CRISPR/Cas9 vector with visual protein fusion antibiotic selection marker as well as construction method and application of CRISPR/Cas9 vector

A technology for screening markers and construction methods, which is applied in the field of genetic engineering, can solve the problems of large transformation vectors and affect transformation efficiency, and achieve the effects of reducing size, improving transformation efficiency, and reducing workload

Pending Publication Date: 2022-07-29
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

Transformation efficiency may be compromised as the plant's resistance selection marker and visualized marker protein genes are driven by different promoters, making the transformation vector too large

Method used

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  • CRISPR/Cas9 vector with visual protein fusion antibiotic selection marker as well as construction method and application of CRISPR/Cas9 vector
  • CRISPR/Cas9 vector with visual protein fusion antibiotic selection marker as well as construction method and application of CRISPR/Cas9 vector
  • CRISPR/Cas9 vector with visual protein fusion antibiotic selection marker as well as construction method and application of CRISPR/Cas9 vector

Examples

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Embodiment 1

[0028] This example provides a method for constructing a CRISPR / Cas9 vector with a visible protein fusion antibiotic selection marker, and the vector is used to construct a gene editing vector for the tomato SlD14 gene. plant.

[0029] 1. Construction of a CRISPR / Cas9 vector with a visible protein fusion antibiotic selection marker

[0030] S1, artificially synthesize the primer sequences shown in SEQ ID NO.2 and SEQ ID NO.3, take the 0380-GN carrier (gifted by Mr. Li Yi, University of Connecticut, USA) as the template, use The Max Super-Fidelity DNAPolymerase kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.) was used to amplify the GUS::NPTII nucleotide sequence; artificially synthesized as shown in SEQ ID NO.4 and SEQ ID NO.5 Primer sequences, using pYLCRISPR-Cas9Pubi-H vector (gifted by Professor Liu Yaoguang of South China Agricultural University) as a template, using The Max Super-Fidelity DNAPolymerase kit (purchased from Nanjing Novizan Biotechnology Co.,...

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Abstract

The invention provides a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) vector with a visual protein fusion antibiotic selection marker as well as a construction method and application of the CRISPR / Cas9 vector, and belongs to the technical field of genetic engineering. An antibiotic selection marker and a visual protein are fused and driven by the same promoter, a new CRISPR / Cas9 vector with a visual protein marker, which is smaller in vector size and higher in conversion rate, is successfully constructed, and the CRISPR / Cas9 vector is successfully used for constructing a gene editing vector of a tomato SlD14 gene, so that the gene editing vector of the tomato SlD14 gene is constructed. Finally, a transgenic tomato plant with the phenotype obviously different from that of wild tomatoes is cultivated, and the effectiveness of the CRISPR / Cas9 vector is proved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR / Cas9 vector with a visible protein fusion antibiotic screening marker and a construction method and application thereof. Background technique [0002] As the acquired immune system of bacteria, the CRISPR / Cas9 system has been transformed into a fast and efficient gene editing technology, and considerable results have been achieved in both animals and plants. CRISPR / Cas9 technology has become the main molecular tool for people to explore gene function and modify genes. Especially applying it to the field of plants allows us to more quickly and accurately improve crops to get the traits we want. [0003] At present, many CRISPR / Cas9 systems suitable for plant gene editing have been developed in the world. However, at present, the CRISPR / Cas9 system is used for gene editing and breeding in crops. Most crops still need to insert the CRISPR / Cas9 comp...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/84C12N15/54C12N15/56A01H6/82A01H5/00
CPCC12N15/66C12N15/52C12N15/8218C12N15/8261C12N9/2402C12N9/1205C12Y302/01031C12Y207/01095
Inventor 吴寒皮颖李治飞
Owner NANJING AGRICULTURAL UNIVERSITY
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