Method for controlling transgene flow by using gene split

A gene splitting and transgenic technology, which is applied in the direction of plant gene improvement, botany equipment and methods, applications, etc., can solve the problems of gene drift that cannot be avoided, can not fundamentally solve the problem of gene drift, and achieve the effect of avoiding environmental risks

Inactive Publication Date: 2011-10-19
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although male sterility can prevent transgenic crops from producing pollen, it can be effectively used for plants that do not need to harvest seeds, such as trees, pastures, flowers, etc., but for crops that use hybrids in production, such as hybrid rice, this method is still Gene flow cannot be avoided, because even if the male parent used to prepare the hybrid does not produce pollen, the hybrid F1 will still have pollen in large-scale production
Therefore generally speaking, the above existing technologies can only limit or reduce gene flow, and cannot fundamentally solve the problem of gene flow

Method used

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  • Method for controlling transgene flow by using gene split
  • Method for controlling transgene flow by using gene split
  • Method for controlling transgene flow by using gene split

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1, gene splitting technology limits and controls the gene flow of transgenic G2-aroA gene tobacco (transformation alone)

[0034] 1) Obtaining genetically modified tobacco

[0035] According to the detachable site F295 / T296 of the G2-aroA gene, the G2-aroA gene was divided into N-terminal and C-terminal two parts by PCR method, and connected with the N-terminal and C-terminal of Ssp DnaE intein respectively to form a fusion gene fragment EnIn and IcEc. Transfer the fusion gene fragments EnIn and IcEc into the plant expression vector pG 2 , constructed into EnIn and I C E. C Plant Expression Vectors pEnIn and pI Fused with Gene Fragments C E. C . The constructed plant expression vector gene expression cassette is as follows figure 1 As shown, the exogenous gene is preceded by the chloroplast guide peptide of the Rubisco small subunit, driven by the Rubisco small subunit promoter, the 3' end is the terminator of the Rubisco small subunit, and the screening ...

Embodiment 2

[0050] Example 2, gene splitting technology to limit and control the gene flow (co-transformation) of transgenic G2-aroA gene tobacco

[0051] 1) Obtaining genetically modified tobacco

[0052] According to the detachable site F295 / T296 of the G2-aroA gene, the G2-aroA gene was divided into N-terminal and C-terminal two parts by PCR method, and connected with the N-terminal and C-terminal of Ssp DnaE intein respectively to form a fusion gene fragment EnIn and IcEc. EnIn and I were constructed separately C E. C Plant Expression Vectors pEnIn and pI Fused with Gene Fragments C E. C . The constructed plant expression vector gene expression cassette is as follows figure 1 As shown, the exogenous gene is preceded by the chloroplast guide peptide of the Rubisco small subunit, driven by the Rubisco small subunit promoter, the 3' end is the terminator of the Rubisco small subunit, and the screening marker gene is nptII. Agrobacterium-mediated co-transformation of tobacco to obt...

Embodiment 3

[0058] Example 3: Combining gene splitting technology and chloroplast transformation technology to limit and control transgenic tobacco gene flow

[0059] 1) Obtaining genetically modified tobacco

[0060] According to the detachable site F295 / T296 of the G2-aroA gene, the G2-aroA gene was divided into N-terminal and C-terminal two parts by PCR method, and connected with the N-terminal and C-terminal of Ssp DnaE intein respectively to form a fusion gene fragment EnIn and IcEc. The nuclear transformation vector pEnIn of EnIn and the chloroplast transformation vector pICEC of ICEC fusion gene fragment were respectively constructed. The EnIn was introduced into the tobacco nuclear genome by Agrobacterium-mediated method, and the ICEC fusion gene fragment was introduced into the tobacco chloroplast genome by the particle gun method.

[0061] 2) Obtaining transgenic tobacco containing two gene segments at the same time

[0062] The transgenic tobacco containing the ICEC fusion g...

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Abstract

The invention discloses a method for controlling transgene flow by using gene split, and belongs to the technical field of genetically modified organism safety. The method is characterized in that: a complete target gene is splited into an N-terminal sequence and a C-terminal sequence; the N-terminal sequence and the C-terminal sequence are respectively ligated with the gene sequence of the N-terminal splicing domain of the Intein gene and the gene sequence of the C-terminal splicing domain of the Intein gene to form a fusion gene A and a fusion gene B; the fusion gene A and the fusion gene B are transferred into a nuclear genome of a receptor plant through technical means of agrobacterium tumefaciens transformation and hybridization, or the fusion gene A is transferred into the nuclear genome while the fusion gene B is transferred into the chloroplast genome to obtain the gene-splited transgenic plant; after the two fusion genes are expressed in the transgenic plant, two inactive protein fragments are reassembled into active and complete protein in the chloroplast genome or the nuclear genome through Intein-mediated protein-splicing function. According to the method provided by the present invention, environment risk due to the transgene flow can be reduced.

Description

technical field [0001] The invention belongs to the technical field of genetically modified organisms safety, and in particular relates to a method for controlling transgene floating by using gene splitting. Background technique [0002] With the rise and rapid development of genetically modified technology, safety issues of genetically modified organisms have emerged, and the safety issues caused by genetically modified organisms have gradually become a hot spot and focus of widespread concern in the international community. Among the safety issues of genetically modified crops (GMC) that the scientific community and the public are concerned about, transgenic drift and its possible environmental consequences and impact on human health are one of the focuses. Although gene flow has always existed, it did not start from GMC, and it is the driving force for the evolution of plant species, but what kind of new changes will occur after genetically distant foreign genes are intro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/82A01H1/02A01H5/00
Inventor 王旭静王志兴唐巧玲
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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