Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Genetically modified soybean lines exhibit less transcriptomic variation compared to natural varieties.

Genetically modified (GM) soybeans provide a huge amount of food for human consumption and animal feed. However, the possibility of unexpected effects of transgenesis has increased food safety concerns. High-throughput sequencing profiling provides a potential approach to directly evaluate unintended effects caused by foreign genes. In this study, we performed transcriptomic analyses to evaluate differentially expressed genes (DEGs) in individual soybean tissues, including cotyledon (C), germ (G), hypocotyl (H), and radicle (R), instead of using the whole seed, from four GM and three non-GM soybean lines. A total of 3,351 DEGs were identified among the three non-GM soybean lines. When the GM lines were compared with their non-GM parents, 1,836 to 4,551 DEGs were identified. Furthermore, Gene Ontology (GO) analysis of the DEGs showed more abundant categories of GO items (199) among non-GM lines than between GM lines and the non-GM natural varieties (166). Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most KEGG pathways were the same for the two types of comparisons. The study successfully employed RNA sequencing to assess the differences in gene expression among four tissues of seven soybean varieties, and the results suggest that transgenes do not induce massive transcriptomic alterations in transgenic soybeans compared with those that exist among natural varieties. This work offers empirical evidence to investigate the genomic-level disparities induced by genetic modification in soybeans, specifically focusing on seed tissues.

Proteomic and Metabolomic Evaluation of Insect- and Herbicide-Resistant Maize Seeds.

Label-free quantitative proteomic (LFQ) and widely targeted metabolomic analyses were applied in the safety evaluation of three genetically modified (GM) maize varieties, BBL, BFL-1, and BFL-2, in addition to their corresponding non-GM parent maize. A total of 76, 40, and 25 differentially expressed proteins (DEPs) were screened out in BBL, BFL-1, and BFL-2, respectively, and their abundance compared was with that in their non-GM parents. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most of the DEPs participate in biosynthesis of secondary metabolites, biosynthesis of amino acids, and metabolic pathways. Metabolomic analyses revealed 145, 178, and 88 differentially accumulated metabolites (DAMs) in the BBL/ZH58, BFL-1/ZH58, and BFL-2/ZH58×CH72 comparisons, respectively. KEGG pathway enrichment analysis showed that most of the DAMs are involved in biosynthesis of amino acids, and in arginine and proline metabolism. Three co-DEPs and 11 co-DAMs were identified in the seeds of these GM maize lines. The proteomic profiling of seeds showed that the GM maize varieties were not dramatically different from their non-GM control. Similarly, the metabolomic profiling of seeds showed no dramatic changes in the GM/non-GM maize varieties compared with the GM/GM and non-GM/non-GM maize varieties. The genetic background of the transgenic maize was found to have some influence on its proteomic and metabolomic profiles.

Integrated proteomics and metabolomics analysis of transgenic and gene-stacked maize line seeds.

Unintended effects of genetically modified (GM) crops may pose safety issues. Omics techniques provide researchers with useful tools to assess such unintended effects. Proteomics and metabolomics analyses were performed for three GM maize varieties, 2A-7, CC-2, and 2A-7×CC-2 stacked transgenic maize, and the corresponding non-GM parent Zheng58.Proteomics revealed 120, 271 and 135 maize differentially expressed proteins (DEPs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that most DEPs participated in metabolic pathways and the biosynthesis of secondary metabolite. Metabolomics revealed 179, 135 and 131 differentially accumulated metabolites (DAMs) in the 2A-7/Zheng58, CC-2/Zheng58 and 2A-7×CC-2/Zheng58 comparisons, respectively. Based on KEGG enrichment analysis, most DAMs are involved in the biosynthesis of secondary metabolite and metabolic pathways. According to integrated proteomics and metabolomics analysis, the introduction of exogenous EPSPS did not affect the expression levels of six other enzymes or the abundance of seven metabolites involved in the shikimic acid pathway in CC-2 and 2A-7×CC-2 seeds. Six co-DEPs annotated by integrated proteomics and metabolomics pathway analysis were further analyzed by qRT-PCR.This study successfully employed integrated proteomic and metabolomic technology to assess unintended changes in maize varieties. The results suggest that GM and gene stacking do not cause significantly unintended effects.

A multiplex and regenerable surface plasmon resonance (MR-SPR) biosensor for DNA detection of genetically modified organisms.

The continuous advancement of analytical technology has provided methods with increasing sensitivity and precision to detect genetically modified organisms (GMOs). Novel analytical strategy-based detection methods are alternatives to conventional polymerase chain reaction (PCR)-mediated assays, which are still the gold standard in this field. However, PCR primers and probes cannot be reused, which makes the technique uneconomical. Surface plasmon resonance (SPR) is an optical and label-free technique for studying ligand-analyte interactions, especially for DNA hybridization, and several SPR biosensors have been described for the detection of nucleic acids. Here, a multiplexed, regenerable and real-time SPR biosensor for the detection of GMOs is described. A biosensor was constructed for qualitative detection of T-nos, CaMV35S and cry1A and had good specificity and sensitivity. The limit of detection (LOD) of this biosensor was 0.1 nM without any signal amplification. Furthermore, our biosensor could be stably regenerated more than 100 times over at least 20 days and showed good reproducibility. This nucleic acid SPR biosensor has potential for application in other types of biological detection.

Proteomic analysis of the seeds of transgenic rice lines and the corresponding nongenetically modified isogenic variety.

BACKGROUND: An isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis was employed to study the seeds of two genetically modified (GM) rice lines, T2A-1 and T1C-19, and their nontransgenic isogenic variety, MH63, to investigate the unintended effects of genetic modification. RESULTS: A total of 3398 proteins were quantitatively identified. Seventy-seven differentially abundant proteins (DAPs) were identified in the T2A-1/MH63 comparison, and 70 and 7 of these DAPs were upregulated and downregulated, respectively. A pathway enrichment analysis showed that most of these DAPs participated in metabolic pathways and protein processing in endoplasmic reticulum and were ribosome components. Some 181 DAPs were identified from the T1C-19/MH63 comparison, and these included 115 upregulated proteins and 66 downregulated proteins. The subsequent pathway enrichment analysis showed that these DAPs mainly participated in protein processing in endoplasmic reticulum and carbon fixation in photosynthetic organisms and were ribosome components. None of these DAPs were identified as new unintended toxins or allergens, and only changes in abundance were detected. Fifty-four co-DAPs were identified in the seeds of the two GM rice lines, and protein-protein interaction analysis of these co-DAPs demonstrated that some interacting proteins were involved in protein processing in endoplasmic reticulum and metabolic pathways, whereas others were identified as ribosome components. Representative co-DAPs and proteins related to nutrients were analyzed using qRT-PCR to determine their transcriptional levels. CONCLUSIONS: The results suggested that the seed proteomic profiles of the two GM rice lines studied were not substantially altered from those of their natural isogenic control. © 2020 Society of Chemical Industry.

Cry80Aa1, a novel Bacillus thuringiensis toxin with mosquitocidal activity to Culex pipiens pallens.

Bacillus thuringiensis (Bt) can synthesize insecticides to efficiently control insects. In this study, Bt strain S3580-1 with mosquitocidal activity was subjected to whole genome sequencing using an Illumina HiSeq 2000 system. A novel toxin, Cry80Aa1, was identified based on the resulting data. A conserved domain analysis revealed that Cry80Aa1 includes the Ricin_B_lectin domain (located at 10-150) and the Toxin_10 domain (located at 155-353). Phylogenetic tree analysis showed that Cry80Aa1 was in a distinct clade significantly distinguished from the known Cry proteins containing the Toxin_10 domain. Bioassays demonstrated that the Cry80Aa1 protein exhibited toxicity to third instar larvae of Culex pipiens pallens (LC50: 71.9 µg/mL; 95% FL: 59.5-122.7 µg/mL).

Development of a sensitive monoclonal antibody-based sandwich ELISA to detect Vip3Aa in genetically modified crops.

OBJECTIVES: To develop a sensitive monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) to detect Vip3Aa in genetically modified (GM) crops and their products. RESULTS: Vegetative insecticidal proteins (Vips) are secreted by Bacillus thuringiensis (Bt) and are known to be toxic to Lepidoptera species. Vip3Aa family proteins, Vip3Aa19 and Vip3Aa20, were successfully applied in GM crops to confer an effective and persistent insecticidal resistance. A sensitive monoclonal antibody-based sandwich ELISA was developed to detect Vip3Aa in GM crops and their products. Two monoclonal antibodies were raised against the overexpressed and purified His-Vip3Aa20, were purified from mouse ascites and characterized. A sandwich ELISA method was developed using the 2G3-1D7 monoclonal antibody for capture and the biotin-labeled 1F9-1F5 monoclonal antibody for detection of Vip3Aa20. The linear detection range of the method was found to be approximately 31.25-500 pg/ml, with a sensitivity of 10.24 pg/ml. CONCLUSIONS: The established ELISA was effective for detecting Vip3Aa family proteins other than Vip3Aa8, and was successfully applied in the detection of Vip3Aa20 and Vip3Aa19 expressed in transgenic maize and cotton.

Bacillus thuringiensis novel toxin Epp is toxic to mosquitoes and prodenia litura larvae.

As a pathogenic bacterium, Bacillus thuringiensis (Bt) has become an alternative to chemical insecticides in commercial agricultural to control forestry pests and mosquitoes. To prevent pest resistance, many novel Bt strains have been isolated. Strain S3580-1 (WGS: VHPX0000000) used in this research and originally isolated from Hainan Qixianling National Forest Park (China) showed significant toxicity to Culex pipiens pallens. Here, using whole genome sequencing, assembly, and bioinformatics analysis, the predicted S3580-1CG_5163 (GenBank Accession No. MK124137) gene-encoded protein was found to share low homology with known toxins designated by the Bt toxin nomenclature system. It was considered to be an ETX/MTX2-type toxin and was designated Epp. Bioinformatics analysis showed that the predicted S3580-1CG_5163 gene-encoded protein Epp shared low identity with other known toxic protein sequences containing Cry-ETX/MTX conserved domains at the amino acid level, but significant similarity at the structural level. In addition, bioassays showed that Epp was toxic against Spodoptera litura (LC50 296.133 µg/mL; 95% FL 200.555-471.318 µg/mL) and Cx. pipiens pallens (LC50 322.193 µg/mL; 95% FL 238.217-477.243 µg/mL). On pathological observation, the peritrophic membrane of Cx. pipiens pallens larvae was degraded causing the midgut structure to become incomplete, resulting in larval death. Further bioassays are required to fully elucidate the insecticidal spectrum of the ETX/MTX2-type toxin Epp, and thereby provide future research directions.

iTRAQ-based quantitative proteomic analysis of two transgenic soybean lines and the corresponding non-genetically modified isogenic variety.

To investigate the unintended effects of genetically modified (GM) crops, an isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic analysis was performed with seed cotyledons of two GM soybean lines, MON87705 and MON87701×MON89788, and the corresponding non-transgenic isogenic variety A3525. Thirty-five differentially abundant proteins (DAPs) were identified in MON87705/A3525, 27 of which were upregulated and 8 downregulated. Thirty-eight DAPs were identified from the MON87701×MON89788/A3525 sample, including 29 upregulated proteins and 9 downregulated proteins. Pathway analysis showed that most of these DAPs participate in protein processing in endoplasmic reticulum and in metabolic pathways. Protein-protein interaction analysis of these DAPs demonstrated that the main interacting proteins are associated with post-translational modification, protein turnover, chaperones and signal transduction mechanisms. Nevertheless, these DAPs were not identified as new unintended toxins or allergens and only showed changes in abundance. All these results suggest that the seed cotyledon proteomic profiles of the two GM soybean lines studied were not dramatically altered compared with that of their natural isogenic control.

Crucial lncRNAs associated with adipocyte differentiation from human adipose-derived stem cells based on co-expression and ceRNA network analyses.

BACKGROUND: Injection of adipose-derived stem cells (ASCs) is a promising treatment for facial contour deformities. However, its treatment mechanisms remain largely unknown. The study aimed to explain the molecular mechanisms of adipogenic differentiation from ASCs based on the roles of long noncoding RNAs (lncRNAs). METHODS: Datasets of mRNA-lncRNA (GSE113253) and miRNA (GSE72429) expression profiling were collected from Gene Expression Omnibus database. The differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs) between undifferentiated and adipocyte differentiated human ASCs were identified using the Linear Models for Microarray Data method. DELs related co-expression and competing endogenous RNA (ceRNA) networks were constructed. Protein-protein interaction (PPI) analysis was performed to screen crucial target genes. RESULTS: A total of 748 DEGs, 17 DELs and 51 DEMs were identified. A total of 13 DELs and 279 DEGs with Pearson correlation coefficients > 0.9 and p-value < 0.01 were selected to construct the co-expression network. A total of 151 interaction pairs among 112 nodes (10 DEMs; eight DELs; 94 DEGs) were obtained to construct the ceRNA network. By comparing the lncRNAs and mRNAs in two networks, five lncRNAs (SNHG9, LINC02202, UBAC2-AS1, PTCSC3 and myocardial infarction associated transcript (MIAT)) and 32 genes (i.e., such as phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), protein tyrosine phosphatase receptor type B (PTPRB)) were found to be shared. PPI analysis demonstrated PIK3R1 , forkhead box O1 (FOXO1; a transcription factor) and estrogen receptor 1 (ESR1) were hub genes, which could be regulated by the miRNAs that interacted with the above five lncRNAs, such as LINC02202-miR-136-5p-PIK3R1, LINC02202-miR-381-3p-FOXO1 and MIAT-miR-18a-5p-ESR1. LINC02202 also could directly co-express with PIK3R1. Furthermore, PTPRB was predicted to be modulated by co-expression with LINC01119. CONCLUSION: MIAT, LINC02202 and LINC01119 may be potentially important, new lncRNAs associated with adipogenic differentiation of ASCs. They may be involved in adipogenesis by acting as a ceRNA or co-expressing with their targets.

iTRAQ-based quantitative tissue proteomic analysis of differentially expressed proteins (DEPs) in non-transgenic and transgenic soybean seeds.

The unintended effects of transgenesis have increased food safety concerns, meriting comprehensive evaluation. Proteomic profiling provides an approach to directly assess the unintended effects. Herein, the isobaric tags for relative and absolute quantitation (iTRAQ) comparative proteomic approach was employed to evaluate proteomic profile differences in seed cotyledons from 4 genetically modified (GM) and 3 natural genotypic soybean lines. Compared with their non-GM parents, there were 67, 61, 13 and 22 differentially expressed proteins (DEPs) in MON87705, MON87701 × MON89788, MON87708, and FG72. Overall, 170 DEPs were identified in the 3 GM soybean lines with the same parents, but 232 DEPs were identified in the 3 natural soybean lines. Thus, the differences in protein expression among the genotypic varieties were greater than those caused by GM. When considering ≥2 replicates, 4 common DEPs (cDEPs) were identified in the 3 different GM soybean lines with the same parents and 6 cDEPs were identified in the 3 natural varieties. However, when considering 3 replicates, no cDEPs were identified. Regardless of whether ≥2 or 3 replicates were considered, no cDEPs were identified among the 4 GM soybean lines. Therefore, no feedback due to GM was observed at the common protein level in this study.

A rock physics modelling algorithm for simulating the elastic parameters of shale using well logging data.

As a high-resolution geophysical method employed by the oil and gas industry, well logging can be used to accurately investigate reservoirs. Challenges associated with shale gas reservoir exploration increase the importance of applying elastic parameters or velocity at the logging scale. An efficient shale rock physics model is the foundation for the successful application of this method. We propose a procedure for modelling shale rock physics in which an appropriate modelling method is applied for different compositions of shale rock. The stiffnesses of the kerogen and fluid (oil, gas or water) mixture are obtained with the Kuster-Toksöz model, which assumes that the fluid is included in the kerogen matrix. A self-consistent approximation method is used to model clay, where the clay pores are filled with formation water. The Backus averaging model is then used to simulate the influence of laminated clay and laminated kerogen. Elastic parameter simulations using well logging data show the importance of treating the volume fractions of laminated clay and kerogen carefully. A comparison of the measured compressional slowness and modelled compressional slowness shows the efficiency of the proposed modelling procedure.

Development and Interlaboratories Validation of Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice G6H1 Event.

The transgenic rice G6H1 was a new event with the traits of herbicide-tolerance and insect-resistant. Herein, we developed one event-specific real-time PCR method with high specificity and sensitivity for G6H1 event quantitative analysis, and validated its performance on practical samples quantification through a collaborative ring trial. A total of eight laboratories participated in this validation and quantified three blind G6H1 powder samples including DNA extraction and real-time PCR analysis. The statistically analyzed results from returned data confirmed its high PCR efficiency and good linearity, trueness, and precision, indicating that the developed G6H1 real-time PCR assay was accurate, reliable, and comparable for G6H1 identification and quantification.

Development of Certified Matrix-Based Reference Material as a Calibrator for Genetically Modified Rice G6H1 Analysis.

The accurate monitoring and quantification of genetically modified organisms (GMOs) are key points for the implementation of labeling regulations, and a certified reference material (CRM) acts as the scaleplate for quantifying the GM contents of foods/feeds and evaluating a GMO analytical method or equipment. Herein we developed a series of CRMs for transgenic rice event G6H1, which possesses insect-resistant and herbicide-tolerant traits. Three G6H1 CRMs were produced by mixing seed powders obtained from homozygous G6H1 and its recipient cultivar Xiushui 110 at mass ratios of 49.825%, 9.967%, and 4.986%. The between-bottle homogeneity and within-bottle homogeneity were thoroughly evaluated with consistent results. The potential DNA degradation in transportation and shelf life were evaluated with an expiration period of at least 12 months. The property values of three CRMs (G6H1a, G6H1b, G6H1c) were given as (49.825 ± 0.448) g/kg, (9.967 ± 1.757) g/kg, and (4.986 ± 1.274 g/kg based on mass fraction ratio, respectively. Furthermore, the three CRMs were characterized with values of (5.01 ± 0.08)%, (1.06 ± 0.22)%, and (0.53 ± 0.11)% based on the copy number ratio using the droplet digital PCR method. All results confirmed that the produced G6H1 matrix-based CRMs are of high quality with precise characterization values and can be used as calibrators in GM rice G6H1 inspection and monitoring and in evaluating new analytical methods or devices targeting the G6H1 event.

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

A transcriptomic analysis for identifying the unintended effects of introducing a heterologous glyphosate-tolerant EPSP synthase into Escherichia coli.

Glyphosate is one of the most commonly used broad-spectrum herbicides with little to no hazard to animals, human beings, or the environment. Some microbial 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase variants are not inhibited by glyphosate, and they provide a powerful tool to engineer glyphosate-tolerant plants. However, the unintended effects of EPSP synthase expression patterns on microbes are not yet clear. Here, we use an Affymetrix GeneChip analysis to study how introduction of a heterologous glyphosate-tolerant EPSP synthase into a model microorganism Escherichia coli (E. coli) affects the global gene expression profile. The profile showed that 161 of 4071 genes were differentially expressed after the introduction of the synthase: 19 (0.47%) were up-regulated and 143 (3.49%) were down-regulated. The microarray results, in combination with BiOLOG substrate utilization and amino acid composition assays, suggested that heterologous EPSP synthase expression had very minor effects on E. coli. Although a small number of genes and metabolites were affected by EPSP synthase expression, no functional correlations were identified among the dataset. This study may shed light on the effect of EPSP synthase expression on microbes, which should help in the assessment of environmental safety.

Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos terminator for rapid detection of genetically modified crops.

Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification and detection technology that enables the amplification of DNA within 30 min at a constant temperature of 37-42 °C by simulating in vivo DNA recombination. In this study, based on the regulatory sequence of the cauliflower mosaic virus 35S (CaMV-35S) promoter and the Agrobacterium tumefaciens nopaline synthase gene (nos) terminator, which are widely incorporated in genetically modified (GM) crops, we designed two sets of RPA primers and established a real-time RPA detection method for GM crop screening and detection. This method could reliably detect as few as 100 copies of the target molecule in a sample within 15-25 min. Furthermore, the real-time RPA detection method was successfully used to amplify and detect DNA from samples of four major GM crops (maize, rice, cotton, and soybean). With this novel amplification method, the test time was significantly shortened and the reaction process was simplified; thus, this method represents an effective approach to the rapid detection of GM crops.

Halomonas huangheensis sp. nov., a moderately halophilic bacterium isolated from a saline-alkali soil.

A novel, Gram-stain-negative, aerobic, rod-shaped, non-motile and moderately halophilic bacterium, designated strain BJGMM-B45(T), was isolated from a saline-alkali soil collected from Shandong Province, China. Growth of strain BJGMM-B45(T) occurred at 10-45 °C (optimum, 30 °C) and pH 5.0-12.0 (optimum, pH 7.0) on Luria-Bertani agar medium with 1-20 % (w/v) NaCl (optimum, 7-10 %). The predominant respiratory quinone was Q-9. The major cellular fatty acids (>5 %) were C18 : 1ω7c, C16 : 0, C19 : 0 cyclo ω8c, summed feature 3, C12 : 0 3-OH and C12 : 0. The genomic DNA G+C content was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BJGMM-B45(T) belonged to the genus Halomonas in the class Gammaproteobacteria. The closest relatives were Halomonas cupida DSM 4740(T) (98.2 % 16S rRNA gene sequence similarity) and Halomonas denitrificans M29(T) (97.8 %). Levels of DNA-DNA relatedness between strain BJGMM-B45(T) and Halomonas cupida CGMCC 1.2312(T) and Halomonas denitrificans DSM 18045(T) were 57.0 and 58.9 %, respectively. On the basis of phenotypic, chemotaxonomic and phylogenetic features, strain BJGMM-B45(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas huangheensis sp. nov. is proposed. The type strain is BJGMM-B45(T) ( = ACCC 05850(T) = KCTC 32409(T)).

Development of a novel reference plasmid for accurate quantification of genetically modified Kefeng6 rice DNA in food and feed samples.

Reference plasmids are an essential tool for the quantification of genetically modified (GM) events. Quantitative real-time PCR (qPCR) is the most commonly used method to characterize and quantify reference plasmids. However, the precision of this method is often limited by calibration curves, and qPCR data can be affected by matrix differences between the standards and samples. Here, we describe a digital PCR (dPCR) approach that can be used to accurately measure the novel reference plasmid pKefeng6 and quantify the unauthorized variety of GM rice Kefeng6, eliminating the issues associated with matrix effects in calibration curves. The pKefeng6 plasmid was used as a calibrant for the quantification of Kefeng6 rice by determining the copy numbers of event- (77 bp) and taxon-specific (68 bp) fragments, their ratios, and their concentrations. The plasmid was diluted to five different concentrations. The third sample (S3) was optimized for the quantification range of dPCR according to previous reports. The ratio between the two fragments was 1.005, which closely approximated the value certified by sequencing, and the concentration was found to be 792 copies/µL. This method was precise, with an RSD of ~3%. These findings demonstrate the advantages of using the dPCR method to characterize reference materials.