转基因水稻BPL9K-2事件特异性检测方法的建立 <sup>*</sup>

doi:10.13523/j.cb.20181105

中国生物工程杂志

2018, Vol. 38

Issue (11): 32-41 DOI: 10.13523/j.cb.20181105

技术与方法

转基因水稻BPL9K-2事件特异性检测方法的建立 ^*

崔帅^1,²,王作平^1,³,于江辉¹,肖国樱^1,^**(

)

^1. 中国科学院亚热带农业生态研究所亚热带农业生态过程重点实验室长沙 410125
^2. 中国科学院大学北京 100049
^3. 北京市农林科学院北京农业生物技术研究中心北京市农业基因资源和生物技术重点实验室北京 100097

Event-specific Detection Methods of Genetically Modified Rice BPL9K-2

Shuai CUI^1,²,Zuo-ping WANG^1,³,Jiang-hui YU¹,Guo-ying XIAO^1,^**(

)

^1. Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture,Chinese Academy of Sciences, Changsha 410125, China
^2. University of Chinese Academy of Sciences, Beijing 100049, China
^3. Beijing Key Laboratory of Agricultural Gene Resources and Biotechnology, Beijing Agro-biotechnology Research Center,Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China

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摘要：

利用hiTAIL-PCR(high efficient thermal asymmetric interlaced PCR)法扩增获得了转基因水稻BPL9K-2的外源基因插入位点的左旁侧序列450bp,与水稻参考基因组数据比对发现其左边界插入在水稻基因组第10号染色体短臂的1 037 765位核苷酸残基之后。根据水稻参考基因组序列和外源基因右边界序列,设计引物扩增得到485bp的特异片段,通过数据库比对发现其右边界插入在水稻基因组第10号染色体短臂的1 037 825位核苷酸残基之前。因为外源基因插入和非正常重组,水稻基因组上缺失了59个核苷酸。基于左右旁侧序列,建立了转基因水稻BPL9K-2的事件特异性定性PCR检测方法,可以分别扩增到片段大小为449bp和485bp的特异条带。该方法特异性好,灵敏度高,能够在BPL9K-2基因组DNA相对含量为0.1%的模板中检测出转基因成分。依据旁侧序列,建立了快速鉴定转基因后代植株外源基因型的三引物PCR检测方法。这些方法的建立,为转基因水稻BPL9K-2的应用和检测提供了技术支持。

关键词： 转基因水稻; 旁侧序列; 事件特异性检测; 基因型鉴定

Abstract:

The hiTAIL-PCR (high-efficiency thermal asymmetric interlaced PCR) was adopted to study the characteristic of insertion site in genetically modified rice BPL9K-2. As a result, a 450bp fragment of left flanking sequence was discovered. By comparison with rice genome database, the insertion site of exogenous gene located on No. 1037765 of chromosome 10 was found. A 485bp fragment of right flanking sequence was amplified using the primers that were designed according to the sequence of integration site on rice genome and right sequence of exogenous gene. The event-specific PCR detection method was developed based on the left and right flanking sequences, which produced 449bp and 485bp fragment respectively in genetically modified rice BPL9K-2, specifically. The event-specific PCR detection method, with high specificity and sensitivity, could detect the genetically modified ingredients in samples containing 0.1% genomic DNA of BPL9K-2. Based on the flanking sequence, a tri-primer PCR method was developed to identify its genotype of exogenous gene in segregation generation quickly and accurately. The above methods established in this research provide technical supports for the utilization and detection of genetically modified rice BPL9K-2.

Key words: Genetically modified rice Flanking sequence Event-specific detection Genotyping

收稿日期: 2018-04-26 出版日期: 2018-12-06

ZTFLH:

Q812

基金资助: * 转基因生物新品种培育科技重大专项资助项目(2016ZX08001003-001)

通讯作者: 肖国樱 E-mail: xiaoguoying@isa.ac.cn

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引用本文:

崔帅,王作平,于江辉,肖国樱. 转基因水稻BPL9K-2事件特异性检测方法的建立 ^*[J]. 中国生物工程杂志, 2018, 38(11): 32-41.

Shuai CUI,Zuo-ping WANG,Jiang-hui YU,Guo-ying XIAO. Event-specific Detection Methods of Genetically Modified Rice BPL9K-2. China Biotechnology, 2018, 38(11): 32-41.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181105 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I11/32

表1 hiTAIL-PCR和事件特异性PCR检测的引物序列

图1 转基因水稻BPL9K-2左旁侧序列hiTAIL-PCR扩增结果

图2 BPL9K-2左旁侧序列及拼接位置特征

图3 BPL9K-2右旁侧序列及拼接位置特征

图4 转基因水稻BPL9K-2的T-DNA插入位置

图5 左右旁侧序列的事件特异性PCR检测

图6 左右旁侧序列的事件特异性检测方法检测限度测定

图7 左右旁侧序列的事件特异性检测 PCR扩增产物相对含量

图8 转基因水稻BPL9K-2基因型鉴定

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