CN114591882A

CN114591882A - Method for transient transformation of acanthopanax cells

Info

Publication number: CN114591882A
Application number: CN202210201087.3A
Authority: CN
Inventors: 范桂枝; 高兴蕾; 王滋涵; 由香玲; 詹亚光
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2022-03-03
Filing date: 2022-03-03
Publication date: 2022-06-07

Abstract

The invention relates to a method for obtaining acanthopanax senticosus callus unicells by an enzymatic hydrolysis method and obtaining a trans-GFP gene by agrobacterium transformation, belonging to the technical field of biology. After 1.5% cellulase R-10 and 0.2% segregation enzyme R-10 are adopted to carry out enzymolysis on acanthopanax senticosus callus for 6 hours, the unicellular yield is the maximum and the vitality is the strongest when the acanthopanax senticosus callus is centrifuged for 5min at 400 Xg, and the unicellular yield is 1.88 X106. g^‑1And 90%. When the agrobacterium is transformed into single cells for 40min under the concentration of 40 percent PEG, the transient transformation efficiency of the single cells reaches 30.34 percent. The invention provides an enzymolysis system of high-yield acanthopanax single cells, and obtains consistent single cells with transferred GFP genes, which researches the functions of genes from the single cell level and realizes the gene function in different types of cellsFunctional analysis provides technical support.

Description

Method for transient transformation of acanthopanax cells

The technical field is as follows:

the invention relates to a method for obtaining acanthopanax senticosus callus unicells by an enzymatic hydrolysis method and obtaining a trans-GFP gene by agrobacterium transformation, belonging to the technical field of biology.

Background art:

acanthopanax senticosus (Acanthopanax senticosus) is perennial deciduous shrub of Araliaceae, and is mainly distributed in Heilongjiang, Jilin, Liaoning, Hebei, Shaanxi provinces, Russia, Korea, Japan, etc. Wu Jia Pi is listed as the superior product in Ben Cao gang mu, Shen nong Ben Cao Jing and Zhong Hua Ming Hui nationality pharmacopoeia, etc., and has the efficacy of tonifying qi and strengthening spleen, and tonifying qi and soothing the nerves. Modern pharmacological research shows that acanthopanax has multiple pharmacological functions of resisting infection, virus, oxidation, fatigue, tumor, immunity, blood sugar and blood fat, and the like. The analysis on the components of the root, stem and leaf of the acanthopanax senticosus shows that the active components mainly comprise saponins, polysaccharides, flavones, lignans, triterpenes, organic acids and the like. At present, the research on acanthopanax is mainly focused on the aspects of growth characteristics, breeding, pharmacology, ecological adaptability, plant tissue culture and the like.

Cells are the basic building blocks of organisms and also the most basic functional units of the body, each of which has a critical role and is unique and inherently heterogeneous in nature. The answer to each bioscience question must therefore be found in the cell. At present, single cell sequencing technology reveals the gene structure and gene expression state of a single cell, identifies the cell type and cell state, reacts the difference and change among cells, analyzes the dynamic change process of the cell and the interaction relation among cells and the like through single cell high-throughput sequencing analysis, can distinguish different cell types, and even reveal new cell types. The single cell sequencing technology is mainly applied to the research of gene expression regulation, immunity, tumor diagnosis, treatment and the like. If the gene function can be researched from the single cell level, more detailed and comprehensive information can be provided for researchers, the researchers can be helped to analyze the cell types and states from different layers, and the regulation mechanism can be deeply excavated, so that the essence and the law of life activities can be deeply researched.

Transient transformation technology refers to transferring a target gene into a receptor cell in a short time, establishing a high-efficiency expression system in the receptor cell, and using the system to verify the function of the target gene or analyze the gene function, such as protein subcellular localization, promoter activity detection, protein interaction and the like.

At present, a acanthopanax transgenic system is not established, the invention aims to explore a method for acanthopanax single cell separation and instantaneous transformation, and the invention lays a foundation for acanthopanax gene function research and genetic improvement thereof.

The invention content is as follows:

the invention provides a method for separating unicellular callus of acanthopanax, which mainly comprises the following steps: after 6h of enzymolysis with 1.5% cellulase R-10 and 0.2% macerozyme R-10, the single cell yield is maximal and the activity is strongest when centrifugation is carried out at 400 Xg for 5min, which are respectively 1.88X 10⁶G^-1And 90%.

The invention provides a unicellular transient transformation method of acanthopanax senticosus callus, which mainly comprises the following steps: when 40min of agrobacterium transformation is carried out under the concentration of 40 percent PEG, the transient transformation efficiency of single cells reaches 30.34 percent. The invention provides technical support for researching the functions of the genes from the single cell level and analyzing the functions of the genes in different types of cells and lays a foundation for screening transgenic single cell lines.

Description of the drawings:

FIG. 1 shows callus of Acanthopanax senticosus;

FIG. 2 shows unicells derived from callus of Acanthopanax senticosus;

FIG. 3 is a detection chart of diacetate Fluorescein (FDA) of Acanthopanax senticosus;

FIG. 4 shows single cells of Acanthopanax senticosus transformed with GFP gene.

The specific implementation mode is as follows:

experimental materials: the callus of Acanthopanax senticosus cultured in the research room is inoculated with seed seedling of Acanthopanax senticosus in the amount of 1.0 mg.L^-12,4-D on MS medium.

Experimental reagent: the formula of the W5 solution is as follows: 2mmol/L MES, 154mmol/L NaCl, 125mmol/L CaCl₂And 5mmol/L KCl, adjusting the pH value of the prepared W5 solution to 5.7 by using 1mol/L KOH, sterilizing at the high temperature of 121 ℃ for 20min under high pressure, and storing at room temperature; PEG-CaCl₂The solution formula is as follows: 0.2mol/L mannitol, 100mmol/L CaCl₂PEG4000 concentration was added according to different treatment requirements. Formulated PEG-CaCl₂Adjusting the pH value of the solution to 5.7 by using 1mol/L KOH, and storing the solution at the temperature of minus 20 ℃; the MMG solution formula is as follows: 0.6mol/L mannitol, 4mmol/L MES and 15mmol/L MgCl₂(ii) a The formula of the WI solution is as follows: 0.5mol/L mannitol, 4mmol/L MES and 20mmol/L KCl.

Example 1: isolation of unicellular Acanthopanax senticosus

In this embodiment, the cellulase R-10 concentration, the macerozyme R-10 concentration, the centrifugal force and the enzymolysis time are used as variables for the test, and a four-factor five-level orthogonal experimental design method is adopted, and is specifically implemented as follows:

1) firstly, soaking mature acanthopanax seeds in 70% ethanol for 1.50min, soaking in 5% sodium hypochlorite for 10min, washing with sterilized distilled water for 3-5 times, taking out complete embryos, inoculating the complete embryos on a callus induction culture medium, culturing for 30-40 days, taking acanthopanax callus, and cutting for later use;

2) preparing an enzymolysis liquid system: 0.5-2% cellulase R-10 and 0.2-1.5% macerase R-10, 0.6mol/L mannitol, 2mol/L KCl, 20mmol/L MES (adjusted to pH 5.7 with KOH), mixing, placing in water bath at 55 deg.C for 10min, cooling to room temperature, adding 10mmol/L CaCl₂0.1% (w/v) BSA, and finally filtering and sterilizing by a 0.45 μm filter membrane to obtain an enzymolysis solution;

3) putting the cut callus obtained in the step 1) into the enzymolysis liquid obtained in the step 2), and carrying out enzymolysis for 1-12h in the dark at room temperature by using a horizontal shaking table at 50rpm to digest cell walls;

4) adding a W5 solution precooled on ice in volume equal to that of the enzymolysis solution into the reaction solution obtained in the step 3), uniformly mixing to terminate digestion, and filtering the digested reaction solution into a 50mL sterilized centrifuge tube through a 325-mesh cell sieve wetted by W5;

5) dripping the prepared acanthopanax single cell suspension on a blood counting chamber to fill the blood counting chamber, observing and counting under a microscope, measuring the yield of the blood counting chamber, and detecting the single cell activity of the prepared acanthopanax single cell suspension by adopting a Fluorescein Diacetate (FDA) staining method;

6) centrifuging at 200 ℃ and 1000 Xg for 5min at 4 ℃ to precipitate single cells, and removing supernatant;

7) carefully adding the W5 solution precooled on ice along the wall of the centrifugal tube by using a pipette, and gently resuspending the acanthopanax single cells;

as shown in Table 1, the analysis revealed that when single cells were extracted from the callus of Acanthopanax senticosus, the single cell yield was the greatest at 1.5% cellulase R-10, 0.2% macerase R-10, 6h for enzymatic hydrolysis, 400 Xg for centrifugation, which was the optimal production condition.

TABLE 1 Effect of different combinations of enzymatic hydrolysates on the yield and viability of Acanthopanax senticosus single cells

Example 2: PEG mediated transient expression and transformation of acanthopanax senticosus single cell

1) Preparing 20-50% PEG solution, and water bathing at 50-60 deg.C until PEG solid is completely dissolved;

2) centrifuging the purified single cell suspension at 4 deg.C for 2min at 200 Xg, and removing supernatant;

3) the W5 solution was added to dilute the cell concentration of the single cell solution to 2X 10⁵Placing the seeds/mL on ice for sedimentation for 30-60 min;

4) w5 was removed as much as possible and the cells were lysed with MMG to a cell concentration of 2X 10⁵Per mL;

5) adding 10 μ L plasmid (about 10-20 μ g) and 100 μ L unicellular-MMG suspension into 2ml centrifuge tube, mixing by gentle inversion, and standing at room temperature for 15 min; wherein the plasmid is an empty vector pNC-Cam1304-SubC with a GFP marker;

6) adding 110 μ L of 20-50% PEG solution; mixing, incubating at room temperature in dark for 10-60 min;

7) adding 440 μ L W5 solution, terminating the conversion reaction, mixing well, centrifuging at 200 × g at 4 deg.C for 2min, and sucking out the supernatant;

8) adding 1mL of WI solution into the precipitate, gently transferring the mixed solution into a porous cell culture plate, culturing for 20 hours at room temperature in a dark place, and observing the gene expression condition under a laser confocal microscope;

as shown in Table 2, the transient transformation efficiency of unicellular Acanthopanax senticosus was 30.34% at 40% PEG concentration and 40min transformation time.

TABLE 2 Effect of different concentrations of PEG and conversion time on conversion efficiency

Claims

1. A method for transient transformation of acanthopanax senticosus cells is characterized by comprising the following steps: after 1.5% cellulase R-10 and 0.2% segregation enzyme R-10 are adopted to carry out enzymolysis on the acanthopanax senticosus callus for 6 hours, single cells with high activity and large yield are obtained.

2. The method of claim 1, wherein: after 40min of agrobacterium transformation single cells at 40% PEG concentration, the transient transformation efficiency was highest.