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Gene site-directed mutation vector as well as construction method and application thereof

A technology of gene site-directed mutagenesis and construction method, which is applied in the field of gene site-directed mutagenesis vector and its construction, and can solve the problems of difficulty in designing mutation technology and the like

Active Publication Date: 2017-06-13
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both of these two gene editing methods need to cut the double strand of DNA and introduce it by repairing the template DNA, so it is very difficult to design mutation technology

Method used

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  • Gene site-directed mutation vector as well as construction method and application thereof
  • Gene site-directed mutation vector as well as construction method and application thereof
  • Gene site-directed mutation vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Method for site-directed mutation of Arabidopsis gene ALS

[0043] (1) Connect the cytosine deaminase gene (rAPOBEC1) to the 5' end of the Cas9 gene (D10A) with XTEN, and the uracil DNA glycosylase inhibitor gene (UGI) to the 3' end of the Cas9 gene (D10A) The nuclear localization signal sequence (NLS) was connected to the 3' end of UGI, and the plant-preferred codon was optimized to obtain the optimized fusion gene CT3, the sequence of which is shown in SEQ ID No.1.

[0044] (2) Replace the Cas9 gene on the carrier PHEE401E with the fusion gene CT3 optimized in step (1), and name it PHEE401CT. The sequence of the carrier PHEE401E is shown in SEQ ID No.2.

[0045] The specific steps are as follows:

[0046] 1) Digest the PHEE401E plasmid and the optimized fusion gene CT3 with XbaI and SacI respectively, and the reaction system (40 μL) is as follows:

[0047]

[0048] 2) recovering and purifying the digested product, and connecting the two DNA fragments a...

Embodiment 2

[0077] Example 2: Detection of site-directed mutations in genes resistant to herbicides in Arabidopsis

[0078] In Example 1, the transgenic Arabidopsis plants were transplanted into the substrate, Arabidopsis DNA was extracted after 3 weeks, primers were designed upstream and downstream of the targeting site, and the extracted DNA was used as a template for PCR amplification, and the PCR products were purified and then sequenced .

[0079] Primer sequences used:

[0080] ALS-1F: 5'-CCTTAACCCGCTCTTCCTCA-3'

[0081] ALS-1R: 5'-CCCCGTAAGTCTCAACAAACC-3'

[0082] The results showed that among the 240 detected Arabidopsis thaliana, there were 4 strains where C was mutated into T in the target region, and 1 strain had a synonymous mutation, and the other 3 strains had mutated target sequences such as SEQ ID No.5, Shown in SEQ ID No.6 and SEQ ID No.7.

Embodiment 3

[0083] Example 3: Edited herbicide-resistant site-directed mutations can stabilize inheritance

[0084] Phenotypic identification of herbicide-resistant T2 plants in the 4 successfully edited T1 generation seeds: The T2 generation seeds of the 4 lines were plated on MS medium containing tribenuron, and a control was set. The results showed that the T1 generation containing the resistance mutation produced a large number of herbicide-resistant offspring ( figure 2 ), indicating that the herbicide resistance traits produced by the T1 generation can be stably inherited.

[0085] For the genotype identification of the herbicide-resistant T2 plants in the 4 successfully edited T1 generation seeds (Table 1), the following primers were used to perform PCR reactions and sequenced. The results showed that the T2 herbicide-resistant offspring contained the resistance mutation of the T1 generation;

[0086] ALS-1F: 5'-CCTTAACCCGCTCTTCCTCA-3'

[0087] ALS-1R: 5'-CCCCGTAAGTCTCAACAAACC-3...

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Abstract

The invention belongs to the technical field of bioengineering and in particular relates to a gene site-directed mutation vector as well as a construction method and application thereof. The invention provides the gene site-directed mutation vector due to lots of optimizations, cytosine deaminase is guided to be close to cytosine through a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, the cytosine is changed into uracil, then uracil is changed into thymine through self-repair in plant cells, and finally, site-specific mutagenesis from C to T is realized in plants so as to generate resistance to herbicides. In addition, point mutation can be produced in a non-sgRNA targeted area, and novel herbicide-resistant great agronomic traits are brought.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a gene site-directed mutation carrier and its construction method and application. Background technique [0002] In vivo gene editing by CRISPR-Cas9 technology is a major breakthrough in the field of life sciences. Under the guidance of the guide RNA (gRNA), the gRNA-Cas9 complex specifically binds to the DNA region complementary to the gRNA, cuts the DNA double strand, and the cell repairs immediately. If there is no DNA template available for repair during the repair process, a large number of random mutations will be introduced during the repair process; if a repair template is provided, it is possible to integrate the repair template into the DNA, thereby introducing the designed mutation type. However, both of these two gene editing methods need to cut the double strand of DNA and introduce it by repairing the template DNA, so it is very difficult to desi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00
CPCC12N15/8274C12N2800/22C12N2810/10
Inventor 姜临建陈其军倪汉文许勇陈易雨王志平
Owner CHINA AGRI UNIV
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