Magnetic nanocarrier-mediated plant transgenic method

A magnetic nano-carrier and plant technology, which is applied in the field of nano-biotechnology and bio-nano materials, can solve the problems that no one has reported on plant pollen research, and achieve the effects of easy promotion, high permeability, and small cell damage

Active Publication Date: 2013-02-13
INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the use of magnetic nanogene vectors

Method used

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  • Magnetic nanocarrier-mediated plant transgenic method
  • Magnetic nanocarrier-mediated plant transgenic method
  • Magnetic nanocarrier-mediated plant transgenic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Nano Fe 3 o 4 / PEI gene carrier-mediated double gene transformation of cotton pollen cells: the cotton insect-resistant plasmid (pGBIF4ABC-α, which contains the transformed Bt and Cpti fusion insect-resistant gene plant expression vector, kanamycin resistance marker) was linked to magnetic nanoparticles After transforming cotton, the specific steps are as follows. a: The recipient material was emasculated one day before flowering, and the stigma was covered with a thin tube for reproductive isolation to prevent other pollen contamination; b: The flowers that opened but did not loose pollen were picked on the same day, and the anthers were taken out; c: The plasmid pGBIF4ABC-α was mixed with nanocarriers Fe 3 o 4 / PEI connection at a mass ratio of 2:1, that is, each 50ul system contains 1ul (1ug / ul) magnetic nanoparticles and 2ug plasmid DNA, and connects at room temperature for 30min; d: Dilute the system in step c by 1000 times, and the Soak the anthers in it, proc...

Embodiment 2

[0031] Nano Fe 3 o 4 / PEI gene carrier mediated single gene transformation of cotton pollen cells: Cotton somatic embryo development-related genes (pK7G-HSERK, kanamycin resistance marker) were connected to magnetic nanoparticles to transform cotton, the transformation and genetic identification steps and implementation Steps a to i of Example 1 are the same. Figure 5 Gel electrophoresis images of nanocarrier / pK7G-HSERK complexes with different mass ratios are shown. The final statistics show that the conversion efficiency in this embodiment is 2%.

Embodiment 3

[0033] Nano Fe 3 o 4 / PEI gene vector mediated multi-gene artificial chromosome transformation of cotton pollen cells: Cotton genomic DNA bacterial artificial chromosome (BAC, containing chloramphenicol marker) was connected with magnetic nanoparticles to transform cotton, the transformation and genetic identification steps were the same as those in Example 1 a to i are the same; the difference is that in step c, BAC is connected to the nanocarrier at a mass ratio of 1:1. Image 6 Gel electrophoresis images of nanocarrier / BAC complexes with different mass ratios are shown. Finally, the conversion efficiency in this embodiment was calculated to be 10%.

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Abstract

The invention provides a magnetic nanocarrier-mediated plant transgenic method. The magnetic nanocarrier-mediated plant transgenic method specifically comprises the following steps: combining a magnetic nanocarrier with an exogenous gene to construct a gene-nano-magnetic particle carrier composite, wherein the magnetic nanocarrier has paramagnetism, and a layer of PEI (polyethyleneimine) thin film is covered on the surface of the magnetic nanocarrier; and performing co-culture on the gene-nano-magnetic particle carrier composite and plant pollen or anther, utilizing the high penetrability of the magnetic nanocarrier to transfer the gene-nano-magnetic particle carrier composite into plant pollen or anther cells under the drive of a magnetic field of 0.1-0.5T and realizing genetic transformation of the exogenous target gene through a pollination and fertilization process. The method disclosed by the invention has the advantages of simplicity in operation, easiness in grasp, convenience in popularization, no limitations by the type and the size of the exogenous gene, capability of breaking the limitations by the genetic background of a receptor material, capability of directly getting transgenic seeds, short time for getting a transgenic material and high transformation efficiency, and can be widely applied to plant genetic transformation. The method disclosed by the invention particularly has great value for genetic improvement of important crops and research of functional genomes.

Description

technical field [0001] The invention relates to the fields of nano-biotechnology and application of bio-nanometer materials, and specifically refers to a plant transgenic method mediated by magnetic nano-carriers. Background technique [0002] Plant genetic engineering is the application of recombinant DNA technology to introduce foreign genes into recipient plant cells, so that recipient plants can obtain new genetic traits. In the 1980s, on the basis of a series of major advances in the fields of cell and tissue culture and molecular biology research, plant genetic engineering has developed rapidly. Cotton is an important economic crop in my country and plays a decisive role in the development of agriculture and cotton spinning industry. However, traditional breeding methods are mostly limited to inter-species or inter-species chromosome recombination, which has great limitations. Therefore, it is of great significance to use genetic engineering technology to breed new co...

Claims

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Application Information

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IPC IPC(8): C12N15/87A01H5/00
Inventor 崔海信孟志刚郭三堆孙长娇王琰赵翔
Owner INST OF ENVIRONMENT & SUSTAINABLE DEV IN AGRI CHINESE ACADEMY OF AGRI SCI
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