Dynamics of Bacterial Community Structure in the Rhizosphere and Root Nodule of Soybean: Impacts of Growth Stages and Varieties.

Bacterial communities in rhizosphere and root nodules have significant contributions to the growth and productivity of the soybean (Glycine max (L.) Merr.). In this report, we analyzed the physiological properties and dynamics of bacterial community structure in rhizosphere and root nodules at different growth stages using BioLog EcoPlate and high-throughput sequencing technology, respectively. The BioLog assay found that the metabolic capability of rhizosphere is in increasing trend in the growth of soybeans as compared to the bulk soil. As a result of the Illumina sequencing analysis, the microbial community structure of rhizosphere and root nodules was found to be influenced by the variety and growth stage of the soybean. At the phylum level, Actinobacteria were the most abundant in rhizosphere at all growth stages, followed by Alphaproteobacteria and Acidobacteria, and the phylum Bacteroidetes showed the greatest change. But, in the root nodules Alphaproteobacteria were dominant. The results of the OTU analysis exhibited the dominance of Bradyrhizobium during the entire stage of growth, but the ratio of non-rhizobial bacteria showed an increasing trend as the soybean growth progressed. These findings revealed that bacterial community in the rhizosphere and root nodules changed according to both the variety and growth stages of soybean in the field.

Chromatic biosensor for detection of phosphinothricin acetyltransferase by use of polydiacetylene vesicles encapsulated within automatically generated immunohydrogel beads.

We developed a simple and sensitive colorimetric biosensor in the form of microparticles by using polydiacetylene (PDA) vesicles encapsulated within a hydrogel matrix for the detection of phosphinothricin acetyltransferase (PAT) protein, which is one of the most important marker proteins in genetically modified (GM) crops. Although PDA is commonly used as a sensing material due to its unique colorimetric properties, existing PDA biosensors are ineffective due to their low sensitivity as well as their lack of robustness. To overcome these disadvantages, we devised immunohydrogel beads made of anti-PAT-conjugated PDA vesicles embedded at high density within a poly(ethylene glycol) diacrylate (PEG-DA) hydrogel matrix. In addition, the construction of immunohydrogel beads was automated by use of a microfluidic device. In the immunoreaction, the sensitivity of antibody-conjugated PDA vesicles was significantly amplified, as monitored by the unaided eye. The limit of detection for target molecules reached as low as 20 nM, which is sufficiently low enough to detect target materials in GM organisms. Collectively, the results show that immunohydrogel beads constitute a promising colorimetric sensing platform for onsite testing in a number of fields, such as the food and medical industries, as well as warfare situations.

Development of a chloroplast DNA marker for monitoring of transgene introgression in Brassica napus L.

Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.

A Review of the Unintentional Release of Feral Genetically Modified Rapeseed into the Environment.

Globally, the cultivation area of genetically modified (GM) crops is increasing dramatically. Despite their well-known benefits, they may also pose many risks to agriculture and the environment. Among the various GM crops, GM rapeseed (Brassica napus L.) is widely cultivated, mainly for oil production. At the same time, B. napus possesses a number of characteristics, including the ability to form feral populations and act as small-seeded weeds, and has a high potential for hybridization with other species. In this review, we provide an overview of the commercialization, approval status, and cultivation of GM rapeseed, as well as the status of the feral rapeseed populations. In addition, we highlight the case studies on the unintentional environmental release of GM rapeseed during transportation in several countries. Previous studies suggest that the main reason for the unintentional release is seed spillage during transport/importing of rapeseed in both GM rapeseed-cultivating and -non-cultivating countries. Despite the fact that incidents of unintentional release have been recorded often, there have been no reports of serious detrimental consequences. However, since rapeseed has a high potential for hybridization, the possibilities of gene flow within the genus, especially with B. rapa, are relatively significant, and considering their weedy properties, effective management methods are needed. Hence, we recommend that specific programs be used for the effective monitoring of environmental releases of GM rapeseed as well as management to avoid environmental and agricultural perturbations.

Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection.

To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

Auto-excision of selectable marker genes from transgenic tobacco via a stress inducible FLP/FRT site-specific recombination system.

Antibiotic resistance marker genes are powerful selection tools for use in plant transformation processes. However, once transformation is accomplished, the presence of these resistance genes is no longer necessary and can even be undesirable. We herein describe the successful excision of antibiotic resistance genes from transgenic plants via the use of an oxidative stress-inducible FLP gene. FLP encodes a recombinase that can eliminate FLP and hpt selection genes flanked by two FRT sites. During a transformation procedure in tobacco, transformants were obtained by selection on hygromycin media. Regenerants of the initial transformants were screened for selective marker excision in hydrogen peroxide supplemented media and both the FLP and hpt genes were found to have been eliminated. About 13-41% of regenerated shoots on hydrogen peroxide media were marker-free. This auto-excision system, mediated by the oxidative stress-inducible FLP/FRT system to eliminate a selectable marker gene can be very readily adopted and used to efficiently generate marker-free transgenic plants.

Development of Selectable Marker-Free Transgenic Rice Plants with Enhanced Seed Tocopherol Content through FLP/FRT-Mediated Spontaneous Auto-Excision.

Development of marker-free transgenic plants is a technical alternative for avoiding concerns about the safety of selectable marker genes used in genetically modified (GM) crops. Here, we describe the construction of a spontaneous self-excision binary vector using an oxidative stress-inducible modified FLP/FRT system and its successful application to produce marker-free transgenic rice plants with enhanced seed tocopherol content. To generate selectable marker-free transgenic rice plants, we constructed a binary vector using the hpt selectable marker gene and the rice codon-optimized FLP (mFLP) gene under the control of an oxidative stress-inducible promoter between two FRT sites, along with multiple cloning sites for convenient cloning of genes of interest. Using this pCMF binary vector with the NtTC gene, marker-free T1 transgenic rice plants expressing NtTC were produced by Agrobacterium-mediated stable transformation using hygromycin as a selective agent, followed by segregation of selectable marker genes. Furthermore, α-, γ-, and total tocopherol levels were significantly increased in seeds of the marker-free transgenic TC line compared with those of wild-type plants. Thus, this spontaneous auto-excision system, incorporating an oxidative stress-inducible mFLP/FRT system to eliminate the selectable marker gene, can be easily adopted and used to efficiently generate marker-free transgenic rice plants. Moreover, nutritional enhancement of rice seeds through elevation of tocopherol content coupled with this marker-free strategy may improve human health and public acceptance of GM rice.

Proteomic identification of a novel toxin protein (Txp40) from Xenorhabdus nematophila and its insecticidal activity against larvae of Plutella xylostella.

For the identification of a novel insecticidal protein, a two-dimensional liquid chromatography (PF-2D) system was used in a quantitative proteomic analysis of Xenorhabdus nematophila CBNU strain isolated from entomophagous nematode Steinernema carpocapsae . Protein patterns obtained from minimum and maximum insecticidal activities during cultivation were contrasted, and a novel toxin protein (Txp40) was identified by MALDI-TOF/MS. The DNA sequence of the cloned toxin gene (1089 bp) has an open reading frame encoding 363 amino acids with a predicted molecular mass of 41162 Da. The txp40 identified in this study is most closely related to the known txp40 cloned from X. nematophila EB (ADQ92844) with 94.4% identical sequence residues. Following the expression of the newly identified toxin gene in Escherichia coli , the insecticidal activity of the recombinant toxin protein was determined against Plutella xylostella larvae; a 56.7% mortality rate was observed within 24 h.