CN-121975718-A - Establishment method and application of low-input high-yield cotton protoplast separation and transient transformation system

Abstract

The invention relates to the technical field of plant molecular biology, in particular to a method for establishing a low-input high-yield cotton protoplast separation and instantaneous conversion system and application thereof, comprising the following steps of (1) taking cotton true leaves of 2 weeks old as materials, carrying out light-proof pretreatment to reduce chlorophyll content in the leaves, reducing interference of autofluorescence on experiments (2) using leaves with extremely low input (not more than 0.1 g), cutting the pretreated leaves, placing the cut leaves in an optimized enzymolysis liquid, carrying out enzymolysis for 8 hours under the low-speed oscillation condition of 27-28 ℃ and 50rpm, (3) terminating enzymolysis, filtering by an optimizing device, centrifuging for 2 times, and washing to obtain purified protoplasts. The cotton leaf protoplast extraction method and the instantaneous conversion system with extremely low input and high extraction rate are low-loss, high-efficiency and wide-application systems, and a feasible scheme is provided for preparing protoplasts by utilizing a very small amount of samples for analysis of gene functions and molecular mechanisms.

Inventors

• WANG KAI
• DAI YAN

Assignees

• 南通大学

Dates

Publication Date

20260505

Application Date

20260330

Claims (4)

1. 1. The method for establishing the cotton protoplast separation system with low investment and high yield is characterized by comprising the following steps: (1) Taking cotton true leaves with the age of 2 weeks as a material, and carrying out light-shielding pretreatment to reduce the chlorophyll content in the leaves and reduce the interference of autofluorescence on experiments; (2) Cutting the pretreated leaves by using low-input leaves, placing the cut leaves in enzymolysis liquid with optimized formula, and carrying out enzymolysis under the low-speed oscillation condition of 27-28 ℃ and 50 rpm; (3) Stopping enzymolysis, filtering by an optimizing device, centrifuging for 2 times, and washing to obtain purified protoplast.
2. 2. The method for establishing a low-investment high-yield cotton protoplast separation system as recited in claim 1, wherein, (1) Using cotton seedlings of 2 weeks old, and pre-treating the cotton seedlings for 5 days in a dark place before extracting protoplasts to reduce autofluorescence of the protoplasts; (2) Weighing pretreated leaves, weighing cotton true leaves with the weight not exceeding 0.1g, cutting the cotton true leaves into filaments with the width not exceeding 0.1mm by using a pair of sharp scissors, immediately loading the filaments into a culture dish containing 10mL of enzymolysis liquid, wherein the diameter of the culture dish is 6cm, adding 0.02% of polyvinylpyrrolidone PVP as a clarifying agent and a stabilizing agent into the enzymolysis liquid to adsorb phenolic substances, obviously reducing the impurity content and greatly improving the yield and activity of protoplasts, and placing the enzymolysis liquid containing cotton leaf filaments into a shaking table to shake and carry out enzymolysis for 8 hours at a low speed and 50rpm in a dark place under the environment of 27-28 ℃; (3) After the reaction was terminated, the mixture was filtered with a 200 mesh stainless steel metal screen plus 2 layers of filter cloth into a square dish, the filtered liquid was transferred to a 50mL centrifuge tube, the supernatant was gently poured into a new 50mL centrifuge tube using a horizontal rotor centrifuge at 4 ℃ 200g x centrifuge for 5min, the pellet was gently resuspended with a suitable amount of W5 sucked by a 1mL blue gun head shear head, the supernatant was removed using a horizontal rotor centrifuge at 4 ℃ 300g x centrifuge for 5min, and the pellet was gently resuspended with a suitable amount of W5 sucked by a 1mL blue gun head shear head.
3. 3. A method for establishing a stable and efficient protoplast transient transformation system of cotton leaves is characterized by comprising the steps of adopting a PEG mediated method to split proper protoplast into 2mL centrifuge tubes, concentrating plasmid DNA obtained by plasmid extraction, finishing transformation by using plasmids with the concentration of not less than 1000 ng/. Mu.L, adding transformation medium with the dosage of 5 mu.g being equal to the total volume of the protoplast and the plasmids, performing transformation at the temperature of 28 ℃ for 20min, adding 1mL of W5 to terminate the transformation reaction after the transformation reaction, performing centrifugation for 350g for 5min to precipitate the protoplast, removing supernatant, performing light and heavy suspension on the protoplast by 1mL of W5, performing fluorescent observation after dark culture for 20-24h at the temperature of 28 ℃, and achieving the transformation efficiency of 90%.
4. 4. A protoplast transient transformation system obtained by the method of claim 3, wherein the application of the transient transformation system in cotton cis-acting element function detection is verified by combining mini 35s: gfp vector.

Description

Establishment method and application of low-input high-yield cotton protoplast separation and transient transformation system Technical Field The invention relates to the technical field of plant molecular biology, in particular to a method for establishing a low-investment high-yield cotton protoplast separation and transient transformation system and application thereof. Background Plant protoplasts are living single cells without cell walls, and thus nucleic acid can be easily introduced into the protoplasts. In contrast to transgenic plants produced by stable transformation, transient expression assays can be performed in a relatively short period of time. Protoplast-based transient expression systems are considered as effective methods for studying gene functions such as subcellular localization, protein-protein interactions and promoter analysis. In the mode plants such as arabidopsis thaliana, brachypodium distachyon and the like, protoplast isolation is mature compared with a transient transformation system. However, cotton leaf cell walls contain a high proportion of lignin and complex polysaccharides, and leaves and other tissues are rich in secondary metabolites, which results in low enzymolysis efficiency, easy browning of tissues, generally poor protoplast yield and activity, thus limiting the application and popularization of protoplast technology in cotton. Conventional plant protoplast isolation and transformation techniques require the use of more plant samples to obtain sufficient amounts of active protoplasts for transformation, and are more costly to isolate and transform, resulting in inefficient protoplast isolation and transformation. In order to solve the above problems, the present application needs to provide a method for establishing a low-investment high-yield cotton protoplast separation and transient transformation system and application thereof. Disclosure of Invention The invention aims to solve the defects in the prior art, and provides a method for establishing a low-input high-yield cotton protoplast separation and transient transformation system and application thereof, wherein cotton leaves with the concentration not exceeding 0.1g are used in the method, cellulase and pectase with the concentration being specific are used in a mannitol environment with the concentration being specific, polyvinylpyrrolidone (PVP) with the concentration being specific is added as a clarifying agent and a stabilizing agent to adsorb phenolic substances, the impurity content is obviously reduced, and the yield and the activity of protoplasts are greatly improved. In order to achieve the above purpose, the present invention adopts the following technical scheme: a method for establishing a low-input high-yield cotton protoplast separation system comprises the following steps: (1) Taking cotton true leaves with the age of 2 weeks as a material, and carrying out light-shielding pretreatment to reduce the chlorophyll content in the leaves and reduce the interference of autofluorescence on experiments; (2) Cutting the pretreated leaves with extremely low input (not more than 0.1 g), placing the cut leaves into enzymolysis liquid with optimized formula, and performing enzymolysis under low-speed oscillation conditions of 27-28 ℃ and 50 rpm; (3) Stopping enzymolysis, filtering by an optimizing device, centrifuging for 2 times, and washing to obtain purified protoplast. Preferably, step (1) comprises the specific steps of pre-treating 2 week old cotton seedlings with a constant temperature climatic chamber at 28℃with a photoperiod of 16h/8h in a dark place for 3-5 days before extracting protoplasts. The removed young and tender true leaves of cotton are washed 3 times with sterile water, and the filter paper is used for sucking the surface moisture. The step (2) comprises the steps of weighing the pretreated leaves, weighing cotton true leaves with the weight not exceeding 0.1g, cutting the cotton true leaves into filaments with the width not exceeding 0.1mm by using a pair of sharp scissors, and immediately placing the filaments into a culture dish containing 10mL of enzymolysis liquid, wherein the diameter of the culture dish is 6cm. The enzymolysis liquid is prepared with MES of pH 5.7, MES of 0.6mol/L, KCl of 20mmol/L, cellulase of 1.5% and isolating enzyme of 0.75%, through heating and dissolving the enzymolysis liquid in water bath of 37 deg.c for 10min while stirring for 2min until the enzymolysis liquid is clarified, cooling to room temperature, adding CaCl 2 of 10mmol/L, BSA of 0.1% and PVP of 0.02% and maintaining the volume with sterile water. And (3) placing the enzymatic hydrolysate containing the cotton leaf shreds in a shaking table at a light-shielding speed of 50rpm under the environment of 27-28 ℃ for oscillating and enzymatic hydrolysis. The step (3) comprises the steps of taking out from a shaking table after enzymolysis time is up, adding 4 ℃ precooled W5 solution equal to the