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An in vitro gene stacking technique compatible with recombinase-mediated gene stacking in vivo and its application

A technology of gene stacking and recombinase, which is applied in recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of increasing examination costs and time, and achieve the effect of fast preparation method and easy application.

Active Publication Date: 2018-11-30
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the old gene and the new gene that have been identified by the review department are re-stacked and retransformed in vitro, additional review costs and time will be added

Method used

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  • An in vitro gene stacking technique compatible with recombinase-mediated gene stacking in vivo and its application
  • An in vitro gene stacking technique compatible with recombinase-mediated gene stacking in vivo and its application
  • An in vitro gene stacking technique compatible with recombinase-mediated gene stacking in vivo and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 In vitro gene stacking system

[0079] The in vitro gene stacking system includes vectors with three substrates, namely, vectors A, B, and C, and the construction process is as follows.

[0080] The conventional recombinant DNA method was used for the construction of the vector in this example. All PCR reactions were performed using Phusion High-Fidelity DNA Polymerase (NEB Beijing, China). Hereinafter, the vector pZH36D uses the replication origin site of plasmid ColE1 and the replication origin site derived from plasmid pVS1 to initiate replication in Escherichia coli and Agrobacterium respectively; the rest of the vectors below include molecules A, B, C, Both D and E use the replication origin site derived from plasmid pBR322 to initiate replication. Escherichia coli DH5α ( F - endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔ M15 Δ(lacZYA-argF)U169 hsdR17(r K - m K + ), λ– ) are applied to the transformation and recovery of recombin...

Embodiment 2

[0103] Example 2 Preparation of Crude Extract of Recombinase and Conditions of Recombination Reaction in Vitro

[0104] 1. Preparation of recombinant enzyme crude extract

[0105] Escherichia coli BL21 (Studier, F.W. and Moffatt, B.A. (1986) Use of bacteriophage-T7 RNA-polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130) were selected as the prokaryotic expression host of the recombinase. Escherichia coli BL21 containing recombinant enzyme (phiC31, Bxb1 or Cre) expression vector was first cultured overnight at 37 degrees Celsius, and then diluted 1:100 into 50 ml of fresh BL medium containing 50 µg / ml kanamycin, and then Incubate at 15°C to OD 600 Reach between 0.3~0.4. Then, IPTG (isopropylthiogalactopyranoside) was added to the bacterial solution to a final concentration of 0.1 mM. Continue to culture the bacterial solution at 15°C for 20 hours and then collect the bacterial cells by centrifugation (4000 rpm, 30 minutes). Su...

Embodiment 3

[0108] Example 3 Application of In Vitro Gene Overlay System

[0109] In this example, 5 exogenous target genes ( DsRed , YFP , GUS , LUC , GFP ) and transfer them into plants as an example, the superposition system described in Example 1 is used for gene superposition, and the specific operation process is as follows.

[0110] 1. The first round of in vitro gene stacking

[0111] Such as figure 1 with figure 2As shown in a, the phiC31 recombinase extract was first used to catalyze the recombination between molecules A and B (recombination products include AB and AB'), and then the recombinant DNA was recovered and transformed into E. coli DH5α. Selection was performed on solid LB medium containing chloramphenicol and ampicillin. Among the 20 clones resistant to both chloramphenicol and ampicillin, primers b+d and a+c detected 4 clones that were consistent with the molecular AB configuration, and primers b+d2 and a2+c detected 2 clones that were consistent with ...

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Abstract

The invention discloses in-vitro gene stacking technology compatible with recombinase-mediated in-vivo gene stacking and application of in-vitro gene stacking technology. The in-vitro gene stacking technology includes element forming of in-vitro gene stacking series vector and an in-vitro gene stacking method. The in-vitro gene stacking method includes: utilizing an irreversible site-specific recombination system for cointegration between two DNA molecules; utilizing a reversible site-specific recombination system to delete unneeded DNA sequences; utilizing a second irreversible site-specific recombination system to transfer multigene vectors which are stacked well in vitro into a binary vector suitable for agrobacterium transformation for plant genetic transformation, or directly integrating the multigene vectors on a specific seat of a genome in a targeted manner through site-specific recombination in vivo. The in-vitro gene stacking technology meets needs of in-vitro gene stacking and is compatible with an in-vivo recombinase-mediated gene stacking system.

Description

technical field [0001] The invention relates to a carrier suitable for in vitro gene stacking and its application. Background technique [0002] With the continuous development of transgenic technology, transgenic crops containing multiple transgenic traits have become the mainstream direction of future development. At present, there are mainly four methods for superimposing multiple transgenic traits into crop varieties: 1. Through genetic crossing of lines containing different transgenic traits, multiple transgenic traits are aggregated into the same strain. 2. Integrating multiple new transgenic traits into the same variety through re-transformation of existing transgenic varieties. In this method, new transgenic traits are integrated into plant chromosomes in a random manner. 3. Site-specific integration of multiple transgenic traits into existing transgenic loci through site-specific recombination or homologous recombination. 4. Retransform all transgenic traits into...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66
Inventor 区永祥陈伟强
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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