Method for site-directed mutagenesis of plant cellulose synthase CESA and application of cesa mutant in preparation of nanocrystals

A plant cellulose and cellulose synthase technology, applied in the field of plant genetic engineering and nanomaterials, can solve the problems of reducing the size of cellulose nanocrystals, changing the structure of natural cellulose microfibrils, etc.

Pending Publication Date: 2022-04-12
HUAZHONG AGRI UNIV
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Problems solved by technology

[0004] Based on the above research background, we found that there is no report so far that directly reduces the size of prepared cellulose nanocrystals by modifying the structure of natural cellulose microfibrils by site-directed mutation of cellulose synthase.

Method used

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  • Method for site-directed mutagenesis of plant cellulose synthase CESA and application of cesa mutant in preparation of nanocrystals
  • Method for site-directed mutagenesis of plant cellulose synthase CESA and application of cesa mutant in preparation of nanocrystals
  • Method for site-directed mutagenesis of plant cellulose synthase CESA and application of cesa mutant in preparation of nanocrystals

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Embodiment 1

[0041] Example 1 Site-directed Mutagenesis of Cellulose Synthase Using Gene Editing Technology

[0042] (1) Find gene editing sites and synthesize spacer sequences

[0043] The OsCESA4 / 7 / 9 gene sequence was obtained from the Rice Genome Database (http: / / rice.uga.edu / ).

[0044] Through the website (http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html) to find the CRISPR / Cas9 editing target site for the OsCESA4 / 7 / 9 gene; design primers and amplify the target site sequence (Space sequence ) was constructed on the vector pRGEB32 or pCSGAPO1.

[0045] Amplification primers are listed in Table 1:

[0046]

[0047] (2) CRISPR / Cas9 vector construction

[0048] 1) Construction of 6 vectors: cesa4-KO, cesa7-KO, cesa9-KO, cesa4-Q, cesa7-PCR, cesa9-ZnF

[0049] Take 1 μL each of the forward and reverse sequences with a concentration of 100 μM, and sequentially add 3 μL ddH 2 O in the PE tube. Place the PE tube in a water bath at 37°C for 60 minutes, then take it out and place ...

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Abstract

The invention discloses a method for site-directed mutagenesis of plant cellulose synthase CESA and application of a cesa mutant in preparation of nanocrystals, and belongs to the field of plant genetic engineering and nanomaterials. The method comprises the following steps: (1) editing a target site according to CRISPR/Cas9 of a cellulose synthase CESA gene, designing a primer, and amplifying to construct a target sequence; (2) constructing the target sequence on a vector to obtain a recombinant vector; and (3) transforming the recombinant vector into a plant to obtain point mutants and frame-shift mutants of the three CESA genes of the secondary cell wall. The structure and the property of the transgenic plant cellulose containing the point mutant or the frameshift mutant are changed, and the cellulose nanocrystal with a smaller size can be prepared by hydrolyzing the transgenic plant straw through an acid hydrolysis method. The rice cellulose nanostructure is improved by utilizing a gene editing technology, and a low-cost and high-quality cellulose raw material is provided for efficiently preparing small-size cellulose nanocrystal particles.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering and nanometer materials, in particular to a method for site-directed mutation of plant cellulose synthase CESA and the application of cesa mutants in preparing nanocrystals. Background technique [0002] Cellulose nanocrystals are one-dimensional rod-shaped nanoparticles with high specific surface area, low density and excellent mechanical properties. It has nano-effects, customizable surface chemical properties, anisotropy and optical properties, and also inherits the excellent properties of native cellulose such as renewability, low toxicity, biocompatibility, easy availability and low cost, making it It becomes an ideal nanomaterial for a variety of applications. The geometric size and structural parameters of cellulose nanocrystals are different due to different material sources, extraction methods and manufacturing conditions. The width is 5-20nm, the length is 100-500nm, and the cr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/54C12N9/10C12N15/113C12N15/55C12N15/11A01H4/00A01H5/00A01H6/46C08B15/02B82Y30/00B82Y40/00
Inventor 彭良才张冉胡振鲁俊王艳婷夏涛王友梅
Owner HUAZHONG AGRI UNIV
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