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Transgenic soybean ZUTS-33 preparation method, detection and application

A technology of ZUTS-33, transgenic soybean, applied in the field of plant genetic engineering technology and breeding

Inactive Publication Date: 2017-03-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Research on transgenic glyphosate-resistant crops has achieved some results, but there is no report of commercialization of glyphosate-resistant genetically modified crops, which is in line with my country's status as the second largest glyphosate production and export country extremely disproportionate

Method used

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  • Transgenic soybean ZUTS-33 preparation method, detection and application
  • Transgenic soybean ZUTS-33 preparation method, detection and application
  • Transgenic soybean ZUTS-33 preparation method, detection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Embodiment 1, vector construction

[0086] Map of target gene and vector construction:

[0087] The transgenic soybeans in this experiment were carried by Agrobacterium such as figure 1 The indicated binary plasmid pSOY19 was transformed. The vector is from the T-DNA region from the right border (T-Border-RB) (sequence as described in SEQ ID NO:1) to the left border (T-Border-LB) (sequence as described in SEQ ID NO:2) region The full length is 6284bp, and this region is mainly composed of 4 parts except the multiple cloning site:

[0088] 1), the promoter (CaMV35Spromoter) of the cauliflower mosaic virus 35 protein that can produce constitutive expression in plants;

[0089] 2), the signal peptide Ch-L from Arabidopsis thaliana, which can guide the protein encoded by the same expression frame to be transported into the chloroplast, the nucleotide sequence of the chloroplast leader peptide encoded by Ch-L is as SEQ ID NO: 13 The deduced amino acid sequence of the chl...

Embodiment 2

[0095] Embodiment 2, soybean conversion

[0096] Genetically modified method:

[0097] Agrobacterium-mediated transformation (Song et al., 2013) is used for soybean transgenesis, in which Agrobacterium integrates the segment between the left and right borders of the binary plasmid T-DNA into plant cells. The recipient cells in transformation are undifferentiated axillary buds between cotyledonary nodes of germinated seeds. The binary vector pSOY19 with g10-epsps constructed in Example 1 is transformed into Agrobacterium, and the plant tissue and Agrobacterium after Agrobacterium infection After 4 days of co-infection, the infected tissues were placed on cluster bud induction medium containing glyphosate and antibiotics for selection and culture for a total of four weeks. Glyphosate and antibiotics in the medium were used to prevent the growth of non-transformed cells and Agrobacterium, respectively.

[0098] After four weeks of cluster bud induction, the soybean cotyledon no...

Embodiment 3

[0099] Embodiment 3, identification and screening of transgenics

[0100] 1. DNA extraction and PCR identification of soybean leaves

[0101] 1.1 SDS method to extract DNA

[0102] 1) Take 0.05g ground leaves or seeds, add 700μl SDS extract and 3μl 10mg / ml proteinase K (no need to add proteinase K to leaf materials), mix well, and bathe in 60°C water bath for 40min-1h (leaf materials should turn brown Take it out later), and mix it upside down every 10 minutes.

[0103] 2) Add an equal volume of phenol / chloroform (1:1) and gently invert and mix for 10 minutes, let stand for more than 30 minutes, centrifuge at 10000g for 10 minutes, and take the supernatant.

[0104] 3) Add an equal volume of phenol / chloroform (1:1) to mix, and let stand for 10 minutes. Centrifuge at 10,000 g for 10 min, take the supernatant, add 2 times the volume of absolute ethanol, precipitate, and let stand for more than 10 min. Centrifuge at 10,000 g for 1 min, discard the supernatant, and wash the pe...

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Abstract

The invention discloses a soybean transformation event ZUTS-33 which comprises an exogenous insertion sequence. A connection sequence formed by a 5'-terminal of the exogenous insertion sequence and endogenous genome DNA is shown as SEQ ID NO:1, and a connection sequence formed by a 3'-terminal of the exogenous insertion sequence and endogenous genome DNA is shown as SEQ ID NO:2. The invention further discloses a detection method for identification of transgenic soybean ZUTS-33, a method for detecting event ZUTS-33 or filial generations thereof in biological samples, a method for detecting exogenous insertion DNA molecules in the event ZUTS-33, a method for detecting event ZUTS-33 derived plant materials and the like.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering technology and breeding. Specifically, the present invention relates to transgenic soybeans and applications thereof, more specifically, the present invention relates to preparation methods, detection techniques and applications of glyphosate-resistant transgenic soybeans. Background technique [0002] Soybean is an important protein and oil crop in my country and plays an indispensable role in agricultural production. Genetic engineering is used to transfer genes related to herbicide resistance into soybeans to breed soybean varieties with high herbicide resistance, which can save the cost of manual weeding in soybean production, reduce weed damage and increase soybean yield. [0003] Glyphosate It is a non-selective broad-spectrum herbicide, which has the advantages of high efficiency, low toxicity, and no residue. It is especially less toxic to humans and animals. It is one of the mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/82C12N15/11C07K16/16G01N33/68G01N33/558A01H5/00
CPCC12Q1/6895C12N9/1092C12N15/8275C12Q2600/13C12Y205/01019G01N33/558G01N33/68G01N2333/415
Inventor 寿惠霞杜娟陆玲鸿李林宋张悦
Owner ZHEJIANG UNIV
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