Preparation method and transient expression transformation method of sorghum protoplasts

A protoplast and sorghum technology, applied in biochemical equipment and methods, using vectors to introduce foreign genetic material, plant cells, etc., can solve the problems of sorghum lacking a protoplast transient expression system, and achieve the effect of a simple method

Active Publication Date: 2020-11-24
湖北索敢科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, sorghum lacks a stable and efficient protoplast transient expression system, so the establishment of sorghum protoplast preparation and transformation system is of great significance for the genetic transformation and gene function research of sorghum

Method used

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  • Preparation method and transient expression transformation method of sorghum protoplasts
  • Preparation method and transient expression transformation method of sorghum protoplasts
  • Preparation method and transient expression transformation method of sorghum protoplasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, disinfection and cultivation of sorghum seeds

[0035] Select plump and clean sorghum seeds, put them in a clean glass bottle, add a small amount of dishwashing liquid, rinse with tap water for 1 hour, transfer to a sterile ultra-clean workbench, disinfect with 75% alcohol for 2 minutes, and wash with sterile water twice , 1 minute each time, soak and disinfect with 0.1% mercury chloride (containing 0.1% Tween-20) for 20 minutes, wash with sterile water 5 times, about 2 minutes each time, try to clean the raw mercury residue on the seeds, wash After completion, the seeds were inoculated into glass bottles containing 1 / 2 MS medium, moved to the tissue culture room, and cultivated for 12 days at 26°C with 16 h of light every day ( figure 1 ).

[0036] 1 / 2 MS medium (1L): 1 / 2 MS powder, 3% sucrose, 1% Agar, pH 5.8.

Embodiment 2

[0037] Example 2. Plasmid (35S::GFP) extraction

[0038] The plasmid used in the present invention adopts QIAGEN Plasmid Midi Kit kit to extract and purify, and specific method is as follows:

[0039]Escherichia coli DH5α (containing the constructed vector) was streaked on the LB medium containing kanamycin, and cultured overnight at 37°C. Take 3 ml of LB medium (containing kanamycin) into a 10 ml centrifuge tube, pick a single colony from step A into the centrifuge tube, and culture overnight at 37°C with shaking at 220rpm. Pipette 1 ml of bacterial liquid into 250 ml of LB medium (containing kanamycin), and culture overnight at 37°C with shaking at 220 rpm. Take the cultured bacterial solution (OD value = 2-4), centrifuge at 6000×g at 4°C for 15 minutes, remove the supernatant, add 10ml P1 (containing RNase A 100 μg / ml) solution to resuspend the bacteria, and use a pipette gun Pipette the solution until the bacteria blocks are evenly dissolved in the P1 solution. Add 10 m...

Embodiment 3

[0040] Embodiment 3, the separation of sorghum protoplast 1

[0041] Select sorghum tissue culture seedlings cultivated for 12 days, quickly remove leaves and roots, take about 1-2 g of stems, and cut them into 0.5-1 mm pieces with a sharp blade (new blades need to be replaced each time, figure 2 ), quickly soaked into a 100ml Erlenmeyer flask filled with 0.6 M mannitol solution, and stood in the dark for 30 minutes for plasmolysis. After removing the mannitol, add 10 ml of enzymatic hydrolysis solution ( image 3 ), put it in a vacuum device in the dark after mixing, place it on a shaker at room temperature to maintain oscillation at 40 rpm, vacuumize for 1 hour and continue enzymatic hydrolysis for 4 hours. After the enzymolysis, adjust the speed of the shaker to 80 rpm for 10 minutes, add an equal volume of W5 solution and continue to shake for 10 minutes, filter the enzymolysis solution with 200-mesh nylon mesh into a 50 ml centrifuge tube, collect the filtrate, and plac...

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Abstract

The invention discloses a method for acquiring a large number of sorghum protoplasts from seedlings of sorghum BTx623 through an enzymolysis method and converting the sorghum protoplasts by using a polyethylene glycol mediated method. The method comprises the following specific steps: 1, selecting full sorghum seeds, disinfecting the sorghum seeds and planting the seeds in a culture medium; 2, selecting sorghum seedlings which grow well, removing leaves and roots, reserving middle leaf sheath parts, cutting the sorghum seedlings into thin strips by using a blade, pretreating the thin strips ina mannitol solution, and then adding enzymatic hydrolysate for enzymolysis; 3, after enzymolysis is finished, enriching protoplasts by using a centrifugal method, and conducting resuspending; and 4,transforming the protoplasts by using the polyethylene glycol mediated method. A stable and efficient sorghum protoplast separation and transformation system is established in the invention; and the method is rapid and efficient in action and high in transformation rate and can be used for target gene expression, protein subcellular localization, protein interaction and the like, and a powerful tool is provided for genetic improvement and gene functional group research of sorghum.

Description

technical field [0001] The invention relates to a preparation method of sorghum protoplast and a transformation method of the prepared protoplast. Background technique [0002] Sorghum is an annual grass C 4 Plants, which can be used as food, feed, brewing and bioenergy, have wide adaptability and good stress resistance. They are widely planted all over the world and in my country, and have high economic value and research value. However, sorghum has problems such as difficult transformation and regeneration. Using conventional genetic transformation techniques, such as the Agrobacterium infection method, has difficulties such as low efficiency, difficult regeneration and long cycle, which limits the genetic improvement and functional genome research of sorghum. development of. [0003] Plant protoplasts are the bare cells of plant cells after removal of the cell wall, which has the property of easily absorbing exogenous nucleic acid fragments. Since 1960, when Cocking et...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12N15/82
CPCC12N5/04C12N15/8206
Inventor 沈祥陵曾弓剑陆业磊周超张德春吕阳韩少鹏
Owner 湖北索敢科技有限公司
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