Method for identifying copy number of T-DAN tandem repeat sequences in transgenic plant through real-time fluorescence quantification PCR method

Abstract

The invention discloses a method for identifying the copy number of T-DAN tandem repeat sequences in a transgenic plant through a real-time fluorescence quantification PCR method. The method comprises the following steps: connecting an HMGA gene to a plant expression vector Pski015 containing a screening gene BAR through a Southern Buddhism mediated plant transformation method; then transfecting wild Col-4 arabidopsis; connecting the vector with two reference plasmids; extracting RNA of glyphosate-screened transgenic arabidopsis and carrying out inverse transcription to obtain cDNA as a detection template; and detecting the copy number of a mutant plant in a way of real-time quantification PCR respectively by virtue of quantitative primers of a glyphosate-resistance gene and the HMGA gene. The method disclosed by the invention has the beneficial effects that the influence of a T-DAN tandem structure on identification of the copy number of a transgenic plant can be effectively shielded; and compared with Southern blot, quantification PCR is more convenient and more efficient.

Description

technical field [0001] The invention relates to a method for identifying the copy number of T-DNA tandem repeat sequences in transgenic plants by means of real-time fluorescent quantitative PCR. Background technique [0002] Agrobacterium-mediated plant genetic transformation is an effective and feasible method for introducing exogenous genes into plant genomes. It has been widely used in genetic engineering of crops such as golden rice, and is an important experimental method in contemporary molecular biology. During the transformation of Agrobacterium, multiple T-DNA insertions into the same site and multiple insertion sites frequently occur. These T-DNA tandem repeat structures often lead to co-suppression of target genes or even gene silencing, thereby affecting follow-up experiments. Therefore, an efficient and simple method for screening single-copy transgenic plants is of great significance for the study of plant genetic improvement and gene function. [0003] Tradi...

AI Technical Summary

Problems solved by technology

However, it is still uncertain whether this method can quickly and accurately quantify the copy number of T-DNA in the case of a large number of transgenic lines

[0004] Although some studies have demonstrated that T-DNA tandem repeats are formed after the plant transformation process, the mechanism of T-DNA tandem repeat formation is not fully understood

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