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  • 2021 Volume 43 Issue 1
    Published: 28 February 2021
      
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  • Current status and prospects of environmental safety standard system for agricultural genetically modified organisms in China
    LIANG Jin-gang, ZHANG Kai-xin, ZHANG Xu-dong, WANG Hao-qian, CHEN Zi-yan, LIU Peng-cheng, ZHANG Xiu-jie
    2021, 43(1): 2. https://doi.org/10.19802/j.issn.1007-9084.2020335
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    Environmental safety standards for agricultural genetically modified organisms (GMOs) has become
    an important technical support for the safety management of GMOs in China. By January 2021, the Ministry of Agri⁃
    culture/Ministry of Agriculture and Rural Affairs had released 47 environmental safety standards for agricultural
    GMOs. This paper summarizes the status quo of China's agricultural GMOs environmental safety standards system,
    and comparatively analyzes the development of agricultural GMOs environmental safety standards in the domestic
    and foreign, and discusses the development direction of agricultural GMOs environmental safety standards system
    construction in the future. We hope to provide reference for further improvement of China's agricultural GMOs envi⁃
    ronmental safety standards, which will provide better technical support for its safety management.
  • Evaluation and supervision of gene editing in context of implementation of the Biosafety Law of the People's Republic of China
    WANG Pan-di, XIONG Xiao-juan, FU Ping, WU Gang, LIU Fang
    2021, 43(1): 15. https://doi.org/10.19802/j.issn.1007-9084.2021020
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    On October 17, 2020, the Biosafety Law of the People's Republic of China (hereinafter referred to
    as the‘biosafety law’) passed the deliberation of the Standing Committee of the National People's Congress, and
    comes into force on April 15, 2021. Application safety of biotechnology is one of the main contents involved in the
    biosafety law. Gene editing, as a research hotspot in field of biotechnology in recent years, has attracted much atten⁃
    tion in its safety evaluation and supervision. This paper summarized the present situation of the application of gene
    editing technology, compared the different national supervision of gene editing technology, discussed the signifi⁃
    cance of biosafety law to gene editing safety assessment and supervision in China, and finally proposed suggestions
    on how to better promote and implement the biosafety law in field of gene editing biosafety.
  • Advances in portable biosensor devices for point-of-care test of genetically modified crops
    CUI Dan-dan, ZHAI Shang-shang, WEN Lu-ke, WU Yu-hua, LI Jun, LI Yun-jing, LI Xiao-fei, ZHU Li, GAO Hong-fei, WU Gang
    2021, 43(1): 22. https://doi.org/10.19802/j.issn.1007-9084.2020346
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    Point-of-care test (POCT) is a necessary technology to implement the supervision of genetically
    modified (GM) crops in resource-limited settings, which plays an important role in GM organisms monitoring. Re⁃
    cently, biosensor provides a promising pathway for a simple, quick and affordable quantification of GM ingredients.
    So far plenty of studies have concentrated on the methodology development of biosensor，however its application
    has rarely been reported. The development of portable biosensors with desirable characteristics of integration and
    miniaturization is highly demand for POCT. This paper reviews recent advances in handheld device-assisted minia⁃
    turized biosensor systems for POCT of GM crops. Miniaturization strategies of biosensors for POCT of GM organisms
    in the future are then proposed. The trends, prospects and challenges of miniaturized biosensors for POCT are fur⁃
    ther described.

  • Application of high-throughput sequencing technology in evaluation of molecular characteristics of transgenic crops: opportunities and challenges
    ZHAO Sheng-bo, LUO Jun-ling, ZENG Xin-hua, YUAN Rong, WU Gang, YAN Xiao-hong
    2021, 43(1): 31. https://doi.org/10.19802/j.issn.1007-9084.2020318
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    Molecular characterization lays a foundation for safety assessment and subsequent monitoring of
    transgenic plants. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods
    cannot comprehensively detect unintended gene insertions, let alone detect unknown GM events. More and more
    newly developed crops by new plant breeding technology challenged classical PCR-based methods. Next-genera⁃
    tion sequencing is expected overcome these limitations, providing rapid and comprehensive molecular feature data.
    This article summarized complex mechanism of T-DNA integration, limitations of traditional molecular identifica⁃
    tion methods, and application of high-throughput sequencing methods in safety evaluation of genetically modified
    molecular characteristics. It also discussed challenges encountered and application strategies, which might provide
    alternatives for safety evaluation of genetic modification.
  • Research progress of digital PCR in quantitative detection of genetically modified organism
    SI Neng-wu, LI Jun, WU Yu-hua, WU Gang, ZHANG Li
    2021, 43(1): 40. https://doi.org/10.19802/j.issn.1007-9084.2020265
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     Labeling of genetically modified organisms (GMOs) is an important part of safety management of
    GMOs. Quantitative detection technology provides important technical support for effective implementation of the
    GM labeling system. Digital PCR does not rely on standard curves and reference materials when performing quanti⁃
    tative detection of nucleic acid molecules, it could directly calculate copy number concentration of the detection tar⁃
    get in sample. Its application in accurate quantification of nucleic acids is becoming more and more extensive. In
    the quantitative detection of GMOs, digital PCR could be used directly to calculate content of exogenous gene in
    template. By comparing of digital PCR and real-time fluorescent quantitative PCR in GMOs detection, factors affect⁃
    ing digital PCR detection results were elaborated, and the possibility of digital PCR results traceable to SI units was
    discussed. This study was expected to provide a reference for application of digital PCR in quantitative detection of
    GMOs and precise quantification of nucleic acids in several fields.

  • Loop-mediated isothermal amplification technology in the detection of genetically modified crops
    LIANG Jin-gang, DU Zai-hui, HUANG Chun-meng, ZHANG Fei-yan, XU Wen-tao, ZHANG Xiu-jie
    2021, 43(1): 51. https://doi.org/10.19802/j.issn.1007-9084.2020336
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     The detection of genetically modified (GM) crops provides important technical support for the safety
    management of agricultural genetically modified organisms (GMOs). Loop-mediated isothermal amplification
    (LAMP) technology is easy to operate, efficient, sensitive, and specific. It has been widely used in the detection of
    GM crops. This article analyzes and summarizes the application of LAMP technology in the detection of GM soy⁃
    bean, maize and rapeseed in recent years, hoping to provide scientific references for further application and stan⁃
    dardization of LAMP technology in the detection of GM crops.
  • Research advances and risk management of gene drives
    WANG Pan-di, XIONG Xiao-juan, FU Ping, WU Gang, LIU Fang
    2021, 43(1): 56. https://doi.org/10.19802/j.issn.1007-9084.2020291
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    Gene drives, a natural phenomenon, means a specific gene can be biased to next generation. This
    phenomenon has recently become one of the new research hotspots in field of biology. It could be used to control
    pests and infectious diseases, and also to clear away invasive species and so on. It was verified in laboratory that
    gene drives based on CRISPR/Cas9 system has been successfully enhanced the heritability of specific genes. Al⁃
    though gene drives has many potential benefits, it faces great challenges in terms of ethics and social security. This
    paper reviewed the principle of gene drives, research progress and applications in recent years, and the technical
    problems faced by this technology including risks and governance of gene drives.

  • Application of nanomaterials in plant genetic transformation
    GAO Hong-fei, FAN Shi-hang, LIU Jing-lin, XIONG Chong-ming, HUA Wei, LI Jun
    2021, 43(1): 64. https://doi.org/10.19802/j.issn.1007-9084.2020206
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    To break the main obstacle that the cell wall preventing delivery of exogenous biomolecules into in⁃
    tact cell during plant genetic transformation, nanomaterial-mediated gene delivery technology was currently suc⁃
    cessfully applied. Traditional transgenic methods require the aid of external force or the mediation by creatures like
    agrobacterium. The nano-carriers for genetic delivery possess outstanding advantages such as excellent biocompati⁃
    bility, ability to protect external DNA/RNA against nuclease degradation, high transfection efficiency, thus provide
    a new promising way for plant genetic engineering. In this paper, we summarized the bottlenecks of plant genetic
    transformation met currently, the drawbacks of the existing methods and the advantages of nanomaterial-mediated
    delivery. And the key techniques and research progresses of nanoparticles-mediated plant transformation were dis⁃
    cussed in detail. Ultimately, the advantages and disadvantages as well as future direction and problems of this newly
    formed technology were also discussed and prospected.

  • Rapid extraction of genomic DNA and its application in detection of genetically modified soybeans
    CHEN Yu, WANG Xiao-fu, CHEN Xiao-yun, PENG Cheng, XU Jun-feng, LI Yue-ying
    2021, 43(1): 70. https://doi.org/10.19802/j.issn.1007-9084.2020301
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     To explore an on-site extraction method for plant genomic DNA during detection of genetically mod⁃
    ified（GMO）soybeans, a rapid extraction of plant DNA was developed by applying silicon membrane to absorb
    DNA, together with filter membrane and syringe. The method was used to extract DNA from leaves and seeds of 5
    major crops, including soybean, cotton, rapeseed, corn, and rice. The conventional PCR and basic RPA（recombi⁃
    nase polymerase amplification）are simultaneously used to amplify endogenous genes from the DNA extracted by
    the rapid extraction and QIAGEN kit methods. Results showed that the rapid extraction method had higher DNA
    yield than the QIAGEN method, while both of the extraction met the needs of conventional PCR and basic RPA am⁃
    plification. By detecting the genetically modified soybean SHZD32-1, the DNA rapid extraction method combined
    with conventional PCR, fluorescence RPA and fluorescence quantitative PCR showed consistent and accurately re⁃
    sults. For this developed rapid DNA extraction method does not require centrifuge and other equipment, and could
    be completed in 3 minutes on site, it was considered to be successfully meeting the requirements on site.

  • Development and application of a gene editing site-specific PCR method
    ZHANG Hong-wen, ZHAO Sheng-bo, YAN Xiao-hong, LI Jun, ZHAI Shan-shan, XIAO Fang, GAO Hong-fei, LI Yun-jing, WU Gang, WU Yu-hua
    2021, 43(1): 77. https://doi.org/10.19802/j.issn.1007-9084.2020314
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    Genome editing technology has been successfully used in agricultural breeding, it is urgent to estab⁃
    lish accurate identification and quantification technology based on molecular characteristics of genome-edited prod⁃
    ucts. In this study, SP1 gene-edited rice was used as materials to develop an editing site-specific PCR method by
    designing universal primer pair upstream and downstream of the editing sites, and gene editing site-specific Taq⁃
    Man probes targeting at the editing sites. The results demonstrated that this method accurately identified specific ge⁃
    nome-edited products with a detection sensitivity of 5-10 copies. The quantitative detection of genome-edited prod⁃
    ucts was performed by both real-time PCR (qPCR) and droplet digital PCR (ddPCR). Since thousands of indepen⁃
    dent droplets of ddPCR might eliminate the competitive consumption of universal primers by wild-type DNA, the
    quantitative results of ddPCR showed higher quantitative accuracy than those of qPCR.

    基因组编辑技术利用位点特异性核酸酶（sitespecific
    nuclease, SSN）切割植物基因组DNA的特定
    位点，产生双链断裂缺口（double stranded break,
    DSB）[
    1,2]，通过非同源末端连接（non-homologous end
    joining, NHEJ）或同源重组（homologous recombina⁃
    tion, HR）[
    3]机制对双链断裂缺口进行修复，导致修复
    位点发生单碱基或寡核苷酸片段的插入、缺失或替
    换等突变，实现对基因组靶基因序列的精确编辑[4]。
    基因组编辑常用的特异性核酸酶主要包括锌指核
    酸酶（zinc finger nucleases, ZFNs）、类转录激活因子
    效应物核酸酶（transcription activator-like effector
    nucleases, TALENs）、以及规则簇状间隔的短回文重
    复相关蛋白（clustered regularly interspaced short pal⁃
    indromic repeat-associated protein，CRISPR/Cas）[
    5]。
    目前，基因组编辑技术已经成功用于多种植物的性
    状改良，如增加千粒重的水稻[6]、低镉含量的籼稻[7]、
    低糖含量的马铃薯[8]、糯性玉米[9]、高油酸大豆[10]、抗
    溃疡病的柑橘[11]等；在北美洲，已经有两种基因组编
    2021，43（1）：77-89
    doi：
    收稿日期：2020⁃11⁃03
    基金项目：
    be completed in 3 minutes on site, it was considered to be successfully meeting the requirements on site.
  • Development and application of MON87427/zSSIIb duplex droplet digital PCR method
    XIAO Fang, ZHANG Xiu-jie, LI Jun, WANG Hao-qian, LI Yun-jing, SHAN Lu-ying, ZHAI Shan-shan, GAO Hong-fei, WU Gang, WU Yu-hua
    2021, 43(1): 90. https://doi.org/10.19802/j.issn.1007-9084.2020322
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    Digital PCR (dPCR), an absolute quantitative method of DNA molecules, has been successfully ap⁃
    plied for genetically modified organisms (GMO) detection and reference material characterization. In this paper, a
    duplex ddPCR method was established for quantification of MON87427 content with reference plasmid pUC57-M
    harboring MON87427-specfic sequence and 5 maize reference gene sequences as quality control. By comparing the
    5 reference genes, zSSIIb was determined to combine with MON87427 for duplex ddPCR assay according to the res⁃
    olution of positive droplets and negative droplets, the number of raindrops with medium signal intensity, and the con⁃
    sistency between measured value and theoretical value. The optimal annealing temperature for MON87427/zSSIIb
    duplex ddPCR was determined at 58.4℃, and the duplex ddPCR displayed good linearity over the range from 10 to
    60 000 copies. The quantitative results of blinded samples by duplex ddPCR were well comparable with those by re⁃
    al-time quantitative PCR (qPCR), indicating that the MON87427/zSSIIb duplex ddPCR might replace the qPCR
    method for quantification of transgenic maize MON87427 and characterization of MON87427 reference materials.
  • Development and application of reference plasmid pYCID-1905 for identification of rapeseed events
    ZHAI Shan-shan, LI Xia-ying, WANG Hao-qian, LI Jun, CHEN Zi-yan, GAO Hong-fei, LI Yun-jing, WU Gang, ZHANG Xiu-jie, WU Yu-hua
    2021, 43(1): 99. https://doi.org/10.19802/j.issn.1007-9084.2019206
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     To monitor and detect the presence of genetically modified rapeseed in China, reference materials
    are strongly needed. This study collected 11 transgenic rapeseed varieties that China had approved for import and
    would approve for import. Target sequences for event-specific identification of 11 transgenic rapeseed varieties
    were determined by analyzing their molecular characteristics and corresponding standard methods. Their sequences
    were fused and inserted into pUC18 vector by gene synthesis as reference plasmid pYCID-1905, which provided a
    general positive control molecule for the identification of transgenic rapeseed events. Results showed that the plas⁃
    mid molecule can be used as positive control for both conventional PCR methods and real-time PCR methods in na⁃

    tional standards (GB/T), declaration of the Ministry of Agriculture and Rural Affairs, entry-exit inspection and quar

    antine industry standards (SN/T), and European Union standards.

  • Development and validation of qPCR method for detection of g10evo-epsps in genetically modified soybean
    XU Xiao-li, JI Yi, CHEN Xiao-yun, WANG Peng-fei, MIAO Qing-mei, LAI Yong-min, XU Jun-feng
    2021, 43(1): 110. https://doi.org/10.19802/j.issn.1007-9084.2020309
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    Herbicide tolerant gene g10evo-epsps was usually used in genetically modified (GM) crops in China.
    To develop a detection method by specific quantitative PCR, g10evo-epsps-specific primers and probe were de⁃
    signed and used for accurate and precise real-time PCR (qPCR) method in house validated. Results supported the
    specific of the method on detecting g10evo-epsps gene from transgenic soybean ZUTS-33. Standard curves construc⁃
    tion showed that the PCR amplification efficiency was over 90% with R2 above 0.99. Limit of detection was assessed
    to be 8 copies and limit of quantification was estimated at 16 copies. The accuracy of qPCR method was verified by
    screening 5 mixed DNA samples with known levels of the ZUTS-33 event (4.5, 2, 0.5, 0.09 and 0.045%, respective⁃
    ly). The average bias between the quantified values and expected values of the 5 samples deviated from 0.00% to
    11.11 %, and relative repeatability standard deviation ranged from 2.30% to 17.10%. The overall data indicated
    that this developed g10evo-epsps-specific qPCR method could meet the requirements of GM soybean screening and
    provide technical support for GM soybean regulation and labeling.

  • Screening and detection of transgenic components in 706 conventional soybean lines
    ZHAO Yang, JIN Long-guo, WANG Bu-jun
    2021, 43(1): 117. https://doi.org/10.19802/j.issn.1007-9084.2020019
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    In order to investigate and analyze whether transgenic soybean lines are mixed into conventional
    soybean breeding in China, 706 soybean lines were selected for PCR amplification of several regulatory elements
    (CaMV35S promoter, FMV35S promoter and NOS terminator), as well as the main herbicide tolerant transgenic soy⁃
    bean transformants (GTS40-3-2 and MON89788) approved for import in China. Meanwhile, a specific qualitative
    PCR detection method for soybean event (MON89788) was established and 5 ′and 3′ flanking sequences were de⁃
    signed. The products of 445 bp and 340 bp were amplified by primers, which can be applied to the detection of
    MON89788 soybean. Finally, of all the samples selected, the proportion of transgenic components containing
    CaMV35S promoter, FMV35S promoter, NOS terminator and herbicide tolerant transgenic line MON89788 was 1%,
    0.85%, 0.71% and 0.85% respectively, so as to evaluate the proportion of transgenic soybeans and provide consum⁃
    ers with the share of transgenic soybeans. To provide a strong basis for the agricultural genetically modified soybean
    regulatory authorities to carry out monitoring work to provide data support.

  • Research on degradation of CP4-EPSPS protein from transgenic soybean
    LI Yun-jing, WAN Dan-feng, LIU Biao, XIAO Fang, LI Xiao-fei, WU Yu-hua, LI Jun, GAO Hong-fei, SHENG Wen-jing, LI Jun, ZHU Li, WU Gang
    2021, 43(1): 124. https://doi.org/10.19802/j.issn.1007-9084.2020214
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    With the development and application of transgenic technology, enrichment and degradation of
    transgnic foreign protein in environment has become a public concern. Detection and monitoring of exogenous pro⁃
    tein in the environment has become a direction of genetically modified organism research and supervision. In this
    study, transgenic soybean (5% GTS40-3-2) seeds containing CP4-EPSPS protein were treated by simulated natural
    degradation and protease K. Western blot, lateral flow test strip and enzyme linked immunosorbent assay (ELISA)
    were selected to detect the presence of CP4-EPSPS. The results showed that CP4-EPSPS was gradually degraded
    under simulated natural degradation conditions, and completely degraded in about 17 weeks. In the condition of pro⁃
    tease K treatment, CP4-EPSPS was rapidly degraded in 9 hours. This study explored the degradation rule of CP4-
    EPSPS in natural environment. And it provided new ideas for the future research of rapid, safe and effective degra⁃
    dation of CP4-EPSPS.

  • Effect of transgenic Brassica napus with mEPSPS gene on diversity and structure of rhizosphere microbial  communities
    DU Kun, WANG Ting, YANG Yang, LI Jin-ping, WANG You-ping
    2021, 43(1): 131. https://doi.org/10.19802/j.issn.1007-9084.2020345
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     Evaluating possible negative effects of alien genes expressed in transgenic plants is essential in en⁃
    vironmental safety assessment of genetically modified（GM）organisms. We compared bacterial communities in rhi⁃
    zosphere soil of Brassica napus overexpressing mEPSPS (JHX6) and the control line J9707. Results showed that Pro⁃
    teobacteria, Bacteroidetes, Acidobacteria and Actinobacteria were the dominant bacterias in the rhizosphere soil of B.
    napus. Neither alpha diversity analysis (e.g. Shannon and Simpson index), PCoA nor MRPP analysis had revealed
    significant differences between the same developmental stage of the GM rape and control line. Meanwhile, Plancto⁃
    mycetes was less enriched in rhizosphere soil of GM rape at flowering stage compared with that of wild type. The en⁃
    richments of few bacterial genera were significantly different at 3 developmental stages of the GM rape and wild
    type. The bacterial diversity and community structure were great difference among the 3 developmental stages, espe⁃
    cially in comparison of seedling stage. In total, mEPSPS overexpression in B. napus plants caused no significant
    variation on bacterial community in rhizosphere soil. While long-term dynamic monitoring is still necessary.
  • Impacts of transgenic herbicide-tolerant soybean ZH10-6 with G2-EPSPS and GAT genes on field biodiversity
    WANG Cheng, YAO Jun-jin, GAO Yue, WANG Ya-si, XIE Mei-xia, ZHAO Xin, LAN Qing-kuo, WANG Yong
    2021, 43(1): 141. https://doi.org/10.19802/j.issn.1007-9084.2020293
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    In order to clarify the safety of transgenic herbicide tolerant soybean ZH10-6 with G2-EPSPS and
    GAT genes in terms of biodiversity, the study reports the biodiversity on arthropod in field, soybean diseases, soy⁃
    bean rhizobia and field weeds. Results showed that there were no significant differences in composition of arthropod
    community between ZH10-6 and non-transgenic control, and neither were the community structure, occurrence of
    major arthropod populations, index of major soybean diseases and number of rhizobia. In addition, the application of
    target herbicides could effectively inhibit weed growth in field. Therefore, the transgenic herbicide tolerant soybean
    ZH10-6 had no negative impact on field biodiversity. It provided data support for the safety of transgenic herbicide
    tolerant soybean ZH10-6.

  • Application of CRISPR/Cas9-mediated multiplex genome editing system in soybean genome editing
    GUAN Bei-bei, WANG Yan-juan, CHEN Hai-feng, CHEN Shui-lian, SHA Ai-hua, CAO Dong
    2021, 43(1): 149. https://doi.org/10.19802/j.issn.1007-9084.2020305
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     To apply CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associ⁃
    ated protein 9) based genome editing system on soybean functional genomics and breeding, CRISPR/Cas9-mediated
    targeted mutagenesis were obtained for more than 5 genes in soybean. One CRISPR/Cas9 plasmid conferred 6
    sgRNA expression cassettes targeting 4 Glycine max ASYMMETRIC LEAVES1 (GmAS1) genes and 3 GmAS2 genes,
    and another plasmid conferred 8 sgRNA expression cassettes targeting 6 G.max AGAMOUS1 (GmAG) genes and 5 G.
    max SHATTERPROOF1 (GmSHP1/2) genes. From the double mutagenesis of 3 GmAS1 genes and 3 GmAS2 genes, a
    phenotype of upward curling leaves and shorten leaf petiole length was discovered. From the double mutagenesis of
    2 GmSHP1/2 genes and 2 GmSTK genes, sterility phenotype was found.
  • Detection of target sites of CRISPR/Cas9 system in soybean mediated by Agrobacterium rhizogenes
    XIONG Yan-ping, XU Chong-jing, SHAN Jin-ming, ZHAO Lin
    2021, 43(1): 161. https://doi.org/10.19802/j.issn.1007-9084.2020337
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    As a new gene-editing technology, CRISPR/Cas system has been widely used in the genome engi⁃
    neering of various organisms. Soybean genetic transformation technology is time-consuming and complicated. The
    CRISPR/Cas9 gene-editing system has a certain possibility of off-target. To ensure the feasibility of selected target,
    the soybean gene GmCO was selected in this study as a target, using the CRISPR/Cas9 gene-editing system to con⁃
    struct a target gene knockout vector. And then plant tissue culture was used to promote soybean explants to grow ad⁃
    ventitious roots by cotyledon explant infection mediated by Agrobacterium rhizogenes K599 strain. The detection of
    adventitious roots showed that there was a four-base deletion at target 2, indicating that the gene-editing of soybean
    adventitious roots was successfully realized. Due to its simple operation and short cycle, this method realizes the fea⁃
    sibility of rapid identification of the selected target in soybean and provides a potential way to study soybean gene
    GmCO function and genetic improvement.
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