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Phloem specific promoter and application thereof

A specific, promoter technology, applied in the field of plant genetic engineering, can solve problems such as plant energy and nutrient waste

Inactive Publication Date: 2013-02-13
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These promoters allow foreign genes to be expressed evenly in various parts of the plant, and cannot be specifically expressed in tissues and organs attacked by certain insects or pathogenic bacteria.
Expressing in non-invasive parts is an extra burden and a waste of energy and nutrients to the plant itself

Method used

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  • Phloem specific promoter and application thereof
  • Phloem specific promoter and application thereof
  • Phloem specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Acquisition of phloem-specific promoter

[0034] 1. Culture of Arabidopsis

[0035] Arabidopsis seeds were placed in a 1.5ml centrifuge tube, first sterilized with 75% alcohol for 1 to 2 minutes, then sterilized with 1% sodium hypochlorite for 3 to 5 minutes, rinsed with sterile water for 5 times, and then sowed on sterilized MS with the tip of a gun. On solid medium (1.5% sucrose, 0.8% agar, pH 5.8), protected from light, vernalized at 4°C for 2 days, then transferred to the light culture room, temperature 22°C / 18°C (day / night), 16h / 8h light cycle, cultivated under incandescent light.

[0036] 2. Using the conventional Trizol method in the art to extract Arabidopsis RNA.

[0037] 3. RT-PCR detection

[0038] 3.1 Reverse transcription reaction (cDNA synthesis)

[0039] MMLV reverse transcriptase is used for reverse transcription reaction, and the reaction system and reaction conditions can refer to the relevant kit instructions.

[0040] 3.2 RT-PCR amplific...

Embodiment 2

[0051] Example 2 Construction of plant phloem-specific promoter (AtGLP15) binary expression vector

[0052] The phloem-specific promoter prepared in Example 1 was connected to the plant expression vector PBI101 to obtain the recombinant expression vector pBAG; pBAG was used to transform Escherichia coli E.coli DH5α, and positive clones were screened out, and the positive clone plasmid was used to transform Agrobacterium A.tumefaciensEHA105, Thus, the desired plant phloem-specific promoter binary expression vector (named pBAG) is prepared, and the specific construction flow chart is as follows: figure 2 shown.

[0053] After the recombinant expression vector pBAG was extracted from the plasmid, it was detected by double enzyme digestion with SalI and SmaI. The electrophoresis results were as follows: image 3 Shown: figure 1 The middle lane M is the molecular weight standard, and lanes 1 and 2 are the products after digestion of the binary expression vector pBAG.

[0054] T...

Embodiment 3

[0055] Example 3 Agrobacterium-mediated genetic transformation of Arabidopsis

[0056] 1. Agrobacterium-mediated genetic transformation of Arabidopsis

[0057] First, sow wild-type Arabidopsis thaliana in a flower pot with a diameter of 10 cm, and remove the terminal inflorescence when the main stem is about 3 cm high, pay attention to avoid damaging the axillary inflorescence, and transform it when the axillary inflorescence grows and the lower flowers begin to pollinate . Before transformation, pollinated flowers and fruit pods were removed. Then, the binary expression vector (positive single colony) prepared in Example 2 was picked and inoculated in 5 ml of LB liquid medium containing antibiotics Kan and Rif, and the bacteria were shaken overnight at 28°C. Pour the cultured bacterial solution into 500ml LB liquid medium containing the same antibiotic, and shake the bacteria overnight at 28°C. Pour the bacterial liquid into six 50ml centrifuge tubes, centrifuge at 4000g f...

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Abstract

The invention discloses a phloem specific promoter. A nucleotide sequence of the phloem specific promoter is shown by SEQ ID NO. 1; a phloem specific gene can ensure that an exogenous target gene is specifically expressed at the phloem, so that harm of injurious insects and pathogenic microorganisms intruded from the phloem is purposely prevented and controlled, the selection pressure of the insects and the pathogenic microorganisms is reduced and the side effect probably produced to the environment thereby is reduced, and positive effects are produced to the aspect of reducing metabolic burden which is brought by expressing insect-resistant and disease-resistant genes to transgenic plants, and the other aspects. In addition, by using the phloem tissue specific promoter different from a combined promoter in the use of bivalent or multivalent insect-resistant and disease-resistant genetic engineering, interaction of homologous sequences of the promoter can be avoided so as not to cause gene silencing.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to the tissue-specific expression of a gene promoter, in particular to a phloem-specific promoter and its application. Background technique [0002] The vascular bundle is an important place for the transportation of water and internal substances and cell proliferation in plants, and the phloem and xylem are the components of the vascular bundle. [0003] Phloem (phloem) is an important complex tissue with transduction function in plants. It not only involves the transportation of nutrients (organic nitrogen, inorganic salts, carbohydrates, etc.), but also includes a wide variety of mRNAs, proteins and other macromolecules, The long-distance transportation of signal molecules in plants, so the function of phloem involves nutrient distribution, signal transduction, system defense and so on. [0004] There have been reports on the cloning of phloem-specific promoter...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N1/21A01H5/00C12R1/19
Inventor 阳成伟杨浪
Owner SOUTH CHINA NORMAL UNIVERSITY
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