Research Progress of Agricultural Genetically Modified Crop Safety Evaluation

The commercial cultivation of genetically modified(GM)crops has eased the global food crisis and brought considerable economic and social benefits to countries.Because of the potential safety problems,it is necessary to make clear the molecular genetic characteristics,edible safety,planting,processing,and other aspects of the safety evaluation of GM crops.The safety problems existing in the cultivation of GM crops,safety evaluation and detection of GM crops were introduced in this paper,which provided the basis for safety evaluation and effective supervision of GM crops and their products.Commercial cultivation and reasonable supervision based on safety evaluation have far-reaching significance for ensuring consumer safety,enhancing the credibility of the national political system and enhancing citizens'confidence in the safety of GM crop products for consumption.

Genetically Modified Doubts Imported GM soybeans impact China’s local farmers and create food safety concerns

The cooking oil made from the imported soybeans does not contain transgenic proteins. So there will be no food safety threat.

Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

Evaluation of Uncertainty in Measuring the Content of CaM V35S Promoter in Genetically Modified Soybean,GTS40-3-2,by Real-time Quantitative PCR

[ Objective ] This study aimed to investigate the major contributors to the measurement uncertainty in quantitative analysis of genetically modified ingreclients and improve the quality of quantitative detection of genetically modified components. [ Method] The content of CaMV35S promoter （parameter） in GTS40- 3-2 soybean powder samples was measured to estimate the measurement uncertainty preliminarily. [ Result] Type A uncertainty （uA） ＇ type B uncertainty （uB） and combined standard uncertainty （Uc） were 0.0 004, 0.002 and 0.002, respectively. At a confidence level ofp = 95% and freedom degree of Voff = 3 251, coverage factor k = 1.96, expanded uncertainty U = 0.004. The final measurement result was C = 0.028 ± 0. 004, which was dose to the conventional true value （0.03）. Thus, the measurement uncertainty was relatively small, indicating a high quality of measurement. In this study, uncertainty evaluation indicated that the deviation of micro liquid transfer made the greatest contribution to the measurement uncertainty. [ Cludusion ] The deviation of micro liquid transfer should be reduced to im- prove the quality of measurement.

Rapid screening of genetically modified ingredients in soybean and cotton processing by-product and waste using direct qPCR

To develop a simple and fast method for screening genetically modified ingredients from processing by-product and waste,direct quantitative PCR(qPCR)kit-Taqman which omitting multi genomic DNA preparing steps was developed in this study.A total of 18 oil crop processing by-products and wastes including 10 soybean and 8 cotton materials were collected from food processing factories.Compared with 2 commercial direct qPCR kits,conditions of DNA releasing procedure and PCR amplification were optimized.Element screening was performed at the initial step of genetically modified(GM)ingredient testing procedure via direct qPCR.GM event identification was carried out in positive samples by initial screening.Totally 5 screening elements(P–35S,T-NOS,Cp4-epsps,bar and pat)for soybean materials and 6 screening elements(P–35S,T-NOS,NPTII,Cry1Ac,bar and pat)for cotton samples were detected.In GM event identification,MON531 and MON1445 were found in cotton materials.Results were further confirmed by real-time PCR with DNA extraction and purification.The direct qPCR system proposed by this research was convenient for rapid screening and identification of GM ingredients in oil crop primary by-product and waste.

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.

Detection of Genetically Modified Crops by Combination of Multiplex PCR and Low-density DNA Microarray

Objective To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray. Methods Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified （GM） soybean. Seventeen capture probes （PCR products） and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean （GTS40-3-2）, maize （MonS10, Nk603, GA21）, canola （T45, MS1/RF1）, and rice （SCK） in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean （Roundup Ready GTS40-3-2） and canola （MS1/RF1）. Results A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS 1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity. Conclusion A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Genetically-Modified Organisms in United States Agriculture: Mandate for Food Labeling

The production of foods with genetically modified organisms (GMOs) has risen rapidly over the past three decades to comprise nearly 90% of crops grown in the United States today. Currently, there are no mandates for labeling foods containing GMOs. GMO agricultural crops contain the insertion of genes encoding for pesticides, pesticide resistance, growth factors, or other substances not normally present. In addition to the foreign genes that are inserted, hundreds to thousands of mutations disrupt normal genes in GMO plants. Recently, animal studies have demonstrated toxicity of GMO foods causing organ failure, infertility, carcinomas and death. The FDA requirement of ingredients added to foods be labeled on the product is not applied to GMO foods, precluding the consumer’s right to know. GMOs provide an economic incentive to companies because the seeds can be patented, driving up costs and creating the potential for monopolies. Herbicide-resistance conferred by GMOs has resulted in higher pesticide applications, which correlate with higher human cancer rates, and the emergence of pesticide-resistant weeds and insects. GMO toxins are spreading into to non-target insects, waterways and aquatic organisms, with toxicity to non-target organisms and resultant contamination of disparate ecosystems in the food chain. The appropriateness of mandatory GMO labeling of foods in the United States is discussed.

Effects of Severe Drought and Glyphosate Stress on Physiological Characteristics and Protein Expression of Photosystem Ⅱ in Genentically Modified Soybean

[Objective] The paper was to investigate effects of glyphosate stress on physiological characteristics and protein expression of photosystem Ⅱ(PSⅡ) in genentically modified soybean GTS 40-3-2 seedlings under severe drought condition. [Method] A pot experiment was carried out in growth chamber to determine the response of genetically modified soybean treated by severe drought stress and different concentrations of glyphosate at the third compound leaf stage. [Result] Severe drought treatment increased the electrolyte leakage(EL), superoxide dismutase(SOD) and peroxidase(POD) activities, and decreased the relative water content(RWC), chlorophyll content, and catalase(CAT) activity. The EL, SOD and POD activities were significantly increased in severe drought and glyphosate treatments, which were related to glyphosate concentrations. The chlorophyll content decreased, which was also related to glyphosate concentrations. But the BWC and CAT activity were not affected by glyphosate concentrations. Western blot displayed that PSⅡ protein Lhcb2 was not affected by stress conditions and stably expressed. D1, D2 and Lhcb4 protein level decreased, and there was no significant change in Lhcb1 expression under severe drought stress. The protein levels of D1, D2, Lhcb1 and Lhcb4 decreased with the increase of glyphosate concentrations under severe drought and glyphosate stress. When the glyphosate concentrations were 0.92 and 1.84 kg·ai/hm^2, the protein levels of D1, D2 and Lhcb4 were slightly higher than those in severe drought stress. When the glyphosate concentrations were 3.68 and 7.36 kg·ai/hm^2, the protein level of D1, D2, Lhcb1 and Lhcb4 decreased sharply. [Conclusion] This research provides a theoretical basis for production of genetically modified soybean.

A Broad Spectrum Identification Approach for Two Generations of epsps GM Soybeans

In order to accurately identify the first and second generations of epsps genetically modified(GM) soybeans and related products,a broad spectrum identification approach was established using the real time Polymerase Chain Reaction(PCR) principle according to the homology of epsps genes of the first and second generations of GM soybeans.A pair of primer and probe was designed to simultaneously identify exogenous gene epsps of two generations of GM soybeans.Besides,evaluation was carried out on this approach from the accuracy,specificity,sensitivity and reproducibility.The experimental results indicated that(ⅰ) although there is certain difference in epsps gene sequence between the first and second generations of epsps genetically modified(GM) soybeans,the established approach can simultaneously detect the epsps genes of the bean curd using two generations of soybean as raw materials;(ⅱ) in the accuracy and specificity experiment,only cp4-epsps genes of two generations of GM soybeans were detected,so this approach has high specificity and accuracy;(ⅲ) in the experiment of 5 copies of epsps genes of 40 repeated identification reaction systems,5 copies of epsps genes can be detected each time,therefore at 100% confidence level,this approach can identify 5 copies of epsps genes,showing that this approach has high sensitivity and reproducibility.

A Novel Approach for Evaluation of Food Functions and Safety Applied in RR GM Soybeans

[Objective] This study aimed to evaluate tbe healthy risk of genetically modified （ GM ） soybeans by using a novel approach for functions and safety of food. [ Me^od] Different from traditional evaluation of substantial equivalence, three great innovations were performed in this study, involving in basic diet, evalu- ation approaches and principle, as well as the clarification of connotation differences between absolute and relative mass of organs. Hence a novel BDI-GS （Bendib Damage Index and General Score） evaluation approach was established and applied in comparative evaluation between RR GM and natural soybeans. Healthy male ICR mice during linear growth were selected; experimental mice were fed with 15% RR GM soybeans and 15% natural soybeans blending maize meal diets, and control mice were fed with single maize meal diet for 13 d; the mice were dissected after collecting blood samples and perfectly obtained nine organs or tissues to re- cord their masses and conduct statistical analyses. [Result] Plenty of matching information was obtained through simple design. The growth performance of treated mice was markedly of individual differences, some mice were thwarted due to regular intake of RR soybeans. Meanwhile, the functions and safety of RR soybeans were markedly lowered in overall nutritional and healthy effects than those of natural soybeans expressed in GS values, and presents some declines in nutrition and health of thymus, pancreas and spermary; especially, it can make thymus immune （P 〈0.05） in markedly lower level than that of natural soybeans. [ Conclusion] Therefore, major troubles and risks of RR soybeans intake are of personal risks in different degrees, in addition, it may increase sub-health and related chronic epi- demics risks, and herein it will presents certain safety issues. The creation of this novel evaluation system provides a simple and available evaluation approach for functions and potential risks revelation of food effects, and will yield far-reaching influences to safety evaluation and healthy development of GM foods, as well as public health.

Advances in the Identification of Genetically Modified Rice with Real-time PCR and Multiplex PCR

In recent years, food security and safety have attracted increasing attention due to the worldwide research and development of genetically modified （GM） rice, and the controversy over the commercialization of GM rice. And the identification of GM rice is of great significance. Therefore, in the present study, the po- tential problems in the identification of GM rice with PCR were analyzed both at a technical level and from a theoretical perspective. In addition, PCR detection on the transgenic elements： promoter, terminator, internal reference gene and target gene was discussed, respectively. The possible solutions were proposed based on the principles of plant virology and genetic engineering.

基于联合验证数据评定转化体定量检测结果测量不确定度

随着转基因生物及产品定量标识制度的不断完善,大批转化体定量检测方法的建立迫在眉睫,为了在实验数据积累不足的情况下,在检测方法建立初期及时评定测量不确定度,本研究以耐除草剂大豆DBN9004定量检测方法为例,在检测实验室积累到足够检测数据之前,在8家实验室对5种转基因含量样品进行联合验证,分析试验数据的再现性标准偏差,形成“自上而下”的不确定度预评定方法。结果显示:经格拉布斯法和科克伦法检验8家实验室数据,实验室5的数据存在离群值和岐离值,故剔除,7家实验室5个含量样品的再现性相对标准差RSD_(jR)为6.025%~11.532%,均小于规定的35%,说明该方法具有良好的精密度。根据上述分析数据绘制回归曲线方程为s_(jR)=0.0783×c/≡_(j)-0.0104,计算的相对标准不确定度分量u_(r)=0.0783,u_(0)=0.012,进而将试样检测结果的平均值(c=)带入公式,计算定量结果的标准不确定度u=√u_(0)^(2)+(u_(r)×c/=)^(2)=√0.012^(2)+(0.0783×c=)^(2)。结果表明基于联合验证数据评定转化体定量检测结果测量不确定度,为转化体定量检测方法建立初期提供了科学有效的实验室自行评定方法,为转基因生物及产品的安全监管提供了技术保障。

转基因抗虫耐除草剂大豆JD321对根际土壤含水量和pH值的影响

为了评价转基因抗虫耐除草剂大豆JD321对土壤生态系统的影响,该实验研究了其对土壤主要理化性质的影响。在大豆苗期、花期、鼓粒期和成熟期,分析了转基因大豆JD321对根际土壤含水量和pH值的影响。与其对应的非转基因大豆相比,转基因大豆JD321的根际土壤含水量和pH值没有明显变化,除草剂处理转基因大豆也没有对土壤含水量和pH值产生明显影响,但土壤含水量和pH值在不同生育期间存在明显差异。研究结果为转基因大豆JD321的环境安全性提供了新的支撑。

基于改进GM(1,1)模型的中国大豆价格预测

中国大豆产业是国家基础性产业之一,国内外大豆市场对大豆价格波动影响极大,对中国大豆波动情况加以研究具有重要的意义。在分析GM(1,1)模型有效性的基础上,讨论了其在农产品预测的可行性,从而采用改进GM(1,1)模型用1990-2014年中国大豆价格预测2015-2017年中国大豆价格分别为4.40,4.57和4.74元·kg-1,在本试验中改进GM(1,1)模型并没有对原始序列进行累加操作,说明原始序列具备准指数规律。检验结果表明后验差比值小于0.5和小误差概率大于0.8,说明采用灰色预测对此数据加以预测合格。2015年价格低于2015年东北三省和内蒙古大豆价格目标价格,说明中国大豆仍处于低迷状态,会导致中国大豆种植面积及农民种植意愿继续降低。据此给出对策与建议,一是继续提高大豆种植补贴和大豆目标价格;二是加大地方大豆产业的政策性扶持,尤其是非转基因大豆产业的扶持,保持国内大豆产业健康发展;三是提高大豆行业与豆农的组织化程度。

Construction of the Plasmid Reference Molecule for Detection of Transgenic Soybean MON89788

[Objective] The aim was to construct a plasmid reference molecule (PRM) for detection of transgenic soybean MON89788. [Method] the lectin gene sequence,3'-junction and 5'-junction sequence between host plant DNA integrated DNA of MON89788 soybean were amplified independently,and the three fragments were cloned into the cloning vector pMD18-T in order through molecular manipulation method to construct pMD-LM3M5,the applicability of the constructed novel PRM was tested. [Result] Sequencing confirmation result showed that the PRM was 3 700 bp in length,containing 1 029 bp of recombined DNA fragment. The limits of qualitative detection of the PRM were 10 copies. [Conclusion] The PRM constructed in this study was suitable for the identification of MON89788 event.

核酸斑点杂交技术快速鉴定转基因大豆品系

为了建立转基因大豆品系高通量检测方法,本研究以转基因大豆DAS-68416-4、DAS-44406-6、DAS-81419、DP305423作为试验对象,根据品系特异的宿主与插入片断间连接区域的基因序列作为检测靶标,设计特异性引物。以转基因标准品为材料,提取DNA作为模板进行定性PCR扩增,尼龙膜点样,生物素探针制备,杂交与显色,并对探针浓度、杂交温度与时间与显色时长进行优化,利用优化后的反应条件对转基因大豆DAS-68416-4、DAS-44406-6、DAS-81419、DP305423及空白对照样品进行检测,验证体系的特异性。将4种转基因大豆的DNA进行10倍系列稀释,利用优化后的反应条件测定灵敏度。对实验室收集的7个品系的转基因大豆标准品和8个能力验证样品进行适用性检测分析,并采用双盲验证法对17份盲样进行核酸斑点杂交测试分析,进一步验证体系的可靠性。结果表明,以转基因大豆DAS-68416-4、DAS-44406-6、DAS-81419、DP305423为模版的扩增产物均有且只有一条清晰明亮的条带,大小均与理论相符,空白对照与内参照基因结果正常。4种转基因大豆特异性探针除对应样品有显色反应,对其他3种样品均未显色,具有方法特异性。样品DAS-68416-4、DAS-81419、DP305423最低检出限为20 pg·μL^(-1),样品DAS-44406-6最低检出限为2 pg·μL^(-1),具有较高的灵敏度。对7个标准品、8个能力验证样品和17份盲样的测试结果与SN/T 1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检测方法》中的结果一致,可用于日常样品检测。本研究建立的定性PCR结合的核酸斑点杂交方法能对4种转基因大豆进行快速准确鉴定,对有序推进生物育种产业化应用,严查非法转基因种子市场流通和田间种植,保障粮食安全具有重要意义。

大豆、玉米及其加工产品中转基因成分筛查

目的:近年来,转基因作物大面积种植,具有抗病、抗虫、耐除草剂等性状的作物及其加工产品广泛的出现在市面上。在我国,只有经过农业部审批的转基因作物才能种植或销售,为加强转基因产品的监管,我国发布了一系列转基因产品检测方法,在对外源基因进行检测时,需要有阳性质控。方法:对SN/T 1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检验方法》中大豆、玉米共34个品系中的15个外源基因进行筛查,为植物及其加工产品中转基因成分的检测提供参考依据。结果:筛选出15个外源基因的阳性对照。结论:可应用于植物及其加工产品中转基因成分的日常检测,为检测机构开展检测时提供依据。

我国转基因玉米大豆应用抗性治理策略

为有序推进我国生物育种产业,保障转基因玉米大豆产业化安全、可持续实施,需建立科学的抗性治理策略,延缓靶标害虫和杂草抗性产生。查阅大量国内外相关靶标害虫和杂草抗性进化研究的文献,总结国际转基因玉米大豆抗性治理的经验教训,并分析我国转基因抗虫棉的相关实践,立足我国当今靶标害虫与杂草实际情况,进行分析讨论。依据玉米种植区域特点及虫情发生规律进行区域划分,并确定各区域推荐的转基因抗虫玉米基因类型和配套庇护所策略,提出适合我国现阶段产业特点的转基因玉米大豆抗性治理策略。在靶标害虫抗性治理方面,应根据我国玉米主产区的靶标害虫发生及迁移扩散为害规律,遵循整体布局、源头治理的原则,在转化体研发、品种审定等环节加强虫源和种源控制;同时,建议因地制宜,采取“一区一类基因一策”的害虫抗性治理措施。在田间杂草抗性治理方面,建议配合轮换使用不同抗性机理的转化体和不同作用机理的除草剂。

耐除草剂转基因大豆SHZD32-1数字PCR绝对定量方法的建立

基于DNA单分子绝对定量的数字PCR技术,建立了该转化体的微滴式数字PCR(ddPCR)双重(转化体特异性基因和大豆内标准基因)定量方法,内容主要包括:优化双重ddPCR引物探针浓度和退火温度,考察特异性,确定线性动力学范围以及检出限(3 copies/μL)和定量限(15 copies/μL)等关键参数。转基因大豆SHZD32-1已获得生产应用安全证书“农基安正字(2019)”第293号,其ddPCR定量检测方法作为一种绝对定量的计量方法,简便高效、精准度高,将为标准物质定值、定量检测及规范生物安全领域数字PCR方法建立等提供技术支撑。