Application of tandem repeat sequence capable of improving expression activity of plant gene

A gene and plant technology, applied in the field of plant transgenic development, can solve problems such as affecting plant growth

Inactive Publication Date: 2012-07-25
JIANGSU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, several recent plant reports on the effect of tandem repeats on gene function have attracted widespread attention: For example, Sureshkumar et al. reported in an article published in Science in 2009: TTC tandem in the intron of the Arabidopsis IIL1 (At4g13430) gene Repeat sequence expansion can significantly affect the expression of the gene, thereby affecting the growth of the plant (Science 2009, 323: 1060-1063)

Method used

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  • Application of tandem repeat sequence capable of improving expression activity of plant gene
  • Application of tandem repeat sequence capable of improving expression activity of plant gene
  • Application of tandem repeat sequence capable of improving expression activity of plant gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction of full-length promoters and deletion promoters

[0021]The leaves of Arabidopsis thaliana (Columbia ecotype) were used as materials, and the total plant DNA was extracted by CTAB method (EMBO J 1987, 6: 3901-3907). Using the total plant DNA as a template, the promoter fragment of zinc finger transcription factor At5g15840 gene was amplified by specific primers. Synthetic primers Z1 (5'-AAGCTTGATCTTAGAAACATGTCCT-3', SEQ ID NO.1, introduced HindIII site), Z2 (5'-GGATCCTCTTGCAGCTAGTTGA-3', SEQ ID NO.2, introduced BamHI site), Z3( 5'-GTCGACATCCTCCTCCTATGCA-3', SEQ ID NO.3, SalI site was introduced), Z4 (5'-GTCGACATAGGCCTTCCCAAAAGCT-3', SEQ ID NO.4, SalI site was introduced). Among them, primer Z1 / Z2 amplifies a 1091bp full-length promoter fragment (named zfP), such as figure 1 ; The 866bp fragment (designated as zfP1) of primer Z1 / Z3 amplification, such as figure 2 ; The 190bp fragment (designated as zfP2) amplified by primer Z2 / Z4, such as im...

Embodiment 2

[0022] Embodiment 2: Construction of GUS fusion expression vector

[0023] The two positive clones and the binary expression vector pBI121 (available from Beijing Tianenze Gene Technology Co., Ltd.) in Example 1 were double-digested with restriction enzymes HindIII and BanHI respectively, and the full-length promoter and deletion The promoter fragments were respectively ligated with pBI121 restriction fragments to construct promoter expression vectors (zfP::GUS and zfPΔ::GUS) ( Figure 4 ), they were respectively transformed into DH5α Escherichia coli competent strains, and multiplied.

Embodiment 3

[0024] Example 3: Agrobacterium Transformation

[0025] The constructed expression vectors (zfP::GUS and zfPΔ::GUS) were transformed into the Agrobacterium host cell EHA105 (available from the National Microorganism Resource Bank www.matrs.com, the product ID is 20110114049). The specific method is as follows: the constructed expression vectors (zfP::GUS and zfPΔ::GUS) Escherichia coli strains were inoculated into 10 ml of Km (50 μg / ml) LB liquid medium, and after culturing overnight at 37°C, the plasmid was extracted for subsequent transformation Agrobacterium spare. The EHA105 Agrobacterium strain was inoculated into 5 ml of YEP liquid medium containing Sm (50 μg / ml), and cultured at 200 rpm at 28° C. for 48 hours. The cells were collected by centrifugation and frozen with 0.1M CaCl 2 Resuspend, place on ice for 20 minutes, centrifuge at 5000rpm for 2 minutes at 4°C, and use 200μl 0.1M frozen CaCl 2 suspended. Then add the prepared expression vector plasmids (zfP::GUS an...

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Abstract

The invention relates to application of a tandem repeat sequence capable of improving expression activity of a plant gene, namely application of a fragment containing five tandem repeat TTTACAC sequences on a zinc finger transcription factor At5g15840 gene promoter of Arabidopsis thaliana in improving expression of a target gene in a transgenic plant. When the tandem repeat sequence is deleted, the activity of the promoter is sharply lowered, and therefore, the tandem repeat sequence has the characteristic of enhancing gene expression. By use of the tandem repeat sequence provided by the invention, the transgene development of a plant is performed, so that the expression of the target gene is improved, and the important economic and social benefits are achieved.

Description

technical field [0001] The invention belongs to the field of plant tandem repeat sequences, in particular to a tandem repeat sequence on a zinc finger transcription factor At5g15840 gene that can improve gene expression activity, and the use of the tandem repeat sequence in the development of plant transgenes. Background technique [0002] A tandem repeat sequence is a segment of DNA composed of several nucleotides, which is ubiquitous and randomly distributed in the genomes of prokaryotes and eukaryotes, and its number of repetitions is highly variable. Tandem repeats usually have the following characteristics: 1. Non-random distribution in the genome; 2. Extensive presence in the genome, and the content of tandem repeats has no correlation with genome size; 3. Tandem repeats can exist in the untranslated region (UTR ), exons, introns or intergenic regions; 4. Tandem repeats are highly variable and polymorphic, so they are widely used in genetic markers; Different regions ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A01H5/00
Inventor 曹军
Owner JIANGSU UNIV
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