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Plant transformation vector, and construction method and application thereof

A carrier and plant technology, applied in the field of plant transformation carrier and its construction, to achieve the effects of large heat production, improved transformation efficiency and integration rate, and low heat production

Inactive Publication Date: 2014-04-23
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Rando et al. found that 3-amino-2,3-diaminobenzoic acid poisons human nerves, inhibits the synthesis of human neurotransmitters, and has poor specificity as a screening reagent
The gene encoding protoporphyrinogen IX oxidase is another screening marker. Li et al. used the mutant gene encoding the enzyme in Arabidopsis thaliana as a screening marker to transform maize, and screened with diphenyl ether herbicides. The transgenic plants appeared green and non-transgenic. Plants are albino, and the harm of herbicide-resistant genes has been described above. Diphenyl ether herbicides have been eliminated because they are not easy to degrade in nature. Several research groups have found that diphenyl ether herbicides are very toxic. cause malignant tumors in humans
[0007]Therefore, there is an urgent need to develop a safe and efficient selection marker that can be used as a source of transgenic plants to replace currently controversial marker genes such as herbicide resistance marker genes, or to As a reporter gene indicating the cytological and tissue-specific localization of the target gene, it replaces the traditional green fluorescent protein gene gfp, red fluorescent protein gene asred and luciferase geneluc, etc., but there are currently no international literature and patent reports

Method used

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  • Plant transformation vector, and construction method and application thereof
  • Plant transformation vector, and construction method and application thereof
  • Plant transformation vector, and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of pC1301Ubi-ZmpBtAD-pUP vector with red fluorescent protein tracer and novel resistance screening marker.

[0040] 1. Reporter gene cobA and resistance selection markers alad gene cloning

[0041] 1. Clone encoding barley ( Hordeum vulgare ) DsRed UPMT cobA Gene

[0042] (1) Growth and cultivation of barley seedlings

[0043] Select barley (Wanmai No. 1) seeds with uniform size and full grains, sterilize the surface with 10% hydrogen peroxide, and place them in a petri dish with moistened gauze for cultivation. During the cultivation process, 0.1% nitrate solution was periodically sprayed, cultured at a constant temperature of 28°C, and illuminated for 16 hours, and seedlings emerged after about one week of growth.

[0044] (2) Extraction of total RNA from young barley leaves

[0045] When it grows to the three-leaf stage, take 1-2g of fresh leaves. The total RNA of barley young leaves was extracted by Trizol method, analyzed by 1% ag...

Embodiment 2

[0097] Example 2 The acquisition of Escherichia coli transformants

[0098] E. coli Preparation of DH5α / BL21 (DE3) Competent Cells:

[0099] (1) Inoculate a single colony of DH5α / BL21 (DE3) into 5 mL of LB liquid medium (see Table 2.1) without antibiotics, shake overnight at 37°C;

[0100] (2) Transfer 1% (v / v) into fresh LB liquid medium, shake at 37°C to) OD 600 =0.3~0.6;

[0101] (3) Take 50-100mL bacterial solution and add it to two sterile centrifuge tubes respectively, place on ice for 30min;

[0102] (4) Centrifuge at 4°C and 4500rpm for 10 minutes, take the supernatant, and suspend the bacteria in 2 mL of pre-cooled 0.1% CaCl 2 The solution was suspended and placed in an ice bath for 30 min.

[0103] (5) Centrifuge at 4°C, 4500rpm for 10min, discard the supernatant, and suspend the bacteria in 2mL of pre-cooled 0.1% CaCl 2 solution, then add 300 μl of sterile glycerol, and mix well. 50-100μl was dispensed into 1.5mL EP tubes and stored at -70℃.

[0104] Ta...

Embodiment 3

[0112] Example 3 Transformation of Agrobacterium tumefaciens

[0113] The plant expression vector constructed in Example 1 can transform rice explants by using methods such as Agrobacterium-mediated method, transient transformation method, particle gun method, electric shock method, pollen tube introduction method or liposome fusion method. Bacteria-mediated method and transient transformation method; the Agrobacterium can be any Agrobacterium tumefaciens or Agrobacterium rhizogenes, and the applicant preferably is Agrobacterium tumefaciens EHA105 and GV3101.

[0114] Preparation of Agrobacterium tumefaciens EHA105 or GV3101 competent cells:

[0115] (1) Pick a single colony of EHA105 or GV3101 from the YEP solid medium (see Table 3.1) plate containing rifampicin (Rif), kanamycin (kana) or gentamicin (Cef), and inoculate it on a plate containing In the YEP liquid medium (according to Table 3.1, without adding agar) of corresponding antibiotics, shake overnight at 28°C and 200...

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Abstract

The invention provides a plant transformation vector, and a construction method and application thereof. In the vector, a red fluorescent protein UPMT (uroporphyrinogen III methyltransferase) and a resistance screening alad gene are used as a reporter gene and a selected marker for plant transfection. The red fluorescent protein UPMT and the resistance screening alad gene have similar functions, are superior to antibiotic and a green fluorescent protein fusion marker for example, and can be used for screening and subcellular organelle positioning in rice chloroplast transformation. Thus, the two genes used as screening markers of a transgenic plant can indicate the positioning of the cytologic and tissue specificity of a target gene, and can also promote the photosynthesis efficiency of plants and the accumulation of nutrient substances. The invention has potential and broad application backgrounds in the transgenic plant research and marketing process, and is hopefully developed into a dominant selected marker system in plant transformation.

Description

technical field [0001] The present invention relates to the fields of plant molecular biology and cell biology, in particular to a plant transformation vector that can be used for specific expression of target genes in plant (sub)cellular localization, and has red fluorescent protein tracer and novel and safe resistance selection markers and its construction method and application. Background technique [0002] At present, about 55 marker genes for transfection and transformation of plant research and growth and development of cereal crops have been confirmed for their effectiveness, safety, scientific research and commercial applications. There are two types of marker genes widely used in transgenic plants: one is a screening marker; the other is to use a marker gene elimination system to remove the screening marker in cells, but this technique is relatively complicated and requires the construction of a new transgenic vector. It takes 2-3 generations of transgenic crops ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66A01H5/00
Inventor 范军李静魏玲玲张宽亮
Owner ANHUI AGRICULTURAL UNIVERSITY
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