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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 system knockout populus alba PDS (phytoene desaturase) gene and application thereof

A technology of systematic knockout and white poplar, applied in application, genetic engineering, recombinant DNA technology, etc., can solve the problems of off-target effect, long generation cycle, poor genetic stability, etc., and achieve the effect of promoting research and promoting gene function.

Active Publication Date: 2020-06-05
CHINESE ACAD OF FORESTRY
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AI Technical Summary

Problems solved by technology

At present, some model tree species have achieved gene editing, but due to the characteristics of perennial trees, long generation cycle, and high genome heterozygosity, there are still poor genetic stability, low genetic transformation efficiency, and serious off-target effects in the gene editing process of forest trees. question

Method used

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  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 system knockout populus alba PDS (phytoene desaturase) gene and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 system knockout populus alba PDS (phytoene desaturase) gene and application thereof
  • CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 system knockout populus alba PDS (phytoene desaturase) gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Experiment preparation stage:

[0025] Plant material: Alba poplar tissue culture seedlings

[0026] Cells and vectors: pYLsgRNA-AtU6-29; pYLCRISPR / Cas9-DH; DH10b Escherichia coli competent; EHA105 Agrobacterium.

[0027] Experimental reagents: Plant RNA Extraction Kit, Reverse Transcription Kit, DNA Rapid Recovery and Purification Kit, pCloneEZ-Blunt-Kan / HCKit, Gold Mix, 2xHeffTMPCCR MasterMix, T4 DNA Ligase, Restriction Enzyme BsaI, Double Distilled Water ( ddH2O), sucrose (Sucrose), agar powder (Agar), acetosyringone (AS), zeatin (ZT), naphthaleneacetic acid (NAA), 6-benzylaminopurine (6-BA), indoleacetic acid (IBA ), Cephalosporin (Cef), Hygromycin (Hyg), Kanamycin (Kan), Rifampicin (Rif), Woody Plant Basal Medium with Vitamins (WPM), Luria-Bertani Culture (LB) Yeast Extract Mannitol Broth(YEB), 1 / 2MS Medium(1 / 2MS)

[0028] Experimental equipment: PCR instrument, constant temperature incubator, shaker, ultra-clean table, ultraviolet spectrophotometer, centrifuge ...

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Abstract

The invention relates to a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 system knockout populus alba PDS (phytoene desaturase) gene and application thereof, and belongs tothe technical field of molecular breeding. The nucleotide sequence of the populus alba PDS gene is disclosed in SEQ ID NO.1. A target site disclosed by the invention can realize the fixed point editing of the PDS gene, and the nucleotide sequence of the target site is disclosed in SEQ ID NO.2. The invention provides a new scheme for researching the gene function of a woody mode plant, a native tree variety populus alba molecular breeding platform in China is established, and technical support is provided for directionally culturing a forest new variety which adapts to Chinese economic development requirements.

Description

technical field [0001] The invention relates to the technical field of molecular breeding, in particular to the application of the PDS gene as a target gene in knocking out endogenous genes of Populus alba to realize directional molecular breeding of Populus alba. Background technique [0002] As a new gene editing tool, CRISPR / Cas9 system is being widely researched and applied. At present, some model tree species have achieved gene editing, but due to the characteristics of perennial trees, long generation cycle, and high genome heterozygosity, there are still poor genetic stability, low genetic transformation efficiency, and serious off-target effects in the gene editing process of forest trees. question. Therefore, it is necessary to continue to transform and optimize the genome editing system for forest trees. Contents of the invention [0003] The purpose of the present invention is to provide the PDS gene as a target gene, and its application in knocking out endoge...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/00C12N15/11C12N15/113
CPCC12N15/113C12N15/8213C12N2310/00
Inventor 曾庆银刘妍婧姜鹏飞王晓霞韩学敏
Owner CHINESE ACAD OF FORESTRY
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