Glycine max transformation event SHZD32-01 and detection method thereof

A technology for transforming events and soybeans, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve problems such as ineffectiveness and inability to analyze

Active Publication Date: 2016-01-06
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These detection methods generally focus on commonly used transgenic elements, such as promoters, terminators, marker genes, etc., but are ineffective for distinguishing different transformation events, especially transformation events using the same DNA vector cannot be analyzed unless inserted Chromosomal DNA sequence near the transgene (flanking DNA) is known

Method used

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  • Glycine max transformation event SHZD32-01 and detection method thereof
  • Glycine max transformation event SHZD32-01 and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] SHZD32-01 genome-specific DNA amplification

[0088] DNA was extracted from tissues of soybean transformation event SHZD32-01, including soybean seeds, vegetative tissues or food. Use Sangon's DNA extraction kit to isolate DNA from the tissue. For the method, refer to the instruction manual of the DNA extraction kit. Or use the CTAB method to extract DNA.

[0089] Amplified from the plant genome containing the soybean transformation event SHZD32-01, including the PCR amplification product of the partial genome flanking sequence inserted into the 5' end of the T-DNA, the DNA product includes SEQ ID NO.3. One of the designed PCR primers hybridizes to the flanking sequence of the 5' end of the transgene insertion (DNA primer A, SEQ ID NO.5; such as FIG1), and the second primer (DNA primer B, SEQ ID NO.6) is located at the terminator of the transgene Region (see SEQ ID NO. 9).

[0090] Amplified from the plant genome containing the soybean transformation event SHZD32-01,...

Embodiment 2

[0102] Transgene / genome sequence determination and Southern hybridization

[0103] The DNA sequence of the PCR product can provide a DNA molecule for designing primers or probes for identifying soybean plants or seeds containing the transformation event SHZD32-01. PCR products representing the expected fragment sizes 5' and 3' of the transgene / flanking sequence were separated by 2.0% agarose gel electrophoresis. The isolated PCR product contained the 5' and 3' insertion-binding regions spanning the transgene insertion into the genome. The PCR products at the 5' and 3' ends of the soybean transformation event SHZD32-01 were purified by agarose gel electrophoresis, and the DNA sequence was separated from the agarose matrix according to the DNA gel recovery kit (SK8131). The DNA fragments were connected with the pMD18-T vector and sent to the sequencing company for sequencing.

[0104] The nucleotide sequence obtained by DNA sequence determination represents the nucleotide sequ...

Embodiment 3

[0113] weed control

[0114] Soybeans containing SHZD32-01 are used to control the growth of weeds in the field. Soybean seeds containing SHZD32-01 are planted in the field, and the seeds germinate to form plants, and the plants in the field are treated with a herbicide formula containing glyphosate. Effective doses of glyphosate formulations range from about 0.25 lbae / A (glyphosate acid balance content, lb / A) to 3 or more in the field. Applications range from approximately 0.75 lbae / A to 1.5 lbae / A and one or more treatments in the field as needed during the growing season to control weed growth. Seeds containing SHZD32-01 were harvested from glyphosate-treated plants.

[0115] The soybean seeds of SHZD32-01 deposited by Shanghai Jiaotong University disclosed above and cited in the claims are deposited in the China Center for Type Culture Collection (CCTCC), in the campus of Wuhan University, Wuhan City, Hubei Province, China, postcode 430072, the preserved organism The cl...

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Abstract

The invention discloses a glycine max plant containing a glycine max transformation event SHZD32-01 and a unique DNA molecule in the glycine max transformation event SHZD32-01 and further provides a detection method for the unique DNA molecule. The glycine max transformation event SHZD32-01 contains a molecule comprising at least one of the following nucleic acid sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.9 as well as complete complementary sequences; the detection method comprises the following steps: 1), mixing samples comprising DNA and primers; 2), carrying out nucleic acid amplification reaction so as to generate an amplification product; 3), detecting the amplification product, wherein the amplification product comprise SEQ ID NO.1 or SEQ ID NO.2. The glycine max plant containing the glycine max transformation event SHZD32-01 shows great glyphosate tolerance, and is beneficial to control over weeds; and the DNA detection of the glycine max transformation event SHZD32-01 has a high value for identifying the glycine max transformation event SHZD32-01 in the sample and breeding of the glycine max containing the DNA.

Description

technical field [0001] The present invention is about a new soybean transformation event, called SHZD32-01. This event showed strong tolerance to the herbicide glyphosate (Glyphoste). The invention also relates to plant parts, seeds and products thereof associated with the transformation event SHZD32-01. The invention proposes the unique DNA molecule of the transformation event SHZD32-01 and proposes a detection method for the SHZD32-01 specific DNA molecule in plant part extracts or seed extracts. Background technique [0002] Soybean (Glycincemax) is an important economic crop. Herbicide tolerance in soybean is an important agronomic trait, especially tolerance to glyphosate herbicide. Glyphosate (N-phosphonomethylglycine) is a broad-spectrum herbicide for plant species. It is the main ingredient in the Roundup (RTM.) herbicide produced by Monsanto. type herbicides. When glyphosate is sprayed on plant surfaces, glyphosate enters the entire plant systematically. The p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 曹越平
Owner SHANGHAI JIAO TONG UNIV
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