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作物学报

作物学报 ›› 2020, Vol. 46 ›› Issue (11): 1703-1710.doi: 10.3724/SP.J.1006.2020.02002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征

马硕1(), 焦悦2(), 杨江涛1, 王旭静1,*(), 王志兴1,*()   

  1. 1 中国农业科学院生物技术研究所 / 农业农村部农业转基因生物安全评价(分子)重点实验室, 北京100081
    2 农业农村部科技发展中心, 北京 100122
  • 收稿日期:2020-01-14 接受日期:2020-06-02 出版日期:2020-11-12 网络出版日期:2020-06-22
  • 通讯作者: 王旭静,王志兴
  • 作者简介:马硕, E-mail:mashuo0801@163.com|焦悦, E-mail:jiaoyue@agri.gov.cn
  • 基金资助:
    本研究由国家转基因新品种培育重大专项(2016ZX08010-003)

Molecular characterization identification by genome sequencing of transgenic glyphosate-tolerant rice G2-7

MA Shuo1(), JIAO Yue2(), YANG Jiang-Tao1, WANG Xu-Jing1,*(), WANG Zhi-Xing1,*()   

  1. 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences / MARA Key Laboratory on Safety Assessment Molecular of Agri-GMO, Beijing 100081, China
    2 Development Center for Science and Technology / MARA, Beijing 100122, China
  • Received:2020-01-14 Accepted:2020-06-02 Published:2020-11-12 Published online:2020-06-22
  • Contact: Xu-Jing WANG,Zhi-Xing WANG
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX08010-003)

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摘要：

外源DNA片段的拷贝数及插入位点的侧翼序列等分子特征信息是转基因植物安全评价过程中必需要提供的信息。本研究利用基因组测序结合生物信息学对耐除草剂转基因水稻G2-7的T-DNA插入位点、拷贝数和侧翼序列进行鉴定。利用Illumina NovaSeq 6000平台对G2-7进行全基因组测序, 共获得47.13 Gb的测序数据, 通过与转基因载体和参考基因组序列的比较, 确定了G2-7中T-DNA在受体基因组中的插入位点。结果显示, 外源DNA片段以单位点单拷贝形式插入到水稻1号染色体的36,189,491~36,189,507位置, 造成水稻基因组16 bp DNA缺失, 无载体骨架的插入。同时我们获得外源基因插入位点5′侧翼序列375 bp和3′端侧翼序列353 bp, 并通过PCR扩增和Sanger测序进一步证明获得的侧翼序列是正确的。研究结果为转基因水稻G2-7的安全评价及转化体特异性检测提供了有效的数据支撑, 同时也证明全基因组测序(WGS)是解析转基因植物分子特征的有效方法。

关键词: 基因组测序, 转基因水稻, 分子特征, 拷贝数, 侧翼序列

Abstract:

Molecular characterization, such as copy number and flanking sequence of foreign DNA fragment insertion site, is the important identity information, provided during safety assessment of genetic modified crop. In this study, the T-DNA insertion site, copy number and flanking sequences were identified in transgenic glyphosate-tolerant rice G2-7 based on whole genome sequencing in combination bioinformatics analysis method. 47.13 Gb clean sequence data for G2-7 was generated on Illumina NovaSeq 6000 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in G2-7 were identified by comparing with sequence of transformation vector and rice reference genome. The results showed that exogenous T-DNA fragments was integrated in the position of Chr. 1 36,189,491-36,189,507 with a single copy, 16 bp rice genome sequence was deleted at the insertion site and no insertion of vector backbone. 375 bp and 353 bp flanking host DNA sequence of 5′-end and 3′-end of the insertion DNA fragment were also obtained, respectively. The putative insertion location and flanking sequences were further confirmed by PCR amplification and Sanger sequencing. The results not only provided data support for safety assessment and event specific detection, but also demonstrated that WGS was an effective technique for identifying molecular characterization in rice.

Key words: genome sequencing, transgenic rice, molecular characterization, copy number, flanking sequence

表1

高通量测序数据质量控制统计"

样品名称
Sample		 原始数据量
Raw bases		 原始读序量
Raw reads		 有效数据量
Clean bases		 有效读序量Clean reads Q20
(%)		 Q30
(%)		 GC含量
GC content (%)
ZH11 41,196,003,900 274,646,422 41,155,766,400 274,371,776 96.22 90.36 43.04
ZH11-p 41,266,820,400 275,112,136 41,228,215,500 274,854,770 97.73 93.69 42.93
G2-7 47,246,859,300 314,990,174 47,125,680,000 314,171,200 96.58 91.15 44.27

图1

G2-7中外源插入片段与受体基因组结合位点分析(部分结合区序列的比对结果)"

图2

测序数据与质粒DNA比对结果的可视化"

图3

载体骨架匹配读序的PCR验证 1: G2-7; 2: ZH11; 3: p13UG2."

图4

外源DNA片段在受体基因组中的整合位点及侧翼序列分析 A: G2-7转化体侧翼序列和整合位点分析; B: G2-7转化体插入序列整合情况示意图。"

图5

PCR电泳图及序列比对 A: 水稻G2-7转化体侧翼序列扩增; 1: G2-7-5F/5R; 2: ZH11-5F/5R; 3: G2-7-3F/3R; 4: ZH11-3F/3R。B: G2-7 5′端序列比对验证。C: G2-7 3′端序列比对验证。"

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