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Detection method of glyphosate-resistant transgenic gossypium hirsutum BG2-7 and flanking sequence

A technology of upland cotton and sequence, applied in the direction of biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc.

Active Publication Date: 2016-12-07
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no relevant reports on the commercial application of transgenic glyphosate-resistant cotton in my country. Therefore, it is of great economic and social significance to introduce G2-aroA gene into cotton and cultivate glyphosate-resistant transgenic cotton.

Method used

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  • Detection method of glyphosate-resistant transgenic gossypium hirsutum BG2-7 and flanking sequence
  • Detection method of glyphosate-resistant transgenic gossypium hirsutum BG2-7 and flanking sequence
  • Detection method of glyphosate-resistant transgenic gossypium hirsutum BG2-7 and flanking sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Acquisition and character identification of glyphosate-tolerant transgenic upland cotton BG2-7

[0067] 1. Agrobacterium-mediated method to obtain transgenic cotton plants

[0068] Using the Agrobacterium-mediated method, the plant expression vector pG2 (pG2) containing the G2-aroA gene (ZL 03826892.2) is shown as figure 1 Shown) The acceptor material Ke 312 is introduced. details as follows:

[0069] (1) Select plump Ke 312 seeds, delint with a small amount of concentrated sulfuric acid and rinse with plenty of water. Sterilize with 75% (volume fraction) alcohol for 5 minutes and rinse with sterilized water three times. Then use 1.8% (volume percentage) sodium hypochlorite solution to disinfect for 30 minutes, and rinse with sterile water 6 times. Finally, the sterilized seeds were incubated at 37°C overnight to accelerate germination.

[0070] (2) Use tweezers to gently peel off the seed coat, soak it in a 500mg / ml cephalosporin sodium solution for 30-45 minutes...

Embodiment 2

[0095] Example 2. Cloning of the flanking sequence of Gossypium hirsutum BG2-7

[0096] 1. Cloning of the 5'flanking sequence of Gossypium hirsutum BG2-7

[0097] 1. Design the following primers F1, F2 and F3 according to the known sequences in the plant expression vector used for cotton transformation. All primers were synthesized by Shanghai Biological Engineering Co., Ltd. The primers were dissolved in water to a final concentration of 100 μM.

[0098] F1: 5’-aacacggcggcatcagagca-3’;

[0099] F2: 5’-taccgaggggaatttatggaacg-3’;

[0100] F3: 5’-gttgcggttctgtcagttccaa-3’;

[0101] 2. Extract DNA from Gossypium hirsutum (Gossypium hirsutum) BG2-7CGMCC No. 10412 leaves by using the plant genomic DNA extraction kit, and the specific operation is performed according to the kit instructions. The extracted DNA was diluted with water to a final concentration of 100ng / μl.

[0102] 3. According to the Genome Walking Kit (TaKaRa, Beijing, Code: D316) instructions, add 10×LA PCR Buffer II(Mg 2+ p...

Embodiment 3

[0115] Example 3. Specific PCR detection of the flanking sequence of glyphosate-tolerant transgenic upland cotton BG2-7

[0116] 1. 5'end flanking sequence specific PCR detection

[0117] 1. According to the flanking sequence (sequence 5) at the 5'end of the insertion site of the foreign gene of Gossypium hirsutum BG2-7, design the primers BG2-7-5-F and BG2- for site-specific PCR detection of the foreign gene. 7-5-R.

[0118] BG2-7-5-F: 5'-CCTATTACACGGCTATGC-3' (sequence 1, which is the 495-512th position of sequence 5);

[0119] BG2-7-5-R: 5'-GTCATAACGTGACTCCCTTAATTCTCC-3' (sequence 2, the reverse complementary sequence of the 1112-1138th position of the sequence 5).

[0120] 2. Use plant genomic DNA extraction kit to extract leaf DNA from Gossypium hirsutum BG2-7, non-transgenic recipient Gossypium hirsutum 312, and Sea island Gossypium 7124. The specific operation is carried out according to the kit instructions. The extracted DNA was diluted with water to a final concentration of ...

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Abstract

The invention discloses a detection method of glyphosate-resistant transgenic gossypium hirsutum BG2-7 and a flanking sequence. A primer pair which is provided by the invention and is used for the detection or the auxiliary detection on whether cotton to be detected is gossypium hirsutum BG2-7 with a preservation number of CGMCC No.10412 or not consists of two single-chain DNA (Deoxyribonucleic Acid) molecules shown by a sequence 1 and a sequence 2 in a sequence table. The primer pair is obtained according to the 5' flanking sequence design of the gossypium hirsutum BG2-7 with a preservation number of CGMCC No.10412. Experiments prove that the primer pair can be used for detecting whether the cotton to be detected is the gossypium hirsutum BG2-7 with a preservation number of CGMCC No.10412 or not by a PCR (Polymerase Chain Reaction) method; and the method has the advantages of high accuracy, high specificity and high sensitivity.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and flanking sequence of glyphosate-resistant transgenic upland cotton BG2-7. Background technique [0002] my country is a big cotton producer, with an annual cotton planting area of ​​about 4.7 million hectares and an annual output of 5 million tons of lint, accounting for about 25% of the world's total cotton production. The rise and fall of cotton production has a decisive influence on the development of our country's economy. However, the ecological type of cotton fields in China is complex and the weeds are more harmful, especially in the cotton areas of the Yangtze River. The early growth period is during the rainy season, and the weeds grow vigorously. In addition to the continuous rain, the weeds cannot be weeded in time. The weed damage is extremely serious, which can cause 14.0 %-16.0% loss, which severely restricts the high-quality and high-efficiency producti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王旭静王志兴唐巧玲张小兵
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI