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全基因组测序：转基因植物中外源基因插入信息分析的有效策略。

Whole-Genome Sequencing: An Effective Strategy for Insertion Information Analysis of Foreign Genes in Transgenic Plants.

作者信息

Wang Xu-Jing, Jiao Yue, Ma Shuo, Yang Jiang-Tao, Wang Zhi-Xing

机构信息

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences/MARA Key Laboratory on Safety Assessment (Molecular) of Agri-GMO, Beijing China.

Development Center for Science and Technology/MARA, Beijing, China.

出版信息

Front Plant Sci. 2020 Dec 1;11:573871. doi: 10.3389/fpls.2020.573871. eCollection 2020.

DOI:10.3389/fpls.2020.573871
PMID:33335534
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7736074/
Abstract

Molecular characterization is a key step in the risk assessment of genetically modified organisms (GMOs) for regulatory approval. Herein, we describe a method for analyzing copy number, insertion loci, and flanking sequences through whole-genome sequencing (WGS) and bioinformatics. Comprehensive molecular characterization of G2-6 transgenic rice was performed using this pipeline. The results showed that one copy of the foreign gene was inserted into rice chromosome 8. There was no vector backbone insertion but an unexpected insertion and DNA rearrangement at the 3' end of the T-DNA. We also obtained the 5' and 3' flanking sequences of the T-DNA. Our results suggested that the use of a combination of WGS and bioinformatics is an effective strategy for the molecular characterization of GMOs.

摘要

分子特征分析是转基因生物（GMO）进行监管审批风险评估的关键步骤。在此，我们描述了一种通过全基因组测序（WGS）和生物信息学分析拷贝数、插入位点及侧翼序列的方法。使用该流程对G2-6转基因水稻进行了全面的分子特征分析。结果表明，外源基因的一个拷贝插入到水稻第8号染色体。没有载体骨架插入，但在T-DNA的3'端存在意外插入和DNA重排。我们还获得了T-DNA的5'和3'侧翼序列。我们的结果表明，结合使用WGS和生物信息学是转基因生物分子特征分析的有效策略。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/305884f2cb40/fpls-11-573871-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/78784c9a2407/fpls-11-573871-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/157394184e9b/fpls-11-573871-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/9d7f683c68fd/fpls-11-573871-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/305884f2cb40/fpls-11-573871-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/78784c9a2407/fpls-11-573871-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/157394184e9b/fpls-11-573871-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/9d7f683c68fd/fpls-11-573871-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0b5/7736074/305884f2cb40/fpls-11-573871-g004.jpg

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Food Chem. 2020 Mar 30;309:125760. doi: 10.1016/j.foodchem.2019.125760. Epub 2019 Oct 23.
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Minimap2: pairwise alignment for nucleotide sequences.
在基于序列的转基因生物数据库的支持下，使用高通量测序技术检测和鉴定授权与未授权的转基因生物。
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Whole-genome sequencing identifies I-SceI-mediated transgene integration sites in Xenopus tropicalis snai2:eGFP line.全基因组测序鉴定 Xenopus tropicalis snai2:eGFP 品系中 I-SceI 介导的转基因整合位点。
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LIFE-Seq: a universal Large Integrated DNA Fragment Enrichment Sequencing strategy for deciphering the transgene integration of genetically modified organisms.LIFE-Seq：一种通用的大型整合 DNA 片段富集测序策略，用于破译基因修饰生物体的转基因整合。
Plant Biotechnol J. 2022 May;20(5):964-976. doi: 10.1111/pbi.13776. Epub 2022 Feb 1.
Minimap2：核苷酸序列的两两比对。
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Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane.利用液滴数字 PCR 技术测定甘蔗内源基因和转基因拷贝数。
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