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The flanking sequence of maroacc transgenic herbicide-resistant maize cc-2 and its application

A herbicide-resistant, CC-2 technology, applied in genetic engineering, recombinant DNA technology, microbial assay/inspection, etc., can solve problems such as the flanking sequence of herbicide-resistant corn CC-2 without maroACC gene, and achieve sensitivity High, accurate and specific effects

Active Publication Date: 2018-04-24
北京粮元生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the analysis of existing patents and documents, it is found that there are no articles or patent reports on the flanking sequence of the foreign insertion fragment of transgenic maroACC gene herbicide-resistant corn CC-2

Method used

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  • The flanking sequence of maroacc transgenic herbicide-resistant maize cc-2 and its application
  • The flanking sequence of maroacc transgenic herbicide-resistant maize cc-2 and its application
  • The flanking sequence of maroacc transgenic herbicide-resistant maize cc-2 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Cloning of the flanking sequence of transgenic maroACC gene herbicide-resistant corn CC-2

[0075] 1. Construction of the BAC library of the herbicide-resistant maize CC-2 transgenic maroACC

[0076] Sample: transgenic maroACC herbicide-resistant corn CC-2.

[0077] BAC vector: plndigoBAC5 produced by Epicentre.

[0078] Escherichia coli competent: DH10B of Invitrogen Company.

[0079] 1. Preparation of large fragments of DNA

[0080] Take 50 g of transgenic corn CC-2 albino seedling leaf samples, quickly grind them into fine powder with liquid nitrogen, add the powder to 500 mL of pre-cooled extraction buffer (containing 0.25% TritonX-100), stir slowly for 15 min, and mix well. The liquid state is first filtered with 1 layer of medical gauze and 2 layers of filter cloth into beakers, and the filtrate is divided into centrifuge tubes, centrifuged at 3500r / min and 4°C for 20min at a low speed, and the supernatant is dried. Add 1 mL of washing buffer to the...

Embodiment 2

[0110] Example 2, Design and synthesis of primer pairs for detection of maroACC gene herbicide-resistant corn CC-2

[0111] According to the 5' flanking sequence (sequence 5) of the maroACC gene transgenic herbicide-resistant corn CC-2 obtained in Example 1, a primer pair 1 for screening specificity was designed; the trans maroACC gene transgenic herbicide-resistant corn CC obtained according to Example 1 The flanking sequence of the 3' end of -2 (SEQ ID NO: 6), the primer pair 2 for screening specificity was designed.

[0112]Primer pair 1 designed for the 5' flanking sequence (SEQ ID NO: 5):

[0113] CC-2 5'aF: 5'-AGGCTTTATCCTGTGCAATGCG-3' (SEQ ID NO: 1);

[0114] CC-2 5'aR: 5'-ATTGAGTATCCGTTTCCCCTCCTTTT-3' (SEQ ID NO: 2).

[0115] Target fragment size: 292bp.

[0116] Theoretically, using this specific primer pair (CC-2 5'aF / CC-2 5'aR) to perform PCR amplification on the genomic DNA of transgenic maroACC gene herbicide-resistant corn CC-2 will obtain the target band with...

Embodiment 3

[0123] The specific detection of embodiment 3, embodiment 2 primer pairs

[0124] 1. Specific detection (1)

[0125] Samples for testing: transgenic maroACC gene herbicide-resistant corn CC-2 (T3, T6, T9 generations), non-CC-2 transgenic maize lines with maroACC gene transferred.

[0126] 1. Extraction of maize genomic DNA

[0127] (1) Take the CTAB solution and put it in a 65°C water bath in advance;

[0128] (2) Take about 0.1g of fresh corn leaves for testing, cut into pieces, put them in a pre-cooled mortar, grind them into powder quickly in liquid nitrogen, and immediately transfer them to a pre-cooled 2ml EP tube (generally not more than 1 / 2 tube volume);

[0129] (3) Quickly add 0.8ml of CTAB buffer in a 65°C warm bath to the EP tube, shake gently evenly, bathe in 65°C water for 30 minutes, and shake gently from time to time;

[0130] (4) Place in a fume hood for about 15 minutes and cool to room temperature;

[0131] (5) Add an equal volume of chloroform: isoamyl ...

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Abstract

The invention discloses a flanking sequence of maroACC transgenetic anti-herbicide corn CC-2 and an application thereof. The invention also discloses a CC-2 specificity detection primer pair 1 and a primer pair 2, the primer pair is designed according to a CC-2 transformant insertion sequence and a flanking sequence, an upstream primer in the primer pair 1 can be designed according to a first to 500 site of a sequence 5, and a downstream primer is designed according to a 501<st> to 1000 site of the sequence 5; an upstream primer of the primer pair 2 is designed according to a first to 500 site of a sequence 6, and a downstream primer is designed according to 501<st> to 1000 site of the sequence 6. The primer pair developed by virtue of the flanking sequence can be used for detecting CC-2 transgenetic corn, and a corn material which is positive in the detection can be used for breeding the anti-herbicide transgenetic corn. The experiment shows that the primer pair designed by virtue of the insertion segments and the flanking sequence can detect whether the corn to be detected is the anti-herbicide corn CC-2 or not by virtue of a PCR method; moreover, the method is high in accuracy, strong in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a flanking sequence for transgenic maroACC gene herbicide-resistant corn CC-2 and application thereof. Background technique [0002] Corn is the cereal crop with the widest planting range and the largest yield in the world, ranking first among the three major grains (corn, wheat, and rice). my country is a big country in corn production and consumption. Its sown area, total output and consumption are second only to the United States, ranking second in the world. In recent years, although my country's corn has a good momentum of development, it still needs to be imported in large quantities, and the import volume is increasing rapidly year by year. With the rapid development of industrialization and urbanization and the continuous improvement of people's living standards, my country has entered a stage of rapid growth in corn consumption. From the perspective of future development, co...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12N15/8213C12Q1/6895C12Q2600/13
Inventor 赖锦盛赵海铭宋伟彬孙思龙
Owner 北京粮元生物科技有限公司