Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method

A genetic transformation method and the technology of Agrobacterium tumefaciens are applied in the field of genetic transformation of flax mustard mediated by Agrobacterium tumefaciens, which can solve the problems of undiscovered patent publications and the like, and achieve the advantages of being suitable for popularization and application, simple equipment, and improved transformation. The effect of efficiency

Inactive Publication Date: 2015-05-13
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Through the patent search, no patent publications related to the patent application for this invention have been found

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  • Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method
  • Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method
  • Agrobacterium tumefaciens-mediated camelina sativa genetic transformation method

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Experimental program
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Effect test

Embodiment 1

[0055] A method for genetic transformation of flax mustard mediated by Agrobacterium tumefaciens, through the construction and identification of engineering strains carrying target genes, cultivation of flax mustard seedlings, control of transformation timing, and preparation of vacuum infiltration devices, so that the engineering strains can be impregnated with flax mustard , co-cultivation, resistance screening, PCR detection, and finally obtain the transgenic plants of linseed mustard.

[0056] The method for genetic transformation of flax mustard mediated by Agrobacterium tumefaciens is carried out as follows:

[0057] (1) Construction and identification of engineering strains: using CaCl 2 Agrobacterium tumefaciens competent cells were prepared by this method, and 100 μL / tube was aliquoted, and used directly for transformation within 24 hours or quickly frozen in liquid nitrogen and stored at -80°C for later use. The carrier plasmid (the carrier plasmid is pBG1100 with k...

Embodiment 2

[0076] A method for the genetic transformation of flax mustard mediated by Agrobacterium tumefaciens, through the construction and identification of engineering strains carrying the target gene, cultivation of flax mustard seedlings, control of transformation timing, preparation of vacuum infiltration device, and then infecting the engineering strains The specific steps of flax mustard, co-cultivation, resistance screening, PCR detection, and finally obtaining the transgenic plants of flax mustard are as follows:

[0077] 1. Construction and identification of engineering strains

[0078] (1) Using CaCl 2 Prepare competent cells of Agrobacterium tumefaciens by using the method, aliquot 100 μL / tube, and use them directly for transformation within 24 hours or freeze them in liquid nitrogen and store them at -80°C for later use;

[0079] (2) Introduce the vector plasmid pBinGlyBar1 into Agrobacterium tumefaciens competent cells by the heat shock method, add 800 μL of YEB medium, ...

Embodiment 3

[0091] A method for the genetic transformation of flax mustard mediated by Agrobacterium tumefaciens, through the construction and identification of an engineering strain carrying a target gene, the specific steps are as follows:

[0092] (1) Using CaCl 2 Prepare competent cells of Agrobacterium tumefaciens by using the method, aliquot 100 μL / tube, and use them directly for transformation within 24 hours or freeze them in liquid nitrogen and store them at -80°C for later use;

[0093] (2) Use the heat shock method to transform the vector plasmid pBinGlyBar1 into Agrobacterium tumefaciens, add 800 μL of YEB medium, mix well and place it in a 28°C environment for 2 hours; centrifuge at 4500r / min for 45s at room temperature, remove most of the supernatant 700μL , take 100 μL of the remaining supernatant to resuspend the bacteria, spread the resuspended bacteria on a YEB solid resistance plate containing 50 mg / L rifampicin and 50 mg / L kanamycin, and culture it upside down at 28°C ...

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Abstract

The invention relates to an agrobacterium tumefaciens-mediated camelina sativa genetic transformation method which comprises the following steps: immersing camelina sativa inflorescences during an early flowering stage into agrobacterium tumefaciens liquid of a vector plasmid carrying a target gene, performing vacuum infiltration dip-dyeing and co-culture in a dark condition, recovering normal illumination culture of camelina sativa until T0-generation seeds are harvested, sowing the T0-generation seeds to obtain T1-generation plants, performing preliminary screening on the seeds or the plants by utilizing a marker gene in the plasmid, and finally identifying a camelina sativa transgenic plant carrying the target gene by utilizing a PCR (polymerase chain reaction) amplification technology and the like. Compared with a conventional dip-dyeing method, the method is relatively high in transformation rate, successfully realizes genetic transformation of the camelina sativa, and not only lays a foundation for application of a new transformed camelina sativa transgenic variety to the fields of functional foods, biofuel and the like but also provides a method and a technical reference for gene transformation of other types of plants.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, in particular to a method for genetic transformation of flax mustard mediated by Agrobacterium tumefaciens. Background technique [0002] Flax mustard (Camelina sativa (L.) Crantz) belongs to Brassicaceae, has excellent characteristics such as drought resistance, cold resistance, barren resistance, salt resistance, disease and insect pest resistance, and is a low-input oil crop. The content of linoleic acid in flax mustard oil is 16%-18%, the content of linolenic acid is 36%-40%, the content of natural vitamin E is 42-48mg / 100g, the total content of polyunsaturated fatty acids exceeds 60%, and the total content of unsaturated fatty acids The content exceeds 90%. Therefore, flax mustard oil is widely used in many fields such as high-grade health care oil, food additives, medicine, and cosmetics because of its natural health care and chemical stability. [0003] At the same time, with the emerg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 王昌禄高瑞飞李贞景陈勉华
Owner TIANJIN UNIV OF SCI & TECH
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