National Center for the Molecular Characterization of Genetically Modified Organisms, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China.
Development Center of Science and Technology, Ministry of Agriculture of People's Republic of China, Beijing, 100025, China.
Commun Biol. 2022 Jul 5;5(1):667. doi: 10.1038/s42003-022-03608-1.
Efficient, accurate molecular characterization of genetically modified (GM) organisms is challenging, especially for those transgenic events transferred with genes/elements of recipient species. Herein, we decipher the comprehensive molecular characterization of one novel GM rice event G281 which was transferred with native promoters and an RNA interference (RNAi) expression cassette using paired-end whole genome sequencing (PE-WGS) and modified TranSeq approach. Our results show that transgenes integrate at rice chromosome 3 locus 16,439,674 included a 36 bp deletion of rice genomic DNA, and the whole integration contains two copies of the complete transfer DNA (T-DNA) in a head-to-head arrangement. No unintended insertion or backbone sequence of the transformed plasmid is observed at the whole genome level. Molecular characterization of the G281 event will assist risk assessment and application for a commercial license. In addition, we speculate that our approach could be further used for identifying the transgene integration of cisgenesis/intragenesis crops since both ends of T-DNA in G281 rice were from native gene or elements which is similar with that of cisgenesis/intrasgenesis. Our results from the in silico mimicking cisgenesis event confirm that the mimic rice Gt1 gene insertion and its flanking sequences are successfully identified, demonstrating the applicability of PE-WGS for molecular characterization of cisgenesis/intragenesis crops.
高效、准确地对转基因生物进行基因特征分析具有挑战性，特别是对于那些采用受体物种基因/元件进行基因转移的转基因事件。在此，我们采用 PE-WGS 和改良 TranSeq 方法，对新型 GM 水稻事件 G281 进行了全面的分子特征分析，该事件转移了内源启动子和 RNA 干扰（RNAi）表达盒。结果表明，外源基因整合到水稻第 3 号染色体 16,439,674 位点，包含一个 36bp 的水稻基因组 DNA 缺失，整个整合体以头对头的方式包含两个完整 T-DNA 拷贝。在全基因组水平上没有观察到非预期的插入或转化质粒的骨架序列。G281 事件的分子特征分析将有助于风险评估和商业许可申请。此外，我们推测我们的方法可进一步用于鉴定顺式/内源作物的转基因整合，因为 G281 水稻中 T-DNA 的两端均来自内源基因或元件，与顺式/内源作物相似。我们对模拟顺式事件的计算机模拟结果证实，成功鉴定了模拟水稻 Gt1 基因插入及其侧翼序列，表明 PE-WGS 适用于顺式/内源作物的分子特征分析。
