Cichorium intybus chloroplast transformation system establishment method

A method for establishing chloroplasts, which is applied in the field of plant genetic engineering, can solve the problems of low efficiency of transgene expression, and achieve the effects of enhanced biological safety, high biological safety, and easy detection

Inactive Publication Date: 2015-07-08
NORTHWEST UNIV
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Problems solved by technology

The current research on genetic improvement through transgenic means adopts the traditional nuclear genome transformation method, the expression efficiency of the transgene is low, and all use antibiotic selection markers

Method used

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  • Cichorium intybus chloroplast transformation system establishment method
  • Cichorium intybus chloroplast transformation system establishment method
  • Cichorium intybus chloroplast transformation system establishment method

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Embodiment Construction

[0043] The present invention is further described below by the embodiment, does not limit the present invention in any way, under the premise of not departing from the technical solution of the present invention, any modification or transformation that the person of ordinary skill in the art that the present invention is done to realize easily will fall into into the scope of the claims of the present invention.

[0044] The chicory variety used in the present invention is "Pune".

[0045] (1) Establishment of chicory leaf explant regeneration system

[0046] Chicory seeds were routinely sterilized and germinated on MS medium to produce sterile seedlings. Take fully extended mature true leaves (25-30 leaf age) as explants, and cut them into 0.5 cm 2 Small pieces of the same size were inoculated in the optimal MS differentiation medium containing 1.5 mg / L 6-BA and 0.4 mg / L NAA, culture conditions: temperature 25°C±2°C, light intensity 30-50 μmol· m -2 ·s -1 , the light time...

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Abstract

The invention discloses a method for establishing a safe and environmentally friendly chicory chloroplast transformation system, comprising the following steps: (1) establishment of a high-frequency regeneration system for chicory leaf explants; (2) a safe and environmentally friendly chicory chloroplast fixed-point integration Construction of expression vector; (3) Transgenic chicory explants were transformed by particle gun bombardment, and chloroplast transgenic lines were obtained through three rounds of resistance selection. The present invention adopts a new type of safe selection marker gene PMI to replace the traditional antibiotic marker gene aadA, constructs a chloroplast fixed-point integration expression vector, and establishes a chloroplast transformation system of chicory. The system has high screening efficiency, high exogenous gene expression level, and transgenic plants. Strong biological security and other advantages. The invention not only broadens the types of receptors for chloroplast transformation, but also builds a technical platform for further genetically improving chicory through the method, or using chicory chloroplasts as a bioreactor to produce animal oral vaccines, medical proteins, and the like.

Description

technical field [0001] The invention relates to a method for establishing a safe and environment-friendly chicory chloroplast genetic transformation system, belonging to the technical field of plant genetic engineering. Background technique [0002] In 1996, the first case of industrial application of genetically modified crops was introduced. Since then, the research and industrial application of global genetically modified technology has developed rapidly. Specifically, it mainly includes two aspects: "gene escape" and the potential safety problems brought by the use of antibiotic marker genes to the environment and organisms. Therefore, it is necessary to establish a brand-new plant transgenic method, which not only has high expression efficiency of foreign genes, but also has no potential biosafety problems. [0003] Plant chloroplasts have their own genetic material, and the chloroplast transformation of plants is based on the chloroplast genome as a platform for genet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00
Inventor 王玉华巩智刚周海鹏徐芳
Owner NORTHWEST UNIV
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