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

A vector plasmid and homologous recombination technology, applied in the biological field, can solve the problems of low deletion efficiency, no dual inducible promoters, no selective marker gene excision, etc., and achieve the effect of high transformation efficiency and high excision rate.

Inactive Publication Date: 2019-10-18
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
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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

Method used

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

Examples

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Effect test

Embodiment 1

[0026] (1) Cloned the 2kb upstream regulatory sequence of the heat shock protein hsp70 (heat shockprotein70) 5' end of tomato, rice, cabbage and Arabidopsis, and designed the regulators of four plants with different lengths and different elements by analyzing the promoter elements The sequence fragments were connected with the GUS (β-glucuronidase) reporter gene, and constructed together with the selection marker gene module of the glufosinate resistance gene bar driven by the CaMV35 promoter to form the hsp70 promoter function detection vector. The four vectors will be used for functional verification of the heat shock-induced activity of the heat shock protein hsp70 promoter in tomato, rice, Chinese cabbage and Arabidopsis.

[0027] (2) Connect the four hsp70 5'-end upstream regulatory sequence fragments to the recombinase gene Cre gene, and connect them in series with the SV40 promoter-bar gene module, and insert them into two reverse loxP sites and two reverse loxP sites resp...

Embodiment 2

[0030] Example 2 The reverse reaction system of point-specific recombination was used to knock out the Arabidopsis SR1 gene.

[0031] SR1 is a lethal protein. Both overexpression and knockout can lead to plant death. This plasmid system uses an inducible promoter to induce the expression of the target gene, and can control the expression of the lethal protein according to the induced expression time and expression abundance. A small RNAi fragment of SR1 gene that regulates SR1 kinase activity was cloned from Arabidopsis thaliana by PCR method. After cloning and sequencing, it was assembled under the Estradiol-induced promoter with a small restriction site, and expressed in the target A tag (HA) is added to the 3-end of the gene to construct an intermediate vector for the expression element. In the T-DNA segment of the plant bivalent expression vector, the 35s: NptⅡ: nos expression element is located between the Lox sites arranged in the same direction, and then a series of int...

Embodiment 3

[0032] Example 3 Applying the reverse reaction system of point-specific recombination to carry out the production of Chinese cabbage-type rapeseed WRKY31 Gene knockout.

[0033] WRKY31 is a transcription factor, both overexpression and knockout result in abnormal silique development and failure to harvest seeds. This plasmid system uses an inducible promoter to induce the expression of the gene of interest, depending on whether the seeds need to be harvested WRKY31 expression. Cloned from Chinese cabbage (Brassica rapa) by PCR WRKY31 After cloning and sequencing, the RNAi gene fragment was assembled under the Estradiol-induced promoter with small restriction sites, and a tag (HA) was added to the 3-end of the target gene to construct an intermediate expression element vector. In the T-DNA segment of the plant bivalent expression vector, the 35s: NptⅡ: nos expression element is located between the Lox sites arranged in the same direction, and then a series of intermediate...

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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 ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/65A01H6/46A01H6/20
Inventor 缪颖伍炳华任育军
Owner FUJIAN AGRI & FORESTRY UNIV
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