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Method for detecting exogenous gene copy number in transgenic tobacco

An exogenous gene, transgenic technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., to meet the needs of screening

Inactive Publication Date: 2014-09-10
GUANGXI VETERINARY RES INST
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  • Abstract
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  • Claims
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Problems solved by technology

[0004] In previous studies, it was found that the copy number results obtained by real-time fluorescent quantitative PCR detection and Southern hybridization were very close, but some results showed that the copy number obtained by real-time fluorescent quantitative PCR was slightly higher than that obtained by Southern hybridization

Method used

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  • Method for detecting exogenous gene copy number in transgenic tobacco
  • Method for detecting exogenous gene copy number in transgenic tobacco
  • Method for detecting exogenous gene copy number in transgenic tobacco

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Embodiment 1

[0059] Example 1, real-time fluorescent quantitative PCR technology detection of exogenous gene copy number in transgenic tobacco

[0060] 1. Primer design

[0061] Select tobacco endogenous single copy gene—ribonucleotide reductase (Ribonucleotide reductase) RNR2 (the copy number of this gene is in literature " Chaboute M E, Combettes B, Clement B, et al. Molecular characterization of tobacco ribonucleotide reductase RNRI and RNR2cDNAs and cell cycle regulated expression in synchronized plant cells.Plant Molecular Biology, 1998, 38:797-806 "disclosed) as an internal reference gene. The primers for the internal reference gene RNR2 and the exogenous gene GFP were designed as follows:

[0062] Upstream primer RNR2U: 5'-TACCCGTCACTGGGAAAC-3' (SEQ ID No.3)

[0063] Downstream primer RNR2D: 5'-GGAAGCCGTAGAAAGCAC-3' (SEQ ID No.4)

[0064] Using RNR2U and RNR2D as primers to amplify the internal reference gene RNR2 in tobacco, the length of the target fragment is 158bp;

[0065] ...

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Abstract

The invention discloses a method for detecting an exogenous gene copy number in transgenic tobacco. A real-time fluorescent quantitation PCR technology is utilized to simply, rapidly and accurately detect the exogenous gene copy number in transgenic tobacco. The invention discloses a pair of primers consisting of DNA molecules shown in (1) and (2), namely (1) a DNA molecule shown in SEQ ID No.3; and (2) a DNA molecule shown in SEQ ID No.4. A single-copy endogenous gene ribonucleotide reductase gene (RNR2) of the tobacco is selected as a reference gene, and the exogenous gene copy number in transgenic tobacco is detected through a SYBR GreenI real-time fluorescent quantitation PCR technology. The method disclosed by the invention provides a new thought for copy number analysis of the transgenic tobacco, and the screening requirement for excellent transformation plants in genetic engineering is met.

Description

technical field [0001] The invention relates to a method for detecting the copy number of an exogenous gene in transgenic tobacco, in particular to a method for detecting the copy number of an exogenous gene in transgenic tobacco by using real-time fluorescent quantitative PCR technology, which belongs to the field of biotechnology. Background technique [0002] In recent years, plant transgenic technology has been widely used in research in the fields of producing medicinal proteins, improving crop quality, and increasing crop yield. At present, the method used for plant transgenesis is mainly Agrobacterium tumefaciens-mediated method, but the T-DNA region containing the target gene carried by Agrobacterium is randomly inserted into the genome of the recipient plant, so the obtained transgenic plants often contain one to two Multiple copies of foreign genes. The copy number of the exogenous gene in the transgenic plant is a key factor affecting the expression of the gene, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2537/16C12Q2545/101
Inventor 谢芝勋王盛谢丽基谢志勤黄莉邓显文罗思思黄娇玲刘加波
Owner GUANGXI VETERINARY RES INST
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