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Cas9 mediated carnation gene editing carrier and application

A gene editing and carnation technology, applied in the field of genetic engineering, can solve the problems of low transgenic efficiency and inability to completely knock out the target gene, and achieve the effect of short time-consuming, shortened time and low cost

Inactive Publication Date: 2016-08-10
FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In carnation, some scholars use RNAi interference technology to study carnation gene function, but the transgenic efficiency is low, and the target gene cannot be completely knocked out

Method used

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  • Cas9 mediated carnation gene editing carrier and application
  • Cas9 mediated carnation gene editing carrier and application
  • Cas9 mediated carnation gene editing carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] (1) Construction of Cas 9-mediated Carnation gene editing vector

[0046] 1) Use overlapping PCR to amplify the sgRNA expression cassette containing 2105 targets.

[0047] The first round of PCR: Using AtU6-26SK as a template, using PrimerF1 and Primer2105R1, Primer2105F2 and PrimerR2 as primers respectively, TransStart TaqDNA Polymerase was used to perform specific PCR to amplify a fragment of more than 500 bp and two fragments of more than 100 bp. The amplification system is 50 microliters,

[0048] The PCR reaction system: 1 microliter of upstream and downstream primers, 4 microliters of dNTP, 1 microliter of template DNA, 1 microliter of TransStartTaqDNA Polymerase, 5 microliters of 10×buffer, and add double distilled water to 50 microliters.

[0049] The reaction conditions were pre-denaturation at 94°C for 10 minutes, 30 cycles at 94°C for 30 seconds, 30 seconds at 64°C, and 30 minutes at 72°C for a total of 35 cycles, and extension at 72°C for 10 minutes.

[00...

Embodiment 2

[0128] A method for directional mutation of carnation gene, using Cas9-mediated carnation gene editing vector, comprising the following steps:

[0129] Step (1), construction of Cas 9-mediated gene editing vector of carnation:

[0130] The expression cassette that AtU6 promoter regulates sgRNA expression and the expression cassette that 2 * 35S promoter regulates Cas9 expression is connected in the PCAMBIA1301 vector by T4 ligase, obtains Cas 9 expression vector; The nucleotide sequence of described Cas 9 expression vector As shown in SEQ ID NO.1;

[0131] Step (2), transform carnation with Agrobacterium:

[0132] The Cas9 expression vector constructed in step (1) was transformed into Agrobacterium by freeze-thaw method, and then the Agrobacterium containing the Cas9 expression vector was mixed evenly with the same volume of 50 V / V % glycerol aqueous solution to obtain the Cas9 expression vector containing Store the Agrobacterium liquid in a -80°C refrigerator;

[0133] Ste...

Embodiment 3

[0146] A method for directional mutation of carnation gene, using Cas9-mediated carnation gene editing vector, comprising the following steps:

[0147] Step (1), construction of Cas 9-mediated gene editing vector of carnation:

[0148] The AtU6-26SK plasmid was digested with KpnI / SalI, the 35S-Cas9-SK plasmid was digested with SalI / EcoRI, and the PCAMBIA1301 plasmid was digested with KpnI / EcoRI. Ligate the three kinds of double-digestion products at ℃ for 16 hours, use the ligation products to transform Escherichia coli competent cells, screen positive clones by PCR, and sequence them to obtain the Cas whose nucleotide sequence is shown in SEQ ID NO.1 9 expression vectors;

[0149] Step (2), transform carnation with Agrobacterium:

[0150] The Cas9 expression vector constructed in step (1) was transformed into Agrobacterium by freeze-thaw method, and then the Agrobacterium containing the Cas9 expression vector was mixed evenly with the same volume of 50 V / V % glycerol aqueou...

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Abstract

The invention relates to a Cas9 mediated carnation gene editing carrier and application. The application comprises the following steps: firstly, establishing a CRISPR-Cas9 system of carnation-containing target gene sites, introducing the Cas9 expression carrier into an agrobacterium tumefaciens C58 strain, putting roots of carnation leaves into a pre-culture medium, culturing with light for 3-4 days at 22+ / -2 DEG C, activating agrobacterium containing the Cas 9 expression carrier, dipping the pre-cultured explant into the activated agrobacterium solution for 20-30 minutes, completely absorbing the agrobacterium solution, transferring into a co-culture medium, performing dark culture for 3-4 days at 22+ / -2 DEG C, further transferring into a screening culture medium to culture, performing light culture at 22+ / -2 DEG C so as to differentiate regeneration buds, further transferring the regeneration buds into a multiplication medium for multiplication screening culture, detecting positive transgenosis regeneration plants, sequencing target sites, and detecting mutation strain systems of carnation target gene sites.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a Cas9-mediated carnation gene editing vector and its application. Background technique [0002] RNAi interference technology and gene editing technology are methods for studying gene function. RNAi-mediated gene downregulation is the most common method to reduce the expression product of target gene in cells. However, RNAi cannot completely remove the target gene. In contrast, genome editing techniques alter the genetic code, causing gene knockouts that result in complete loss of gene function. Currently, there are two technologies that can be used to efficiently form double-strand breaks: TALEN (Transcription Activator-like Effector Nucleases) and CRISPR-Cas (Clustered Regularly Interspaced Palindromic Repeat Associated) proteins. TALEN is a chimeric protein fused with a DNA binding protein with a specific binding site and a restriction endonuclease Fok...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/84C12N15/66A01H5/00
CPCC12N15/8205C12N15/66C12N15/8218C12N2800/80
Inventor 周旭红朱健康苏艳王继华李姝影田敏赵丹丹
Owner FLOWER RES INST OF YUNNAN ACAD OF AGRI SCI
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