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Plant binary expression vectors containing LoxP-FRT recombinase sites

A loxp-frt, carrier technology, applied in the biological field, can solve problems such as limitations

Inactive Publication Date: 2013-10-23
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This limits its application in commercial production of plant genetic transformation

Method used

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  • Plant binary expression vectors containing LoxP-FRT recombinase sites
  • Plant binary expression vectors containing LoxP-FRT recombinase sites
  • Plant binary expression vectors containing LoxP-FRT recombinase sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1, Construction of Plant Expression Binary Vectors pYBA100, pYBA200, pYBA300

[0055] Wild-type Arabidopsis Columbia (Col-0) was purchased from ABRC (Arabidopsis Biological Resource Center, Columbus, OH, USA), the product catalog number is CS28166, and it is referred to as wild-type Arabidopsis for short in the examples;

[0056] Escherichia coli strain DH5α was purchased from Tiangen Company, the product catalog number is CB101;

[0057] Agrobacterium tumefaciens GV3101 (pMP90) can be obtained from the Beijing Agricultural Biotechnology Research Center; reference: Koncz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396.

[0058] The construction method of the pBBBasta vector is as follows: 1. The plasmid pDHB321.1 was digested with SacI, and a fragment of about 2100 bp was recovered (including LB-bar expression c...

Embodiment 2

[0226] Example 2, application of pYBA100, pYBA200, pYBA300 binary vectors

[0227] 1. Transformation and identification of Arabidopsis

[0228] The pYBA100, pYBA200, and pYBA300 vectors obtained in Example 1 were transformed into the Agrobacterium tumefaciens GV3101:pMP90 strain by electric shock method, respectively, and GV3101:pMP90 / pYBA100, GV3101:pMP90 / pYBA200, and GV3101:pMP90 / pYBA300 were obtained respectively.

[0229] 1) GV3101:pMP90 / pYBA100 was used to infect 32 wild-type Arabidopsis plants by inflorescence dipping method, which was the T0 generation.

[0230] After disinfection, 32 transgenic Arabidopsis seeds harvested from the T0 generation were sown on Hoagland / 2 solid medium containing 100mg / L Kan, placed at 25°C, and the photoperiod was 16h (light) / 8h (dark). After about 2 weeks, 56 surviving T1 generation transgenic Arabidopsis green seedlings were selected and transplanted to the artificial climate chamber.

[0231] 36 transgenic Arabidopsis thaliana plants ...

Embodiment 3

[0245] Example 3, Verification of deletion of screening marker gene expression cassettes in pYBA100, pYBA200, pYBA300 binary vectors

[0246] In order to detect whether the recombinase site can accurately delete the expression frame of the plant screening marker gene, an in vitro deletion experiment was designed. Take 0.5 μg of pYBA100, pYBA200, pYBA300 plasmid DNA obtained in Example 2, mix with 1 μl Cre recombinase, 3 μl 10x Cre Buffer, and make up to 30 μl with water. 60min at 37°C, 5min at 70°C. Take 5 μl of the recombinant product to transform Escherichia coli. 12 clones were randomly selected to extract plasmid DNA, which was verified by EcoRI enzyme and 1% agarose electrophoresis.

[0247] The result is as Figure 9 As shown, 2 of the 12 clones of pYBA100 (lanes 5 and 13) were recombined with Cre, and the target fragment (1.45kb) was deleted, and the plasmid was reduced to 3.91kb.

[0248] The result is as Figure 10 As shown, one of the 12 clones of pYBA200 (lane ...

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Abstract

The invention discloses plant binary expression vectors containing LoxP-FRT recombinase sites. The plant binary expression vectors are cyclic double-strand deoxyribonucleic acid (DNA) molecules and comprise a replication initial original point Co1E1 of escherichia coli, a replication initial original point Rep-ori of agrobacterium tumefaciens, a regulatory protein Rep expression frame, a transferred-deoxyribonucleic acid (T-DNA) ultra-drive area, a T-DNA right margin, a T-DNA left margin and a kanamycin (Kan) resistance gene expression frame sequentially from a 5' tail end to a 3' tail end. Experiments prove that the plant expression binary vectors pYBA100, pYBA200 and pYBA300 are constructed, the vectors are small, and a large number of multi-clone sites (MCS) are obtained in the T-DNA area; and in-vitro deletion experiments prove that a vector pYBA series can delete an expression frame of a plant marker gene by Cre recombinase, so that the requirement for producing transgenic plantssafely can be met.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a plant binary expression vector containing a LoxP-FRT recombinase site. Background technique [0002] With the development of technology, new methods of plant genetic transformation are constantly increasing. However, among the numerous plant genetic transformation methods, the transformation method mediated by Agrobacterium is the most researched and the mechanism is the most thorough. Agrobacterium-mediated transformation has become the first choice for plant genetic transformation due to its unique advantages such as simple operation and low cost. Agrobacterium tumefaciens mediated method is one of the methods to obtain stable plant genetic transformation. [0003] Agrobacterium tumefaciens, a Gram-negative soil bacterium, is a plant pathogen that can cause crown gall tumors in plants. Agrobacterium is able to integrate the T-DNA region of its Ti plasmid into the plant genome ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 姚磊闫晓红王慧马荣才
Owner BEIJING AGRO BIOTECH RES CENT