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Method for converting prunus avium L. through target genes and application thereof to instantaneous conversion of prunus avium L. protoplasts

A technology of protoplasts and sweet cherries, applied in the fields of application, horticultural methods, botanical equipment and methods, etc., can solve the problems affecting the function verification and research of important candidate genes of sweet cherries, and achieve crisp texture, light yellow color, multiplication powerful effect

Active Publication Date: 2016-08-17
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, no efficient and stable genetic transformation system has been established in the genus of sweet cherry, which affects the functional verification and research of important candidate genes of sweet cherry

Method used

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  • Method for converting prunus avium L. through target genes and application thereof to instantaneous conversion of prunus avium L. protoplasts
  • Method for converting prunus avium L. through target genes and application thereof to instantaneous conversion of prunus avium L. protoplasts
  • Method for converting prunus avium L. through target genes and application thereof to instantaneous conversion of prunus avium L. protoplasts

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Embodiment 1

[0078] Transient expression of target gene in embodiment 1, sweet cherry protoplast

[0079] The transient expression of the target gene in the sweet cherry includes transferring the target gene to the sweet cherry by using the sweet cherry transformation method, specifically infecting the sweet cherry protoplast with an infection solution containing the target gene to complete the sweet cherry transformation. The experiment was repeated three times, and the specific operation steps of each repeated experiment were as follows:

[0080] 1. Preparation of sweet cherry protoplasts

[0081] 1. Preparation of sweet cherry pulp callus

[0082] 1.1 Disinfection of explants

[0083] The young fruit of 34DAFB (Days after full bloom, Day after full bloom) 'red light' sweet cherry (P.aviumL.cv.Hong Deng) is cleaned, and with 75% (volume percentage concentration) ethanol aqueous solution surface disinfection 30s, then Sterilize with an aqueous solution of sodium hypochlorite having a m...

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Abstract

The invention discloses a method for converting prunus avium L. through target genes and application thereof to instantaneous conversion of prunus avium L. protoplasts. The prunus avium L. converting method comprises the following steps: performing suspension subculture on prunus avium L. fruit pulp calluses to obtain suspension cells; treating the suspension cells with CPW13M to obtain suspension cells treated by the CPW13M, wherein the CPW13M is a solution with a mannitol mass percentage of 11-15 percent obtained by adding mannitol into cell-protoplast cleaning fluid; performing enzymatic hydrolysis on the suspension cells treated by the CPW13M by using cellulase and pectinase to obtain the prunus avium L. protoplasts; performing prunus avium L. conversion through taking the prunus avium L. protoplasts as receptors. An experiment proves that with the adoption of the prunus avium L. converting method disclosed by the invention, the target genes can be transiently expressed in the prunus avium L. protoplasts, the functional verification of the target genes and the positioning of proteins and organelles can be realized, and the protein interaction and the cell metabolism can also be detected.

Description

technical field [0001] The invention relates to a method for transforming sweet cherry with a target gene in the field of biotechnology and its application in transiently expressing the target gene in sweet cherry protoplasts. Background technique [0002] Sweet cherry (Prunus avium L.) is a popular fruit with high nutritional value and health care function. The color of fruit is one of the important factors affecting the commerciality and value of fruit. The research on the regulation genes that affect the formation of fruit color will help to reveal the molecular mechanism of fruit coloring. Embryogenic callus, which is widely used to regenerate plants, is a good test material for free protoplasts. Protoplast cells without cell walls are basically the same as intact plant cells or tissues in terms of signal transduction pathways and metabolic processes. As an important tool of modern biotechnology, plant protoplasts are ideal recipients for genetic transformation. They c...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N5/04A01H4/00
Inventor 李天红姚立萍郭萧赵迪王彦涛
Owner CHINA AGRI UNIV
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