Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method

A technology of transgenic rice and flanking sequences, which is used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2010-09-08
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country has not yet approved the commercial production of genetically modified rice, but some varieties (strains) have been approved for environmental release and productive experiments

Method used

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  • Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method
  • Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method
  • Flanking sequence of transgenic rice Kefeng No. 6 and qualitative PCR detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning of flanking sequences of transgenic rice line Kefeng No. 6 exogenous insertion vector

[0029] 1. Experimental materials

[0030] 1. Plant material: transgenic rice Kefeng 6 and its receptor variety Minghui 86, provided by Fujian Academy of Agricultural Sciences;

[0031] 2. Reagents: Taq DNA polymerase, 10×PCR Buffer (100mM Tris-HCl pH8.3, 500mMKCl, 15mM MgCl 2), dNTP were purchased from Treasure Bioengineering Dalian Co., Ltd.; 100bp ladder DNA Marker was purchased from Beijing Quanshijin Biotechnology Co., Ltd. PCR product purification kit (Cat.NO.SK1141) and PCR product cloning kit (Cat.NO.SK2213) were purchased from Shanghai Sangon Bioengineering Co., Ltd. Primer synthesis and clone sequencing were completed by Invitrogen. Other biochemical reagents are imported subpackages or domestic analytical pure.

[0032] 3. Experimental instrument: PCR amplification instrument: PTC-200 (produced by Bio-Rad)

[0033] Biological spectrophotometer 6131 (...

Embodiment 2

[0054] Example 2: Line-specific qualitative PCR detection based on two exogenous insertion flanking sequences of the transgenic rice line Kefeng 6

[0055] 1. Experimental materials: transgenic rice line Kefeng 6 and its derivative variety II Youkefeng 6 were provided by Fujian Academy of Agricultural Sciences; other transgenic rice Huahui 1 and Bt63 were provided by Huazhong Agricultural University; The non-transgenic rice pairs Zhaominghui 86 and II Youming 86 were provided by Fujian Academy of Agricultural Sciences, Xianyou 63 was provided by Huazhong Agricultural University, and Xianyou 10 was provided by Zhejiang University. The enzymes and reagents used in the experiment are the same as in Example 1.

[0056] 2. Experimental process and methods:

[0057] 1. Extraction and detection of DNA: same as Example 1.

[0058] 2. Specific detection based on two different insertion sites: According to the two sequences determined in Example 1, two pairs of PCR primers were design...

Embodiment 3

[0064] Example 3: Sensitive detection of strain-specific detection

[0065] 1. Experimental materials: transgenic rice line Kefeng 6 and control recipient variety Minghui 86. The enzymes and reagents used in the experiment are the same as in Example 1.

[0066] 2. Experimental process and methods:

[0067] 1. Extraction and detection of DNA. After grinding the seeds of Kefeng 6 and its non-transgenic control recipient Minghui 86 into powder, a series of mixed samples were prepared. The content of Kefeng 6 in each sample was 10% and 5.0% respectively. , 2.5%, 1.25%, 0.5%, 0.1%, 0.05% and 0.01% (W / W), after mixing, the DNA extraction method is the same as in Example 1.

[0068] 2. Sensitivity detection based on two different insertion sites: the primers used are shown in Table 4, and the PCR reaction system is 1×PCR buffer, 200umol / L dNTP, 0.2umol / L specific primer, and 0.025U / uLTaq enzyme. The reaction program was 95°C, 5min; 35 cycles of 94°C for 30s, 59°C for 45s, and 72°C...

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Abstract

The invention provides a flanking sequence of transgenic rice Kefeng No. 6, which comprises a carrier RB side flanking sequence and a carrier LB side flanking sequence, wherein the RB side flanking sequence is a sequence shown by SEW ID NO.1, and the LB side flanking sequence is a sequence shown by SEQ ID No.2. The invention provides other two copied flanking sequences of Kefeng No. 6 and a conversion event specificity detection method established on the basis of the two copied flanking sequences, and provides importance data for the molecular characteristics of the transgenic rice Kefeng No. 6.

Description

(1) Technical field [0001] The invention relates to a side sequence of the transgenic rice Kefeng 6 and a qualitative PCR detection method of the transgenic rice Kefeng 6. (2) Background technology [0002] Rice (Oryza sativa) is a major food crop, and it is also an early successful application of transgenic technology for genetic improvement. Transgenic rice varieties containing different traits have been bred so far. my country has not yet approved the commercial production of genetically modified rice, but some varieties (strains) have been approved for environmental release and productive experiments. Effective supervision of genetically modified crops is the prerequisite and guarantee for the safe use of genetically modified crops. The flanking sequence of the exogenous insert in a transgenic plant is one of the most important molecular characteristics of a transgenic plant line. Therefore, the flanking sequence of the exogenous insert is an important technical data f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 王渭霞
Owner CHINA NAT RICE RES INST
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