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A method for quantitative detection of mon757 transformant content in cotton material population

A MON757, quantitative detection technology, applied in biochemical equipment and methods, microbial measurement/inspection, recombinant DNA technology, etc., to achieve high sensitivity, high practical value, and good specificity

Active Publication Date: 2019-03-22
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at present, there is no report on quantitative PCR method of MON757 transformant at home and abroad

Method used

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  • A method for quantitative detection of mon757 transformant content in cotton material population
  • A method for quantitative detection of mon757 transformant content in cotton material population
  • A method for quantitative detection of mon757 transformant content in cotton material population

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Experimental program
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Effect test

Embodiment 1

[0037] Example 1. Primer and probe design

[0038] The present invention accidentally found through sequence comparison that the flanking sequence of Ezamian No. 1 (its full-length sequence Genbank accession number is AR656168, full-length 4973bp) is highly similar to the MON757 transformant sequence disclosed in the patent US 6893826, so it can be determined that the Ezamian The transgenic transformant of Zamian 1 was derived from the MON757 transformant.

[0039] Design of qualitative detection primers:

[0040] In the present invention, primers are designed on both sides of the genome of the MON757 transformant insertion site, and primers are designed on the insertion sequence. The three primers are as follows:

[0041] 757-GF: 5'-CTAACCTCAATGAGAGCTACC-3' (Seq ID No.1),

[0042] 757-TF: 5'-TCATGTAGCAATGTCCTGCC-3' (Seq ID No.2),

[0043] 757-GR: 5'-GTAATAACTTGATGATGATTTGAG-3' (Seq ID No.3), such as figure 1 shown.

[0044] Among them, the 757-GF / 757-TF amplified region ...

Embodiment 2

[0052] Example 2. Qualitative PCR detection method of transgenic insect-resistant cotton MON757 transformant

[0053] 1 Experimental materials

[0054] 1.1 Transgenic insect-resistant cotton material

[0055] Ezamian No. 1 (Runfu Cotton 839) (manufacturer: Hubei Zhongxiang Rice Industry Co., Ltd., production batch number: 2009-10, approval number: Eexamination cotton 001-2000), purchased from cotton seeds in Hubei Province in 2010 market. Planted in the greenhouse of the Plant Protection Institute of the Chinese Academy of Agricultural Sciences, the homozygous and heterozygous materials of MON757 transformants were obtained through single plant screening and identification, self-breeding and so on.

[0056] Ezamian No. 7 F1 (alias Hongzamian 071) (manufacturer: Hubei Cangguman Seed Industry Co., Ltd., approval number: Ecenmian 2004003), purchased from the cotton seed market of Hubei Province in 2014.

[0057] Huahui No. 4 (manufacturer: Hubei Huimin Agricultural Science and...

Embodiment 3

[0080] Example 3. Quantitative PCR detection method of transgenic insect-resistant cotton MON757 transformant

[0081] 1 Experimental materials

[0082] 1.1 Transgenic insect-resistant cotton material

[0083] Ezamian No. 1 (Runfu Cotton 839) (manufacturer: Hubei Zhongxiang Rice Industry Co., Ltd., production batch number: 2009-10, approval number: Eexamination cotton 001-2000), purchased from cotton seeds in Hubei Province in 2010 market.

[0084] Homozygous material of MON757 transformant: Ezamian No. 1 was planted in the greenhouse of Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and homozygous and heterozygous materials of MON757 transformant were obtained through single plant screening and identification, self-propagation, etc., for quantification Preparation of detection method standard curve.

[0085] Ezamian No. 7 F1 (alias Hongzamian 071) (manufacturer: Hubei Cangguman Seed Industry Co., Ltd., approval number: Ecenmian 2004003), purchased...

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Abstract

The invention relates to a 'method for quantitatively detecting MON757 transformant content in a cotton material group' and belongs to the technical field of biology.The method comprises the following steps: (1), acquiring a curve equation of specific fragment sequences of MON757 transformant; (2), acquiring a standard curve equation of ACP1 gene in cotton endogenous reference genes; (3), randomly selecting multiple materials from the cotton material group to be detected, grinding, and mixedly extracting DNA to be detected; (4), carrying out MON757 transformant fluorescent quantitative PCR (polymerase chain reaction) with the DNA to be detected as a template to obtain a copy number of MON757 transformant in the DNA to be detected; carrying out fluorescent quantitative PCR on the ACP1 gene to obtain a copy number of the ACP1 gene in the DNA to be detected; (5) acquiring MON757 transformant content = (MON757 transformant copy number / ACP1 gene copy number)*2.The specific identification for the existence and content of MON757 transformant in transgenic insect-resistant cotton in a group is facilitated, and the method is high in efficiency and speed, high in sensitivity, good in specificity and low in required experimental condition and has very high practical value.

Description

technical field [0001] The invention belongs to the field of biotechnology. In particular, it relates to a method for quantitatively detecting the content of MON757 transformants in cotton material populations. Background technique [0002] Transgenic cotton is one of the main transgenic crops planted in the world, and it is also the transgenic crop with the largest planting area in my country. In 2014, 7.1 million farmers in my country planted 3.9 million hectares of genetically modified cotton, accounting for 93% of the total cotton planting area, higher than the proportion of 90% in 2013. At present, the transgenic cotton approved for commercial planting in my country mainly includes the cry1Ab / Ac fusion gene transgenic insect-resistant cotton developed in China and the Monsanto transgenic cry1Ac gene transgenic insect-resistant cotton MON531 transformant (http: / / www.moa.gov.cn / ztzl / zjyqwgz / spxx / ). [0003] The transgenic cotton MON757 transformant was developed by Mon...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6851C12Q1/6895C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/13C12Q2600/158C12Q2545/101C12Q2545/113C12Q2563/107
Inventor 谢家建彭于发杨阳王叶
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI