CRISPR/Cas9 vector for sugarcane and construction method and application thereof

A construction method and carrier technology, applied in the field of genetic engineering, can solve problems such as polyploidy and low genetic transformation efficiency, large gene copy number, gene redundancy, etc., to improve lodging resistance, efficient site-directed mutation, and reduce plant height Effect

Active Publication Date: 2020-09-29
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Claims
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Problems solved by technology

[0003] Sugarcane is the largest tropical crop in my country. 80% of China's total sugar production comes from sugarcane. Sugarcane is an allopolyploid plant (rice and corn are diploid), with many gene copies and serious gene redundancy. The sugarcane genome Editing platforms lag behind other crop plants due to their polyploidy and low genetic transformation efficiency
In order to better carry out functional genomics and molecular breeding research, it is very necessary to establish accurate site-directed mutation sugarcane lines. At present, there is no system for site-directed mutation in sugarcane. Therefore, an efficient CRISPR / Cas9 system for sugarcane was established and applied. The gene editing system for sugarcane is particularly important

Method used

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  • CRISPR/Cas9 vector for sugarcane and construction method and application thereof
  • CRISPR/Cas9 vector for sugarcane and construction method and application thereof
  • CRISPR/Cas9 vector for sugarcane and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Acquisition of Target Sequence

[0042] According to the sorghum gene RNA series (XM_021462448.1), use Primer primer 5.0 to design and amplify the target sequence primers ZL-F and ZL-R (see the sequence table SEQ8 and SEQ9 for details), amplify the DNA template of Xintai Sugar No. 22, and amplify About 870bp target gene fragment ( Figure 5 ), the obtained PCR product was recovered from the gel, connected to the pMD18-T vector, and sequenced to obtain the target sequence (see SEQ7 for details).

[0043] The PCR amplification reaction system is as follows:

[0044] dna 0.5μL ZL-F (10μM) 1.0 μL ZL-R (10μM) 1.0 μL 2XTaq Master Mix 12.5μL ddH2O 10 μL Total Volume 25.0 μL

[0045] Amplification program: 95°C for 3.5min; 94°C for 30s, 62°C for 30s, 72°C for 50s, 33 cycles; 72°C for 7.5min.

Embodiment 2

[0046] Example 2: Construction method of CRISPR-Cas9 sugarcane target sequence editing vector

[0047] (1) Design the base sequence of the expression DNA of the specific sgRNA, as shown in SEQ ID NO.1, which is synthesized by the solid-phase phosphite amine triester method: first, the 3′ terminal nucleoside of the oligonucleotide chain to be synthesized is (N1) is coupled to a solid support with its 3'-OH through a long alkyl arm, and the 5'-OH of N1 is protected with dimethoxytrityl (DMTr); then treated with benzenesulfonic acid Nucleosides with protective groups, removing the DMTr at the 5' end, exposing the 5'-OH, the 5'-OH base and the 3'-OH of the next base form a phosphite triester, which is oxidized to triphosphate by iodine ester to obtain the second base, add dichloroacetic acid to remove the protective agent DMTr on the 5'-OH of the second base, and carry out the synthesis of the third base, and obtain the target fragment by repeating this step. The synthesis of thi...

Embodiment 3

[0058] Example 3: Genetic Transformation of Sugarcane Target Sequence Editing Vectors with CRISPR-Cas9

[0059] (1) With the Agrobacterium containing the target sequence CPB-ZmUbi-hspCas9-U6-PDS carrier constructed in Example 1, pick positive single clones in YEP liquid medium (containing 50ng / mL rifampicin, 70ng / mL kanamycin (100 μm / mL acetosyringone) at 28°C and 180 r / min for overnight shaking, and the next day, a small amount of bacterial liquid was added to fresh YEP liquid medium (containing 50 ng / mL rifampicin, 70 ng / mL kanamycin, 100 μm / mL acetosyringone) at 28°C and 180 r / min until the OD600 was about 0.3, centrifuged to remove the supernatant, and the bacteria were resuspended in MS liquid medium containing acetosyringone (100 μm / mL) at 90 rpm / Shake on a shaker for 1 min to activate the strains for the next step of callus infection.

[0060] (2) Collect the cultured sugarcane callus with pointed tweezers into a sterile petri dish with sterilized filter paper, about ...

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Abstract

The invention provides a CRISPR / Cas9 vector for sugarcane. The CRISPR / Cas9 vector is formed by connecting a fragment obtained after HindIII enzyme digestion of a pUC57-simple-sgRNA vector with a fragment obtained after HindIII enzyme digestion of a CPB-ZmUbi-hspCas9 vector, wherein the sequence of the pUC57-simple-sgRNA vector is shown in SEQ ID NO. 2. The CRISPR / Cas9 vector for sugarcane is constructed for the first time, sugarcane genes can be subjected to efficient site-directed mutagenesis rapidly and easily, directional gene editing of specific sites is achieved, and therefore permanent deletion of a target gene sequence is caused, and technical support is provided for research of sugarcane gene functions and cultivation of new varieties through a molecular breeding technology. The DNA sequence of sgRNA is constructed directly through a chemical synthesis method, construction of a CRISPR-Cas9 system can be completed rapidly and conveniently, and development and application of a sugarcane gene editing system are promoted.

Description

technical field [0001] The invention belongs to genetic engineering, and specifically relates to a CRISPR / Cas9 vector for sugarcane and its construction method and application. Background technique [0002] The genome editing technology CRISPR / Cas9 (clustered regularly interspaced shortpalindromic repeats / CRISPR associated 9) system mainly includes single guide RNA (sgRNA) and Cas9 protein. Cas9 and sgRNA form a complex, and the successfully expressed sgRNA forms a complex with Cas9 protein through its own Cas9 handle (Cas9 handle); then, the complex starts to search and match the PAM (mainly NGG) sequence, and then the base of the sgRNA is complementary The paired region sequence recognizes the target sequence of the target marker through the principle of complementary base pairing, and the Cas9 protein uses its own endonuclease activity to cut the target sequence, forming a DNA double-strand break (DSB) and generating a DNA site. Specific DNA changes. In 2013, after the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/113A01H5/00A01H6/46
CPCC12N15/8213C12N15/8261C12N15/8205C12N15/113C12N2310/20
Inventor 王俊刚王文治赵婷婷张树珍冯翠莲沈林波冯小艳熊国如
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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