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CN-121992014-A - Method for realizing instantaneous expression of exogenous target genes in rhododendron molle plants by using vacuum agrobacterium infection method

CN121992014ACN 121992014 ACN121992014 ACN 121992014ACN-121992014-A

Abstract

The invention discloses a method for realizing instantaneous expression of exogenous target genes in rhododendron molle plants by using a vacuum agrobacterium infection method, belonging to the technical field of forest genetic engineering. The invention comprises the following steps of carrying out ultrasonic pretreatment on rhododendron molle seedlings, immersing plants in agrobacterium infection liquid containing target genes, adopting cyclic vacuum treatment with specific parameters to enable the infection liquid to efficiently infiltrate into tissues, and finally finishing transient expression through shading and normal culture. The invention solves the problems of low efficiency, complicated operation and incapability of batch treatment of the traditional injection method by optimizing ultrasonic treatment conditions and combining a circulating negative pressure vacuum infiltration technology. The method remarkably improves the transformation efficiency and the expression uniformity of the exogenous gene in the whole plant level of the rhododendron molle, has simple and convenient operation, short period and good repeatability, and provides a high-efficiency and reliable technical platform for gene function research and molecular breeding of the rhododendron and rhododendron plants.

Inventors

  • GENG XINGMIN
  • XU SHIDA
  • MAO LINGFENG
  • ZENG FANYU
  • PENG JIEYU

Assignees

  • 南京林业大学

Dates

Publication Date
20260508
Application Date
20251231

Claims (10)

  1. 1. A method for realizing the instantaneous expression of exogenous target genes in rhododendron molle plants by utilizing a vacuum agrobacterium infection method is characterized by comprising the following steps: (1) Sample treatment is carried out on rhododendron molle seedling plants, wherein the sample treatment comprises stopping watering before infection and immersing the whole seedling into water for ultrasonic treatment before infection; (2) Immersing the rhododendron molle seedling plant treated in the step (1) into agrobacterium infection solution containing target genes, and carrying out cyclic vacuum treatment, wherein the cyclic vacuum treatment comprises vacuumizing to below-0.07 MPa, maintaining for a period of time, and recovering normal pressure, and repeating the process at least twice; (3) And (3) carrying out shading culture on the plant subjected to vacuum infection, and transferring the plant into normal culture to realize the transient expression of the target gene.
  2. 2. The method according to claim 1, wherein the specific conditions of the cyclic vacuum treatment are that the vacuum pressure is-0.07 MPa, the normal pressure is recovered after 3-7 minutes, and the cyclic vacuum treatment is circulated for 2-5 times.
  3. 3. The method according to claim 2, wherein the specific conditions of the cyclic vacuum treatment are that the vacuum pressure is-0.07 MPa, the normal pressure is recovered after 5 minutes, and the cyclic vacuum treatment is circulated for 4 times.
  4. 4. The method according to claim 1, wherein in the step (1), the rhododendron molle seedlings are seedlings within 1 year of each other.
  5. 5. The method according to claim 1 or 4, wherein in the step (1), the ultrasonic treatment is performed at a frequency of 35 to 45 KHz, a power of 150 to 250W, and a treatment time of 20 to 60 seconds.
  6. 6. The method of claim 5, wherein the ultrasonic treatment has a frequency of 40 KHz, a power of 200W, and a treatment time of 30 seconds.
  7. 7. The method of claim 1, wherein the agrobacterium infection solution in step (2) is prepared by re-suspending agrobacterium with MMA-assisted infection solution, and the MMA-assisted infection solution comprises 5-15 mM magnesium chloride, 5-15 mM 2-morpholinoethanesulfonic acid and 100-300 μm acetosyringone.
  8. 8. The method according to claim 7, wherein the MMA co-infestation liquid comprises 10mM magnesium chloride, 10mM 2-morpholinoethanesulfonic acid and 200. Mu.M acetosyringone, and the OD 600 value of the post-resuspension infestation liquid is 0.8-1.2.
  9. 9. The method according to claim 1, wherein in the step (3), the time of the light-shielding culture is 1 to 2 days, and the expression of the target gene is detected after the light-shielding culture for 5 to 9 days in normal culture.
  10. 10. The method according to claim 9, wherein the time of the light-shielding culture is 1 day, and the expression of the target gene is detected 7 days after the normal culture.

Description

Method for realizing instantaneous expression of exogenous target genes in rhododendron molle plants by using vacuum agrobacterium infection method Technical Field The invention relates to the technical field of forest genetic engineering, in particular to a method for realizing instantaneous expression of exogenous target genes on rhododendron molle plants by a vacuum agrobacterium infection method Background Azalea is one of ten flowers in China, and has higher ornamental value and cultural value. Rhododendron molle (Rhododendron molle) is a few species of yellow flower in azalea, and is worth more attention both for ornamental value and medicinal value. At present, related researches on azalea are concentrated in the fields of resource distribution, morphological classification, stress physiology and the like, the research progress on molecular biology is limited, and one of the limiting reasons is the lack of a convenient and effective genetic transformation method. At present, the research of azalea genetic transformation mainly focuses on stable genetic transformation, namely, the path of infection explants to differentiate callus through tissue culture, and the transient genetic transformation adopts an injection method. However, the above methods have the defect of low efficiency, long culture period is required for tissue culture, injection infection is time-consuming and labor-consuming, and sometimes even the risk of incapacitation of smooth injection occurs. Therefore, a set of efficient genetic transformation methods for rhododendron molle is needed in the art. The invention utilizes the principle of vacuum infiltration to uniformly and efficiently infiltrate the dyeing liquor into rhododendron molle plants, can simultaneously process a large number of samples, saves time and labor, and provides a more effective tool for the research of the molecular biology field of azalea. Disclosure of Invention The invention provides a method for realizing instantaneous expression of exogenous target genes on rhododendron molle plants by a vacuum agrobacterium infection method, which aims at solving the problems of low efficiency, long time consumption and the like of the current rhododendron genetic transformation method. According to the method, the actual seedlings are directly used for vacuum infection, so that the development difficulty of genetic transformation of rhododendron molle is greatly reduced, tens of seedlings can be simultaneously transformed at one time, the time is also greatly shortened, no special culture is needed after infection is finished, and the cost is further reduced. In order to achieve the above purpose, the present invention adopts the following technical scheme. A method for realizing the instantaneous expression of exogenous target genes in rhododendron molle plants by utilizing a vacuum agrobacterium infection method comprises the following steps: (1) Sample treatment is carried out on rhododendron molle seedling plants, wherein the sample treatment comprises stopping watering before infection and immersing the whole seedling into water for ultrasonic treatment before infection; (2) Immersing the rhododendron molle seedling plant treated in the step (1) into agrobacterium infection solution containing target genes, and carrying out cyclic vacuum treatment, wherein the cyclic vacuum treatment comprises vacuumizing to below-0.07 MPa, maintaining for a period of time, and recovering normal pressure, and repeating the process at least twice; (3) And (3) carrying out shading culture on the plant subjected to vacuum infection, and transferring the plant into normal culture to realize the transient expression of the target gene. In some embodiments, the cyclic vacuum treatment is performed under conditions that the vacuum pressure is-0.07 MPa, and the cyclic vacuum treatment is performed for 3-7 minutes and then the normal pressure is recovered for 2-5 times. In some embodiments, the specific conditions of the cyclic vacuum treatment are that the vacuum pressure is-0.07 MPa, and the cyclic vacuum treatment is carried out for 4 times after maintaining the normal pressure for 5 minutes. In some embodiments, in step (1), the rhododendron molle seedlings are seedlings within 1 year of age. In some embodiments, in the step (1), the ultrasonic treatment has a frequency of 35-45 KHz, a power of 150-250W, and a treatment time of 20-60 seconds. In some embodiments, the ultrasonic treatment has a frequency of 40 KHz, a power of 200W, and a treatment time of 30 seconds. In some embodiments, the agrobacterium infection solution in the step (2) is prepared by re-suspending agrobacterium bacteria with MMA co-infection solution, wherein the MMA co-infection solution comprises 5-15 mM magnesium chloride, 5-15 mM 2-morpholinoethanesulfonic acid and 100-300 mu M acetosyringone. In some embodiments, the MMA co-infestation liquid comprises 10 mM magnesium chloride, 10 mM 2-morpholinoethanesulfonic a