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Method for effectively reducing gene drift of transgene plant through pollen mediation

A technology of transgenic plants and plants, applied in the biological field, can solve the problems of barnase leakage, changes in agronomic traits, instability, etc., and achieve the effect of reducing environmental risks and high efficiency

Active Publication Date: 2012-05-30
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Also, there is evidence that barnase is toxic to animal and human cells, so the part that requires plant-specific expression of barnase is not the part that is eaten
In addition, there are still many problems in the application of Barnase, such as: male sterility traits are temperature sensitive, easily affected by the environment and unstable, even under the pollen-specific promoter, the toxicity of barnase may leak expression in plants other parts, thus causing changes in various important agronomic traits, instability of barnase in offspring segregation, etc.
Therefore, this method is also not ideal for controlling transgene drift

Method used

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  • Method for effectively reducing gene drift of transgene plant through pollen mediation
  • Method for effectively reducing gene drift of transgene plant through pollen mediation
  • Method for effectively reducing gene drift of transgene plant through pollen mediation

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Construction of expression vectors

[0025] The name of the expression vector used for transformation in the present invention is pSPT05 (attached figure 1 ). The vector is artificially constructed on the basis of pPZP. There are 2 gene expression cassettes on the expression vector: ZM-AA1 Gene expression cassette, which is driven by the late-stage pollen-specific promoter PG47 from maize (see SEQ ID NO.5 for its nucleotide sequence), and terminates transcription by the terminator IN2-1 from maize (the nucleoside acid sequence, see SEQ ID NO.6), and the transit peptide ZM-BT1 is fused between the ZM-AA1 gene and the PG47 promoter, and the nucleotide sequence of ZM-BT1 is shown in SEQ ID NO.7, reporter / screening marker gene RP The gene expression cassette is driven by the callus and seed-specific promoter END2 from maize (see SEQ ID NO.8 for its nucleotide sequence), and is terminated by the terminator PINⅡ from potato. The glycoside of PINⅡ See SEQ ID...

Embodiment 2

[0026] Embodiment 2: obtain transgenic rice plant

[0027] The transformation vector in Example 1 was introduced into the Agrobacterium strain AGLO by electric shock method, and used to transform the callus induced by wild type Zhonghua 11. Transformed callus was screened by red fluorescent protein, attached figure 2 In order to express the callus of exogenous gene, it was further differentiated to regenerate transformed plants expressing exogenous gene (attached image 3 ).

Embodiment 3

[0028] Embodiment 3: PCR detection of transgenic plants

[0029] The leaves of the transgenic rice plants in Example 2 were taken, and the total DNA was extracted. by DsRED2 The gene sequence is used as a template to design primers, for T 0 Genomic DNA of transgenic rice was amplified by PCR. The fragment of the amplified product is 789bp. The amplification program was: 94°C 10min; 94°C 1min, 60°C 1min, 72°C 1min; 37 cycles; 72°C 10min. The forward primer sequence is 5'-GGACTTGAACTCCACCAGG-3'; the reverse primer sequence is: 5'-ATAATGCCAATACGACACC-3'. attached Figure 4It is the PCR amplification result of some transgenic plants, indicating that the exogenous gene has been integrated into the rice recipient genome.

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Abstract

The invention provides a method for effectively reducing environmental risk caused by pollen propagation of transgene plant. In the method, two tightly linked gene expression cassettes of 1) a pollen lethal gene ZM-AA1 driven by a pollen growth anaphase singular promoter PG47 and 2) a fluorescence screening mark gene FP driven by a callus / seed coat singular promoter END 2 are transferred to a Zhonghua 11 wild type material. During selfing fructification of a single plant carrying a single copy transgene, a pollen grain without carrying the transgene can be fertilized with a female gamete normally, but a pollen grain carrying the transgene is abortive in pollen growth anaphase and can not be fertilized with a female gamete, so as to reduce risk of transgene drift in the environment.

Description

technical field [0001] The invention provides a method for stopping transgenic plants from transferring exogenous genes to other plants through pollen, thereby effectively reducing the environmental risk of transgenic plants and belonging to the field of biotechnology. Background technique [0002] In recent years, with the rapid increase of genetically modified plant species and planting area, the large-scale environmental release of genetically modified organisms and the possible environmental biosafety issues have become one of the most concerned and controversial areas in the world. Foreign genes in transgenic plants are transferred by gene drift (or natural hybridization) to non-transgenic varieties or their wild relatives (including weed species) in the environment, and through genetic introgression in populations of wild relatives Retention or further spread, thus causing environmental biosafety problems. Pollen-mediated gene flow widely exists among plant species wi...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00
Inventor 邓兴旺王海洋万向元周君莉
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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