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Codon phytochemically modified nmecas9 gene and its application

A plant genome and plant-based technology, applied in the fields of biotechnology and plant genetic engineering, can solve problems affecting expression efficiency, affecting DNA double-strand cutting efficiency, safety concerns, etc., and achieve the effect of improving cutting efficiency

Active Publication Date: 2021-12-21
RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, direct use of NmeCas9 without artificial optimization design will affect its expression efficiency in eukaryotic cells, thereby affecting its cutting efficiency of DNA double strands
In addition, since NmeCas9 is derived from bacteria, it may adversely affect the genome of the transformed recipient and may also raise concerns about its safety

Method used

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  • Codon phytochemically modified nmecas9 gene and its application
  • Codon phytochemically modified nmecas9 gene and its application
  • Codon phytochemically modified nmecas9 gene and its application

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Effect test

Embodiment approach

[0019] According to a specific embodiment of the present invention, the nucleotide sequence of the plant NmeCas9 gene is shown in SEQ ID No: 1.

[0020] In the second aspect, the present invention also provides an expression cassette, which contains the NmeCas9 gene of the above-mentioned codon phytochemical transformation.

[0021] In the third aspect, the present invention also provides an expression vector, which is inserted with the NmeCas9 gene of the above-mentioned codon phytochemical transformation or the above-mentioned expression cassette.

[0022] According to the present invention, the construction method of the expression vector can be carried out according to conventional methods in the art, for example, use the same restriction endonuclease to digest the plant NmeCas9 gene and the vector to be inserted, and then use the ligase to insert the plant The NmeCas9 gene is connected into the vector to obtain the expression vector of the present invention.

[0023] Whe...

Embodiment 1

[0051] Example 1—Construction of plant targeting vector containing plant NmeCas9 gene 1. Entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the nucleotide sequence shown in SEQ ID No: 1, which is the same as the original NmeCas9 nucleotide sequence (Neisseria meningitidis 402733~405931 in 053442) comparison picture as shown Figure 1-1 to Figure 1-6 As shown, it was connected to the PUC57-AMP vector to form a PUC57-AMP-plant NmeCas9 vector, and loaded into E. coli XL-blue strain.

[0052] 2. Extract the plasmid from the Escherichia coli XL-blue containing the PUC57-AMP-plant NmeCas9 vector with the Axygen plasmid extraction kit, digest with NotI / SacI, and recover the plant NmeCas9 fragment. At the same time, NotI / SacI enzymes were used to linearize pHUN900, and pHUN900 was recovered, and the above-mentioned plant NmeCas9 fragment and pHUN900 fragment were connected with T4 ligase (purchased from TaKaRa Company) to obtain the plant expression vector pHUN900-plant Nme...

Embodiment 2

[0054] Example 2—the genetic transformation of rice using pHUN711-PDS as a targeting vector and the acquisition of mutants.

[0055] 1. Induction and pre-culture of mature embryo callus

[0056] The mature seeds of Nipponbare are shelled, and the seeds with normal appearance and cleanness without mildew are selected, shaken for 90 sec with 70% alcohol, and pour off the alcohol; then use 50% sodium hypochlorite containing Tween20 (the available chlorine concentration of the stock solution is greater than 4%, wherein, 50% sodium hypochlorite is the solution after diluting the stock solution by 1 times, every 100 milliliters adds 1 drop of Tween20) solution to clean the seeds, and shakes on a shaker for 45min (180r / min). Pour off the sodium hypochlorite, wash with sterile water 5-10 times until there is no smell of sodium hypochlorite, finally add sterile water, soak overnight at 30°C. The embryos were separated along the aleurone layer with a scalpel blade, placed on the callus...

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Abstract

The invention relates to the technical fields of biotechnology and plant genetic engineering, and discloses a NmeCas9 gene transformed with codons into plants and an application thereof. The NmeCas9 gene modified by codon plantization has the nucleotide sequence shown in SEQ ID No:1. The NmeCas9 gene transformed by codon phytochemical transformation provided by the present invention is obtained by transformation based on the codon of the model crop rice, that is, under the premise of keeping the encoded amino acid sequence unchanged, the inventor uses the codons selected from the codons related to rice genes Replace the original codons with the extracted codons to obtain the plant-modified NmeCas9 gene, which is then chemically synthesized. Using the gene of the invention can obviously improve the cutting efficiency.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the present invention relates to a NmeCas9 gene modified by codon plantization, an expression cassette containing the NmeCas9 gene modified by codon plantization, an expression vector, a targeting vector, a transgenic cell, and applications thereof. Background technique [0002] Genome editing technology is a powerful tool for the study of plant gene function and crop improvement. It mainly relies on artificial endonucleases (SSNs) to generate double-strand breaks (DSBs) at the target genomic location. and homologous recombination (HDR) for repair. Repair by NHEJ is prone to errors, causing small fragment deletions or insertions at the break position, resulting in gene mutations; in the presence of donor DNA, it is possible to repair the break position through HDR, resulting in precise gene insertion or replace. The most widely used genome edi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/55C12N15/82C12N5/10A01H5/00A01H6/46
CPCC12N9/22C12N15/8213C12N2510/00C12N2800/22
Inventor 秦瑞英魏鹏程李娟李浩杨亚春杨剑波
Owner RICE RES ISTITUTE ANHUI ACAD OF AGRI SCI