LIU Dong-bo,XING Guo-jie,ZHAO Qian-qian,et al.Efficient Identification of T-DNA Insertions and Qualitative Analysis in Two Virus-resistant Transgenic Soybean Events[J].Soybean Science,2020,39(01):23-29.[doi:10.11861/j.issn.1000-9841.2020.01.0023]
抗病毒转基因大豆事件外源T-DNA序列分析及定性检测
- Title:
- Efficient Identification of T-DNA Insertions and Qualitative Analysis in Two Virus-resistant Transgenic Soybean Events
- Keywords:
- Insertion site; Flanking sequence; Whole genome sequencing; Virus-resistant transgenic soybean; Time-specific detection
- 文献标志码:
- A
- 摘要:
- 转基因作物的T-DNA插入及其与宿主基因组的连接序列构成的侧翼区域，是转基因事件检测的关键组成部分，也是转基因作物开发、评估和监管所必需的。前期研究得到2个携带大豆花叶病毒P3（soybean mosaic virus P3）基因干扰片段的转基因大豆事件，即L13和L104，可显著提高大豆对大豆花叶病毒、大豆花叶坏死病毒和西瓜花叶病毒的抗性，具有较强的光谱性。为了便于对该转基因事件进行监管以及建立事件特异性检测方法，本研究通过Illumina Xten平台，对L13和L104进行全基因组测序（whole genome sequencing，WGS），并结合生物信息学分析和PCR技术，对转基因事件L13和L104的旁侧序列进行了分析。通过全基因组测序，每一个事件获得了超过12.13 Gb的原始数据，测序深度为11×，与大豆参考基因组(Wm82.a2.v1)和转化载体序列做比较，初步识别出这两个转化事件T-DNA的边界及其旁侧序列。转化事件L13和L104的T-DNA分别位于Chr04和Chr11染色体上。进一步的PCR检测和序列测定表明转基因事件L13的T-DNA为单位点双拷贝整合到大豆基因组Chr04:46747946位点；转基因事件L104的T-DNA为单拷贝插入，整合位点为Chr11:30461895位点。结果证明了全基因组测序在识别转基因作物T-DNA插入和旁侧序列区域方面的有效性和稳定性。此外，插入位点的鉴定和事件特异性检测的建立将有助于广谱抗病毒转基因大豆品种的应用和发展。
- Abstract:
- Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported two transgenic soybean events, that were L13 and L104, harboring the reverted repeats of soybean mosaic virus P3 gene, exhibiting significantly enhanced and broad-spectrum resistance to soybean mosaic virus, soybean mosaic necrosis virus, watermelon mosaic virus. To facilitate safety assessment and event-specific detection establishment, we identified the transgenic insertion sites and flanking sequences of the event L13 and L104 using whole genome sequencing(WGS) through Illumina Xten platform combined with bioinformatics analysis and PCR method. More than 12.13 Gb sequence data (11 coverage) were generated of each event through WGS. The sequence reads corresponding to boundaries of inserted T-DNA and associated native flanking sequences were identified by comparison with soybean reference genome(Wm82.a2.v1) and sequence of the transformation vector. Inserted T-DNAs were mapped on Chromosome 04 and 07 for the events L13 and L104, respectively. The putative insertion loci and flanking regions were further confirmed by PCR amplification and sequencing. The T-DNAs of the transgenic event L13 were two copies inserted in Chr04:46747946 site. And the T-DNA of the L104 was inserted in Chr11:30461895 with one copy. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established with specific PCR primers. Our results demonstrate the efficacy and robustness of whole genome sequencing in identifying the T-DNA insertions and flanking regions of GM crops. Moreover, characterization of insertion loci and establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.
参考文献/References:
［1］Latham J R, Wilson A K, Steinbrecher R A. The mutational consequences of plant transformation［J］. Journal of Biomedicine and Biotechnollogy, 2006(2): 25376.［2］ Nacry P, Camilleri C, Courtial B, et al. Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis［J］. Genetics, 1998, 149(2): 641-650.［3］ Wilson A K, Latham J R, Steinbrecher R A. Transformation-induced mutations in transgenic plants: Analysis and biosafety implications［J］. Biotechnolnoly and Genetic Engineering Reviews, 2006, 23(1):209-238.［4］ Windels P, Sylvie D B, Erik V B, et al. T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences［J］. Plant Physiology, 2003, 133(4): 2061-2068.［5］ Codex Alimentarius Commission. Guide line for the conduct of food safety assessment of foods derived from recombinant-DNA plants［S］. FAO/WHO Joint Publications, 2003:1-14. 〖HJ1.5mm〗［6］ European Food Safety Authority Panel on Genetically Modified Organisms. Guidance on the environmental risk assessment of genetically modified plants［J］. European Food Safety Authority Journal,2010, 8(11):1879. ［7］ Kok E J, Pedersen J, Onori R, et al. Plants with stacked genetically modified events: To assess or not to assess［J］. Trends Biotechnology, 2014, 32(2): 70-73. ［8］ Ochman H, Gerber A S, Hartl D L. Genetic applications of an inverse polymerase chain reaction［J］. Genetics, 1998, 120(3): 621-623.［9］ Liu Y G, Mitsukawa N, Oosumi T, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR［J］. Plant Journal, 8(3):457-463. ［10］O′Malley R C, Ecker J R. Linking genotype to phenotype using the Arabidopsis unimutant collection［J］. Plant Journal, 61(6): 928-940.［11］Ji J B, Braam J. Restriction site extension PCR: A novel method for high-throughput characterization of tagged DNA fragments and genome walking［J］. PLoS ONE, 2010, 5: e10577.［12］Spalinskas R, Bulcke M V D, Eede G V D, et al. LT-RADE: An efficient user-friendly genome walking method applied to the molecular characterization of the insertion site of genetically modified maize MON810 and rice LLRICE62［J］. Food Analytical Method, 2013, 6(2): 705-713.［13］Babekova R, Funk T, Pecoraro S, et al. Development of an event-specific real-time PCR detection method for the transgenic Bt rice line KMD1［J］. European Food Research and Technology, 2009, 228(5): 707-716.［14］Milcamps A, Rabe S, Cade R, et al. Validity assessment of the detection method of maize event Bt10 through investigation of its molecular structure［J］. Journal of Agricural and Food Chemistry, 2009, 57(8):3156-3163. ［15］Wang J, Wang W, Li R, et al. The diploid genome sequence of an Asian individual［J］. Nature, 2008, 456(7218): 60-65.［16］Ajay S S, Parker S C, Abaan H O, et al. Accurate and comprehensive sequencing of personal genomes［J］. Genome Research, 2011, 21(9): 1498-1505. ［17］Lepage E, Zampini E, Boyle B, et al. Time and cost-efficient identification of T-DNA insertion sites through targeted genomic sequencing［J］. PLoS One, 2013, 8(8): e70912.［18］Inagaki S, Henry I M, Lieberman M C, et al. High-throughput analysis of T-DNA location and structure using sequence capture［J］. Plos One, 2015, 10(10): e0139672.［19］Wahler D, Schauser L, Bendiek J, et al. Next-generation sequencing as a tool for detailed molecular characterisation of genomic insertions and flanking regions in genetically modified plants: A pilot study using a rice event unauthorised in the EU［J］. Food Analytical Methods, 2013, 6(6): 1718-1727.［20］Park D, Kim D, Jang G, et al. Efficiency to discovery transgenic loci in GM rice using next generation sequencing whole genome re-sequencing［J］. Genomics & Informatics, 2015, 13(3):81. ［21］Williams-Carrier R, Stiffler N, Belcher S, et al. Use of illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy mutator lines of maize［J］. The Plant Journal, 2010, 63(1):167.［22］Kovalic D. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology［J］. Plant Genome, 2012, 5(3):149-163.［23］Guo B, Yong G, Hong H, et al. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genomes equencing method［J］. Frontiers in Plant Science, 2016, 7: 1009.［24］Yang X D, Niu L, Zhang W, et al. RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean［J］. Plant Cell Reports, 2018, 37(1): 103-114.［25］Porebski S, Bailey L G, Baum B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components［J］. Plant Molecular Biology Reporter, 1997, 15(1): 8-15.［26］马阔, 仲晓芳, 牛陆, 等. 耐盐转基因大豆FA8015旁侧序列分离及定性PCR检测［J］. 大豆科学, 2018, 37（6）:854-859.（Ma K, Zhong X F, NIU L, et al. Sequence separation and qualitative PCR detection of salt-tolerant transgenic soybean FA8015［J］. Soybean Science, 2018, 37(6): 854-859.）［27］仲晓芳, 杨静, 贺红利, 等. 基于基因组重测序的高含量油酸转基因大豆T-DNA旁侧序列分析及事件特异性PCR检测［J］. 农业生物技术学报, 2018, 26（12）:2017-2026.（Zhong X F, Yang J, He H L, et al. Sequence analysis and event specific PCR detection of high content oleic acid transgenic soybean T-DNA based on genomic resequencing［J］. Journal of Agricultural Biotechnology, 2018, 26(12): 2017-2026.）［28］Langmead B and alzberg S L. Fast gapped-read alignment with Bowtie 2［J］. Nature Methods, 2012, 9:357-359.［29］Yang L T, Wang C M, Jensen A H, et al. Characterization of GM events by insert knowledge adapted re-sequencing approaches［J］. Scientific Reports, 2013, 3:2839. ［30］Campbell P J, Stephens P J, Pleasance E D, et al. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing［J］. Nature Genetics, 40(6):722-729. ［31］Hormozdiari F, Hajirasouliha I, Mcpherson A, et al. Simultaneous structural variation discovery among multiple paired-end sequenced genomes［J］. Genome Research, 2011, 21(12):2203-2212. ［32］DuBose A J, Lichtenstein S T, Narisu N, et al. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene［J］. Nucleic Acids Research, 2013, 41(6):e70. ［33］Kovalic D, Garnaat C, Guo L, et al. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology［J］. The Plant Genome, 2012, 5(3): 149-163.［34］Urbanski D F, Malolepszy A, Stougaard J, et al. Genome wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus［J］. The Plant Journal, 2012, 69(4):731-741.［35］Daniela W, Leif S, Joachim B, et al.Next-generation sequencing as a tool for detailed molecular characterization of genomic insertions and flanking regions in genetically modified plants: A pilot study using a rice event unauthorized in the EU［J］. Food Analytical Method, 2013, 6(6):1718-1727. ［36］Pauwels K, DeKeersmaecker S C J, DeSchrijver A, et al.Next-generation sequencing as a tool for the molecular characterization and risk assessment of genetically modified plants: Add value or not?［J］. Trends in Food Science and Technology, 2015, 45(2):319-326.
相似文献/References:
[1]马阔,仲晓芳,牛陆,等.耐盐转基因大豆事件FA8015旁侧序列分离及定性PCR检测 [J].大豆科学,2018,37(06):854.[doi:1011861/jissn1000-98412018060854]
MA Kuo,ZHONG Xiao-fang,NIU Lu,et al.Flanking Sequence Isolation of Salt-Tolerant Transgenic Event FA8015 of Soybean and Quantitative PCR Test [J].Soybean Science,2018,37(01):854.[doi:1011861/jissn1000-98412018060854]
[2]闫伟,马月,谢彦博,等.大豆转化体E8A7027外源T-DNA分析及特异性检测体系建立[J].大豆科学,2023,42(05):579.[doi:10.11861/j.issn.1000-9841.2023.04.0579]
备注/Memo
?收稿日期：2019-06-19