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Backbone plasmid vector and application thereof

A plasmid vector and backbone technology, applied in the field of crop genetic engineering, can solve the problem of no crop CRISPR/Cas9 vector optimization and transformation, and achieve the effects of stable and efficient shearing, improving efficiency, and improving targeting efficiency.

Active Publication Date: 2014-08-13
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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Problems solved by technology

However, in the research on rice, the current limited reports mainly focus on the mechanism research, and there is no report on the engineering and optimization of the crop CRISPR / Cas9 vector to make it suitable for simple and rapid operation

Method used

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  • Backbone plasmid vector and application thereof
  • Backbone plasmid vector and application thereof
  • Backbone plasmid vector and application thereof

Examples

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Embodiment Construction

[0033] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0034] Design of backbone plasmid vectors

[0035] The present invention designs such a backbone plasmid vector, which includes a guide RNA expression frame and a Cas9 nuclease expression frame, and the guide RNA expression frame includes: wheat TaU3p promoter, spectinomycin resistance gene SpR, artificially synthesized sgRNA backbone sequence and Poly -T terminator. The Cas9 nuclease expression cassette includes the ZmUBI promoter, the Cas9 coding sequence engineered with plant-preferred codons, and the 35s terminator. Optionally, the backbone plasmid vector also includes left and right border sequences of T-DNA; the guide RNA expression box and the Cas9 expression box are located between the left border and the right border. It sh...

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Abstract

The invention discloses a backbone plasmid vector. In the backbone plasmid vector, a guide RNA (Ribonucleic Acid) expression cassette and a Cas9 nuclease expression cassette are located in the same binary vector; the guide RNA expression cassette is sequentially composed of a wheat TaU3p promoter, a spectinomycin resistance gene, an artificially-synthesized sgRNA framework sequence and a Poly-T terminator; the Cas9 nuclease expression cassette is sequentially composed of a ZmUBI promoter, a plant preferred codon modified Cas9 coding sequence and a 35s terminator. The invention also provides a target-sequence-contained recombinant vector constructed by using the backbone plasmid vector and used for crop gene targeting.

Description

technical field [0001] The invention belongs to the field of crop genetic engineering, and in particular relates to a backbone plasmid carrier for crop gene targeting and its application in genetic modification. Background technique [0002] my country is a large agricultural country, and the sustained and stable increase in the production of major agricultural products is of great strategic significance to ensure my country's food security. Seeds are the source of food production, and excellent varieties are an important basis for ensuring high, high-quality, and stable agricultural yields. With the continuous development of the national economy, the continuous increase of the population and the frequent occurrence of extreme weather, it is very important to accelerate the improvement of varieties to stabilize the output of agricultural products and meet the needs of modern agriculture in the development of the times. Since the middle of the last century, traditional breed...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/66C12N1/21A01H5/00C12R1/01
Inventor 魏鹏程杨剑波秦瑞英李莉李浩马卉陆徐忠杨亚春倪大虎倪金龙宋丰顺
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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