Perennial ryegrass breeding method based on CRISPR/Cas system

A technology of ryegrass and strains, applied in the field of plant genetic engineering, can solve the problems of rigid requirements for the richness of germplasm resources, distant incompatibility, etc.

Inactive Publication Date: 2016-01-06
TIANJIN GENOVO BIOTECH CO LTD
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Problems solved by technology

Traditional crossbreeding has strict requirements on the abundance of germplasm resources and technical bottlenecks of distant incompatibility. How to obt

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  • Perennial ryegrass breeding method based on CRISPR/Cas system

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Embodiment Construction

[0017] The present invention will be further described below in conjunction with embodiment:

[0018] The breeding method of the perennial ryegrass based on the CRISPR / Cas system provided by the invention comprises the following steps:

[0019] (1), screening functional genes, the functional genes are key enzyme genes LpCCR and LpCOMT of lignin synthesis;

[0020] (2), optimize Streptococcus pyogenes Cas9 gene, and add NLS nuclear localization signal and restriction endonuclease site respectively at the two ends of gene coding sequence;

[0021] (3), digest the optimized Cas9 gene with a restriction endonuclease, obtain the gene fragment, connect it into the vector, and obtain the recombinant plasmid pA;

[0022] (4), amplify the U3 or U6 RNA promoter of perennial ryegrass, connect it before the gRNA backbone sequence, and construct the U3 or U6 promoter and gRNA on the pB vector;

[0023] (5), design and synthesize target site primers with cohesive ends, and connect to the ...

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Abstract

The invention provides a perennial ryegrass breeding method based on a CRISPR/Cas system. A CRISPR/Cas genome-editing technique is applied to breed improvement of perennial ryegrass, gene site-directed knock-out is conducted on lignin synthesized key enzyme genes LpCCR and LpCOMT, homozygous and transformation carrier deleted mutants are selected out, the breeding material of the perennial ryegrass of which the lignin content is decreased is bred, and important significance for breeding research and breed improvement of pasture is achieved.

Description

Technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a perennial ryegrass breeding method based on a CRISPR / Cas system. Background technique [0002] Ryegrass is one of the most important temperate forage grasses in the world. Excellent ryegrass varieties can improve the nutritional balance of animals, increase milk production and quality, increase the interests of the dairy industry, and even improve artificial turf, windbreak and sand fixation, water and soil conservation, It plays an important role in improving the living environment of human beings. Ryegrass grows fast, has many tillers, and is capable of grazing. It is a high-quality pasture grass for grazing, and it is also one of the forage grasses with the highest digestible matter yield among grasses. Ryegrass has high nutritional value and is rich in protein, minerals and vitamins. Among them, the crude protein content of hay is as high as 25%, and the ...

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Application Information

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IPC IPC(8): C12N15/82C12N15/87A01H5/00
Inventor 高崑
Owner TIANJIN GENOVO BIOTECH CO LTD
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