A double-induced homologous recombination marker-free vector plasmid and its application

Abstract

The invention belongs to the technical field of biology, and provides a bi-induction homologous recombination mark-free vector plasmid and an application thereof. The sequence of the plasmid is shown as SEQ ID NO.1. A point-specific-recombination reverse reaction system is applied, the plasmid can be inserted into a target genome in a positioned mode, the expression of homologous recombination enzymes is induced through temperature, the antibiotic gene, for converter screening, in an original plasmid vector is cut, the expression vector of plant specific character genes and the vector of small amiRNA are built accordingly, and possible pollution, caused when transgenic plants carry antibiotic genes harmful to people, to the ecological environment is avoided.

Description

technical field [0001] The invention relates to a double-induced homologous recombination marker-free vector plasmid and its application, belonging to the field of biotechnology. Background technique [0002] The application of high and new technologies such as biotechnology in agricultural production will revolutionize the entire agriculture. In crop breeding, using recombinant gene technology to introduce exogenous beneficial genes into crops, and through antisense RNA technology or amiRNA technology, Oriented modification of a certain (some) target traits of plants while retaining other original traits is an ideal that cannot be achieved by conventional hybrid breeding. At home and abroad, protoplast fusion, microinjection, electroporation, laser and gene guns are used to transfer disease-resistant , insect resistance or high protein genes, and flower color genes are introduced into plants and expressed, such as tomatoes that do not soften when they mature, potatoes with ...

AI Technical Summary

Problems solved by technology

In the 1990s, there were laboratories in the world that started this work. Others have reported some plasmid vectors that use site-specific recombination systems and heat shock promoters to delete marker genes, mainly using Cre / loxP Mediated transgenic safety plant expression vectors, the existing Cre / loxP site-specific recombination system removes marker genes in transgenic plants. Because the Cre / loxP site-specific recombination system is reversible, its deletion efficiency is reduced The problem, and the FLP / frt site-specific recombination system, when the recombinase FLP plant expression vector was used to transform the selection marker-resistant tobacco plants containing the frt site for the second time, 17% of the co-transformed plants had selection marker gene excision events; When using the plant expression vector containing the frt site and the recombinase FLP plant expression vector to co-transform tobacco, the co-transformation efficiency was the highest when the concentration ratio of the two infected tobacco leaf discs was 3:1, which was 21%. The excision efficiency was 5%; and when the transgenic tobacco plants containing the recombinase FLP gene were transformed with the plant expression vector containing the frt site for the second time, the transformation efficiency was only 1.6%, and no selectable marker gene was excised

None of them used dual inducible promoters to control the expression efficiency of the recombinase, and they did not use the marker gene excision efficiency of plants transformed with the vector in different plants (Arabidopsis thaliana, rice, Chinese cabbage), at different developmental stages, and at different induction temperatures Conduct systematic research; others have reported some plasmid vectors with site-specific recombination system, low-temperature-inducible promoter, piggyBac transposase, and Gateway cloning system, but they did not use site-specific Site-specific dual-inducible recombination system and heat-shock promoter construction vector

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