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Method for site-specific mutagenesis of taraxacum kok-saghyz or dandelion genes by using CRISPR/Cas9 system

A technology of dandelion and rubber grass, which is applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve the problems of not yet efficient, and achieve the effect of improving construction efficiency and editing efficiency

Active Publication Date: 2021-01-29
RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stable hereditary homozygous mutants have been successfully obtained through CRISPR / Cas9 technology in Arabidopsis, rice, wheat, corn, soybean and other species, but efficient CRISPR / Cas9 gene editing has not yet been achieved in rubber grass

Method used

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  • Method for site-specific mutagenesis of taraxacum kok-saghyz or dandelion genes by using CRISPR/Cas9 system
  • Method for site-specific mutagenesis of taraxacum kok-saghyz or dandelion genes by using CRISPR/Cas9 system

Examples

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Effect test

Embodiment 1

[0051] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1 Construction of the CRISPR / Cas9 gene editing vector targeting the AtPAP1 gene in transgenic rubber grass overexpressing the AtPAP1 gene

[0052] 1. Design two targeting sgRNA sequences based on CRISPR / Cas9 for the AtPAP1 gene in rubber grass transgenic plants, wherein the nucleotide sequence of target 1 (AtPAP1 / sgRNA1) is 5'-CTTCGCCTTCATAGGCTTCT-3' (SEQ ID NO.3), The nucleotide sequence of target 2 (AtPAP1 / sgRNA2) is 5'-TACGCCCATTCCTACAACAC-3' (SEQ ID NO.4).

[0053] 2. Design and assemble primers according to the targeting sgRNA1 and sgRNA2 sequences of the target gene AtPAP1:

[0054] CmYLCV (SEQ ID NO. 7):

[0055] 5'-TGCTCTTCGCGCTGGCAGACATACTGTCCCAC-3';

[0056] Csy-gRNA1 (SEQ ID NO.8):

[0057] 5′-TCGTCTCC ATGAAGGCGAAG CTGCCTATACGGCAGTGAAC-3';

[0058] rep-gRNA1 (SEQ ID NO.9):

[0059] 5′-TCGTCTCA TCATAGGCTTCT GT...

Embodiment 2

[0069] The preparation method of embodiment 2 gene site-directed mutation rubber grass plant

[0070] 1, the acquisition of rubber grass aseptic seedlings: adopt the rubber grass T2 generation transgenic plant carrying exogenous AtPAP1 gene as acceptor material, concentration is the sodium hypochlorite solution (stock solution presses 100%) of 10-15% (v / v), Add 1 drop (20μL) / 200mL of TritonX-100 and mix to make a disinfectant solution. Use the disinfectant solution to sterilize the T2 generation transgenic seeds for 10-15 minutes, wash them with sterile water for 3-5 times, and then sow them in the seedlings containing 50-100mg / L Germinate in the 1 / 2MS medium of kanamycin, transfer once 5 days later to 1 / 2MS medium containing 50-100mg / L kanamycin and cultivate for about 20 days to obtain sterile seedlings with good root growth. Both root and root are purple.

[0071] 2. Take the roots of sterile seedlings in good growth state, wash them with sterile water for 2-3 times, and d...

Embodiment 3

[0079] Example 3 Detection of gene site-directed mutation rubber grass

[0080] 1. Phenotype observation of AtPAP1 gene site-directed mutation lines

[0081] Observing the adventitious buds induced after the site-directed mutation of the AtPAP1 gene in Example 2, it was found that adventitious buds of green and purple colors were differentiated on the purple root segment, and the purple adventitious buds indicated that the AtPAP1 gene in this part of the root segment The expression is still normal without site-directed mutation, and the green or light-colored adventitious buds indicate that the AtPAP1 gene has been inactivated. The results are shown in figure 1 . From figure 1 It can be seen that the green adventitious buds have no AtPAP1 expression in the circled area, indicating that the AtPAP1 gene has been successfully edited and inactivated in the transformed cells.

[0082] 2. Gene sequencing verification of mutant strains

[0083] (1) DNA extraction: Genomic DNA of ...

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Abstract

The invention relates to the technical field of plant gene editing, in particular to a method for site-specific mutagenesis of taraxacum kok-saghyz or dandelion genes by using a CRISPR / Cas9 system. According to the gene site-specific mutagenesis method, a CRISPR / Cas9 based targeting sgRNA sequence is designed for a target gene, multiple sgRNAs can be driven by using one CmYLCV promoter, differenttargeting sgRNAs are constructed at intervals by adopting a 20bp Csy4 hairpin sequence, a CRISPR / Cas9 carrier is transferred into an acceptor material carrying the target gene, and a target gene site-specific mutated mutant plant is obtained. The method provided by the invention greatly improves the efficiency and gene mutation rate of multi-target simultaneous knockout vector construction, has the characteristics of short experimental period, simple operation and the like, lays a technical foundation for gene mutation of taraxacum kok-saghyz and dandelion, and provides a new method for improved breeding of taraxacum kok-saghyz and dandelion.

Description

technical field [0001] The invention relates to the technical field of plant gene editing, in particular to an expression cassette and vector for CRISPR / Cas9 gene editing of rubber grass or dandelion, and a method for site-directed mutation of rubber grass or dandelion genes using the CRISPR / Cas9 system. Background technique [0002] CRISPR / Cas9 (Clustered regularly interspaced short palindromicrepeats / CRISPR-associated protein 9) genome editing system is composed of Cas9 nuclease and sgRNA (Single guide RNA). The Cas9 protein is responsible for cutting the target sequence and forming a DNA double-strand break structure, thereby activating two repair mechanisms in the cell, namely non-homologous end joining (NHEJ) or homologous recombination (HR), The loss, insertion and substitution of bases at the break are induced by these two repair mechanisms to obtain mutants. CRISPR / Cas9 technology has been widely used in genome editing of various species due to its advantages of sim...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/14
CPCC12N15/8218C07K14/415
Inventor 覃碧刘实忠王锋张立群杨玉双聂秋海张继川王肖肖
Owner RUBBER RES INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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