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Construction method of red grape PEPCK gene knockout vector

A gene knockout and construction method technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problem that the effect is not completely satisfactory, and achieve the effect of high application value

Pending Publication Date: 2022-03-18
ZHEJIANG WANLI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

The existing regulation of gluconic acid content and ratio is mainly based on exogenous regulation, and the effect is not completely satisfactory

Method used

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  • Construction method of red grape PEPCK gene knockout vector
  • Construction method of red grape PEPCK gene knockout vector
  • Construction method of red grape PEPCK gene knockout vector

Examples

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Embodiment 1

[0025] Example 1: Design the insertion site according to the original vector pAtU6-26-sgRNA-35S-EGFP-Cas9 sequence and the design principles of CRISPR-Cas9 gene editing technology, and insert the target fragment between the AtU6-26 promoter of the original vector and gRNAscaffold ( figure 1 Indicated by two black straight arrows in the middle), to realize the construction of the pAtU6-26-sgRNA-35S-EGFP-Cas9-PEPCK gene knockout vector, the construction method specifically includes the following steps:

[0026] S1. Preparation of linearized pAtU6-26-sgRNA-35S-EGFP-Cas9 vector

[0027] For the two BsaI restriction sites between the AtU6-26 promoter and the gRNA scaffold of the original circular plasmid vector pAtU6-26-sgRNA-35S-EGFP-Cas9, FastDigest Eco31I (type IIs, purchased from Thermo Fisher Scientific Company) restriction endonuclease to complete the linearization of the circular plasmid vector, the reaction system is (20 μL): 10×FastDigestbuffer 2 μL, Eco31I 2 μL, plasmid ...

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Abstract

The invention discloses a construction method of a red grape PEPCK gene knockout vector, which comprises the following steps: designing an insertion site according to a design principle of an original vector pAtU6-26-sgRNA-35S-EGFP-Cas9 sequence and a CRISPR-Cas9 gene editing technology, and inserting a target fragment between an AtU6-26 promoter and a gRNA scaffold of the original vector, so as to realize the construction of the pAtU6-26-sgRNA-35S-EGFP-Cas9-PEPCK gene knockout vector. According to the construction method of the gluconeogenesis key gene PEPCK knockout vector based on the CRISPR-Cas9 gene editing technology, through endogenous gene regulation and control, technical support can be provided for further research and cultivation of new varieties with different flavors on the basis of red grapes through a reverse genetic technology, and the construction method has high application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a PEPCK gene knockout vector of the red grape. Background technique [0002] Grape (Vitis vinifera L.) is one of the oldest fruit tree species in the world, and it is also one of the main fruits planted in my country. Its planting area and output rank among the top in the world. Due to its good economic benefits, "Yinhong" grapes have been widely cultivated in Ningbo City, Zhejiang Province. It has rich nutritional value, fresh and sweet unique taste, and is widely loved by people. With the development of the market economy and the improvement of consumer demand, whether it is table grapes or wine grapes, the quality of grapes directly affects their economic value. Fruit flavor is one of the main factors affecting the quality of grapes. It is affected by the content of sugar and acid in the fruit. In order to increase the market competitiveness, it is an im...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113C12N15/55C12N15/66C12N15/65A01H5/00A01H6/88
CPCC12N15/8218C12N15/8245C12N15/1137C12N15/65C12N15/66C12N9/22C12N9/88C12Y401/01032C12N2310/20
Inventor 肖旭腾吴月燕贾永红
Owner ZHEJIANG WANLI UNIV
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