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Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermediary

A technology of Caragana intermedia and Agrobacterium rhizogenes is applied in the field of plant genetic engineering to achieve the effects of high gene expression level, convenient detection and simple operation.

Pending Publication Date: 2021-11-16
INNER MONGOLIA AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of transforming the hairy root of Caragana intermedia in one step is also rarely reported

Method used

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  • Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermediary
  • Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermediary
  • Agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermediary

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A step of transformation of a hairy root of Agrobacteria, including the following steps:

[0039] S1. Gas-converted plasmid electrochemical transformation of the E-GFP expression vector in Agrobacterium K599 sensitive state cells

[0040] S11. Take out the PEGAD plasmid and frozen, placed on ice;

[0041] S12. From the -80 ° C refrigerator to remove the root Agrobacterium K599 sensitive cells, placed on ice;

[0042] S13. Cleaning Electrode Cup: Soak the electrode cup for 10 min with 75% ethanol, then soak 10 min with 100% ethanol, washed with water, and put it into a fume hood and blow it.

[0043] S14. Add 1 μlpegad plasmid to the Range Agrobacterium K599 to feel cells, mix well, then use the rigging of the peculider K599 to add all the hair rooted Agrobacterium K599 to the cleaned pre-cooling electrode cup with a tip of the rifle. (U = 1400V, T = 5.4 ~ 5.8ms) Get transformant;

[0044] S15. The transformant in step S14 was sued out, and in a 1.5 MLEP tube containing 800 μ...

Embodiment 2

[0058] Unlike the first embodiment, the method of preparing the spontaneous Agrobacterium bacteria suspension in step S2 is as follows: S21. Type the above-mentioned pestinal Agrobacterium K599 containing a particulate carrier PEGAD-E-GFP to 4 ml LB liquid medium (Containing 50 μg / ml kanamycin and 50 μg / ml streptomycin), at 28 ° C, the oscillation velocity at 180 rpm was oscillated overnight to the OD600 nm value of about 0.9, and the initiatra suspension was obtained.

[0059] S22. The next day, a part of the initiatra suspension is guaranteed, and the step of cleavage is: Take 1 mL of the rapids of the rapids to the 1.5MLEP tube. After centrifugation of 4000 rpm, the supernatant is discarded, and 800 μl of fresh LB liquid medium and 200 μl of 75% glycerol, mix, liquid nitrogen quick freeze and placed in a -80 ° C refrigerator for a long time, another part of the raised resuspension, transfer 1 ml of the initiator suspension to 100 ml of fresh LB liquid The medium (containing...

Embodiment 3

[0062] Unlike Example 1, the method of preparing the root apex bacteria suspension in step S2 is as follows:

[0063] S21. The above identification was inoculated into a 4 mL LB liquid medium (containing 50 μg / ml kanamycin and 50 μg / ml streptomycin) in 5 ° C. , With 180 rpm oscillation speed overnight rock, oscillate overnight to OD 600nm The value is about 0.9, resulting in a rapid suspension;

[0064] S22. The next day, a part of the initiatra suspension is guaranteed, and the step of cleavage is: Take 1 mL of the rapids of the rapids to the 1.5MLEP tube. After centrifugation of 4000 rpm, the supernatant is discarded, and 800 μl of fresh LB liquid medium and 200 μl of 75% glycerol, mix, liquid nitrogen quick freeze and placed in a -80 ° C refrigerator for a long time, another part of the raised resuspension, transfer 1 ml of the initiator suspension to 100 ml of fresh LB liquid The medium (containing 50 μg / ml kanamycin and 50 μg / ml streptomycin), at 28 ° C, racefius at 1...

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Abstract

The invention discloses an agrobacterium rhizogenes-mediated one-step transformation method for hairy roots of caragana intermediary. The method comprises the following steps: S1, electrically transforming plasmids containing e-GFP expression vectors into agrobacterium rhizogenes K599 competent cells; S2, preparing an agrobacterium rhizogenes bacterial suspension with the OD600nm of 0.8 to 0.95, and adding acetosyringone into the agrobacterium rhizogenes bacterial suspension; S3, culturing bacterial paste for infection; and S4, selecting a caragana intermedia seedling, obliquely cutting a part which is 1cm below a cotyledonary node of the seedling, discarding the lower part, infecting a cut, then transplanting the seedling back into soil, pouring a 1 / 4B5 culture medium to the seedling, and putting the seedling into an illumination incubator for culturing. According to the method, the caragana intermediary hairy roots are converted through a one-step method, so that the genes are over-expressed in the hairy roots, and compared with a tissue culture method for converting the caragana intermediary hairy roots, the method is simpler to operate, does not need to prepare sterile seedlings, and is shorter in detection time, high in gene expression level and convenient to detect.

Description

Technical field [0001] The present invention relates to the field of plant gene engineering, and in particular, to a method of transformation of a macrophocyte-mediated intermediate rocket of Agrobacterium. Background technique [0002] The concept of horses was earlier than 1900 by Stewart. In 1907, Smith and Townsend found that Peter bacteriobacterium can invade plants to produce hair roots. Agrobacterium cemented method is an efficient conversion method in plant gene engineering. Most of the transgenic plants are obtained from the Agrobacterium Ti Plasmid of Root Cancer. In 1982, Chilton reported that the horsid root was caused by Gas of Mycobacterium bacteria. At this point, the hair root technology entered a new era. The related studies of RI plasmid-mediated plant-like roots genetic transformation are carried out in many plant species. Moisting root culture technology is mainly used in the synthesis and promoting gene expression of plant secondary metabolism, as well as an ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65A01H4/00A01H5/06A01H6/54
CPCC12N15/8205C12N15/8212C12N15/65A01H4/005A01H4/008
Inventor 万永青杨闯柳金华李国婧王瑞刚
Owner INNER MONGOLIA AGRICULTURAL UNIVERSITY
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