Safety assessment of transgenic Bacillus thuringiensis rice T1c-19 in Sprague-Dawley rats from metabonomics and bacterial profile perspectives

Bacillus thuringiensis rice is facing commercialization as the main food source in the near future. The unintended effects of genetically modified (GM) organisms are the most important barriers to their promotion. We aimed to establish a new in vivo evaluation model for genetically modified foods by using metabonomics and bacterial profile approaches. T1c-19 rice flour or its transgenic parent MH63 was used at 70% wt/wt to produce diets that were fed to rats for ～ 90 days. Urine metabolite changes were detected using (1)H NMR. Denaturing gradient gel electrophoresis and real-time polymerase chain reaction (RT-PCR) were used to detect the bacterial profiles between the two groups. The metabonomics was analyzed for metabolite changes in rat urine, when compared with the non-GM rice group, where rats were fed a GM rice diet. Several metabolites correlated with rat age and sex but not with GM rice diet. Significant biological differences were not identified between the GM rice diet and the non-GM rice diet. The bacteria related to rat urine metabolites were also discussed. The results from metabonomics and bacterial profile analyses were comparable with the results attained using the traditional method. Because metabonomics and bacterial profiling offer noninvasive, dynamic approaches for monitoring food safety, they provide a novel process for assessing the safety of GM foods.     

Metabolic profiling based on LC/MS to evaluate unintended effects of transgenic rice with cry1Ac and sck genes

As a primary characteristic of substantial equivalence, the evaluation of unintended effects of genetically modified plants has been evolving into an important field of research. In this study, a metabolic profiling method for rice seeds was developed using rapid resolution liquid chromatography/quadrupole time-of-flight mass spectrometry. The analytical properties of the method, including the linearity, reproducibility, intra-day precision and inter-day precision, were investigated and were found to be satisfactory. The method was then applied to investigate the differences between transgenic rice and its native counterparts, in addition to the differences found between native rice with different sowing dates or locations. Global metabolic phenotype differences were visualized, and metabolites from different discriminated groups were discovered using multivariate data analysis. The results indicated that environmental factors played a greater role than gene modification for most metabolites, including tryptophan, 9,10,13-trihydroxyoctadec-11-enoic acid, and lysophosphatidylethanolamine 16:0. The concentrations of phytosphingosine, palmitic acid, 5-hydroxy-2-octadenoic acid and three other unidentified metabolites varied slightly due to gene modification.     

Metabonomics study of transgenic Bacillus thuringiensis rice (T2A-1) meal in a 90-day dietary toxicity study in rats

Rice is one of the most important staple foods in the world. The Cry2A gene was inserted into the rice genome to help the plant combat insects. As the unintended effects of the genetically modified (GM) organisms are the most important barriers to the promotion of GM organisms, we have carried out a useful exploration to establish a new in vivo evaluation model for genetically modified foods by metabonomics methods. In this study, the rats were fed for 90 days with the GM and NON-GM rice diets. The changes in metabolites of the urine were detected using (1)H-NMR. The metabonomics were analyzed to see whether the GM rice can induce the metabolite changes in the rats' urine when compared with the NON-GM rice group. The multivariate analysis and ANOVA were used to determine the differences and the significance of differences respectively, and eventually we concluded that these differences did not have a biological significance. The conclusion of the metabonomics was comparable with that from the traditional method. As a non-invasive and dynamic monitoring method, metabonomics will be a new way of assessing the food safety of GM foods.     

Proteomics insight into the biological safety of transgenic modification of rice as compared with conventional genetic breeding and spontaneous genotypic variation

The potential of unintended effects caused by transgenic events is a key issue in the commercialization of genetically modified (GM) crops. To investigate whether transgenic events cause unintended effects, we used comparative proteomics approaches to evaluate proteome differences in seeds from 2 sets of GM indica rice, herbicide-resistant Bar68-1 carrying bar and insect-resistant 2036-1a carrying cry1Ac/sck, and their respective controls D68 and MH86, as well as indica variety MH63, a parental line for breeding MH86, and japonica variety ZH10. This experimental design allowed for comparing proteome difference caused by transgenes, conventional genetic breeding, and natural genetic variation. Proteomics analysis revealed the maximum numbers of differentially expressed proteins between indica and japonica cultivars, second among indica varieties with relative small difference between MH86 and MH63, and the minimum between GM rice and respective control, thus indicating GM events do not substantially alter proteome profiles as compared with conventional genetic breeding and natural genetic variation. Mass spectrometry analysis revealed 234 proteins differentially expressed in the 6 materials, and these proteins were involved in different cellular and metabolic processes with a prominent skew toward metabolism (31.2%), protein synthesis and destination (25.2%), and defense response (22.4%). In these seed proteomes, proteins implicated in the 3 prominent biological processes showed significantly different composite expression patterns and were major factors differentiating japonica and indica cultivars, as well as indica varieties. Thus, metabolism, protein synthesis and destination, and defense response in seeds are important in differentiating rice cultivars and varieties.     

Metabolic profiling of transgenic rice with cryIAc and sck genes: An evaluation of unintended effects at metabolic level by using GC-FID and GC–MS

The cryIAc and sck genes were introduced to the rice for the purpose of improving the insect resistance. Metabolic profiles of wild and transgenic rice were compared to assess the unintended effects related to gene modification. Wild samples with different sowing dates or sites were also examined to determine the environmental effects on metabolites. The polar compounds of grains were extracted, trimethylsilylated and analyzed by gas chromatography-flame ionization detection (GC-FID). Partial least squares-discriminant analysis (PLS-DA) and principal component analysis (PCA) were applied to differentiate transgenic and wild rice grains. The significantly distinguishable metabolites were picked out, and then identified by gas chromatography–mass spectrometry (GC–MS). It was found that both the environment and gene manipulation had remarkable impacts on the contents of glycerol-3-phosphate, citric acid, linoleic acid, oleic acid, hexadecanoic acid, 2,3-dihydroxypropyl ester, sucrose, 9-octadecenoic acid (Z)-, 2,3-dihydroxypropyl ester and so on. Sucrose, mannitol and glutamic acid had a significant increase in transgenic grains in contrast to those in non-genetically modified (GM) rice.     

Difference in cesium accumulation among rice cultivars grown in the paddy field in Fukushima Prefecture in 2011 and 2012

After the accident of the Fukushima 1 Nuclear Power Plant in March 2011, radioactive cesium was released and paddy fields in a wide area including Fukushima Prefecture were contaminated. To estimate the levels of radioactive Cs accumulation in rice produced in Fukushima, it is crucial to obtain the actual data of Cs accumulation levels in rice plants grown in the actual paddy field in Fukushima City. We herein conducted a two-year survey in 2011 and 2012 of radioactive and non-radioactive Cs accumulation in rice using a number of rice cultivars grown in the paddy field in Fukushima City. Our study demonstrated a substantial variation in Cs accumulation levels among the cultivars of rice.     

转基因水稻外源基因向近缘种群漂流和渐渗的研究进展

水稻是我国最重要的粮食作物之一,我国有8亿以上的人口以稻米作为主食。但在水稻生产中,由于病、虫、草害及不良气候等逆境因子的影响,严重制约了水稻的高产、稳产。转基因生物技术的迅速发展,为水稻抗性育种提供了新途径。自20世纪80年代以来,我国全方位地开展了转基因水稻的研发,目前已经培育出大量的抗病、抗虫、抗除草剂和抗逆的转基因水稻品种,这将为提高我国水稻的生产力和确保粮食安全做出重要的贡献。但转基因水稻的基因漂流及其可能带来的生物安全问题备受关注。已有报道证明,外源转基因可以通过异交向非转基因品种和野生近缘种漂流。在不同的试验条件下,抗除草剂基因有0．05％-0．53％逃逸的可能,其向不育系的最大漂移频率可达4．518％。抗虫基因向相邻非转基因水稻的平均漂移频率最高为0．875％。因此,本文对水稻与其近缘野生种的杂交情况,转基因水稻外源基因向非转基因品种、野生近缘种以及野生非近缘种的漂流和渐渗及其潜在的生态环境风险等方面进行了简要分析,并对转基因水稻的发展进行了展望,以期为转基因水稻的安全应用提供参考。     

Gene flow from herbicide-tolerant GM rice and the heterosis of GM rice-weed F2 progeny

Gene flow from genetically modified (GM) crops to non-GM cultivars or weedy relatives may lead to the development of more aggressive weeds. We quantified the amount of gene flow from herbicide-tolerant GM rice (Protox GM, derived from the cultivar Dongjin) to three cultivars (Dongjin, Aranghyangchal and Hwaseong) and a weedy rice line. Gene flow frequency generally decreased with increasing distance from the pollen donor. At the shortest distance (0.5 m), we observed a maximum frequency (0.039%) of gene flow. We found that the cultivar Dongjin received the greatest amount of gene flow, with the second being weedy rice. Heterosis of F2 inbred progeny was also examined between Protox GM and weedy rice. We compared growth and reproduction between F2 progeny (homozygous or hemizygous for the Protox gene) and parental rice lines (GM and weedy rice). Here, transgene-homozygous F2 progeny was significantly taller and produced more seeds than the transgene-hemizygous F2 progeny and parental lines. Although the gene flow frequency was generally low, our results suggest that F2 progeny between GM and weedy relatives may exhibit heterosis.     

Analysis of metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei

ABSTRACT

In sake brewing, the steamed rice is used in two ways, added to sake-mash (as kake-mai) and making koji. The rice is an important determinant for the quality of sake, as the metabolites in sake affect its taste/aroma. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by genetically crossing two sake rice, Gohyakumangoku and Yamadanishiki. However, the metabolites in sake from KOS have not been analyzed. Here, to investigate the characteristic metabolites in sake from KOS, we performed two types of small-scale sake-fermentation tests changing only the rice used for kake-mai or total rice (both kake-mai and koji) by these three rice cultivars and examined the effect of KOS on sake metabolites by the metabolome analysis method using UPLC-QTOF-MS. We identified the peaks/metabolites, whose intensity in sake from KOS was higher/lower than those from the other cultivars. The brewing properties of KOS were partially characterized by this analysis.     

Metabolic changes in transgenic maize mature seeds over-expressing the <Emphasis Type="Italic">Aspergillus niger phyA2</Emphasis>

Key message

Non-targeted metabolomics analysis revealed only intended metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2.

Abstract

Genetically modified (GM) crops account for a large proportion of modern agriculture worldwide, raising increasingly the public concerns of safety. Generally, according to substantial equivalence principle, if a GM crop is demonstrated to be equivalently safe to its conventional species, it is supposed to be safe. In this study, taking the advantage of an established non-target metabolomic profiling platform based on the combination of UPLC-MS/MS with GC–MS, we compared the mature seed metabolic changes in transgenic maize over-expressing the Aspergillus niger phyA2 with its non-transgenic counterpart and other 14 conventional maize lines. In total, levels of nine out of identified 210 metabolites were significantly changed in transgenic maize as compared with its non-transgenic counterpart, and the number of significantly altered metabolites was reduced to only four when the natural variations were taken into consideration. Notably, those four metabolites were all associated with targeted engineering pathway. Our results indicated that although both intended and non-intended metabolic changes occurred in the mature seeds of this GM maize event, only intended metabolic pathway was found to be out of the range of the natural metabolic variation in the metabolome of the transgenic maize. Therefore, only when natural metabolic variation was taken into account, could non-targeted metabolomics provide reliable objective compositional substantial equivalence analysis on GM crops.

     

实质等同性原则和转基因食品的安全性评价

讨论转基因食品安全性评价中的一些热点话题：现代生物技术与传统育种比较,整体食物的动物毒性实验,实质等同性原则及其局限性,食品成分安全性评价,食品成分的预料外改变,抗生素抗性标记基因,过敏性问题,标签问题等。     

Response and relation of allantoin production in different rice cultivars to competing barnyardgrass

The production of secondary metabolites and biomass of plants may be altered by coexistence and competition. Rice has coexisted with barnyardgrass in paddy systems for centuries and represents a model system for understanding plant–plant interactions. Despite increasing knowledge of allelochemicals produced and released by rice involved in coexistence of these two species, relatively little is known about the role and production of beneficial chemicals of rice in response to competing barnyardgrass. The purpose of this study was to quantify the growth-stimulating allantoin (5-ureidohydantoin) and its effect on barnyardgrass growth in a rice–barnyardgrass system. In this system, rice produced and released allantoin but barnyardgrass did not. The production of allantoin by rice responded to competition from barnyardgrass, and its concentration varied among rice cultivars tested. At 1:1 barnyardgrass and rice mixture proportion, allantoin concentration in allelopathic cultivars was obviously lowered by competing barnyardgrass, while no significant variation of allantoin concentration was observed in non-allelopathic cultivars. Barnyardgrass biomass was reduced in mixed-culture with rice cultivars, in particular, allelopathic cultivars. There was a positive relationship between allantoin concentration in rice cultivars and barnyardgrass biomass. Furthermore, allantoin stimulated the growth of barnyardgrass once released from rice or added to soil. The results suggest that rice plants of allelopathic cultivars appear to be able to detect the presence of competing barnyardgrass and respond by decreasing production of growth-stimulating allantoin, regulating the growth of barnyardgrass. In this manner, given rice cultivars may alleviate the competition of barnyardgrass and provide greater benefit to their own growth.     

Transgene flow to hybrid rice and its male-sterile lines

Gene flow from genetically modified (GM) crops to the same species or wild relatives is a major concern in risk assessment. Transgenic rice with insect and/or disease resistance, herbicide, salt and/or drought tolerance and improved quality has been successfully developed. However, data on rice gene flow from environmental risk assessment studies are currently insufficient for the large-scale commercialization of GM rice. We have provided data on the gene flow frequency at 17 distances between a GM japonica line containing the bar gene as a pollen donor and two indica hybrid rice varieties and four male-sterile (ms) lines. The GM line was planted in a 640m² in an isolated experimental plot (2.4 ha), which simulates actual conditions of rice production with pollen competition. Results showed that: (1) under parallel plantation at the 0-m zone, the transgene flow frequency to the ms lines ranged from 3.145 to 36.116% and was significantly higher than that to hybrid rice cultivars (0.037–0.045%). (2) Gene flow frequency decreased as the distance increased, with a sharp cutoff point at about 1–2 m; (3) The maximum distance of transgene flow was 30–40 m to rice cultivars and 40–150 m to ms lines. We believe that these data will be useful for the risk assessment and management of transgenic rice lines, especially in Asia where 90% of world's rice is produced and hybrid rice varieties are extensively used. Shirong Jia, Feng Wang and Lei Shi contributed equally to this investigation.     

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.     

云南高原粳稻品质特性分析

采用表型主成分及聚类分析法,对76个云南高原粳稻的11个品质性状进行了分析。结果表明,（1）直链淀粉含量的平均值达食用稻品种品质1级标准,糙米率、透明度、碱消值和胶稠度达2级标准,精米率达3级标准,其余品质性状的平均值均在3级以下。品种间变异最小的品质性状是糙米率和碱消值,变异最大的是垩白度,其次是垩白粒率。（2）稻米品质性状间存在复杂的相关关系。（3）在选出的6个影响云南稻米品质的主因子中,精米率、垩白粒率和垩白度因子对稻米品质的累积贡献率为49.6%。（4）76个云南粳稻聚为4类,其中第一类包括52个品种,表明遗传距离近,遗传基础狭窄。在今后的云南粳稻品质育种中,应降低垩白粒率和垩白度,提高精米率和整精米率,同时拓宽遗传基础。     

Effect of koji starter on metabolites in Japanese alcoholic beverage sake made from the sake rice Koshitanrei

ABSTRACT

In sake brewing, the steamed rice is used in 2 ways, added to sake-mash and making rice-koji. Rice-koji is made from the steamed rice by using koji starter, and its quality is an important determinant of the aroma/taste of sake. The sake rice Koshitanrei (KOS) was developed in Niigata Prefecture by crossing 2 sake rice varieties, Gohyakumangoku and Yamadanishiki. Recently, we reported the characteristic components/metabolites in sake made from KOS by conducting metabolome analysis using UPLC-QTOF-MS. In this study, to investigate the effect of koji starter and sake rice cultivars on the sake metabolites, we performed small-scale sake-making tests using the above 3 rice cultivars and 3 koji starters. Finally, we demonstrated that some of the characteristic components/metabolites of sake from KOS are affected by the koji starter. Thus, in addition to rice cultivar, koji starter plays an important role for establishment/maintenance of the quality of the final product.     

Genome-wide searching of single-nucleotide polymorphisms among eight distantly and closely related rice cultivars (Oryza sativa L.) and a wild accession (Oryza rufipogon Griff.).

We searched the genomes of eight rice cultivars (Oryza sativa L. ssp. japonica and ssp. indica) and a wild rice accession (Oryza rufipogon Griffith) for nucleotide polymorphisms, and identified 7805 polymorphic loci, including single-nucleotide polymorphisms (SNPs) and insertions/deletions (InDels), in predicted intergenic regions. Polymorphisms are useful as DNA markers for genetic analysis or positional cloning with segregating populations of crosses. Pairwise comparison between cultivars and a neighbor-joining tree calculated from SNPs agreed very well with relationships between rice strains predicted from pedigree data or calculated with other DNA markers such as p-SINE1 and simple sequence repeats (SSRs), suggesting that whole-genome SNP information can be used for analysis of evolutionary relationships. Using multiple SNPs to identify alleles, we drew a map to illustrate the alleles shared among the eight cultivars and the accession. The map revealed that most of the genome is mono- or di-allelic among japonica cultivars, whereas alleles well conserved among modern japonica paddy rice cultivars were often shared with indica cultivars or wild rice, suggesting that the genome structure of modern cultivars is composed of chromosomal segments from various genetic backgrounds. Use of allele-sharing analysis and association analysis were also tested and are discussed.     

Low frequency of transgene flow from Bt/CpTI rice to its non-transgenic counterparts planted at close spacing

Crop-to-crop transgene flow will affect seed purity of non-GM rice varieties, leading to unwanted consequences. To assess the maximum probability of transgene outflow in rice (Oryza sativa), gene flow experiments were conducted with three cultivation patterns with different mixed-planting proportions of adjacent GM and non-GM rice at two sites in Fujian and Hainan Provinces of China. Three GM rice lines containing two insect-resistance genes (Bt/CpTI) and their non-GM counterparts were used in the experiments to allow natural hybridization to occur. A hygromycin resistance gene was used as a selective marker for identifying hybrids. Based on the examination of > 645 700 geminated seeds, the result showed low frequencies (0.05-0.79%) of transgene flow from GM to non-GM rice at close spacing, although with significant variation among mixed-planting proportions. It is concluded that rice transgene flow will occur at a very low frequency (< 1.0%), even if the GM rice is planted at close spacing with non-GM rice, and high densities of GM rice cultivated in the neighborhood of non-GM rice will increase the probability of outcrossing with the non-GM rice.     

Genetic diversity among cultivars, landraces and wild relatives of rice as revealed by microsatellite markers

Genetic diversity among 35 rice accessions, which included 19 landraces, 9 cultivars and 7 wild relatives, was investigated by using microsatellite (SSR) markers distributed across the rice genome. The mean number of alleles per locus was 4.86, showing 95.2% polymorphism and an average polymorphism information content of 0.707. Cluster analysis based on microsatellite allelic diversity clearly demarcated the landraces, cultivars and wild relatives into different groups. The allelic richness computed for the clusters indicated that genetic diversity was the highest among wild relatives (0.436), followed by landraces (0.356), and the lowest for cultivars. Allelic variability among the SSR markers was high enough to categorize cultivars, landraces and wild relatives of the rice germplasm, and to catalogue the genetic variability observed for future use. The results also suggested the necessity to introgress genes from landraces and wild relatives into cultivars, for cultivar improvement.