCR was performed using different primer sets for actin Sacl, 35S promoter and CP4 EPSPS. The actin gene was detectable in both non-genetically modified and glyphosate-tolerant soybeans as a constant endogenous gene. Target DNAs for the 35S promoter, and CP4 EPSPS were detected in samples containing 0.01-0.1% glyphosate-tolerant soybean, although there were variations depending on primers by multiplex PCR. Soybean seeds from five plants of non-genetically modified soybean were co-cultivated for six months with those of genetically modified soybean, and they were analyzed by PCR. As a result, they were not positive for 35S promoter, nos3' or CP4 EPSPS. Therefore, these results suggest there was no natural crossing of genes between glyphosate-tolerant and non-genetically modified soybean during co-cultivation, which indicates that gene transfer between these plants is unlikely to occur in nature.

• Korean Journal of Food Science and Technology
• /
• v.33 no.5
• /
• pp.521-524
• /
• 2001

A method using PCR was developed for the monitoring of glyphosate tolerant soybean (GTS) produced by the DNA recombination technique. We designed 3 pairs of specific oligonucleotide primers based on the gene sequences inserted in soybean and in lectin and ferritin genes as internal standards. Template DNAs were isolated from soybeans by the modified hexadecyl trimethyl ammonium bromide (CTAB)method and used for PCR with different primer sets. PCR, used with specific primer sets for GTS detection, showed the amplified DNA fragments with GTS template DNA but no product showed with non-GTS template. PCR amplified products were confirmed by DNA sequencing and were detected for up to 0.05% of GTS template DNA.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.9
• /
• pp.1162-1169
• /
• 2014

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene ($aroA_{R.\;leguminosarum}$) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that $aroA_{R.\;leguminosarum}$ is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the $aroA_{R.\;leguminosarum}$ gene was obtained to prove its potential use in developing glyphosate-resistant crops.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.3
• /
• pp.561-572
• /
• 2017

The global commercial cultivation of transgenic crops, including glyphosate-tolerant soybean, has increased widely in recent decades with potential impact on the environment. The bulk of previous studies showed different results on the effects of the release of transgenic plants on the soil microbial community, especially rhizosphere bacteria. In this study, comparative analyses of the bacterial communities in the rhizosphere soils and surrounding soils were performed between the glyphosate-tolerant soybean line NZL06-698 (or simply N698), containing a glyphosate-insensitive EPSPS gene, and its control cultivar Mengdou12 (or simply MD12), by a 16S ribosomal RNA gene (16S rDNA) amplicon sequencing-based Illumina MiSeq platform. No statistically significant difference was found in the overall alpha diversity of the rhizosphere bacterial communities, although the species richness and evenness of the bacteria increased in the rhizosphere of N698 compared with that of MD12. Some influence on phylogenetic diversity of the rhizosphere bacterial communities was found between N698 and MD12 by beta diversity analysis based on weighted UniFrac distance. Furthermore, the relative abundances of part rhizosphere bacterial phyla and genera, which included some nitrogen-fixing bacteria, were significantly different between N698 and MD12. Our present results indicate some impact of the glyphosate-tolerant soybean line N698 on the phylogenetic diversity of rhizosphere bacterial communities together with a significant difference in the relative abundances of part rhizosphere bacteria at different classification levels as compared with its control cultivar MD12, when a comparative analysis of surrounding soils between N698 and MD12 was used as a systematic contrast study.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.35 no.4
• /
• pp.470-475
• /
• 2006

In order to elucidate the differences of protein profiles among soybean cultivars, the protein composition of three conventional domestic soybean cultivars and two imported ones including glyphosate-tolerant HS2906 was analyzed by total nitrogen measurement, amino acid analysis and PAGE/densitometry. There were no statistically significant differences in the levels of any amino acid, including aromatic amino acids, between glyphosale-tolerant soybean and the conventional soybean WS82. In the extraction of protein, the SDS/buffer system was more efficient than the defatting/water system. The SDS-PAGE/densitometry analysis showed that there was a similar profile of proteins among cultivars, although the amount of total protein ranged from 380.2 mg/g to 423.9 mg/g. In addition, there was no discernable difference of protein profile between glyphosate- tolerant soybean (total protein amount, 380.2 mg/g) and the conventional soybean WS82 (390.2 mg/g), although the amount of ${\beta}$-conglycinin (55 kDa) was lower in glyphosate-tolerant soybean. Meanwhile, the amount of 25 kDa protein was greater in domestic soybean cultivars than imported ones. Thus, normal PAGE/ densitometry method would be useful to analyze the difference in protein profiles of soybean proteins, and furthermore Evaluate the protein profile of proteins between GMO and conventional soybean.

• Weed & Turfgrass Science
• /
• v.6 no.4
• /
• pp.350-354
• /
• 2017

Conyza canadensis is the weed species which most frequently develops resistance to glyphosate in many agricultural crop fields. The continuous use of glyphosate has resulted in the spontaneous occurrences of resistant biotypes. This research was conducted to investigate the response of suspected C. canadensis biotypes to glyphosate. Seeds of C. canadensis were collected from 18 sites in tangerine orchards in Jeju province of Korea. In the preliminary screening, 6 resistant and 12 susceptible biotypes were found at the recommended glyphosate rate ($3.28kga.i.ha^{-1}$). The susceptible biotypes were completely killed at the field application rate whereas the resistant biotypes were initially injured but recovered 14 days after glyphosate application. This is the first case of glyphosate resistance found in Korea despite the national ban on genetically modified glyphosate tolerant crops cultivation. Extended monitoring should be conducted to understand how widely spread the glyphosate resistant C. canadensis is and to estimate the severity of this weed problem in the tangerine orchards of Korea.

• Horticultural Science & Technology
• /
• v.28 no.4
• /
• pp.648-655
• /
• 2010

This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.11
• /
• pp.1473-1483
• /
• 2014

Reports of herbicide resistance events are proliferating worldwide, leading to new cultivation strategies using combinations of pre-emergence and post-emergence herbicides. We analyzed the impact during a one-year cultivation cycle of several herbicide combinations on the rhizobacterial community of glyphosate-tolerant Bt-maize and compared them to those of the untreated or glyphosate-treated soils. Samples were analyzed using pyrosequencing of the V6 hypervariable region of the 16S rRNA gene. The sequences obtained were subjected to taxonomic, taxonomy-independent, and phylogeny-based diversity studies, followed by a statistical analysis using principal components analysis and hierarchical clustering with jackknife statistical validation. The resilience of the microbial communities was analyzed by comparing their relative composition at the end of the cultivation cycle. The bacterial communites from soil subjected to a combined treatment with mesotrione plus s-metolachlor followed by glyphosate were not statistically different from those treated with glyphosate or the untreated ones. The use of acetochlor plus terbuthylazine followed by glyphosate, and the use of aclonifen plus isoxaflutole followed by mesotrione clearly affected the resilience of their corresponding bacterial communities. The treatment with pethoxamid followed by glyphosate resulted in an intermediate effect. The use of glyphosate alone seems to be the less aggressive one for bacterial communities. Should a combined treatment be needed, the combination of mesotrione and s-metolachlor shows the next best final resilience. Our results show the relevance of comparative rhizobacterial community studies when novel combined herbicide treatments are deemed necessary to control weed growth.

• Journal of Microbiology
• /
• v.45 no.2
• /
• pp.153-157
• /
• 2007

5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EC 2.5.1.19) is the sixth enzyme in the shikimate pathway which is essential for the synthesis of aromatic amino acids and many secondary metabolites. The enzyme is widely involved in glyphosate tolerant transgenic plants because it is the primary target of the nonselective herbicide glyphosate. In this study, the Dunaliella salina EPSP synthase gene was cloned by RT-PCR approach. It contains an open reading frame encoding a protein of 514 amino acids with a calculated molecular weight of 54.6 KDa. The derived amino acid sequence showed high homology with other EPSP synthases. The Dunaliella salina EPSP synthase gene was expressed in Escherichia coli and the recombinant EPSP synthase were identified by functional complementation assay.