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Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants

A transgenic plant, transgenic technology, applied in the field of molecular biology and biology, to achieve important economic and social benefits

Inactive Publication Date: 2011-06-01
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, methylation-sensitive amplified polymorphism technology has not been used to analyze whether transgenic plants have unexpected toxicity

Method used

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  • Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants
  • Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants
  • Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Obtaining of Differential Fragments of Methylation Sensitivity Polymorphisms in Transgenic Plants

[0023] 1. Extraction of genomic DNA from transgenic plants

[0024] Under the same cultivation conditions, transgenic Arabidopsis thaliana and wild-type Arabidopsis seedlings with fhy3 and GUS genes were cultured, and their genomic DNA was extracted by traditional CTAB method.

[0025] 2. Enzyme digestion and ligation of genomic DNA of transgenic plants

[0026] Entrust Shanghai Sangon Bioengineering Technology Service Co., Ltd. to synthesize the following primer sequences: EcoR I linker 1 (5'-CTCGTAGACTGC GTACC-3'), EcoR I linker 2 (5'-AATTGGTACGCAGTC-3'), MspI / Hpa II linker 1 (5'-GATCATGAGTC CTGCT-3'), Msp I / Hpa II linker 2 (5'-CGAGCAGGACTCATGA-3'). EcoR I linker 1 and EcoR I linker 2 were dissolved in distilled water and mixed uniformly, placed at 95 °C for 5 min, and then cooled at room temperature to prepare 5 pmol of EcoR I linker. MspI / HpaII adapter 1...

Embodiment 2

[0033] Example 2: Recovery, cloning and sequencing of differentially methylated DNA fragments in transgenic plants

[0034] 1. Recovery of differentially methylated DNA fragments

[0035] The methylation-sensitive polymorphic differential fragments excavated from transgenic plants were placed in 30 μl of double-distilled water and boiled for 5 minutes. After slowly cooling down to room temperature, centrifuge and recover the supernatant. Take 4 μl of the supernatant as a template for PCR amplification, and the PCR reaction conditions are consistent with the pre-amplification PCR reaction. The PCR product was recovered and purified according to the instructions of the DNA recovery and purification kit (product of Shanghai Sangon Bioengineering Technology Service Co., Ltd.).

[0036] 2. Cloning of differentially methylated DNA fragments

[0037]According to the instructions of the ligation kit (product of Shanghai Sangon Bioengineering Technology Service Co., Ltd.), the recov...

Embodiment 3

[0040] Example 3: Analysis of Unexpected Toxicity of Transgenic Plants

[0041] 1. Open the KEGG database, input the DNA sequence without methylation in the transgenic Arabidopsis but methylated in the wild-type Arabidopsis, and analyze whether the metabolic pathway involved in the sequence is related to the synthesis of toxic substances. If the metabolic pathway involved in the sequence is related to the synthesis of toxic substances, it indicates that the transgenic plants may have unexpected toxicity.

[0042] 2. Southern hybridization verification

[0043] Genomic DNA of transgenic Arabidopsis and wild-type Arabidopsis were extracted by traditional CTAB method. If the metabolic pathway involved in the sequence of the differentially methylated DNA fragment of the transgenic plant is related to the synthesis of toxic substances, the sequence is labeled as a probe according to the instructions of the digoxin labeling and detection kit (product of Roche Company), and then res...

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Abstract

The invention relates to application of a methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants, in particular provides a method for analyzing and predicting non-expected toxicities of the transgenic plants, which comprises the following steps of: 1 acquiring methylation sensitive amplification polymorphism difference segments of the transgenic plants; 2 recovering, cloning and sequencing methylation difference DNA segments of the transgenic plants; and 3 analyzing the non-expected toxicities of the transgenic plants by utilizing the methylation difference DNA segments of the transgenic plants. In the invention, the methylation sensitive amplification polymorphism technique is used for analyzing the non-expected toxicities of the transgenic plants so that a technical system for analyzing whether the transgenic plants have the non-expected toxicities or not is established, and therefore, the invention contributes to monitoring and generalization application of the transgenic plants and has important economic and social benefits.

Description

(1) Technical field [0001] The invention uses methylation-sensitive amplified polymorphism technology to analyze the unexpected toxicity of transgenic plants, and belongs to the field of molecular biology and biotechnology. (2) Background technology [0002] Multiple exogenous genes are often introduced into the new generation of transgenic plants, which are transgenic plants with complex traits. There may be interactions between exogenous genes and between exogenous genes and endogenous genes in wild-type (recipient) plants, so that some DNA sequences that are methylated in wild-type (recipient) plants are not found in the new generation of transgenic plants. Demethylation, and then the genes in these DNA sequences are expressed in the new generation of transgenic plants, resulting in unexpected toxicity in the new generation of transgenic plants, so it is necessary to establish an analysis and prediction method for the unexpected toxicity of transgenic plants. Methylation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 白吉刚林荣呈代爱华李倩张静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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