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A kind of method and application of alfalfa overexpression squalene epoxidase gene

A technology of squalene epoxidase and alfalfa, which can be applied in application, genetic engineering, oxidoreductase and other directions, and can solve problems such as reducing SE expression.

Active Publication Date: 2020-09-25
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology

Studies have shown that when the expression of SE gene is increased by in vitro inducers, the biosynthesis of triterpenoid saponins also increases, while the expression of SE is reduced, and the synthesis of triterpenoid saponins is inhibited.

Method used

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  • A kind of method and application of alfalfa overexpression squalene epoxidase gene
  • A kind of method and application of alfalfa overexpression squalene epoxidase gene
  • A kind of method and application of alfalfa overexpression squalene epoxidase gene

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Embodiment Construction

[0041] 1 Materials and methods

[0042] 1.1 Plant material

[0043] Medicago sativa L.'Zhongmu No.3' was preserved and provided by the Forage Genetics and Cultivation Team of the Institute of Animal Husbandry, Chinese Academy of Agricultural Sciences. Alfalfa No. 3 seeds germinated on wetted filter paper, transplanted into Hoagland's nutrient medium, and placed in a light incubator (16h day / 8h night, 24°C during the day, 20°C at night, relative Humidity 40%), change the nutrient solution every 7 days. After 25 days of cultivation, the seedlings were irradiated with 15W ultraviolet light for 30min, dark treatment, MeJA (200μM), ABA (100μM), GA 3 (50μM) and other stress treatments, the roots, stems, and leaf tissues were collected at different time points for the analysis of the expression of the target gene and the determination of the saponin content. After the seeds of No.3 alfalfa were sterilized by 75% alcohol solution and 0.1% HgCl solution, they were washed 7-8 times w...

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Abstract

The invention discloses an alfalfa squalene epoxidase gene overexpression method and application thereof. The alfalfa squalene epoxidase gene overexpression method comprises the following steps of (1)cloning and sequence analysis of alfalfa SE1 gene, (2) bioinformatics analysis, (3) subcellular localization, (4) prokaryotic expression, (5) expression of alfalfa squalene epoxidase MsSE1, and (6) MsSE1 gene overexpression carrier establishment and alfalfa genetic transformation. By establishing a high-efficiency genetic transformation system to transfer alfalfa squalene epoxidase genes into alfalfa for overexpression, the alfalfa squalene epoxidase gene overexpression method can improve the content of total saponins in transgenic alfalfa and provide breeding materials for culturing high-saponin-content alfalfa.

Description

technical field [0001] The invention relates to the field of rate-limiting enzyme genes related to plant saponin synthesis, in particular to a method and application for overexpressing squalene epoxidase gene in alfalfa. Background technique [0002] Squalene epoxidase, also known as squalene monooxygenase, catalyzes the epoxidation reaction of the C=C double bond of squalene in the mevalonate (MVA) pathway to generate 2,3-oxidized squalene. This reaction generates precursors of sterols and triterpenoids, and is the first oxidation reaction in the synthesis pathway of triterpene saponins, so the activity and content of squalene epoxidase determines the output of downstream products. In the metabolic pathway of triterpenes, the role of squalene epoxide (SE) is very important. Squalene epoxidase has a binding site for flavin adenine dinucleotide (FAD), an important auxiliary factor required for redox reactions, which is an important binding site for key enzymes in the saponin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66C12N15/53A01H5/00A01H6/54
CPCC12N9/0083C12N15/66C12N15/8243C12Y114/99007
Inventor 康俊梅张铁军张俏燕王珍龙瑞才杨青川
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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