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CN-121975845-A - Method for transfecting plant receptor by using cell penetrating peptide

CN121975845ACN 121975845 ACN121975845 ACN 121975845ACN-121975845-A

Abstract

The invention discloses a method for transfecting plant receptors by using cell penetrating peptide, which comprises the following steps of (1) constructing an expression vector of cell penetrating peptide fusion protein CPPs-Cys-mCherry, (2) transforming the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry into a protein expression system, (3) inducing the expression of the cell penetrating peptide fusion protein CPPs-Cys-mCherry by using IPTG to obtain a crude protein sample, (4) purifying the crude protein sample obtained in the step (3), then regulating the concentration of the cell penetrating peptide fusion protein CPPs-Cys-mCherry to 1-200 mu g/ml, and (5) transfecting the solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (4) into different types of plant receptors.

Inventors

  • WU HAN
  • WANG YANQIU
  • DUAN YOUHOU
  • ZHANG ZHIPENG
  • ZHU KAI
  • ZOU JIANQIU
  • ZHAO JIAMING
  • LIU ZHIQIANG
  • ZHANG FEI
  • KE FULAI
  • Zhang Kuangye
  • WANG JIAXU
  • CHEN BAIZHI
  • YANG LINLIN
  • ZHAO ZEYANG
  • LU FENG

Assignees

  • 辽宁省农业科学院

Dates

Publication Date
20260505
Application Date
20260213

Claims (7)

  1. 1. A method for transfecting a plant receptor with a cell penetrating peptide comprising the steps of: (1) Constructing an expression vector of a cell penetrating peptide fusion protein CPPs-Cys-mCherry; (2) Transforming the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry constructed in the step (1) into a protein expression system; (3) Inducing the CPPs-Cys-mCherry expression of the cell penetrating peptide fusion protein by using IPTG to obtain a crude protein sample; (4) Purifying the crude protein sample obtained in the step (3), obtaining a solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry after the purification is completed, and then regulating the concentration of the cell penetrating peptide fusion protein CPPs-Cys-mCherry to 1-200 mug/ml; (5) And (3) transfecting the solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (4) into different types of plant receptors, and finishing.
  2. 2. The method for transfecting a plant receptor with a cell penetrating peptide according to claim 1, wherein in the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry in step (1), CPPs represents a cell penetrating peptide which is a short peptide consisting of 5 to 30 amino acids, and the cell penetrating peptide enters cells through an endocytic mechanism or creates a cell membrane pore, carrying a macromolecular substance; cys represents cysteine; mCherry represents a red fluorescent protein.
  3. 3. The method of claim 1, wherein the protein expression system in step (2) is e.coli BL21 (DE 3).
  4. 4. A method of transfecting a plant receptor with a cell penetrating peptide according to claim 3, wherein step (2) comprises the specific steps of: (21) E.coli BL21 (DE 3) competent cells were removed from-80℃and thawed on ice; (22) Taking melted competent cells of escherichia coli BL21 (DE 3), adding 1-100 mu L of the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (1) into the competent cells of escherichia coli BL21 (DE 3), sucking and uniformly mixing, and transferring the expression vector to a metal sheet at the bottom of an electric conversion tube of an electric converter; (23) Opening the electric converter, setting the working voltage of the electric converter to be 0.36-36V, setting the working time to be 2-30 seconds, starting the electric converter to perform short-time high-voltage pulse, and entering the step (24) after the completion; (24) Adding 50-5000 mu L of LB culture medium into the electrotransformation tube, suspending competent cells of escherichia coli BL21 (DE 3) containing cell penetrating peptide fusion protein CPPs-Cys-mCherry, transferring the competent cells into a centrifuge tube, and incubating at 28-40 ℃ for 0.1-10 h to obtain bacterial liquid after the completion; (25) Taking 5-100 mu L of the bacterial liquid after the incubation in the step (24), coating the bacterial liquid on an LB plate containing 25mg/L kanamycin, and incubating at 37 ℃ overnight; (26) Selecting a monoclonal on the next day, placing the monoclonal in a 50mL centrifuge tube containing 10mL of LB culture medium, incubating at 37 ℃ overnight, extracting plasmids, performing enzyme digestion detection, preparing 20% glycerol bacteria from bacterial liquid for later use if the enzyme digestion detection is correct, returning to the step (22) if the enzyme digestion detection is incorrect, and re-executing the steps (22) - (26).
  5. 5. The method of claim 4, wherein the specific step of step (3) is as follows: (31) Adding 3-20 mu L of 20% glycerol bacteria obtained in the step (2) into 5-30 mL of LB medium containing 0.5% glucose and 100mg/L carbenicillin, and performing overnight preculture at 28 ℃ to obtain preculture bacteria liquid; (32) Sucking 1-5 mL of the preculture bacterial liquid obtained in the step (31) in the next day, adding the preculture bacterial liquid into 15-30 mL of LB (LB) medium containing 0.5% glucose and 100mg/L carbenicillin, continuing to culture at 28 ℃ to obtain the culture bacterial liquid, and entering the step (33) until the OD value of the culture bacterial liquid reaches 0.1-0.8; (33) Adding 0.1-10 mM IPTG into the culture bacterial liquid obtained in the step (32), collecting a sample after inducing for 1-10 hours, centrifuging, discarding the supernatant, and re-suspending the bacterial liquid in an equilibrium buffer solution of HisPur nickel-NTA Spin Columns kit to obtain a suspension bacterial liquid, wherein: the volume ratio of the bacterial liquid to the equilibrium buffer liquid of the HisPur nickel-NTA Spin Columns kit is 1 (10-100); (34) And (3) crushing the suspension obtained in the step (33) on compact ice by using 200W ultrasonic waves for 5-20 seconds, standing on the ice for 5-20 seconds, completing one cycle, repeating the operation, centrifuging for 10-35 minutes at 15,000Xg after completing 3-30 cycles, removing residual cells, and taking the supernatant to obtain a crude protein sample.
  6. 6. A method of transfecting a plant receptor with a cell penetrating peptide according to claim 1, wherein step (4) comprises the steps of: (41) Taking the nickel column refrigerated in the refrigerator at the temperature of 4 ℃ out of the refrigerator, placing the nickel column in a room temperature environment for balancing for 5-30 minutes, and entering the step (42) after finishing; (42) Removing the storage liquid in the nickel column, balancing the nickel column by using a balancing liquid, enabling the balancing liquid to be in full contact with the nickel column, centrifuging the nickel column for 2-5 minutes at 600-800 g, and removing the balancing liquid; (43) Adding the crude protein sample obtained in the step (3) into a nickel column, fully and uniformly mixing, incubating for 1-50 minutes at room temperature, centrifuging the nickel column for 2-5 minutes at 600-800 g, and collecting and storing liquid in a collecting pipe; (44) Washing the nickel column with a washing buffer solution with the volume of 1-2 times that of the nickel column, centrifuging 600-800 g for 2-5 minutes, collecting and storing the liquid in a collecting pipe, and repeatedly washing at least twice; (45) Adding a dissolution buffer solution with the volume of 1 time of the nickel column, eluting the cell penetrating peptide fusion protein CPPs-Cys-mCherry from the nickel column, centrifuging the nickel column for 2-10 minutes at 600-800 g, repeating the operation for at least two times, and combining and collecting filtrate, wherein the filtrate is a solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry; (46) Detecting the OD280nm absorbance value of the filtrate obtained in the step (45) by using a spectrophotometer, determining the concentration of the eluted cell penetrating peptide fusion protein CPPs-Cys-mCherry, and then adjusting the concentration to 1-200 mug/ml for later use.
  7. 7. The method of claim 1, wherein the different type of plant receptor in step (5) is one of embryogenic callus of arabidopsis mesophyll cells, arabidopsis thaliana root tip tissue, chinese cabbage microspores, and sorghum mature seeds.

Description

Method for transfecting plant receptor by using cell penetrating peptide Technical Field The invention belongs to the technical fields of plant biotechnology and plant gene editing, and particularly relates to a method for transfecting a plant receptor by using a cell penetrating peptide. Background Sweet sorghum is a widely planted feed crop, is difficult to genetically transform, and limits the industrialized application of genome editing. Delivery systems with low cytotoxicity, high transfection efficiency are a prerequisite for genetic transformation. Genetic transformation of plants generally relies on the natural infection process of Agrobacterium or the physical penetration process of a gene gun (biolistic). For example, chinese patent application CN 101906434A discloses a method for producing large-scale sympodial bamboos by using agrobacterium-mediated method, belonging to the field of plant transgenic technology. The method comprises the steps of callus induction, preculture, activation and resuspension of agrobacterium containing a target gene, infection and co-culture, screening of resistant callus, differentiation and growth of cluster buds, identification of transformed plants, cultivation of the transformed plants, transplanting of seedlings and the like, and the formulation of special culture medium and induction liquid in each transformation process. However, the technical scheme has the following defects that 1, exogenous genes are difficult to remove after genetic transformation, so that public worry about the safety of transgenic foods is continuously present, 2, the regeneration differentiation rate is low after transfection of many crops, the genotype dependence is obvious, successful reports are mostly concentrated on individual transformation lines (such as Tx430 and P8980112 in the sorghum industry) and the like, and the exogenous genes are difficult to integrate into the existing breeding system. Disclosure of Invention The invention provides a method for transfecting a plant receptor by using a cell penetrating peptide, which aims to solve the problem that exogenous genes are difficult to remove after genetic transformation in the prior art. Cell penetrating peptides (CELL PENETRATING PEPTIDES, CPPs) are an ideal alternative transfection technique, are poorly cytotoxic, do not affect post-receptor transfection regeneration, and are easily co-transfected with a variety of gene editing elements. The invention congeals the transfection mechanism mediated by the cell penetrating peptide and the technical flow of gene editing, takes the model plant arabidopsis somatic cells and sorghum embryogenic callus as receptors, and obtains the positive transfection result of the cell penetrating peptide fusion protein. The technical scheme is that the method for transfecting plant receptors by using cell penetrating peptide comprises the following steps: (1) Constructing an expression vector of a cell penetrating peptide fusion protein CPPs-Cys-mCherry; (2) Transforming the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry constructed in the step (1) into a protein expression system; (3) Inducing the CPPs-Cys-mCherry expression of the cell penetrating peptide fusion protein by using IPTG to obtain a crude protein sample; (4) Purifying the crude protein sample obtained in the step (3) by using a nickel column, obtaining a solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry after the purification is completed, and then regulating the concentration of the cell penetrating peptide fusion protein CPPs-Cys-mCherry to 1-200 mug/ml; (5) And (3) transfecting the solution containing the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (4) into different types of plant receptors, and finishing. Further, in the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry in the step (1), CPPs represent cell penetrating peptides, wherein the cell penetrating peptides are short peptides composed of 5-30 amino acids, and the cell penetrating peptides carry macromolecular substances into cells through an endocytic mechanism or create cell membrane holes; cys represents cysteine; mCherry represents a red fluorescent protein. Further, the protein expression system in the step (2) is escherichia coli BL21 (DE 3). Further, the specific steps of the step (2) are as follows: (21) E.coli BL21 (DE 3) competent cells were removed from-80℃and thawed on ice; (22) Taking melted competent cells of escherichia coli BL21 (DE 3), adding 1-100 mu L of the expression vector of the cell penetrating peptide fusion protein CPPs-Cys-mCherry obtained in the step (1) into the competent cells of escherichia coli BL21 (DE 3), sucking and uniformly mixing, and transferring the expression vector to a metal sheet at the bottom of an electric conversion tube of an electric converter; (23) Opening the electric converter, setting the