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A kind of universal primer and detection method for detecting expression of target gene in transgenic plants

A transgenic plant and purpose technology, applied in the field of general primers and detection of gene expression, can solve problems such as misjudgment, achieve good specificity, high-efficiency and high-throughput detection, and save costs

Active Publication Date: 2020-11-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Zhong Boxiong and others from Zhejiang University invented a method for predicting the expression of exogenous genes and screening transgenic organisms by using marker genes (application number: 201410678673.2, publication number: CN104404072A), but marker genes and exogenous genes are sometimes inserted into the host genome separately This will lead to misjudgment; most importantly, because marker genes may bring some potential threats, non-selectable marker transgenics have become the focus of transgenic technology research

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  • A kind of universal primer and detection method for detecting expression of target gene in transgenic plants
  • A kind of universal primer and detection method for detecting expression of target gene in transgenic plants
  • A kind of universal primer and detection method for detecting expression of target gene in transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1. Discovery of a small segment of carrier sequence connected to the 3' end of transgenic sample cDNA and the establishment of amplification primers and detection methods

[0055] 1. The discovery of a small vector sequence connected to the 3' end of the cDNA of the transgenic sample

[0056] 1. Extraction of RNA from transgenic plants

[0057] All the corn transgenic plants used in this patent were created by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University.

[0058] 1) Preparation of transgenic corn

[0059] First, the target gene was connected to the pBCXUN vector by TA cloning method to obtain an overexpression vector, and the constructed plasmid was transformed into the Agrobacterium EHA105 strain by the heat shock method, and then introduced into the corn B73-329 variety by the Agrobacterium-mediated method. The transgenic T2 homozygous line was finally identified.

[0060] The pBCXUN vector replaces t...

Embodiment 2

[0136] Embodiment 2, detect the expression of target gene in the transgenic plant

[0137] In the following examples, the specific primer pair designed for the target gene was used as the control, and the universal primer pair obtained in Example 1 was used as the experimental group.

[0138] 1. Detect the expression of the target gene in the transgenic plant, and the host target plant of the transgenic plant itself expresses the target gene

[0139] The transgenic maize to be tested is CAUB0084 transgenic maize, which is the transgenic maize obtained by introducing the target gene (GRMZM2G117633; http: / / ensembl.gramene.org / Zea_mays / Info / Index) into the maize variety B73-329 through the pBCXUN vector backbone.

[0140] test group:

[0141] The three methods in Example 1 were used to extract RNA from 4 CAUB0084 transgenic maize lines and non-transgenic wild-type maize B73-329, and reverse transcribed to obtain cDNA. Using the above cDNA as a template, the real-time fluorescen...

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Abstract

The invention discloses a general primer and a detection method for detecting target gene expression in transgenic plants. The present invention provides a substance for detecting the expression of an exogenous target gene introduced into a transgenic plant, which is as follows: 1) a DNA fragment shown in sequence 1 or positions 1-93 of sequence 1; 2) used to amplify 1) the primer pairs for the indicated DNA fragments; 3) PCR reagents or kits containing the indicated primer pairs for 2). By performing 3' RACE on the cDNA of the transgenic material, the inventor proved for the first time that the exogenous gene has a short vector sequence (see Sequence Table 1), that is, the partial sequence of the Tnos terminator, which is closely linked after transcription. Since the Tnos terminator is a commonly used terminator in transgenic research, based on the research results, the present invention designs a universal primer for the vector sequence fragment, and predicts the expression level of exogenous genes according to the expression level of the vector sequence, which can quickly extract from a large number of transgenic individuals. Screen out transgenic individuals or varieties that express foreign genes with high efficiency.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a general primer and a detection method for detecting the expression of a target gene in a transgenic plant. Background technique [0002] Since the first industrial application of genetically modified crops in 1996, global research and industrial application of genetically modified technology has entered a new stage of large-scale integrated application. With the advancement of the industrialization of transgenics, the corresponding transgenic detection methods have also developed from the original detection of a single target gene to the simultaneous detection of multiple species and multiple genes, and continue to develop in the direction of high-throughput detection. [0003] In most cases, exogenous genes are generally randomly inserted into the host genome, and the expression of exogenous genes at different insertion sites will vary. Therefore, in transgenic research, it i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6895C12Q2600/158C12Q2600/13C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 王喜庆刘芳庄军红
Owner CHINA AGRI UNIV