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Plant expression vector without antibiotic marker gene and construction method and application thereof

A technology for plant expression vectors and transgenic plants, which is applied in the field of plant expression vectors and their construction, can solve the problems of reduced expression levels and high difficulty, and achieve the effects of promoting plant growth and development, improving safety, and improving stress resistance

Inactive Publication Date: 2011-09-14
BEIJING NORMAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This technology is more difficult to construct the above-mentioned carrier
[0005] (3) Among the co-expressed genes, the ipt gene is used as the second gene, and its expression level is reduced
At present, there is no research report on using the ipt gene as a marker gene in the way of second gene expression

Method used

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  • Plant expression vector without antibiotic marker gene and construction method and application thereof
  • Plant expression vector without antibiotic marker gene and construction method and application thereof
  • Plant expression vector without antibiotic marker gene and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the cloning of ipt gene and 35S promoter

[0020] (1) ipt gene cloning

[0021]According to the ipt gene sequence (GenBank accession number: NC_003065) on the Agrobacterium tumefaciens C58 nopaline type Ti plasmid reported by Wood et al. 3') and the antisense primer sequence (5'-GAGCTCCTAATACATTCCGAATGGAT-3') have XmaI and SacI restriction sites at the 5' end respectively, and the length of the amplified product fragment is 735bp. The Agrobacterium tumefaciens C58 nopaline-type Ti plasmid was used as a template. Add 2U of high-fidelity pfu polymerase, 20ng of Ti plasmid, 200μmol / L of dNTP, 20ρmol / L of primers and 5μl of 10×pfu amplification buffer into the 50μl reaction system, and make up to 50μl with ultrapure water. The amplification conditions were: pre-denaturation at 94°C for 4 minutes, followed by denaturation at 94°C for 1 minute, annealing at 55°C for 1 minute, extension at 72°C for 2 minutes, a total of 30 cycles, and finally incubation at 72°C...

Embodiment 2

[0025] Example 2, constructing plant expression vectors without antibiotic markers

[0026] (1) Digest the T-ipt plasmid and the binary vector pBI121 plasmid with Xma I and Sac I nucleic acid restriction endonucleases. The specific method is to use Xma I and Sac I restriction endonucleases from Fermentas Company, add 0.5 μg of T-ipt plasmid DNA, 2 μl of 10×Tango double digestion reaction buffer and Xma I and Sac I to the 20 μl reaction system Each 10U, enzyme digestion reaction in 37 ℃ constant temperature water bath for 4 to 6 hours. Electrophoresis enzyme digestion products, under long-wave ultraviolet light, quickly cut out the gel containing DNA fragments, and purify the ipt gene fragments with Shanghai Shenergy Biotechnology Co., Ltd. 3S Nucleic Acid Purification Kit. The pBI121 fragment with a length of 12 868 bp was obtained by the same digestion and purification method.

[0027] (2) The ipt gene was connected with the binary vector pBI121 plasmid. In 10 μl ligation ...

Embodiment 3

[0030] Example 3, Agrobacterium-mediated genetic transformation and screening of transgenic regenerated plants

[0031] (1) Genetic transformation of Agrobacterium LBA4404. Mix 0.1 μg of pBI201 plasmid carrying the target gene iaaM with 100 μl of Agrobacterium LBA4404 competent cells, and transform Agrobacterium by freeze-thaw method. Then, culture on YEB medium containing 80mg / L rifampicin and 100mg / L kanamycin for 24 to 48 hours, take single colonies and expand them in 2ml of YEB liquid medium, and extract the plasmid in a small amount by alkaline lysis method. The Agrobacterium cell clone carrying the pBI201 plasmid containing the iaaM gene was confirmed by electrophoresis observation and identification after double digestion with Bsu 36I and Xba I enzymes.

[0032] (2) Tobacco K326 variety was used as material to determine the highest concentration of cytokinin growth regulators at which leaf explants could not differentiate into adventitious buds. Specifically, 0, 0.02,...

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Abstract

The invention discloses a plant expression vector without antibiotic marker genes and a construction method thereof. The T-DNA region of the vector contains a gene cassette which comprises CaMV 35S promoter, Xba I, Bam HI and XmaI restriction endonuclease enzyme sites, isopentenyl transferase (ipt) gene and nos terminator sequence. The construction method of the vector includes: using a binary vector pBI121 as a base, replacing gus gene with ipt gene, deleting npt II gene through Bsu 36 I and Xba I enzyme sites, reconnecting the CaMV 35S promoter to the T-DNA region. With the technical scheme provided by the invention, transgenic plants can be induced and regenerated on a culture medium with a proper amount of low-concentration cytokinin growth regulators, and the obtained transgenic plants are the transgenic plants expressed by the target genes, which can reduce steps for identifying whether the target gene is expressed, and can avoid potential risks of antibiotic marker genes.

Description

technical field [0001] The invention relates to a plant transgenic technology in plant biotechnology, in particular to a plant expression vector without antibiotic markers and its construction method and application. Background technique [0002] In plant genetic engineering, selectable marker genes on plant expression vectors can be divided into negative selectable marker genes and positive selectable marker genes. Negative selection marker genes are mainly a class of antibiotic markers and herbicide marker genes. Although the existing data have not found that these marker genes are transferred at a detectable frequency or cause harm between related species, people still pay attention to antibiotics and herbicides. Potential risks of agent marker genes in transgenic plants. Positive selection markers mainly use toxic and non-toxic drugs, metabolic analogs or carbon sources as selection agents, and generally belong to safety selection markers. Positive selection markers al...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 肖尊安何艳李朝霞殷娴王春城肖桔清张彦潘芳
Owner BEIJING NORMAL UNIVERSITY
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