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

doi:10.13523/j.cb.20181105

China Biotechnology

2018, Vol. 38

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

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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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

Received: 26 April 2018 Published: 06 December 2018

ZTFLH:

Q812

Corresponding Authors: Guo-ying XIAO E-mail: xiaoguoying@isa.ac.cn

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Cite this article:

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.

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

Table 1 The primer sequence for hiTAIL-PCR and event-specific PCR

Fig.1 The hiTAIL-PCR results of left flanking sequence of BPL9K-2 M: DL 2000 DNA marker; 1: The secondary TAIL-PCR products of 9K19-5 (AC1/LB-1a); 2: The tertiary TAIL-PCR products of 9K19-5 (AC1/LB-2a); 3: The secondary TAIL-PCR products of BPL9K-2 (AC1/LB-1a); 4: The tertiary TAIL-PCR products of BPL9K-2 (AC1/LB-2a); LAD1-LAD4: Arbitrary primers

Fig.2 The left flanking sequence of BPL9K-2 and characteristics of junction region(a) The left flanking sequence of BPL9K-2. The underlined part is the left sequence of T-DNA (235-450bp), and the rest is rice genome sequence (b) Characteristics of junction region at the left border Vector: The left sequence of T-DNA including its left border; BPL9K-2: The obtained left flanking sequence of BPL9K-2; AP014966.1: The rice genomic sequence on chromosome 10 where the T-DNA was inserted into; The arrowheads indicate the splicing site of T-DNA and rice genome

Fig.3 The right flanking sequence of BPL9K-2 and characteristics of junction region(a) The right flanking sequence of BPL9K-2. The underlined part is the right sequence of T-DNA (310-485bp), and the rest is rice genome sequence (b) Characteristics of junction region at the right border Vector: The right sequence of T-DNA including its right border; BPL9K-2: The obtained right flanking sequence of BPL9K-2; AP014966.1: The rice genomic sequence on chromosome 10 where the T-DNA was inserted into; The arrowheads indicate the splicing site of T-DNA and rice genome

Fig.4 The insertion site of T-DNA in genome of genetically modified rice BPL9K-2

Fig.5 Event-specific qualitative PCR detection based on the left and the right flanking sequence(a) Event-specific qualitative PCR detection based on the left flanking sequence of BPL9K-2 (b) Event-specific qualitative PCR detection based on the right flanking sequence of BPL9K-2 M: DL 2000 DNA marker; B: Blank control; 3: Genetically modified rice BPL9K-2; 1, 2, 6-11: Conventional rice 9K19-1, 9K19-5, 11C2277, 11C2292, 7001S, Guangzhan63S, Ke108A, and R106; 4, 5, 12-17: Genetically modified rice BPL9K-1, BPL9K-4, EB7001S-5, B1C893, EB185BS, B2A68, BarKasalath-01, and Bar9K29-9-2

Fig.6 Detection limit of event-specific detection method based on the left and the right flanking sequences(a) Detection limit of event-specific detection method based on the left flanking sequences (b) Detection limit of event-specific detection method based on the right flanking sequence M: DL 2000 DNA marker; 0: Blank control; CK: Non-transgenic rice 9K19-5; 100%, 20%, 5%, 2%,1%, 0.5%, and 0.1%: The relative content of genetically modified BPL9K-2 genomic DNA in the template (V/V)

Fig.7 The relative content of PCR amplification products of event-specific detection based on the left and the right flanking sequences(a)The relative content of PCR amplification products of event-specific detection based on the left flanking sequences (b) The relative content of PCR amplification products of event-specific detection based on the right flanking sequences Marker: The relative content of DNA in 750bp brand of DL 2000 marker; 0: Blank control; CK: Non-transgenic rice 9K19-5; 100%, 20%, 5%, 2%, 1%,0.5% , and 0.1 %: The relative content of genomic DNA of genetically modified rice BPL9K-2 in the template (V/V)

Fig.8 Genotyping of genetically modified rice BPL9K-2 M: DL 2000 marker; 0: Blank control; CK: Non-transgenic rice 9K19-5; 1: Homozygote of genetically modified rice BPL9K-2; 2: Heterozygote of genetically modified rice BPL9K-2


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