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Transposon vector and method for obtaining selective marker-free transgenic offspring of transposon vector

A technology for selecting markers and transgenes, which is applied in the field of transgenic research and can solve problems such as laborious efficiency

Inactive Publication Date: 2021-03-12
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

However, the identification of stable Ds insertion lines often relies on Southern blot hybridization analysis of a large number of transgenic progeny, which is laborious and inefficient

Method used

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  • Transposon vector and method for obtaining selective marker-free transgenic offspring of transposon vector
  • Transposon vector and method for obtaining selective marker-free transgenic offspring of transposon vector
  • Transposon vector and method for obtaining selective marker-free transgenic offspring of transposon vector

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Embodiment

[0033] Example: Taking the RNAi expression cassette of the rice blast resistance gene Pi21 as an example, through the Ac / Ds transposable vector, the transgenic blast resistance rice germplasm materials without selection markers were efficiently screened.

[0034] Vector construction: First, the green fluorescent protein gene (GFP), red fluorescent protein gene (mCherry), hygromycin resistance selectable marker gene (HPT), Ac transposase from maize and Ds transposable element were assembled in series in Together, the transposable element universal vector pBDL11 was constructed, and then the target gene Pi21-RNAi expression cassette was introduced into the Ds element to form the final vector pBDL23. Similarly, the target gene Pi21-RNAi expression cassette was introduced into the Ds element of the single fluorescent universal vector pLJ26 to form the control vector pBDL22.

[0035] The dual fluorescent and single fluorescent universal vectors, pBDL11 and pLJ26, the transposable v...

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Abstract

The invention relates to a transposon vector and a method for obtaining a selectable marker-free transgenic offspring of the transposon vector, and the vector is constructed by the following steps: 1)assembling a green fluorescent protein gene (GFP), a red fluorescent protein gene (mCherry) hygromycin resistance selection marker gene (HPT), an Ac transposase gene (AcTPase) from corn and a Ds transposon in series, constructing a transposon factor vector pBDL11, wherein the Ds factor does not have any selection marker, and designing an AscI single restriction enzyme cutting site inside the Ds factor in advance; 2) constructing an intermediate vector pBDL11 containing AscI restriction enzyme cutting sites on two sides of the multiple cloning sites so as to assemble any target gene, and thencutting off the target gene and cloning the target gene into the Ds factor. According to the invention, a negative selection technology based on GFP and mCherry double fluorescence labeling is combined with a positive selection technology based on PCR, and individuals containing TDNA and individuals without selective markers containing target genes are sequentially eliminated from transgenic offspring, so that the screening efficiency of the transgenic offspring is improved.

Description

technical field [0001] The invention relates to the field of transgene research, and mainly relates to a transposable element carrier and a method for obtaining its non-selectable marker transgenic offspring. Background technique [0002] When DNA vectors are used in the field of plant genetic transformation research, they usually contain a selection marker gene that can express antibiotic resistance or herbicide resistance, and are used to screen transformed cells during plant genetic transformation. However, the long-term retention of the marker gene in the transgenic plant may cause potential harm to human health and safety and the surrounding environment. After the plant transformation is completed, the selectable marker gene needs to be eliminated. Creating transgenic plants without selectable markers can avoid these risks and facilitate the commercial cultivation of transgenic plants. Therefore, in recent years, the establishment of efficient and reliable transgenic t...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/65G01N21/64
CPCC12N15/8212C12N15/65G01N21/6428C12N2800/80
Inventor 瞿绍洪李莘潘龙玉毕冬玲李利华田旭丹王岚岚龙文莉许赵蒙刘中来赵建华邹小维高晓庆杨海河曲海艳
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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