A kind of backbone plasmid carrier and application for genetic engineering

Abstract

The invention discloses a backbone plasmid carrier for genetic engineering. In the backbone plasmid carrier, a guide RNA (Ribonucleic Acid) expression frame and a Cas9 nuclease expression frame are positioned in one same double-element carrier; the guide RNA expression frame sequentially consists of a rice U6 promoter, a spectinomycin resistance gene, an artificially synthesized sgRNA backbone sequence and a Poly-T terminator; the Cas9 nuclease expression frame sequentially consists of a ZmUBI promoter, a rice preference codon modified Cas9 coded sequence and a 35s terminator. The invention further discloses a recombinant carrier which is established by using the plasmid carrier and contains a target sequence, and an application of the recombinant carrier in rice gene targeting.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a genetic engineering carrier for plant gene targeting and its application in rice genetic modification. Background technique [0002] Rice is one of the most important crops in the world. Rice production is closely related to national economic life, and continuous cultivation of varieties with better agronomic shapes is crucial for rice production. Compared with traditional breeding methods, the gene targeting technology developed in the past ten years provides a convenient and efficient means for improving key shapes such as rice yield, quality and resistance. Traditional gene targeting methods include homologous recombination technology, zinc finger nuclease technology (ZFN) and transcription factor activator-like technology (TALEN), among which homologous recombination technology has a very low targeting efficiency in plants, while ZFN and TALEN technolog...

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Problems solved by technology

However, the current limited reports mainly focus on mechanism research, and the rice genetic engineering vector has not been optimized, mainly due to the limited efficiency of the combination of sgRNA and Cas9 used in the existing vector (Shan et al, 2013 Nature Biotechnology article reported that through stable genetic transformation The targeting efficiency of CRISPR / Cas9 is only 4.0%~9.4% by the method; the operation is complicated, and it is necessary to construct two vectors of sgRNA and Cas9 for co-transformation (Xie et al., MolecularPlant in 2013) or two clones to combine the expression cassettes of sgRNA and Cas9. Integrated into a single plasmid (Miao et al., 2013, CellResearch); single applicability, most existing plant CRISPR / Cas9 system vectors are too large for stable expression vectors for transient transformation (Miao et al., 2013, CellResearch) , or the lack of T-DNA borders that cannot be used for transient vectors for Agrobacterium-mediated transformation; and the lack of suitable rice endogenous promoters and coding methods suitable for rice expression, etc. (Mao et al., MolecularPlant 2013), great Limitations of the advantages of CRISPR / Cas9-mediated gene targeting technology in rice variety improvement

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