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Compiling vector of kiwi fruit gene AcPDS based on CRISPR-Cas9 as well as construction method and application thereof

A technology of kiwi fruit, cas9p-35s-n, applied in the field of genetic engineering

Active Publication Date: 2017-12-08
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to better study gene function and genetic improvement, it is necessary to accurately establish gene mutation lines for site-directed mutation. At present, there is no system for site-directed mutation of kiwifruit. Therefore, an efficient CRISPR / Cas9 system for kiwifruit was established and applied to Gene editing of kiwifruit appears particularly important

Method used

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  • Compiling vector of kiwi fruit gene AcPDS based on CRISPR-Cas9 as well as construction method and application thereof
  • Compiling vector of kiwi fruit gene AcPDS based on CRISPR-Cas9 as well as construction method and application thereof
  • Compiling vector of kiwi fruit gene AcPDS based on CRISPR-Cas9 as well as construction method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] The construction method of the kiwifruit gene AcPDS editing vector based on CRISPR-Cas9, the specific steps are as follows:

[0044] (1) Pick a single colony containing pYLCRISPR / Cas9P-35S-N and pYLsgRNA-AtU6-1 plasmids, and inoculate them in 50 mL LB liquid medium containing 50 ng / mL Kan and 50 ng / mL Amp respectively, at 37 °C, 200 r / mL overnight culture on a constant temperature shaker;

[0045] (2) The cells were collected by centrifugation, and the plasmids pYLCRISPR / Cas9P-35S-N and pYLsgRNA-AtU6-1 were extracted by alkaline lysis, and the concentration of the plasmids was determined by Nanodrop 2000.

[0046] (3) Use AscI, BamHI and HindIII to carry out enzyme digestion verification on pYLCRISPR / Cas9P-35S-N and pYLsgRNA-AtU6-1 plasmids respectively, and use primers SP-DL / SP-R to carry out enzyme digestion on pYLCRISPR / Cas9P-35S-N plasmids PCR and sequencing verification;

[0047] (4) Use the plasmid pYLsgRNA-AtU6-1 as a template, and use primers U6-1-F and U6-1-C...

Embodiment 2

[0060] The kiwifruit gene AcPDS editing vector based on CRISPR-Cas9 performs site-directed mutation on the kiwifruit gene AcPDS, and the specific steps are as follows:

[0061] (1) The pHLW-gRNA-Cas9-U6-1-PDS vector constructed in Example 1 containing target sequence 1 and target sequence 2 is designated as A1.

[0062] (2) Take 5 μL of the obtained vector A1, use the standard electric shock transformation method to transform the Agrobacterium strain EHA105 competent cells, and carry out screening and identification, and then pick a single colony and inoculate it in a medium containing 50 ng / mL rifampicin and 50 ng / mL Kan Shake the bacteria in LB liquid medium at 28°C and 180r / min overnight, and in the next morning, take the seed solution and add it to fresh LB liquid medium containing 50ng / mL rifampicin and 50ng / mL Kan at a ratio of 1:100 at 28°C, Shake the bacteria at 180r / min until the OD600 is about 0.6, centrifuge to remove the supernatant, add liquid MS medium containing...

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Abstract

The invention discloses a compiling vector of a kiwi fruit gene AcPDS based on CRISPR-Cas9 as well as a construction method and application thereof. The compiling vector of the kiwi fruit gene AcPDS based on CRISPR-Cas9 established can quickly and simply perform efficient site-specific mutagenesis on the kiwi fruit gene, so that the blank of a gene fixed point compiling technology in kiwi fruit is filled. Experimental results verify that by means of agrobacterium-mediated kiwi fruit genetic transformation, the compiling vector of the kiwi fruit gene AcPDS based on CRISPR-Cas9 performs site-specific mutagenesis on two target spots of the kiwi fruit gene AcPDS successfully, and meanwhile, an albefactedphenotype is induced.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CRISPR-Cas9-based kiwifruit gene editing vector and its construction method and application. Background technique: [0002] Creating mutants is the most critical step in the study of gene function and genetic improvement of crops. Traditional methods of inducing gene mutations include random mutagenesis, T-DNA / transposon insertion. Site-specific nucleases developed in recent years, including zinc fingers (ZFs) and transcription activator-like effector nucleases (TALENs), have successfully induced site-directed mutagenesis. Traditional mutagenesis methods are blind and costly. Time consuming and very inefficient. At the same time, since both zinc finger ribonuclease (ZFN) and transcription activator-like effector nuclease (TALENs) need to redesign the corresponding endonuclease for specific genes and finally fuse with FokI endonuclease, this constructi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N1/21C12N15/66C12N9/22C12N15/82A01H5/00
CPCC12N9/22C12N15/113C12N15/66C12N15/8213C12N2310/10
Inventor 汪祖鹏刘义飞黄宏文李大卫张琼
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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