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Agrobacterium-induced strawberry leaf transient gene expression method and application of same

An Agrobacterium-mediated gene expression technology, which is applied in the fields of application, genetic engineering, botanical equipment and methods, etc., can solve the problems of stable genetic transformation of strawberry, such as heavy workload, difficult high-throughput verification of gene function, and low transformation efficiency , to achieve rapid evaluation of gene function, high transformation efficiency, and short transformation cycle

Inactive Publication Date: 2018-10-23
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the shortcomings of the stable genetic transformation of strawberry, such as heavy workload, complicated operation, time-consuming and laborious, low transformation efficiency and difficulty in high-throughput verification of gene function, the progress has been slow.

Method used

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  • Agrobacterium-induced strawberry leaf transient gene expression method and application of same
  • Agrobacterium-induced strawberry leaf transient gene expression method and application of same
  • Agrobacterium-induced strawberry leaf transient gene expression method and application of same

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1: A fast and efficient method for transient gene expression in strawberry leaves mediated by Agrobacterium

[0043] A rapid and efficient method for transient gene expression in strawberry leaves mediated by Agrobacterium, the method flow chart is shown in figure 1 , the detailed steps are as follows:

[0044] 1. Material selection: select the plant binary expression vector C15 containing the CaMV 35S promoter carrying the YFP tag, and introduce it into the Agrobacterium tumefaciens strain GV3101 by electroporation to obtain the empty vector bacterial liquid, which contains the plant expression vector The positive bacterial liquid of the carrier C15; the selected strawberry material is the tissue culture seedling of wild strawberry Heilongjiang-3.

[0045] 2. Preparation of transformed bacteria solution: pipette 200 μl of empty carrier bacteria solution into 75 mL LB liquid medium containing 25 mg / L rifampicin, 25 mg / L gentamicin and 75 mg / L kanamycin at 28°C,...

Embodiment 2

[0049] Embodiment 2: the influence of different strawberry leaf seedling ages on transformation efficiency

[0050] The method is the same as in Example 1, except that the wild strawberry Heilongjiang-3 materials selected in step 1 are: tissue culture seedlings, transplanted seedlings in one month, transplanted seedlings in two months, transplanted seedlings in half a year and one year Transplant seedlings. The results showed that different strawberry leaf seedling ages significantly affected the transformation efficiency, and the fluorescence signal of the tissue-cultured seedlings of the wild strawberry Heilongjiang-3 was the strongest. Correspondingly, the results of WesternBlot showed that the relative expression of YFP protein in the tissue cultured seedlings was the highest, up to 80%. The specific instantaneous conversion results are shown in Figure 2.

Embodiment 3

[0051] Embodiment 3: the influence of the position of different strawberry leaves on the instantaneous transformation efficiency of leaves

[0052] The method is the same as in Example 1, except that the leaves of the wild strawberry Heilongjiang-3 tissue culture seedlings clipped in step 3 are respectively: the first leaf, the second leaf, the third leaf, the fourth leaf and Fifth leaf. The results showed that the first leaf of wild strawberry Heilongjiang-3 had the strongest fluorescence signal and the highest relative expression of YFP protein. For specific instantaneous conversion results, see image 3 .

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Abstract

The invention discloses an agrobacterium-induced strawberry leaf transient gene expression method and an application of same, wherein the method is established through a vacuum-infection technology. In the method, a strawberry leaf serves as an infection material, and variety of the strawberry, seedling age of the strawberry leaf, leaf position, vacuum infection time, and growth culture medium type are optimized with yellow fluorescent protein YFP being a report gene, thereby obtaining the optimum conditions for expressing foreign proteins. The method is rapid and high-effective and can be applied to the fields of foreign protein expression, gene sub-cell positioning, promoter activity detection, resistance gene function verification, etc.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to an Agrobacterium-mediated rapid and high-efficiency strawberry leaf transient gene expression method and its application. Background technique [0002] Transient expression technology is a technology that transfers a target gene into target cells in a short period of time and obtains a transient high-level expression of the gene. Compared with the stable transformation system with complicated operation, high price and long transformation period, plant transient expression technology has the advantages of high expression efficiency, short experimental period, simple operation and less restriction on materials. Common methods of transient expression techniques include particle gun bombardment, protoplast transformation, and Agrobacterium-mediated transient transformation. Among them, the Agrobacterium-mediated transient transformation method is more e...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/74
CPCC12N15/8205
Inventor 冯嘉玥崔孟媛未魏
Owner NORTHWEST A & F UNIV
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