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A method for the establishment of a high-efficiency onion transient expression system mediated by Agrobacterium

An Agrobacterium-mediated and transient expression technology, applied in the field of plant genetic engineering, can solve the problem of low transformation efficiency, achieve accurate subcellular localization research, improve transformation efficiency, and achieve high transformation efficiency

Inactive Publication Date: 2019-09-06
麟州(巨野)孵化器有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current conversion efficiency is not very high, and there are no successful reports on protein-protein interaction in the application of the system.

Method used

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  • A method for the establishment of a high-efficiency onion transient expression system mediated by Agrobacterium
  • A method for the establishment of a high-efficiency onion transient expression system mediated by Agrobacterium
  • A method for the establishment of a high-efficiency onion transient expression system mediated by Agrobacterium

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Effect test

example 1

[0035] Optimization of Agrobacterium-mediated Subcellular Localization System of Onion Epidermal Cells

[0036](1) Select the onion bulbs that grow vigorously and have strong vitality, and remove the outermost 1-2 layers of scale leaves ( image 3 a), soak the onion bulbs in 70-75% ethanol for 10 minutes for disinfection, then wash the bulbs 3-5 times with sterile water in an ultra-clean workbench, and cut open the onion bulbs longitudinally with a sterile scalpel ( image 3 b) Take the thicker scale leaves in the middle layer, and use a scalpel to lightly draw about 1cm on the inner epidermis (concave surface) of these scale leaves 2 A number of cross squares, tear off the inner skin of these onions ( image 3 c);

[0037] (2) Using GFP as a reporter gene, optimize the transformation system. The GFP reporter gene was connected into the pCAMBIA3300 vector to construct the expression vector pCAMBIA3300-ubi-gfp binary expression vector ( figure 1 ). The vector was introduce...

Embodiment 2

[0048] Application of Agrobacterium-mediated transformation of onion epidermal cells to the study of protein subcellular localization

[0049] (1) Select the onion bulbs that grow vigorously and have strong vitality, and remove the outermost 1-2 layers of scale leaves ( image 3 a), soak the onion bulbs in 70-75% ethanol for 10 minutes for disinfection, then wash the bulbs 3-5 times with sterile water in the ultra-clean workbench, and cut the onion bulbs longitudinally with a sterile scalpel ( image 3 b) Take the thicker scale leaves in the middle layer, and use a scalpel to lightly draw about 1cm on the inner epidermis (concave surface) of these scale leaves 2 A number of cross squares, tear off the inner skin of these onions ( image 3 c);

[0050] (2) Using mCherry as the reporter gene, CWIN10B was connected into the pCAMBIA1300-mCherry vector to construct the expression vector pCAMBIA1300-CWIN10B-mCherry binary expression vector ( figure 1 ). The vector was introduced...

Embodiment 3

[0054] Agrobacterium-mediated transformation of onion epidermal cells applied to the study of protein-protein interactions

[0055] (1) Select the onion bulbs that grow vigorously and have strong vitality, and remove the outermost 1-2 layers of scale leaves ( image 3 a), soak the onion bulbs in 70-75% ethanol for 10 minutes for disinfection, then wash the bulbs 3-5 times with sterile water in the ultra-clean workbench, and cut the onion bulbs longitudinally with a sterile scalpel ( image 3 b) Take the thicker scale leaves in the middle layer, and use a scalpel to lightly draw about 1cm on the inner epidermis (concave surface) of these scale leaves 2 A number of cross squares, tear off the inner skin of these onions ( image 3 c);

[0056] (2) Connect CYCH and CDKD; 2 into pSPYNE-YCHA and pSPYNE-YNEE vectors respectively to form pSPYNE-CYCH-YCHA / CDKD; 2-YNEE vectors, pSPYNE-YCHA and pSPYNE-YNEE empty loads as negative controls ( figure 2 ). All vectors were introduced in...

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Abstract

The invention discloses a method for building an agrobacterium-mediated efficient onion transient expression system. The method includes: placing onion epidermal cells into an infecting culture medium IM containing agrobacterium, infecting for 10-20 minutes under 20-22 DEG C, and cultivating under light for 24 hours. The method has the advantages that the method is fast and convenient and low in cost, and the conversion efficiency of the method reaches up to more than 90%; the method can be accurately used for the subcellular localization of genes and the research of the protein and protein interaction.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and is an agrobacterium-mediated and efficient transient transformation method for studying the subcellular localization of exogenous genes in the inner epidermis of onions and the interaction between proteins. Background technique [0002] Transient expression of target genes is a simple and useful method to analyze biological functions (Zhou et al. 2009). Transient expression of genes provides very useful information in studying protein functions such as protein activity analysis, subcellular localization, and protein-protein interactions (Lee et al. 2008; Ueki et al. 2009; Hollender and Liu 2010). Compared with the complex, expensive and time-consuming stable transformation system (Scott et al.1999), the transient transformation method is fast and flexible without affecting the position of chromosomes and can be used in highly differentiated plant tissues (Fischer et al.1999)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82
Inventor 张玉苗景海春徐涵王君许卉吴涛王燕范延辉刘涛
Owner 麟州(巨野)孵化器有限公司
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