Agrobacterium mediated onion epidermis cell conversion method

A technology mediated by Agrobacterium and epidermal cells, applied in the direction of introducing foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of high cost, high equipment requirements, and difficulty in onion epidermal cells, and achieve high-efficiency transformation and low cost. cost effect

Inactive Publication Date: 2008-01-30
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to overcome the problems of the existing particle bombardment method of transforming onion epidermal cells, such as difficulty, high cost, and high requirements for equipment, the purpose of the present invention is to provide a method for transforming onion epidermal cells mediated by Agrobacterium

Method used

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  • Agrobacterium mediated onion epidermis cell conversion method
  • Agrobacterium mediated onion epidermis cell conversion method

Examples

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Embodiment 1

[0020] Example 1: Agrobacterium-mediated onion epidermal cell transformation method for promoter activity identification

[0021] (1) Select fresh and well-growth onion bulbs, remove 3-5 layers of scale leaves on the outer layer of the onion bulb in the aseptic environment of the superfloating workbench, soak the bulbs in 75% ethanol for 10 minutes, and use Wash with sterile water 3 times. Use a sterile scalpel to cut the onion bulb in a cross shape, take the bulb leaf in the middle layer close to the inside of the bulb, and use a blade to gently scratch out an area of ​​about 1cm on the inner epidermis (concave surface) 2 Then use tweezers to gently tear off the single-layer inner skin of the onion.

[0022] (2) Taking the promoter pasr as the research object, detecting the promoter activity of the promoter sequence. The promoter sequence and the ORF sequence of the GFP reporter gene were connected into the pCAMBIA1391Z vector, and the promoter plant expression vector pCAMB...

Embodiment 2

[0025] Example 2: Agrobacterium-mediated onion epidermal cell transformation method is used to study the subcellular localization of proteins

[0026] (1) Select fresh and well-growth onion bulbs, remove 3-5 layers of scale leaves on the outer layer of the onion bulb in the aseptic environment of the superfloating workbench, soak the bulbs in 75% ethanol for 10 minutes, and use Wash with sterile water 3 times. Use a sterile scalpel to cut the onion bulb in a cross shape, take the bulb leaf in the middle layer close to the inside of the bulb, and use a blade to gently scratch out an area of ​​about 1cm on the inner epidermis (concave surface) 2 Then use tweezers to gently tear off the single-layer inner skin of the onion.

[0027](2) Inoculate the Agrobacterium LBA4404 identified as positive carrying the pCAMBIA1301-MpASR-GFP plasmid into 50ml liquid YEB liquid culture medium (containing 125ug / ml streptomycin, 50ug / ml kanamycin Suspension, 100mMol / L acetosyringone), cultured ...

Embodiment 3

[0030] Example 3: Combined use of Agrobacterium-mediated onion epidermal cell transformation and GUS reporter gene

[0031] (1) Select fresh and well-growth onion bulbs, remove 3-5 layers of scale leaves on the outer layer of the onion bulb in the aseptic environment of the superfloating workbench, soak the bulbs in 75% ethanol for 10 minutes, and use Wash 2 times with sterile water. Use a sterile scalpel to cut the onion bulb in a cross shape, take the bulb leaf in the middle layer close to the inside of the bulb, and use a blade to gently scratch out an area of ​​about 1cm on the inner epidermis (concave surface) 2 Then use tweezers to gently tear off the single-layer inner skin of the onion.

[0032] (2) A plant expression vector pCAMBIA1391Z-pasr-gus was constructed by linking the gus reporter gene with the promoter pasr, and transformed into Agrobacterium EHA105 strain. In YEB liquid medium (containing 50 mg / L kanamycin, 125 mg / L streptomycin and 100 mMol / L acetosyringo...

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Abstract

The invention discloses an onion epidermal cell transformation method mediated by agrobacterium. Onion monolayer endocuticula is dipped and floated in an infiltration medium containing with agrobacterium and is fully contacted with agrobacterium for 5-30min for transformation and then is picked up and laid in an MS solid culture medium for culture for 16-24 hours under a temperature of 25 DEG C, 16-hour lighting and 8-hour darkness. The invention needs no expensive instrument such as a gene gun, etc, no expensive experimental carrier materials such as gold powder, etc. but a super clean working platform and relative agrobacterium strains can realize high effective transformation of onion epidermal cell in the simplest experimental environment. The whole course only requires four days with low cost and the transformation rate thereof can reach 80 percent.

Description

technical field [0001] The invention relates to a method for genetic transformation of onion epidermal cells, in particular to a simple and efficient method for transforming exogenous genes into epidermal cells in onion bulb leaves mediated by Agrobacterium tumefaciens. Background technique [0002] The inner epidermis of onion bulb leaves has the advantages of convenient material collection, obvious cell structure, transparency, no chloroplast interference, easy observation, and in vivo detection. It is used as a plant genetic transformation receptor and is widely used in protein subcellular localization and promoter activity. In recent years, with the rapid development of cell biology and molecular biology, it has become increasingly important to use onion epidermal cells as receptors to study the functions and characteristics of genes, proteins, promoters, etc. A hotspot in molecular biology research. [0003] At present, the genetic transformation of onion epidermal cel...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 刘海燕梁炫强
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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