Rice BC20 mutant gene, recombinant vector, transformant and application of rice BC20 mutant gene, recombinant vector and transformant in preparation of rice brittle stem mutant

A technology of mutant gene and recombinant vector, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of increased lignin content, decreased cellulose content, abnormal cell expansion, etc., to change grain shape, increase crop yield, The effect of improving lodging resistance

Active Publication Date: 2021-12-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the BC1 gene encodes a glycosylphosphatidylinositol-anchored protein, which is related to the synthesis of cell walls, and its mutation leads to abnormal cell swelling, decreased cellulose content, and increased lignin content; BC3 gene encodes a membrane-associated dynein, which may be involved in Disrupts the endocytic pathway and post-Golgi membrane trafficking, and its mutations interfere with normal cellulose biosynthesis and membrane trafficking of secondary cell walls

Method used

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  • Rice BC20 mutant gene, recombinant vector, transformant and application of rice BC20 mutant gene, recombinant vector and transformant in preparation of rice brittle stem mutant
  • Rice BC20 mutant gene, recombinant vector, transformant and application of rice BC20 mutant gene, recombinant vector and transformant in preparation of rice brittle stem mutant
  • Rice BC20 mutant gene, recombinant vector, transformant and application of rice BC20 mutant gene, recombinant vector and transformant in preparation of rice brittle stem mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1, the acquisition of rice knockout mutant bc20

[0051] In order to study the function of the rice fragility gene, we used CRISPR Cas9 technology to construct mutants of a large number of rice genes. One of the dwarf mutants caught our attention. The transformed plants of the T0 generation of this mutant were detected by PCR sequencing, and two BC20 mutant materials were screened out, which we named bc20-1 and bc20-2 respectively.

[0052] Among them, the material editing type of mutant bc20-1 is the deletion of CC after the 220th base, and its protein has a frameshift mutation after the 73rd amino acid; the material editing type of mutant bc20-2 is the deletion of the 223rd base to the 336th base Among the 114 bases, the protein has deletion mutations in 38 amino acids between the 75th amino acid and the 112th amino acid.

[0053] The specific method for constructing rice mutant bc20 material by CRISPR Cas9 system is as follows:

[0054] 1. Design sgRNA

...

Embodiment 2

[0087] Example 2, field phenotype observation of wild type and bc20-1 and bc20-2 mutant plants

[0088] In order to observe the phenotypes of rice wild-type plants and bc20-1 and bc20-2 mutant plants under field growth conditions, we conducted an experimental field in Wuhan City, Hubei Province, China (central China, northern China) from June to October 2019 to 2020. subtropical monsoon humid climate) field experiments were carried out, and the test material was T 2 Generation and T 3Generation of homozygous mutant seeds. In each plot, 20 rice seedlings of three kinds of materials were transplanted on the plot of 10 plants in each row, and the distance between the rows and the plants was 20 centimeters. Three plots were planted as biological replicates. In sampling and measurement, boundary rows are excluded. The application of fertilizers, the use of pesticides and all other field management measures are done in the same way.

[0089] The field phenotypes of the wild typ...

Embodiment 3

[0090] Embodiment 3, comparison of wild type and bc20-1 and bc20-2 mutant plant grain type

[0091] We take the field test materials in Example 2 for analysis. The wild-type and mutant bc20-1 and bc20-2 plants at the grain maturity stage were selected, and 5 plants were randomly selected from each of the three plots as biological replicates. After threshing each plant, dry it in the sun. The 1000-grain weight data was obtained using a digital rice seed testing machine (YTS-5D, Gufeng Optoelectronics, China). Then randomly take 10 plump seeds and arrange them neatly and take pictures. The result is as Figure 4 As shown, it can be seen that compared with the wild type, the mutant bc20-1 and bc20-2 plants have little change in rice grain shape and lighter dry grain weight.

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Abstract

The invention provides a rice BC20 mutant gene, a recombinant vector, a transformant and application of the rice BC20 mutant gene, the recombinant vector and the transformant in preparation of a rice brittle stem mutant. The mutant gene comprises a mutant bc20-1, wherein the nucleotide sequence of the mutant bc20-1 is as shown in SEQ ID NO: 5; and a mutant bc20-2, wherein the nucleotide sequence of the mutant bc20-2 is as shown in SEQ ID NO: 6. The mutant bc20-1 and bc20-2 plants show that the plants are short and small, the tillering is reduced, the mechanical strength is reduced, the stalks are easy to break, the rice grain shape is reduced, and the like. The further observation of wild type and mutant bc20-1 and bc20-2 plant stem thick-wall cells through a transmission electron microscope finds that the cell wall thickness of the mutant stem thick-wall cells is obviously thinner than that of the wild type plant stem thick-wall cells. Straw of the mutants is easy to smash, and the mutant has important significance in agricultural production, renewable resource utilization and the like in China.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rice BC20 mutant gene, a recombinant vector, a transformant and the application thereof in preparing a rice brittle stem mutant. Background technique [0002] The cell wall is a natural nanoscale network structure, mainly composed of polysaccharide polymers such as cellulose, hemicellulose and pectin, lignin and a small amount of glycoproteins and minerals. This complex structure envelops plant cells and provides them with different shapes, sizes and physicochemical properties that allow them to function in different organs and stages of development. At the same time, the cell wall has a variety of biological functions, including determining plant morphogenesis and structure, providing mechanical support, transferring water and nutrients, and resisting biotic and abiotic stresses, etc. In addition, plant cell walls can also be used in animal feed, building materials, biofuels, tex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/113C12N15/84C12N15/55C12N1/21A01H5/04A01H6/46
CPCC12N9/1051C12N15/8218C12N15/8261C12N15/8205C12N9/22
Inventor 侯昕岳晓虹匡琪王玉坤王业涛柯祥生刘宇翰
Owner WUHAN UNIV
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