Analysis method of unexpected effect of transgenosis plants

A technology of unexpected effects and transgenic plants, applied in the field of genetic engineering, can solve problems such as the methylation status of endogenous genes with low homology, and achieve important economic and social benefits

Inactive Publication Date: 2016-07-06
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At home and abroad, this technology has not been used to analyze the methylation status of endoge

Method used

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  • Analysis method of unexpected effect of transgenosis plants
  • Analysis method of unexpected effect of transgenosis plants
  • Analysis method of unexpected effect of transgenosis plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Extraction and Bisulfite Treatment of Genomic DNA

[0026] 1. Extraction of genomic DNA from transgenic plants

[0027] Under the same cultivation conditions, wild-type Arabidopsis (WT), Arabidopsis fhy3 gene deletion mutant (fhy3), and Arabidopsis thaliana transfected with fhy3 and GUS genes ( FHY3-GUS) seedlings, wild-type cucumber (WT2) and GUS-transgenic cucumber (GUS) seedlings, the genomic DNA of their leaves were extracted using the traditional CTAB method.

[0028] 2. Bisulfite treatment of genomic DNA

[0029] Arabidopsis and cucumber genomic DNA were subjected to bisulfite treatment according to the EZDNAMethylationGold kit (ZymoResearch) instructions.

Embodiment 2

[0030] Example 2: Cloning and sequencing of PCR amplification products

[0031] 1. PCR amplification of endogenous genes

[0032] Select Arabidopsis thaliana endogenous gene AT4G16835 and cucumber endogenous gene Csa026131 that are not homologous to fhy3 and GUS genes, and use the online MethyPrimer software to design primers for a CpG island in its coding region as follows: The forward primer of the AT4G16835 gene is 5'- TTTGAGGAAGGAATTAGGTTTAATT-3', its nucleotide sequence is shown in SEQNo.1, the reverse primer is 5'-AAAACTTCCAAACATCACCAAAT-3', its nucleotide sequence is shown in SEQNo.2; the forward primer of Csa026131 gene is 5' -TAGTTTTTTTAGTAGTTGAAGTTGA-3', the nucleotide sequence of which is shown in SEQNo.3, and the reverse primer is 5'-AACTTTTAAATAAAAAATCCTTTTT-3', the nucleotide sequence of which is shown in SEQNo.4.

[0033] PCR amplification was performed using bisulfite-treated genomic DNA as a template. 20μL PCR reaction system includes: 0.4μL MgCl 2 (25mM), ...

Embodiment 3

[0044] Embodiment 3: Analysis of unexpected effects of transgenic plants

[0045] 1. According to the sequencing results of the PCR amplification products, calculate the ratio of unmethylated cytosine to the total cytosine in each clone, and obtain the average methylation level of each test material (20 clones). The experimental data are expressed in the form of mean ± standard error, using the statistical software SPSS13.0, through the one-way analysis of variance and TukeyHSD test for significant difference analysis, when P<0.05, there is a significant difference between the data.

[0046] Such as figure 1 showed that the average methylation level of endogenous gene AT4G16835 was higher in Arabidopsis fhy3 gene deletion mutant (fhy3), lower in wild-type Arabidopsis (WT), and was higher in fhy3 gene deletion mutants. The average methylation level was the lowest in the receptor, transfhy3 and GUS transgenic Arabidopsis (FHY3-GUS). Therefore, although the endogenous gene AT4G...

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Abstract

The invention relates to the technical field of genetic engineering, and provides an analysis method for unexpected effects of transgenic plants. For the first time, bisulfite sequencing technology is used to analyze endogenous genes in transgenic plants that are not highly homologous to transformed exogenous genes To study the unintended effects of transgenic plants, establish a technical system for analyzing whether transgenic plants have unintended effects, which will help the monitoring and popularization of transgenic plants, and have important economic and social benefits.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and provides an analysis method for unexpected effects of transgenic plants. Background technique [0002] With the development of transgenic technology, the safety of transgenic organisms has attracted great attention at home and abroad. It is of great significance to study the unexpected effects of transgenic plants for the monitoring and popularization of transgenic plants. DNA methylation can cause transgene silencing in plants. In transgenic plants, internal factors and environmental conditions can affect the methylation status of the transformed exogenous gene, thus changing the phenotype of the transformed exogenous gene. On the other hand, after the virus infects plants, it can induce the silencing of genes homologous to the virus in plants through mechanisms such as DNA methylation; The homology is high, and the transformed exogenous gene can cause expression silencing of t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q1/686C12Q2600/154C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 白吉刚王秀娟李雪张静
Owner SHANDONG AGRICULTURAL UNIVERSITY
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