Optimized dual T-DNA expression vector obtaining marker-free genetically modified organisms (GMOs) and applications thereof

Abstract

The invention discloses an optimized dual T-DNA expression vector obtaining marker-free genetically modified organisms (GMOs) and applications thereof. The dual T-DNA expression vector A provided by the invention is prepared by inserting a dual T-DNA region into an expression vector; wherein the dual T-DNA region comprises two individual T-DNA regions: a T-DNA A region and a T-DNA B region; the right boundary of the T-DNA A region is next to the right boundary of the T-DNA B region, and the left boundary of the T-DNA A region is far away from the left boundary of the T-DNA B region. Experiment results have shown that: in the constructed dual T-DNA plasmid, two right boundaries of the two T-DNA regions get close to each other in a plasmid ring so as to form a head-to-head structure (LB-RB-RB-LR type); thus readthrough in the transcription process is avoided, the ratio of interlocked transforming genes and selective marker genes is reduced, and finally the probability that marker-free transgenic strains are separated from the T1 generation is improved, so the expression vector has a wide application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an optimized double T-DNA expression vector for obtaining non-selectable marker transgenic organisms and its application. Background technique [0002] Since its introduction in 1983, plant transgenic technology has made great progress. It can break the biological isolation between different species, make gene exchange between species possible, and allow transgenic crops to develop in the desired direction. At present, researchers have found a variety of different transgenic methods, such as Agrobacterium-mediated method, gene gun method, PEG method, electric shock method, microinjection method, liposome method, etc. (Sawahel W A, et al.1992.Biotech Advances .10:393-412). When these transformation methods are used to introduce exogenous target genes into plant cells, usually only some cells can become stable transformed cells, and the application of selectable marker genes enables t...

AI Technical Summary

Problems solved by technology

T 0 Generation co-transformation frequency was 53.6%, for co-transformation T 1 Genetic analysis of transgenic lines showed that 41.4% of the lines segregated and produced non-selectable marker transgenic plants, but T 1 Among the 18 strains in the generation, 6 strains (33.3%) were linked with double TDNA insertions, which affected the segregation ratio of the offspring.

The reason for the analysis is that because the two T-DNA regions are close to each other, the double border of the two T-DNA regions is homeopathic (the border type is LB-RB-LB-RB type), which may form a read-through during transcription, resulting in two Two T-DNA chain insertions at the same site severely reduced the offspring segregation marker genes

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