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Method for improving gene grouped detection of transgenic plant GUS report

A transgenic plant and genome technology, applied in the field of plant genetic engineering, can solve problems affecting the accuracy of test results, missed detection, etc., to avoid damage, reduce missed detection rate, and improve accuracy.

Inactive Publication Date: 2006-03-22
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This may cause missed detection and affect the accuracy of the detection results.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The transgenic plant GUS reporter genome detection in this embodiment is used in cotton transgenic research, and can quickly screen and identify transgenic positive strains. The specific process is as follows:

[0020] 1. Optimize the preparation of GUS histochemical detection solution: 80mM potassium phosphate buffer (pH7.0), 2mM K 4 Fe(CN) 6 , 2mM K 3 Fe(CN) 6 , 10 mM EDTA, 0.1% Triton X-100, 1.0 mg / ml X-Gluc. Filter sterilize with a 0.45 μm filter. Packed and stored in a refrigerator at 20°C, the validity period is 6 months.

[0021] 2. Peel the harvested seeds and soak them in H 2 o 2 Sterilize in 30% solution for 20 minutes, wash 2-3 times with sterile water, and then sow on MS medium. When the radicle grows to about 3 cm, cut 5-7 tissue blocks with a thickness of 1 mm from the meristem at the top of the root tip in an ultra-clean workbench, transfer the tissue blocks to a 0.5 ml sterile tube, and add 20 μl of GUS for histochemistry detection solution.

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Embodiment 2

[0025] At present, transgenic insect-resistant cotton has been popularized and applied in agricultural production. Since the insect-resistant cotton contains the insecticidal Bt gene and the GUS reporter gene, the method of the invention can be used to identify the purity of the insect-resistant cotton seeds and identify the authenticity of the cotton field plants.

[0026] Seed purity identification steps are basically the same as in Example 1. The steps to identify the authenticity of cotton plants are different: Step 2 For plant tissues such as leaves taken from the cotton field, they should be rinsed with tap water first, then sterilized with 75% alcohol for 30 seconds, and then disinfected with 2.5% hypochlorite. Calcium acid solution for 15 minutes, take it out and wash it with sterile water for 3 times, absorb the surface moisture with sterile filter paper, cut into 2mm 2 Tissue pieces of about the size are transferred to a sterile tube, and then added with GUS histoch...

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PUM

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Abstract

The present invention relates to a method for improving transgenic plant GUS reporter gene histochemical detection, belonging to the field of plant gene engineering technology. It includes the following steps: A. preparing GUS histochemical detection liquor; 75-85 mM potassium phosphate buffer solution (pH 7.0), 1.5-2.5 mM K4Fe(CN)6, 1.5-2.5 mM K3Fe(CN)6, 8-12 mMEDTA, 0.08-0.012% Triton X-100, 0.8-1.2 mg / ml X-Glue; B. under the sterile condition cutting tissue block of plant to be tested, soaking said tissue block in the detection liquor; C placing the reaction system in the environment with 29-31 deg.C, heat-insulating for 40-60 hr.; and D. observing tissue block colour in reaction system, if said tissue block is blue, the sample is GUS transgenic positive. It can obviously raise the accuracy for screening and identifying transgenic positive plant.

Description

technical field [0001] The invention relates to a method for screening and identification of transgenic plants, in particular to an improved method for the histochemical detection of a GUS reporter gene of transgenic plants, which belongs to the technical field of plant genetic engineering. Background technique [0002] Histochemical detection of β-glucuronidase was originally used for the tissue localization of this enzyme as an endogenous enzyme in mammals. Higher plants do not contain β-glucuronidase. Jefferson first cloned the gene (GUS) encoding the enzyme from Escherichia coli, constructed it into a plant expression vector for transformation of higher plants, obtained plants transfected with the GUS gene, and detected the expression product of the gene in plant tissues - beta-glucuronidase. Since then, GUS has been widely used as a reporter gene in plant genetic engineering to screen transgenic positive plants. The method is simple, and as long as the detection cond...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 张震林周宝良陈松张香桂
Owner JIANGSU ACAD OF AGRI SCI