Plutella xylostella (diamondback moth; DBM) is a significant pest of Brassica crops, causing billions of dollars in annual global damage and developing resistance to many insecticides. Climate change is increasing the frequency and severity of infestations by influencing the moth's reproduction and expanding its range, leading to increased crop losses. In this study, we examined the early metabolomic responses of four Brassica napus accessions to DBM infestation, focusing on identifying the metabolic basis of tolerance. Phenotypic analysis showed that R4220 and R4415 were highly susceptible, with remaining leaf areas of 27 and 38%, respectively, while the tolerant accessions R4637 and R5064 retained 85 and 91% of their leaf area post-infestation. Metabolomic profiling revealed a distinct separation between tolerant and sensitive accessions under both control and infested conditions. Notably, tolerant accessions exhibited differential accumulation of metabolites, with abundant metabolites belonging to lipid and lipid-like molecules, organic acids and derivatives, and benzenoids. Additionally, 31 metabolites were found to be consistently expressed at higher levels in tolerant accessions as compared to sensitive ones, notably tridecanedioic acid, 3,5-dihydroxyphenylglycine and benzoxazine-6-carboxylic acid. Furthermore, KEGG analysis revealed that pathways such as phenylpropanoid biosynthesis, aminoacyl-tRNA biosynthesis and ABC transporters were enriched, indicating their critical roles in the defense mechanisms. This comprehensive analysis of metabolomic alterations provides valuable insights into the biochemical pathways underpinning insect tolerance in rapeseed, potentially guiding the development of more resilient cultivars and leading a pathway to improve crop farming for sustainable agriculture.

Genetically modified food has come under suspicion because of the potential safety risks. Only a limited number of target genes could be detected by traditional methods. Here, customized-fast DNA extraction methods and a LAMP (Loop-mediated isothermal amplification) system were developed combining a hand-held chip, portable analyzer, and an Android app for the qualitative detection of genetically modified crops. DNA of seeds and leaves was obtained within 5minutes. Primers were designed with high specificity and sensitivity (10 copies) for later LAMP amplification. Crude DNA was added to the chip pre-embedded with customizable primers by quantitative dropper and the whole detection process was less than 40minutes without any large-scale instrument. The limit of detection was approximately 0.1% (w/w). The detection system with its low workload, high accuracy, and stability offers a novel, effective approach for the rapid detection of GM corps and many other food safety areas in the field.

Societal Impact Statement Advancements in science and technology enable us to address the challenges of the times: hunger, malnutrition, climate change, and dwindling natural resources. One of the most significant innovations in food and agriculture is the advent of genetic engineering, which may help us address the aforementioned challenges. Millions of people could benefit from the application of genetically engineered crops in agriculture and food. However, it is vital that biosafety assessments of unintended effects are carried out to identify any potential risks to humans, the environment, or society. Summary The study compared the extent of siRNA, transcriptomic and metabolomic changes in the leaves of four RNAi‐based genetically engineered rice lines to evaluate whether RNAi‐based genetically engineered rice that is resistant to black‐streaked dwarf virus experiences changes not seen in the non‐genetically engineered counterpart. siRNA, transcriptome, and metabolome profiling were conducted. siRNAs with different lengths and abundance were highly enriched in the transcript of rice; however, relative expression analysis of eight potential off‐target genes revealed that there was no decrease in gene expression in the RNAi‐based genetically engineered lines, meaning that no off‐target phenomena were caused as a result of siRNA derived from invert repeat sequences of rice black‐streaked dwarf virus in this study. This study identified some differentially expressed genes and differentially accumulated metabolites in the RNAi‐based genetically engineered lines; however, they were not enriched in pathways detrimental for humans, animals and the environment. Only 30 differentially expressed genes were significantly enriched in the plant hormone signal transduction pathway via a combined analysis using two‐way orthogonal partial least squares model. These genes belonging to salicylic acid, jasmonic acid, cytokinin, abscisic acid, and brassinosteroid pathway involved in pathways related to virus resistance or infection response were significantly changed. Taken together, there was no off‐target in RNAi‐based genetically engineered rice lines and may have a positive effect on virus resistance overall in this study.

A multi-chromatic and multi-component lateral flow immunoassay (MCMC-LFIA) was developed for simultaneous detection of CP4 EPSPS, Bt-Cry1Ab, Bt-Cry1Ac, and PAT/bar proteins in genetically modified (GM) crops. Captured antibodies specific to these exogenous proteins were separately immobilized on a nitrocellulose membrane as test zones. Multi-colored microspheres, used as visible multi-probes, were conjugated with corresponding antibodies and sprayed on the conjugate pad. The assay results can be visually interpreted within 10 min by observing the appearance of colored bands. The MCMC-LFIA demonstrated high sensitivity, with detection of limits of 7.8 ng/mL for CP4 EPSPS and 2.5 ng/mL for Bt-Cry1Ab, Bt-Cry1Ac, and PAT/bar proteins, significantly improving the performance of previously reported LFIAs. The MCMC-LFIA exhibited excellent specificity and was validated for practical use in field-based applications. The proposed MCMC-LFIA offers a rapid, sensitive, and user-friendly tool for the on-site large-scale screening of GM materials.

Background In recent years, genome editing technology represented by clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9? has been developed and applied in transgenic research and development, and transgenic products have been developed for a variety of applications. Gene editing products, unlike traditional genetically modified crops, which are generally obtained by target gene deletion, insertion, base mutation, etc., may not differ significantly at the gene level from conventional crops, which increases the complexity of testing. Objective We established a specific and sensitive CRISPR/Cas12a-mediated gene editing system to detect target fragments in a variety of transgenic rice lines and commercial rice-based processing products. Methods In this study, the CRISPR/Cas12a visible detection system was optimized for the visualization of nucleic acid detection in gene-edited rice. The fluorescence signals were detected by both gel electrophoresis and fluorescence-based methods. Results The detection limit of the CRISPR/Cas12a detection system established in this study was more precise, especially for low-concentration samples. In addition to achieving single-base detection in gene-edited rice, we showed that different base mutations in the target sequence have different detection efficiencies by sitewise variant compact analysis. The CRISPR/Cas12a system was verified via a common transgenic rice strain and commercial rice sources. The results proved that the detection method could not only be tested in samples with multiple mutation types but could also effectively detect target fragments in commercial rice products. Conclusion We have developed a set of efficient detection methods with CRISPR/Cas12a for gene-edited rice detection to provide a new technical basis for rapid field detection of gene-edited rice.

对转基因生物产品实行标识管理,既是规范相关产品生产与销售行为的需要,也是保护消费者知情权和选择权的需要.为适应我国不断推进转基因技术研发及产品应用的发展趋势,建议为转基因生物、种子以及相关食品建立统一的转基因标识管理制度,及时根据转基因产品应用情况调整标识目录,将定性标识改为定量标识管理,统一违反标识管理行为应承担的法律责任,明确可以使用"非转基因"标识和禁止使用"非转基因"标识的具体情形,以维护公平的市场竞争秩序,保障消费者的合法权益.

基因编辑技术自诞生以来,在作物育种、基因功能分析中得到了广泛的应用,其中CRISPR/Cas9系统是目前使用最多的基因编辑技术.基于荧光定量PCR技术,针对编辑位点设计特异性探针,结合基因组快速提取方法和成熟的水稻内参基因PLD引物,对自行研发的CRISPR/Cas9系统编辑水稻的Os11N3基因进行检测,并通过合成质粒模拟不同突变情况对该方法的特异性进行了验证.研究所建立的体系可在节约时间和成本的同时获取满足qPCR检测所需的基因组,能够区分1 bp基因编辑突变体与野生型,具有良好的特异性和灵敏度.检测方法可为作物育种筛选节省时间和成本,为基因编辑产品检测监管提供技术支持.

The use of omics techniques to analyze the differences between genetic engineering organisms and their parents can identify unintended effects and explore whether such unintended effects will have negative consequences. In order to evaluate whether genetic engineering will cause changes in crops beyond the changes introduced by conventional plant breeding, we compared the extent of transcriptome and metabolome modification in the leaves of three lines developed by RNA interference (RNAi)-based genetic engineering and three lines developed by conventional breeding. The results showed that both types of plant breeding methods can manifest changes at the short interfering RNA (siRNA), transcriptomic, and metabolic levels. Relative expression analysis of potential off-target gene revealed that there was no broad gene decline in the three RNAi-based genetic engineering lines. We found that the number of DEGs and DAMs between RNAi-based genetic engineering lines and the parental line was less than that between conventional breeding lines. These unique DEGs and DAMs between RNAi-based genetic engineering lines and the parental lines were not enriched in detrimental metabolic pathways. The results suggest that RNAi-based genetic engineering do not cause unintended effects beyond those found in conventional breeding in maize.

随着全球经济贸易一体化发展,转基因产品商业化为多边国际贸易带来无限的机遇和挑战.随之而来的转基因低水平混杂(LLP)问题作为不可规避的现实问题,成为国际贸易中的前沿议题.转基因阈值制度作为国际社会缓解转基因低水平混杂问题的重要因素,逐渐引起各国政府关注.以转基因低水平混杂问题现状为立足点,从技术和政策层面分析了建立转基因阈值制度对于我国完善转基因低水平混杂法律制度及配套长效机制的必要性,从标识制度和LLP阈值制度两个角度概述了转基因阈值制度在维护本国生物安全和贸易顺畅等方面的双重作用,旨在为我国完善转基因低水平混杂法律制度和开展定量检测标准制定工作提供参考.

BACKGROUND:The high sensitivity of droplet digital PCR (ddPCR) contributes to its excellent performance in animal and microorganism identification, but the utilization of ddPCR is limited in plant adulterant identification of highly processed products for which effective methods are lacking. PURPOSE:This study investigated the feasibility of ddPCR in the identification of plant adulterants in Chinese patent medicine (CPM) as groundwork to develop ddPCR assays for other highly processed goods. METHODS:The original plant, processed and highly processed products of Mutong (Akebiae Caulis) and its two adulterants were used to analyze the specificity, sensitivity, and practical performance of the developed singleplex and triplex ddPCR assays. RESULTS:The results revealed that the limit of detection (LOD) and limit of quantification (LOQ) for the selective ddPCR assays developed to identify Mutong and its adulterants were 0.00002 ng/μl and 0.00016 ng/μl, respectively, and that the regression equations representing the relationships between DNA concentration and target copy number all exhibited good linearity. Furthermore, the common adulterant of Mutong in three samples of Longdan Xiegan pills was successfully identified through ddPCR assays and confirmed by Sanger sequencing. CONCLUSION:This work comprehensively revealed the great ability of ddPCR technology in detecting plant adulterants in traditional Chinese medicine (TCM), providing a method for the quality control of highly processed plant products with complex components for commonly used goods.

Deciphering the molecular mechanisms of insect resistance to Bacillus thuringiensis (Bt) based biotechnology products including Bt sprays and Bt crops is critical for the long-term application of Bt technology. Previously, we established that down-regulation of the ABC transporter gene PxABCG1, trans-regulated by the MAPK signaling pathway, contributed to high-level resistance to Bt Cry1Ac toxin in diamondback moth, Plutella xylostella (L.). However, the underlying transcriptional regulatory mechanism was unknown. Herein, we identified putative binding sites (PBSs) of the transcription factor (TF) POUM1 in the PxABCG1 promoter and used a dual-luciferase reporter assay (DLRA) and yeast one-hybrid (Y1H) assay to reveal that POUM1 activates PxABCG1 via interaction with one of these sites. The expression of POUM1 was significantly decreased in the midgut tissue of Cry1Ac-resistant P. xylostella strains compared to a Cry1Ac-susceptible P. xylostella strain. Silencing of POUM1 expression resulted in reduced expression of the PxABCG1 gene and an increase in larval tolerance to Bt Cry1Ac toxin in the Cry1Ac-susceptible P. xylostella strain. Furthermore, silencing of PxMAP4K4 expression increased the expression of both POUM1 and PxABCG1 genes in the Cry1Ac-resistant P. xylostella strain. These results indicate that the POUM1 induces PxABCG1 expression, while the activated MAPK cascade represses PxABCG1 expression thus reducing Cry1Ac susceptibility in P. xylostella. This result deepens our understanding of the transcriptional regulatory mechanism of midgut Cry receptor genes and the molecular basis of the evolution of Bt resistance in insects.

随着转基因产品商业化种植面积不断增加、国际贸易日趋频繁,对转基因生物安全管理提出了更高的要求.转基因产品检测技术作为安全评价的关键环节,逐渐引起了各国政府的关注.目前,针对转基因产品的快速检测方法层出不穷,但这些检测方法对于设备、试剂和专业的实验人员均有较高的要求.因此,为了有效支撑转基因相关产业的发展和管理,亟需建立一种高灵敏度、高特异性及高效的转基因检测技术.基因组编辑技术是近年来迅速发展的一类遗传修饰技术,其代表技术——CRISPR/Cas技术,更是极大地推动了生物技术的发展.CRISPR/Cas技术除了被应用于基因编辑领域,也逐渐被应用于核酸分子检测领域.基于此,以转基因产品检测技术为立足点,从CRISPR/Cas的检测原理、检测效果等技术层面分析了CRISPR/Cas检测技术发展的必然性,并对其在转基因产品检测上的应用前景进行展望,旨在为我国转基因产品快速检测和有效监管工作提供资料,对于保障我国转基因产品贸易的顺利进行具有重要意义.

转基因技术作为现代生物技术的核心之一,在保障粮食安全、保护生态安全、拓展农业功能等方面发挥了重要作用,但同时,也带来了潜在的安全性问题.为此,世界上主要国家和国际组织都制定了与转基因生物安全管理相关的法律法规,加强管理.通过对国际食品法典委员会(Codex Alimentarius Commission,CAC)、经济合作与发展组织(Organization for Economic Co-operation and Development,OECD)的转基因食品安全评价体系与评价政策进行介绍分析,总结其现实积极作用及价值,指出其缺陷或不足,以期为健全我国转基因食品安全评价体系、完善监管措施,提供理论政策及法律规范方面的参考性建议.

Background: With the commercialization of genetically modified organisms (GMOs) in the market, laboratories have undergone a significantly increased workload. A universal analytical approach was designed to achieve cost-efficient and high-throughput GMOs screening with high specificity and accuracy. The approach provides accurate qualification of authorized and unauthorized GMOs. Objective This article describes the assessment of this analytical approach developed to detect the majority of commercialized GMOs over the world. Method: Seven elements and three events were detected by qPCR in a single laboratory to detect 59 commercialized GMOs. Certificated reference materials and food/feed samples from the Chinese market were also evaluated for the specificity, conformity, and robustness of this approach and were challenged in the interlaboratory study. Results: The results showed that elements and events selected can best detect GMO presence with good specificity and sensitivity. The results showed a concordance between 97.5 and 99.56% and the variance between 0.65 and 12.88%, which is in line with the minimum requirement of analytical methods of GMO testing. Conclusions: The approach validated here can be used to manipulate GMO presence in food and feed and showed the capacity to manipulate GMO trace in the trade and domestic agriculture market in China.

Genetic engineering (GM) has great potential to improve maize productivity, but rises some concerns on unintended effects, and equivalent as their comparators. There are some limitations through targeted analysis to detect the UE in genetically modified organisms in many previous studies. We here reported a case-study on the effects of introducing herbicides and insect resistance (HIR) gene cassette on molecular profiling (transcripts and metabolites) in a popular maize variety Zhengdan958 (ZD958) in China. We found that introducing HIR gene cassette bring a limited numbers of differential abundant genes (DAGs) or differential abundant metabolites (DAMs) between transgenic events and non-transgenic control. In contrast, averaged 10 times more DAGs and DAMs were observed when performed comparison under different growing environments in three different ecological regions of China than the numbers induced by gene effects. Major biological pathways relating to stress response or signaling transduction could explain somehow the effects of growing environments. We further compared two transgenic events mediated ZD958 (GM-ZD958) with either transgenic parent GM-Z58, and other genetic background nonGM-Z58, nonGM-ZD958, and Chang7-2. We found that the numbers of DAGs and DAMs between GM-ZD958 and its one parent maize variety, Z58 or GM-Z58 is equivalent, but not Chang7-2. These findings suggest that greater effects due to different genetic background on altered molecular profiling than gene modification itself. This study provides a case evidence indicating marginal effects of gene pleiotropic effects, and environmental effects should be emphasized.

转基因玉米是目前全球商业化种植的4大转基因作物之一,也是转化体获批数量最多的转基因作物,随着全球转基因玉米栽培面积的不断增加,其安全性也越来越被社会公众广泛关注.各国都非常重视转基因玉米产品监管和检测,转基因标识制度的不断发展和完善也使转基因检测技术从定性发展到定量,数字PCR技术因具有绝对定量、灵敏度高、精准度高、特异性强等特点,在玉米及其产品中转基因成分检测领域具有极大的应用前景.文中介绍了数字PCR技术的基本原理,综述了其在转基因玉米及其产品检测领域的应用.

RNA干扰(RNA interference,RNAi)是指由双链RNA(double-stranded RNA,dsRNA)诱发同源mRNA高效特异性降解的现象,在真核生物中普遍存在且进化保守.RNAi技术作为21世纪初的重大科学成就,目前被广泛应用于疾病防治、基因功能研究、植物改良育种等领域.RNAi技术常与转基因技术结合用于植物改良育种,通过不同的载体设计或作用途径来研发满足生产需要的农业生物技术产品.为了明确现阶段基于RNAi技术的转基因植物育种技术进展,综述了RNAi现象的发现和作用机制、转基因载体设计、小RNA(small RNA,sRNA)的递送方式等方面的研究进展,并阐述了基于RNAi技术的转基因植物的研究实例和商业化情况,以期为相关研究提供参考,从而发挥RNAi技术的最大应用价值,使之服务于新时代的农业发展.

RNA干扰(RNA interference,RNAi)在植物、动物和真菌的生长、发育、病毒防御和转座子失活中起着至关重要的作用.目前已成功利用RNAi技术培育出抗病虫和品质改良等具有优良性状的生物技术产品,为农业绿色发展提供了强有力的支撑.然而,目前RNAi的相关机制尚未完全明确,基于RNAi技术的转基因植物面临着一些亟待解决的问题,同时,对于RNAi转基因植物应用的安全高效监管也需进一步完善.基于此,对RNAi转基因植物的监管现状及其面临的育种上的挑战进行了综述,并对存在的问题提出了解决建议,以期为RNAi技术进一步应用于农业植物改良育种提供新思路,并为其监管评价提供依据.

我国是转基因大豆(Glycine max)主要消费国之一,转基因大豆安全性受到各界广泛关注,针对大豆转基因成分开展快速、准确的检测方法的研究势在必行.本研究基于TaqMan探针技术,建立了同时检测MON87701及MON87708两种转基因大豆品系的双重实时荧光PCR检测方法,并对该方法的特异性、灵敏度及适用性进行了分析.结果显示,该方法能准确地从18种转基因样品和5种非转基因样品中检出靶基因,表明其具有良好的特异性;灵敏度可达0.01％;利用该方法对10批实际大豆样品进行检测,结果与欧盟标准方法一致,表明其具有良好的适用性.该方法适用于各口岸实验室对转基因大豆MON87701和MON87708品系同时进行快速、准确检测.本研究为转基因产品的监管提供了参考依据.

利用微滴式数字PCR(droplet digital PCR,ddPCR)平台建立针对MON87705、MON87769、DP356043三种转基因大豆中外源基因的双重PCR检测方法.利用双重数字PCR方法检测特异性、定量范围等参数,优化所用引物探针组合及实验体系程序,检测外源基因与内标准基因的拷贝数.结果表明,所用引物探针组合在数字PCR方法中仅对目标大豆品系有荧光信号,具有特异性,可用于转基因大豆品系的筛选与鉴别.检测了大豆的转基因成分含量,结果与材料标准品参数基本一致,并根据结果设定定量检测限为0.5％,定性检测限为0.05％,可满足低纯度样品检测的需求.双重数字PCR体系能够准确且稳定的满足实际检测需要,在实际应用上具有良好的发展前景.