Welcome to Chinese Journal of Tropical Crops,

Chinese Journal of Tropical Crops ›› 2021, Vol. 42 ›› Issue (9): 2468-2477.DOI: 10.3969/j.issn.1000-2561.2021.09.005

• Omics & Biotechnology • Previous Articles     Next Articles

Analysis of the T-DNA Flanking Sequence and Event-specific Detection for Insect-resistant Transgenic Sugarcane BtG-2

FENG Cuilian1, WAN Yue2, FENG Xiaoyan1, WANG Jungang1, ZHAO Tingting1, WANG Wenzhi1, SHEN Linbo1, ZHANG Shuzhen1,2,*()   

  1. 1. Institute of Tropical Bioscience and Biotechnology, Sugarcane Research Center, Chinese Academy of Tropical Agricultural Sciences / Key Biotechnology Laboratory for Tropical Crops, Ministry of Agriculture and Rural Affairs, Haikou, Hainan 571101, China
    2. College of Life Science, Nanjing Agriculture University, Nanjing, Jiangsu 210095, China
  • Received:2020-08-28 Revised:2020-12-18 Online:2021-09-25 Published:2021-11-01
  • Contact: ZHANG Shuzhen

Abstract:

Sugarcane BtG-2 is an insect resistance transgenic sugarcane strain, developed by introducing the Cry1Ac- 2A-gna fusion gene into ‘ROC22’ with the Agrobacterium-mediated method. It has strong insect resistance and excellent agronomic traits. In order to clarify the molecular characteristics and detection of transgenic sugarcane BtG-2, and promote biological safety evaluation, the T2 generation of BtG-2 was selected, and the copy number of foreign genes in the transgenic sugarcane genome was detected by Southern hybridization. The flanking sequence of the insertion site of the foreign gene was isolated using the chromosome walking technology, and an efficient specific PCR detection method of the strain was established. The results showed that the foreign T-DNA insertion of BtG-2 strain was a single copy. After three times amplifications of thermal asymmetric interlaced PCR, 984 bp of the left flanking sequence and 705 bp of the right flanking sequence of the foreign gene T-DNA were obtained. According to the flanking sequences, three pairs of detection primers were designed respectively, then the event-specific PCR detection for transgenic sugarcane BtG-2 was established. The primer pairs with the highest amplification efficiency were LS011/LA451 and RS160/RA588, with 440 bp and 428 bp specific amplified fragments respectively. Among them, the pair of primers LS011/LA451 designed on the left side of T-DNA had high sensitivity and specificity for detection, and this method could detect the genetically modified ingredients in samples containing 0.1% genomic DNA of sugarcane BtG-2. This study completed the molecular characteristics and event-specific detection of the transgenic strain BtG-2, which provided a technical basis for the detection and identification of the transgenic sugarcane and its derivatives.

Key words: Insect-resistant transgenic sugarcane, T-DNA flanking sequence, event-specific detection, chromosome walking

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