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Establishment and application of melon transient expression system

A technology of transient expression, melon, applied in the field of cell engineering

Active Publication Date: 2021-04-20
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, studies on the isolation of melon protoplasts and the establishment of transient transformation systems in melons have not been reported

Method used

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  • Establishment and application of melon transient expression system
  • Establishment and application of melon transient expression system
  • Establishment and application of melon transient expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1 provides a kind of method for preparing the muskmelon transient expression system, comprising:

[0076] (1) Material cultivation: seeds of the melon variety M7 were placed in a 30° C. incubator and soaked in the dark for 24 hours. Select well-germinated seeds and cultivate them for about 2 weeks in a culture room with a relative air humidity of 60% and a photoperiod of 16h light / 8h dark.

[0077] (2) Sampling: under the condition of good growth, the fully stretched cotyledon or the first true leaf is clipped to prepare protoplasts. Use a blade to cut the leaves into thin strips about 1 mm wide in the direction perpendicular to the midrib, and immediately put them into the enzymatic hydrolysis solution [comprising: 1.5% (w / v) cellulase R-10, 0.4% (w / v) dissociating enzyme R-10, 20mM 2-morpholinoethanesulfonic acid (MES) and 0.4M mannitol] (add 0.5g true leaf or cotyledon per 10mL enzymolysis solution).

[0078] (3) Dark enzymolysis: Vacuumize the enzymolys...

Embodiment 2

[0088] Usually young plant tissue is used for isolating protoplasts. With reference to the steps shown in above-mentioned embodiment 1, the sampling site has been changed, and embodiment 2 has compared the impact of different sampling sites on the obtained melon protoplast, and its research content is as follows:

[0089] In this example, the cotyledon of about 1 week and the first true leaf of about 2 weeks are used to compare the influence of the sampling site on the protoplast.

[0090] Such as figure 1 as shown, figure 1 A is the cotyledon and first true leaf of melon M7 on the left and right, respectively. The cotyledons and first true leaves were harvested using the same razor blade shredding method and immediately digested in an enzymatic solution. First, after 30 minutes of vacuuming in the dark, and then through low-speed constant temperature enzymatic hydrolysis (28 ° C, 40 r min -1 ). in figure 1 B and figure 1 C shows the enzymolysis solution after vacuum in...

Embodiment 3

[0092] With reference to the steps and conditions shown in the above-mentioned Example 1, the enzymatic hydrolysis time was changed, and Example 3 compared the impact of different enzymatic hydrolysis times on protoplasts.

[0093] To optimize the enzymatic hydrolysis time, the protoplast yield and vigor of the cotyledon and first true leaf were analyzed at 2 h, 4 h, 6 h and 8 h, respectively.

[0094] The result is as figure 2 2A is the graph showing the effect of enzymolysis time on cotyledon protoplast yield and vigor, and 2B is the graph showing the effect of enzymolysis time on the protoplast yield and vigor of the first true leaf. figure 2 A and figure 2 The abscissa in B is hour, figure 2 A and figure 2 The histogram in B represents the result graph of protoplast yield, and the line graph represents the result graph of protoplast viability. The results showed that the protoplast yield of cotyledon and true leaf increased with the increase of enzymatic hydrolysi...

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Abstract

The invention relates to the technical field of cell engineering, and in particular relates to establishment and application of a melon transient expression system. A construction method comprises the following steps of: (1) performing enzymolysis on melon tissues by utilizing enzymatic hydrolysate in a dark environment; (2) performing termination treatment on a solution after enzymolysis by using a first termination solution, performing filtering, and centrifuging the filtrate to remove supernatant to obtain a protoplast; (3) performing ice bath on the protoplast, performing centrifuging to remove the supernatant, and performing resuspending by using a resuspension solution to obtain the purified protoplast; (4) continuing to mix with plasmid DNA, adding a PEG4000-Ca<2+> solution, and performing transfecting in a dark environment; (5) adding a second termination solution into a transfection product, and centrifuging a termination product to obtain a protoplast containing the plasmid DNA; and (6) continuing to incubate in a dark environment, and performing centrifuging to remove the supernatant to complete transient expression. The system is stable and can be used for various functional analysis, such as gene expression analysis, subcellular localization, intracellular protein transport analysis and protein interaction.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to the establishment and application of a muskmelon transient expression system. Background technique [0002] Compared with stable genetic transformation, transient expression system is a fast and efficient expression method. Its convenient operation and high-throughput transformation can be used for a variety of functional analysis, such as gene expression analysis, subcellular localization, and intracellular protein transport analysis , protein interaction, and promoter activity analysis. Agrobacterium tumefaciens-mediated injection, biolistic bombardment, and PEG-mediated protoplast transfection are the most commonly used means for transient transformation. Compared with bio-bombardment and Agrobacterium infection, PEG-mediated protoplast transformation is more convenient and efficient. A protoplast is a cell that has had its cell wall removed but retains ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/12A01H6/34
Inventor 许昕阳沈佳寿伟松张跃建
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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