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A kind of crispr/cas9 carrier for sugarcane and its construction method and application

A sugarcane and transgenic sugarcane technology, applied in the field of genetic engineering, can solve the problems of gene redundancy, polyploidy, low genetic transformation efficiency, and large gene copy number, so as to reduce plant height, improve lodging resistance, and promote development and Applied effect

Active Publication Date: 2022-02-22
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Description
  • 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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  • A kind of crispr/cas9 carrier for sugarcane and its construction method and application
  • A kind of crispr/cas9 carrier for sugarcane and its construction method and application
  • A kind of crispr/cas9 carrier for sugarcane and its construction method and application

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 10μL 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, shake to OD600 about 0.3, centrifuge to remove the supernatant, and resuspend the bacteria with 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 3-5g, dry...

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Abstract

The present invention provides a CRISPR / Cas9 carrier for sugarcane, which is connected with the fragment digested by HindIII of pUC57-simple-sgRNA carrier whose sequence is shown in SEQ ID NO.2 and the fragment digested by HindIII of CPB-ZmUbi-hspCas9 vector made. The present invention constructs a CRISPR / Cas9 vector for sugarcane for the first time, which can quickly and simply perform efficient site-directed mutation on sugarcane genes, and realize directional gene editing at specific sites, thereby causing permanent deletion of the target gene sequence, which is the key to sugarcane gene functions. Research and use molecular breeding technology to provide technical support for cultivating new varieties, and this application directly uses chemical synthesis to construct the DNA sequence of sgRNA, which can quickly and easily complete the construction of the CRISPR-Cas9 system, and promote the development and application of the sugarcane gene editing system .

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 Patents(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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