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Simultaneous Knockout of 1-sst and 1-fft Genes in Rubbergrass Using the CRISPR/Cas9 System

A technology of rubber grass and genes, which is applied in the field of rubber and rubber, can solve the problem of simultaneous mutation of multiple target sites, and achieve the effect of avoiding long cycles

Active Publication Date: 2022-08-02
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

At present, there is no report on the use of CRISPR / Cas9 technology to achieve simultaneous mutation of multiple target sites in rubber grass.

Method used

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  • Simultaneous Knockout of 1-sst and 1-fft Genes in Rubbergrass Using the CRISPR/Cas9 System
  • Simultaneous Knockout of 1-sst and 1-fft Genes in Rubbergrass Using the CRISPR/Cas9 System
  • Simultaneous Knockout of 1-sst and 1-fft Genes in Rubbergrass Using the CRISPR/Cas9 System

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 sgRNA design of rubber grass inulin synthase genes 1-SST and 1-FFT and construction of CRISPR / Cas9 recombinant expression vector SST-FFT-Cas9

[0045] 1. Screening of sgRNA target sites

[0046] Since rubber grass is a self-incompatible plant, there are differences in gene sequences among different strains. By comparing the 1-SST and 1-FFT gene sequences in different germplasms, the PAM sequence (NGG) was found in the conserved regions of the two genes, respectively. The 20 bp sequence at the 5' end of the PAM position is the sgRNA sequence, and the published rubber grass genome sequence was searched to analyze the specificity of the target.

[0047] The present invention has determined two sgRNAs (sgRNA-SST1 and sgRNA-SST2) targeting the 1-SST gene through a large number of screenings, and their sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 (both are located in the fourth exon), and 2 sgRNAs (sgRNA-FFT1 and sgRNA-FFT2) targeting the 1-FFT gene, the sequ...

Embodiment 2

[0057] Example 2 The recombinant expression vector SST-FFT-Cas9 transformed rubber grass to obtain regenerated plants

[0058]1. Obtainment of aseptic seedlings of rubber grass: The seeds of rubber grass strain 20112 are used as materials, and a sodium hypochlorite solution with a concentration of 10-15% (v / v) (the original solution is used as 100%) is used, and 1 drop (20 μL) / 200 mL is used. Add TritonX-100 and mix well to make a disinfectant, disinfect the seeds with the disinfectant for 10-15 minutes, rinse with sterile water for 3-5 times, then sow them in 1 / 2MS medium for germination, and transfer to 1 / 2MS after 5 days The medium was cultivated for about 20 days to obtain sterile seedlings with good root growth.

[0059] 2. Take the roots of sterile seedlings in good growth condition, wash them with sterile water for 2-3 times, and dry them with filter paper for infection and transformation.

[0060] 3. Agrobacterium infection and transformation: Agrobacterium AGL1 conta...

experiment example 3

[0064] Experimental Example 3 Detection of Mutation in Transgenic Plants and Their Phenotypes

[0065] 1. Screening and detection of transformed plants

[0066] DNA extraction: The genomic DNA of the T0 generation plants obtained in Example 2 was extracted with a DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0067] 2. 1-SST and 1-FFT gene PCR amplification

[0068] Use KOD-FX (TOYOBO, cat.no.KFX-101, Shanghai) to carry out PCR amplification on the T0 generation plants. The reaction system is: genomic DNA 80-100ng, upstream and downstream primers (SST-390F / SST-1355R primer pair Amplify 1-SST gene target site; FFT-338F / FFT-1322R primer pair to amplify 1-FFT gene target site) (10μmol / L) each 1.5μL, 2×PCRBuffer 25μL, dNTP Mix 10μL, KOD Polymerase1. 0μL, add ddH 2 0 to 50 μL. The PCR amplification program was as follows: denaturation at 98°C for 2 min; denaturation at 98°C for 10s, annealing at 55°C for 30s, extension at 68°C for 30s, a ...

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Abstract

The invention relates to the technical field of rubber grass gene editing, in particular to a method for simultaneously knocking out 1-SST and 1-FFT genes of rubber grass by using a CRISPR / Cas9 system. The sgRNAs specifically targeting the inulin synthase genes 1-SST and 1-FFT provided by the present invention can efficiently guide the endonuclease protein Cas9 to cut at the target site and generate double-stranded DNA breaks, combined with resistance screening and DNA sequencing Accurate and efficient simultaneous knockout of the rubbery inulin synthase genes 1-SST and 1-FFT. The present invention provides a convenient and efficient method for creating a rubber grass inulin synthase gene mutant, which effectively avoids the problems of long period, mutation uncertainty, high screening difficulty and high cost in conventional mutagenesis breeding. Breeding of grass elite germplasm provides a new method.

Description

technical field [0001] The invention relates to the technical field of rubber grass gene editing, in particular to a sgRNA, an expression cassette, a vector, a host cell and a CRISPR / Cas9 system for simultaneously targeting and knocking out rubber grass inulin synthase genes 1-SST and 1-FFT. A method for the simultaneous targeted knockout of the rubbery inulin synthase genes 1-SST and 1-FFT by the Cas9 system. Background technique [0002] Natural rubber is an important strategic material and industrial raw material, mainly produced from the Brazilian rubber tree. Rubber grass (Taraxacum kok-saghyz Rodin) is a perennial herb of the genus Asteraceae, the root of which can synthesize high-quality natural rubber, which is similar in structure and performance to the natural rubber produced by the Brazilian rubber tree. It is a rubber-producing crop with great development prospects. . In recent years, the rubber grass germplasm obtained through the investigation and collection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/65A01H5/06A01H6/00
CPCC12N15/1137C12N15/8245C12N15/8246C12N15/8218C12N15/65C12N9/1051C12Y204/01099C12Y204/011C12N2310/20
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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