Crops ›› 2016, Vol. 32 ›› Issue (1): 69-75.doi: 10.16035/j.issn.1001-7283.2016.01.013

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Analysis of the Flanking Sequence and Event-Specific Detection of Transgenic G2-EPSPS Line of Maize

Guo Cui1,2,Zhang Wei2,Yu Guirong3,Zhou Zhengfu2,Li Liang2,Feng Shuai1,2,Chen Ming2,Wang Jin1,2   

  1. 1 Life Science and Engineering College,Southwest University of Science and Technology,Mianyang 621010,Sichuan,China
    2 Biotechnology Research Institute,Chinese Academy of Agriculture Sciences,Beijing 100081,China
    3 Institute of Biological & Nuclear Technology,Sichuan Academy of Agricultural Sciences,Chengdu 610066,Sichuan,China;
  • Received:2015-10-20 Revised:2015-12-04 Online:2016-02-15 Published:2018-08-25
  • Contact: Jin Wang

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Abstract:

By hiTAIL-PCR(3'-terminal high-efficiency thermal asymmetric interlaced PCR) and LD-PCR(long distance PCR),the complete sequence and flanking sequence of T-DNA in transgenic maize D-3 were obtained. A specific PCR detection method for transformant was also established. The results revealed that the total length of T-DNA was 4318 bp,which consisted of a G2-EPSPS gene expression cassette. The primers had been designed based on the 3' and 5' terminal flanking sequence,followed by specific PCR to detect the sensitivity as well as the detection limit. The results showed that 3'-qualitative PCR detection method was highly specific and sensitive,with a sensitivity of 0.05% per 100ng DNA template.This research provides the methods for the detection of exogenous gene in transgenic maize and the risk assessment on bio-safety.

Key words: Transgenic maize, G2-EPSPS, Flanking sequence, Event-specific detection

Table 1

The degenerate prime AD1-n and AD-C used in hiTAIL-PCR"

引物Primer 引物序列（5'-3'）Primer sequence（5'-3'）
AD1-1
AD1-2
AD1-3
AD1-4
AD-C		 ACGATGGACTCCAGAGCGGCCGCVNVNNNGGAA
ACGATGGACTCCAGAGCGGCCGCBNBNNNGGTT
ACGATGGACTCCAGAGCGGCCGCVVNVNNNCCAA
ACGATGGACTCCAGAGCGGCCGCBDNBNNNAGGT
ACGATGGACTCCAGAG

Fig.1

Amplification and verification of the flanking sequence A: 3'-terminal hiTAIL-PCR of transgenic maize D-3; M: Trans 2K plusⅡ marker; 1-3: The result of 3' terminal hiTAIL-PCR amplification; B: The 3' terminal flanking sequence; The black body part is the T-DNA sequence; The italic part is 3' terminal flanking sequence; C: The 5' terminal flanking sequence; The black body part is the T-DNA sequence; the italic part is 5' terminal flanking sequence; D: Verificate flanking sequence by PCR; M:Trans 2k plusⅡDNA Marker; 1: The amplification products of LB-F1/LB-R;2: The amplification products of LB-F2/LB-R;3: The amplification products of LB-F3/LB-R; 4: The amplification products of RB-F1/RB-R; E: The putative transgenic structures of integrated structure and the primer design sketch"

Fig.2

Specificity detection with different primers by PCR M: Trans 2k plusⅡDNA Marker; 1: D-3; 2: 14sT133; 3: 255; 4: Negative control; 5: Blank."

Fig.3

Sensitivity detection for event-specific PCR M: Trans 2k plus ⅡDNA Marker; 1: Blank; 2: Negative control; 3-10: The content of DNA template: 100.00%, 50.00%, 10.00%, 5.00%, 1.00%, 0.50%, 0.10%, 0.05%."

Fig.4

The limit test for the event-specific detection A,B,and C.The amplification result of samples containing 0.10%,0.05%,and 0.01%,respectively; M: Trans 2k plus ⅡDNA Marker; 1~10: 10 paralles."

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