Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof

A herbicide-tolerant, G6H1 technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Inactive Publication Date: 2014-07-16
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there is a lack of a qualitative PCR detection method with strong specificity and h

Method used

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  • Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof
  • Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof
  • Qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1 Primer screening and optimization of PCR reaction conditions for the qualitative PCR detection method of transgenic insect-resistant and herbicide-resistant rice G6H1 and its derivatives

[0049] 1. Primer design and screening

[0050] According to the insertion sequence of G6H1 rice ( figure 1 ) to design primers, design 8 primers according to the side sequence of the 5' end of the inserted sequence, design 9 primers according to the side sequence of the 3' end of the inserted sequence, and pair the above primers respectively, and the length of the target sequence is between 100 and 400 bp , the primer sequences are shown in Table 1.

[0051] Table 1 Design primer information table

[0052]

[0053] G6H1 rice and non-transgenic rice DNA with mass fractions of 100%, 1%, and 0.1% were used as templates, and double-distilled water was used to replace the template of the PCR reaction system in the blank control. Conventional PCR reaction conditions and react...

Embodiment 2

[0061] Example 2 Establishment of Qualitative PCR Reaction System and Reaction Program of Transgenic Insect-resistant and Herbicide-resistant Rice G6H1 and Its Derivatives

[0062] According to the Tm value of the detection primer screened in Example 1 and the stability of the amplification result, determine the qualitative PCR reaction system (see Table 3) and reaction program of the transgenic insect-resistant and herbicide-resistant rice G6H1 and its derivative varieties: denaturation at 94°C for 5 minutes ; Denaturation at 94°C for 30s, annealing at 58°C for 45s, extension at 72°C for 60s, a total of 35 cycles; extension at 72°C for 7min.

[0063] Table 3 Qualitative PCR reaction system of transgenic insect-resistant and herbicide-resistant rice G6H1 and its derivatives

[0064]

experiment example 1

[0065] Experimental example 1 Specificity test of qualitative PCR detection method for transgenic insect-resistant and herbicide-resistant rice G6H1 and its derivative varieties

[0066] 1. Plant material

[0067] Transgenic rice: G6H1, Kefeng 6, Kefeng 8, Kemo rice, M12, TT51;

[0068] Other transgenic plants:

[0069] Corn: Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, 59122, MIR604, MON88017, 3272, MON87460;

[0070] Soybean: 356043, 305423, CV127, MON89788, A5547-127, A2704-12, GTS40-3-2;

[0071] Cotton: MON1445, MON531, MON15985, LLCOTTON25, MON88913; Rapeseed: MS1, MS8, RF1, RF2, RF3, T45, Oxy235, Topas19 / 2;

[0072] Non-transgenic rice Xiushui 110;

[0073] Transgenic corn, soybean, cotton and rape were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; transgenic rice and non-transgenic rice Xiushui 110 were collected in this experiment; among them, the seeds of transgenic rice G6H1 were provided by the developer, Professor Zhicheng Shen...

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Abstract

The invention discloses a qualitative PCR detecting method for transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof, and belongs to the qualitative detecting field of transgenic rice varieties. The invention firstly discloses 8 pairs of specific PCR detecting primer pairs for detecting the transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof. Based on this, the invention establishes a qualitative PCR detecting method for the transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof, which is strong in specificity and high in sensitivity. The invention further discloses a qualitative PCR detecting kit for the transgenic insect-resistant herbicide-tolerant rice G6H1 and derived varieties thereof. An experiment for specificity, sensitivity and detection limit proves that the qualitative PCR detecting method for the transgenic insect-resistant herbicide-tolerant rice G6H1 variety is strong in specificity, high in sensitivity and good in detection limit, and applicable to qualitative detection of the transgenic G6H1 rice and derived varieties thereof.

Description

technical field [0001] The present invention relates to a qualitative PCR detection method for transgenic rice varieties, in particular to qualitative PCR detection primers, a qualitative detection method and a detection kit for transgenic insect-resistant and herbicide-resistant rice G6H1 and its derivative varieties, belonging to transgenic insect-resistant and herbicide-resistant rice G6H1 and The field of qualitative PCR detection of its derivatives. Background technique [0002] The transgenic insect-resistant and herbicide-tolerant rice variety G6H1 is obtained by introducing the codon-optimized insecticidal gene HJC-1 and glyphosate-resistant gene G6 into the cultivated rice variety "Xiushui 110". The total length of the inserted sequence is about 10.2kb ( figure 1 ). HJC-1 is a fusion gene of Cry1Ab and Vip3H. The fusion of these two genes has a broader spectrum of insecticidal ability against Lepidoptera pests. G6 is a novel 5-enolpyruvate 3-phosphate synthase ge...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 徐俊锋汪小福陈笑芸沈志诚刘慧
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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