Journal of Integrative Agriculture ›› 2014, Vol. 13 ›› Issue (9): 1865-1876.DOI: 10.1016/S2095-3119(13)60628-0

• 论文 • 上一篇    下一篇

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

 WU Yu-hua, ZHANG Li, WU Gang, NIE Shu-jing , LU Chang-ming   

  1. 1、Oilcrops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,
    Ministry of Agriculture, Wuhan 430062, P.R.China
    2、College of Life Science, South-Central University for Nationalities,Wuhan 430074, P.R.China
  • 收稿日期:2013-05-08 出版日期:2014-09-22 发布日期:2014-09-24
  • 通讯作者: LU Chang-ming, Tel: +86-27-86728186, Fax: +86-27-86711573, E-mail: cmlu@oilcrops.cn
  • 作者简介:WU Yu-hua, Tel: +86-27-86711501, E-mail: wuyuhua@oilcrops.cn; ZHANG Li, E-mail: zhangli0624@gmail.com;
  • 基金资助:

    This work was supported by the grant from the National Major Special Project for the Development of Transgenic Organisms, China (2013ZX08012-003 and 2011ZX08012-005) and the Special Funds of the State Environmental Protection Public Welfare Industry, China (201109028).

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

 WU Yu-hua, ZHANG Li, WU Gang, NIE Shu-jing , LU Chang-ming   

  1. 1、Oilcrops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops,
    Ministry of Agriculture, Wuhan 430062, P.R.China
    2、College of Life Science, South-Central University for Nationalities,Wuhan 430074, P.R.China
  • Received:2013-05-08 Online:2014-09-22 Published:2014-09-24
  • Contact: LU Chang-ming, Tel: +86-27-86728186, Fax: +86-27-86711573, E-mail: cmlu@oilcrops.cn
  • About author:WU Yu-hua, Tel: +86-27-86711501, E-mail: wuyuhua@oilcrops.cn; ZHANG Li, E-mail: zhangli0624@gmail.com;
  • Supported by:

    This work was supported by the grant from the National Major Special Project for the Development of Transgenic Organisms, China (2013ZX08012-003 and 2011ZX08012-005) and the Special Funds of the State Environmental Protection Public Welfare Industry, China (201109028).

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摘要： To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border (RB) and left border (LB) regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head (RB-to-RB) concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.

关键词: transgenic rapeseed , junction fragment , pre-insertion site , DNA rearrangement

Abstract: To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border (RB) and left border (LB) regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head (RB-to-RB) concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.

Key words: transgenic rapeseed , junction fragment , pre-insertion site , DNA rearrangement