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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 transgene expression efficiency, and achieve the effects of enhancing biological safety, improving expression efficiency, and being easy to detect

Inactive Publication Date: 2014-02-05
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 Regeneration System of Chicory Leaf Explants

[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...

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Abstract

The present invention discloses a safe and environmentally friendly cichorium intybus chloroplast transformation system establishment method, which comprises: (1) establishing a high frequency regeneration system of a cichorium intybus leaf explant; (2) constructing a safe and environmentally friendly cichorium intybus chloroplast site-specific integration expression vector; and (3) adopting a gene gun bombardment method to transform the cichorium intybus explant, and carrying out three rounds of resistance screening to obtain the chloroplast transgene strain. According to the present invention, the novel safe selection marker gene PMI is adopted to replace the traditional antibiotic marker gene aadA to construct the chloroplast site-specific integration expression vector and establish the cichorium intybus chloroplast transformation system, wherein the system has advantages of high screening efficiency, high exogenous gene expression level, strong transgenic plant bio-safety and the like; and receptor types of chloroplast transformation are broadened, and technical platforms are built for genetic improvement on cichorium intybus, or application of cichorium intybus chloroplast 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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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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