Application of Cre/loxP recombinant enzyme system in transgenic breeding of chrysanthemum

A transgenic and recombinase technology, used in applications, recombinant DNA technology, angiosperms/flowering plants, etc.

Inactive Publication Date: 2012-07-11
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, ground cover chrysanthemum is mainly used in Beijing, Tianjin and the "Three North" regions of China; in the south, due to the hot and humid climate, it has not been widely introduced and cultivated.

Method used

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  • Application of Cre/loxP recombinant enzyme system in transgenic breeding of chrysanthemum
  • Application of Cre/loxP recombinant enzyme system in transgenic breeding of chrysanthemum
  • Application of Cre/loxP recombinant enzyme system in transgenic breeding of chrysanthemum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1Pksb-rd29A-Cre / lox plant expression vector construction

[0027] First, the promoter rd29A was amplified from Arabidopsis thaliana by PCR, digested with XbaI and BamHI, and connected with the large fragment of the intermediate vector pUC18 also digested with XbaI and BamHI to obtain the intermediate vector pUC18-rd29A. The target gene Cre was amplified by PCR from the pCre plasmid, and double-digested with NcoI and PstI. At the same time, pUC18-rd29A was double-digested with NcoI and PstI, ligated to obtain the intermediate vector pUC18-rd29A-Cre, and then PCR was used to obtain the Puc18 -The rd29A-Cre-nos reading frame sequence was amplified from the rd29A-Cre vector, and double-digested with SacI and StuI, and connected with the expression vector pksb that was also double-digested with SacI and StuI to obtain a plant expression vector that eliminated the selectable marker system Pksb-rd29A-Cre / lox.

[0028] (1) PCR amplification of rd29A promoter

[002...

Embodiment 2

[0037] Example 2 The acquisition of gene gun-mediated transgenic chrysanthemums

[0038] 1. Pre-cultivation of explants

[0039] Select the upper young leaves, middle leaves and lower old leaves of the aseptic chrysanthemum seedlings of 'Beilinhuang' which are relatively consistent in size and nutritional growth for about 30 days, cut them into small pieces, and put them in MS+0.2mol / L sorbitol+0.2mol / L Mannitol hyperosmotic medium cultured for 12h.

[0040] 2. Gold powder and bullet preparation

[0041] The preparation of particle bullets refers to the method of Sanford et al.

[0042] Preparation of gold powder suspension:

[0043] 1) Weigh 30 mg of gold powder particles with a diameter of 1.0 μm into a 1.5 mL centrifuge tube.

[0044] 2) Add 1 mL of absolute ethanol, vortex for 3-5 minutes, let stand for 15 minutes, and centrifuge at 15000 rpm for 5 minutes.

[0045] 3) Carefully remove the supernatant, add 1 mL of sterile distilled water, vortex fully for 1 min, let s...

Embodiment 3

[0070] The PCR detection of embodiment 3 transgenic plants

[0071] (1) Chrysanthemum genomic DNA extracted by CTAB method

[0072] A. Cultivate the chrysanthemum seedlings in dark for 2-3 days before extraction to reduce the content of starch in the material;

[0073] B. Cut the resistant buds and the young leaves of the non-transgenic control chrysanthemum. As for the pre-cooled mortar, pour liquid nitrogen into the powder and quickly grind it into powder. Take an appropriate amount of powder and put it into a pre-cooled 2mL centrifuge tube with a small key. Add 650 μL of preheated 2×CTAB extraction buffer (with 2% mercaptoethanol added in advance), and mix quickly;

[0074] C. Keep warm in a water bath at 65°C for 30-50 minutes. Gently turn it upside down 2-4 times by hand to fully mix the extract and the material;

[0075] D. After cooling the extract to room temperature, add an equal volume of 24:1 chloroform:isoamyl alcohol and mix gently. Centrifuge at 12000rpm for ...

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Abstract

The invention relates to the application of a Cre / loxP recombinant enzyme system in transgenic breeding of chrysanthemum. According to the invention, a particle bombardment method is adopted to convert plant expression vectors that respectively carry exogenous P5CS (Pyrroline 5 Carboxylate Synthetase), NP-1 (Nuclear Protein-1) genes and Cre / loxP to together into ground-cover chrysanthemum, and transgenic lines adopting polygenic integration transformation are determined and obtained through hybridization detection of PCR (Polymerase Chain Reaction) and Southern; and then low temperature treatment is performed at the temperature of 4 DEG C, and Cre can express, so that a selectable marker gene positioned between two equidirectional loxP sites is deleted, detection is carried out through a molecular method to prove that the obtained results are correct, and the ground-cover chrysanthemum transgenic lines that do not contain marker genes npt-II are obtained.

Description

technical field [0001] The invention belongs to the field of plant breeding, and in particular relates to a method for using a Cre / loxP recombinase system to delete a selection marker gene and obtain a transgenic chrysanthemum without the selection marker gene. Background technique [0002] In plant transgenic research, marker genes are generally used to screen positive transformants from a large number of transformed cells, and antibiotic resistance genes or herbicide resistance genes are currently the most used. Although the use of marker genes in ornamental plants will not have an impact on food safety, with the continuous development and breakthrough of transgenic technology, more and more attention has been paid to the environmental pollution caused by transgenic plants. Currently, the Cre / loxP system is the most complete and widely used method for deleting marker genes, which can be used to delete marker genes in transgenic plants. Chakraborti et al. applied the Cre / l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 张启翔赵伶俐张秀海高亦珂程堂仁
Owner BEIJING FORESTRY UNIVERSITY
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