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Plant recombination expression vector and transformant containing same

An expression vector, plant technology, applied in the field of genetic engineering, can solve the problems of promoter conflict, unable to achieve simultaneous regulation of key enzyme genes, etc.

Inactive Publication Date: 2009-12-30
SHANGHAI HAITAI PHARMA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since transcription factors are specific to the regulation of key enzymes in metabolic pathways, the construction of vectors containing only transcription factors sometimes cannot achieve the purpose of simultaneously regulating all key enzyme genes in metabolic pathways, and the construction of vectors with transcription factors plus key enzyme genes There is also the problem of promoter conflicts

Method used

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  • Plant recombination expression vector and transformant containing same
  • Plant recombination expression vector and transformant containing same
  • Plant recombination expression vector and transformant containing same

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1. The transcription factor orca3, the specific promoter tdc promoter and the key enzyme gene g10h in periwinkle were obtained by the method of gene cloning.

[0025] 1. Isolation

[0026] Soak periwinkle seeds in 75% ethanol for 1 min, then soak in 2% NaCl for 10 min, rinse with sterile water for 3-4 times, blot the surface moisture with sterile absorbent paper, and inoculate in hormone-free MS (Murashige and Skoog, 1962 ) in solid medium, 25°C, 12h / 12h light culture, to obtain sterile periwinkle seedlings, after the seedlings grow to about 5cm, cut leaves and stems for RNA extraction.

[0027] 2. RNA isolation (RNA isolation)

[0028] Weigh 0.5 g of the periwinkle aseptic test-tube seedlings, quickly freeze them with liquid nitrogen, grind them quickly with a mortar, add them to a 1.5 mL Eppendorf tube filled with 1 mL TRIzol (TRIzol Reagents, GIBCO BRL, USA), shake them fully Afterwards, stand at room temperature for 5 minutes, add 200 μL of chloroform, sha...

Embodiment 2

[0037] Embodiment 2. includes the construction of the plant expression vector comprising orca3 (transcription factor), tdc (specific promoter), g10h (key enzyme gene).

[0038] 1. Construction of intermediate vector pCAMBIA1304+

[0039] Select pBI121 and pCAMBIA1304 as the basic elements to construct the binary plant expression vector pCAMBIA1304+.

[0040] The construction process is as follows: pBI121 and pCAMBIA1304 were digested with HindIII and EcoRI; the pBI121GUS expression cassette was recovered, and the pCAMBIA1304 large fragment was recovered; the two recovered products were connected, transformed into DH5α, screened on LB / kan+ plate to obtain a single clone, and then the plasmid was extracted , Enzyme digestion verification (HindIII and EcoRI double digestion), that is, the successful construction of the intermediate vector pCAMBIA1304+.

[0041] 2. Construction of the intermediate vector pCAMBIA1304+-O comprising the transcription factor orca3

[0042] The trans...

Embodiment 3

[0050] Example 3. Agrobacterium rhizogenes mediated construction of a plant expression vector comprising orca3 (transcription factor), tdc (specific promoter), g10h (key enzyme gene) Genetic transformation of periwinkle to obtain transgenic hairy roots

[0051] 1. Acquisition of plant expression vector Agrobacterium rhizogenes engineering bacteria containing transcription factor orca3, tdc promoter and key enzyme g10h

[0052] The plant expression vector containing transcription factor orca3, tdc promoter and key enzyme g10h in Example 2 was transformed into Agrobacterium rhizogenes C58C1.

[0053] 2. Agrobacterium rhizogenes mediates transcription factor orca3, tdc promoter and key enzyme g10h to transform periwinkle

[0054] 2.1. Preculture of explants

[0055] Cut periwinkle aseptic seedling blade (0.5cm * 0.5cm) and stem segment (0.5cm) explant in embodiment 1, inoculate on the pre-cultivation medium (MS+AS 100 μ mol / L), 25 ℃ of dark cultures 2d.

[0056] 2.2. Co-cultiv...

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Abstract

The invention discloses a plant recombination expression vector, belongs to the technical field of genetic engineering, and is characterized in that the provided plant recombination expression vector comprises a fusion sequence of a transcription factor, a promoter and a key enzyme gene; the promoter comprises an element specifically responding to the transcription factor; and the promoter can be constitutive, inducible or tissue specificity expression promoter, preferably a promoter with jere element, most preferably tdc promoter. The invention also provides a transformant of the plant recombination expression vector, wherein the preparation method of the transformant comprises the following steps: preparing the plant recombination expression vector; transforming a plant material; and screening a transgenic plant material.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a plant recombinant expression vector and a transformant comprising the vector. Background technique [0002] Transcription factors (transcription factor, TF), also known as trans-acting factors, are a group of cis-acting elements that can specifically bind to eukaryotic gene promoter regions, thereby ensuring that the target gene interacts with the target gene at a specific intensity and at a specific time. Spatial expression of egg self-plasma molecules. When plants experience external drought, high salinity, hormones, and diseases, they activate transcription factors through a series of signal transmissions. After the transcription factors bind to the corresponding cis-acting elements, they activate the RNA polymerase II transcription complex, thereby initiating the activation of specific genes. Transcription and expression, and finally the regulatory ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H1/00
Inventor 张磊邸鹏肖莹陈军峰唐克轩龚一富方平孙宇
Owner SHANGHAI HAITAI PHARMA
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