CN115354013A - Preparation method of tobacco protoplast - Google Patents
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Abstract
The application belongs to the technical field of tobacco tissue and organ dissection, and particularly relates to a preparation method of a tobacco protoplast. The method aims at the preparation and separation application of protoplasts of tobacco root cells or tobacco leaf cells, and comprises the following steps: preparing enzymolysis liquid, pretreating materials, extracting by enzymolysis, separating protoplast and the like. On the basis of the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco leaves cells and the research requirement of generating metabonomics by secondary metabolism, further optimizes and designs the type and the dosage of biological enzymes required in the process of preparing the protoplast by an enzymolysis method, and constructs the protoplast preparation method suitable for the root tip source of tobacco or the glandular hair source of tobacco. Preliminary experiment results show that the prepared protoplast has a large quantity and high activity, and meanwhile, the related extraction method is simple, quick and short in time consumption, so that the method has good scientific research practical value.
Description
Technical Field
The application belongs to the technical field of tobacco tissue and organ dissection, and particularly relates to a preparation method of a tobacco protoplast.
Background
As a special economic crop, the tobacco is not only highly related to the national economic development, but also is a common model plant in the plant research because the growth period is relatively short and the cultivation and management are relatively simple.
In the research of botany, when research is carried out on various metabolites or cell levels, the preparation and the acquisition of protoplasts are the basis of the development of related tests. Plant protoplasts are naked cells that have been removed from their cell walls and are surrounded by the plasma membrane.
In the prior art, the common methods for preparing and separating the protoplast include a mechanical method, a chemical method, an enzymatic hydrolysis method and the like, wherein the enzymatic hydrolysis method is the mainstream method for preparing the plant protoplast at present due to the advantages of safety, effectiveness, relative controllability, rapidity and the like. However, in an objective aspect, due to the large difference of the cell structures and the different purposes of research, there is no general preparation method for preparing plant protoplasts, and the most suitable method for separating and preparing protoplasts can be respectively explored only according to the differences of different plants, different plant tissues and organs, and different cells.
Disclosure of Invention
The application aims to provide a method for preparing and separating protoplast for researching secondary metabolites of tobacco, thereby laying a certain technical foundation for researching tobacco cell metabonomics.
The technical solution adopted in the present application is detailed as follows.
A tobacco protoplast preparation method is suitable for the protoplast preparation and separation application of root cells or tobacco leaf (glandular hair) cells in the tobacco metabonomics research, and specifically comprises the following steps:
preparing enzymolysis liquid
Taking tobacco root tips or tobacco leaves (glandular hairs) as a source of protoplast preparation materials (raw materials);
aiming at the tobacco root tip material, the enzymatic hydrolysate for the protoplasm preparation is as follows: MES buffer containing cellulase celllast, pectase pectolyase (or isolation enzyme Macerozyme); the specific formula comprises:
MES、5 mM + CaC1 2 10 mM + NaCl, 10 mM + mannitol Manmitol (pH = 5.7), 0.35M + 1.5% cellulase cellulose + 1.0 to 1.5% pectinase pectolyase (or eductase macrozyme) + 0.1% BSA;
aiming at tobacco leaf (glandular hair) materials, the enzymatic hydrolysate for the protoplast preparation is as follows: a CPW buffer containing cellulase celllast and an eductase Macerozyme; the specific formula comprises the following components:
buffer CPW (ph 5.8) + mannitol (ph 5.7), 0.4M + 1% cellulase celllast + 0.5% macerase macrozyme;
the buffer CPW (ph 5.8): KH (Perkin Elmer) 2 PO 4 、27.2 mg/L + KNO 3 、101.0 mg/L + CaCl 2 •2H 2 O、 1480.0 mg/L + MgSO 4 •7H 2 O、246.0 mg/L + KI、0.16 mg/L + CuSO 4 •5H 2 O、0.025 mg/L；
The cellulase cellulose is, for example, cellulose Onozuka R-10 with the enzyme activity of 10000U/g;
the pectase pectolyase is pectolyase Y-23 with 1000U/g of enzyme activity;
the macerozyme is, for example, a macrozyme R-10 with the enzyme activity of 3000U/g;
(2) Pretreatment of materials
Taking aseptically cultured tobacco or fresh tobacco leaves (glandular hairs) as a raw material source for preparing protoplasts; during pretreatment:
taking root tip tissue of tobacco which germinates and grows for about 7-10 days, cleaning with sterile water (not less than 3 times), drying with filter paper, and cutting with blade in the growth direction of root tip for 0.5cm;
aiming at the tobacco leaf tissue, selecting the tobacco leaves which are completely stretched in the leaf period of 4-6 after the tobacco germination and growth, placing the tobacco leaves on ice, and quickly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
tobacco, such as K326, nicotiana benthamiana or Nicotiana lutescens;
(3) Enzymolysis extraction
Placing the root tip or the tobacco leaf filament pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment at 26-30 ℃ for 1-5h (for example, oscillation treatment at 80rpm for 3 h);
the specific dosage aspect is as follows:
the dosage of the enzymolysis liquid is 5mL per 100 root tips; the dosage of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the oscillation treatment in the step (3) is finished, filtering the mixed solution by a 250-mesh cell sieve;
centrifuging the filtrate of the root tip, retaining the precipitate, and re-suspending the precipitate with MES buffer solution (without lyase) to obtain tobacco root tip protoplast extract;
and (3) centrifuging the filtrate of the tobacco leaves at 500rpm for 5-10min, and reserving the supernatant to obtain the tobacco glandular hair cell protoplast extracting solution with higher activity and purity.
On the basis of the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco leaves cells and the research requirement of generating metabonomics by secondary metabolism (the root is the synthetic part of tobacco alkaloid, and the tobacco gland is the main synthetic part of terpenes, especially diterpenes), further optimizes and designs the type and the dosage of the biological enzyme required in the process of preparing the protoplast by an enzymatic hydrolysis method, and constructs the protoplast preparation method suitable for the root tip source of the tobacco or the gland source of the tobacco leaves.
After the optimization of relevant extraction process parameters, preliminary experiment results show that the prepared protoplast has a large quantity and high activity, wherein the quantity of the protoplast in the tobacco root extracting solution can reach 1.0 multiplied by 10 6 The number of protoplasts in the tobacco glandular hair cell leaf extract can reach 5.3 multiplied by 10 per ml 5 Per ml, and the viability of the protoplasts preparedHigh sexual activity (>85%). Meanwhile, the related extraction method is simple and rapid, and consumes short time, so that the method has good scientific research practical value, and can lay a good technical foundation for related cytology research and metabonomics research.
Drawings
FIG. 1 shows root tip protoplasts obtained with different combinations of K326 enzymes;
FIG. 2 shows root tip protoplasts obtained with different enzyme concentrations of K326;
FIG. 3 shows the root tip protoplasts obtained by treating K326 at different treatment times;
FIG. 4 shows root tip protoplasts obtained after root tip treatment with K326 at different growth times;
FIG. 5 shows the results of activity detection of root protoplasts of different tobacco varieties; in the figure: a: benshi tobacco; b: k326; bright yellow under color view: a living cell; blue (cyan): dead cells or cell debris;
FIG. 6 shows protoplasts of tobacco leaves (glandular hairs) obtained by K326 treatment at different times;
FIG. 7 shows tobacco leaf (glandular hair) protoplast cells in supernatant and pellet after 3 hours of K326 treatment;
FIG. 8 shows tobacco leaf (glandular hair) protoplasts under different mannitol treatment conditions of K326;
FIG. 9 shows tobacco leaf (glandular hair) protoplasts obtained after lysis of K326 and yellow tobacco leaves.
Detailed Description
The present application is further illustrated by the following examples. Before describing the specific embodiments, a brief description will be given of some experimental background cases in the following embodiments.
Experimental materials:
cellulose: cellula Onozuka R-10 (enzyme activity 10000U/g), macerozyme Macerozyme: macerozyme R-10 (enzyme activity 3000U/g), pectinase pectolyase: pectolyase Y-23 (enzyme activity 1000U/g), all products of Yakult Japan company;
tobacco material:
culturing tobacco K326 and daylily in a greenhouse, picking the leaves which are completely spread in the 4-6 leaf stage, and preparing glandular hair protoplasts;
the root tissue material adopts root tissue which germinates (K326, nicotiana benthamiana) under aseptic condition and grows for 7-10 days;
the experimental method comprises the following steps:
when the number of protoplasts is read by a cell counting method: firstly, taking 20 muL of protoplast suspension, adding 20 muL of trypan blue and uniformly mixing; then adding 10 mu L of suspension into a cell counting plate, standing for 1 minute, and inserting into a cell counter for counting; cell concentration N (counts/mL) was read and cell number was calculated:
cell number = N × cell volume (mL).
Example 1
This example is a tobacco root tissue example, and the preparation of a protoplast of interest is briefly described below.
(I) preparing an enzymatic hydrolysate
In combination with the summary of the prior art and the analysis of the tobacco root tissue structure by the inventor (compared with tobacco flake tissue, the tobacco root has high fibrosis degree and high cellulose content), the inventor considers that the preparation of root tissue protoplast mainly depends on the enzymolysis of cellulase and pectinase, but in order to determine the appropriate dosage ratio of the biological enzyme, and in combination with the relevant preliminary test results, the inventor conducts further screening on a plurality of better biological enzyme liquid combinations, specifically:
enzymolysis liquid 1:
MES、5 mM + CaC1 2 10 mM + NaCl, 10 mM + mannitol Manmitol (pH = 5.7), 0.35M + 1.5% cellulase cellulast + 1.5% macerase macrozyme + 0.1% BSA;
enzymolysis liquid 2:
MES、5 mM + CaC1 2 10 mM + NaCl, 10 mM + mannitol Manmitol (pH = 5.7), 0.35M + 1.5% cellulase celllast + 1.5% pectinase pectolyase + 0.1% BSA;
enzymolysis liquid 3:
MES、5 mM + CaC1 2 10 mM + NaCl, 10 mM + mannitol Manmitol (pH = 5.7), 0.35M + 1.5% cellulase celllast + 1.0% pectinase pectolyase + 0.1% BSA;
(2) Pretreatment of materials
Taking the tissue of the root tip of the tobacco, cleaning the tissue with sterile water (3 times), sucking water by using filter paper, and cutting the root tip with the length of 0.5cm by using a blade along the growth direction of the root tip;
(3) Enzymolysis extraction
Placing the root tips pretreated in the step (2) into a container, respectively adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out vibration treatment at 28 ℃;
the specific dosage aspect is as follows:
the dosage of the enzymolysis liquid is 5mL per 100 root tips;
(4) Protoplast isolation
After the oscillation treatment in the step (3) is finished, filtering the mixed solution by a 250-mesh cell sieve;
centrifuging the filtrate at 700rpm for 10min, retaining the precipitate, and resuspending the precipitate with 500. Mu.L buffer;
the buffer solution is a solution without biological enzymes in the step (I) (i.e. the enzymolysis solution in the step (I) does not contain residual components of cellulase, pectinase and eductase, and during heavy suspension, the corresponding buffer solution is adopted for heavy suspension, i.e. the protoplast obtained by treating the group with the enzymolysis solution 1 adopts the buffer solution without the corresponding biological enzymes in the enzymolysis solution 1);
counting the protoplasts obtained from different treatment groups by using a cell counter, and performing activity detection by using a fluorescence microscope. The detection results of the protoplasts obtained from different material sources and different enzymolysis processing parameters are shown in FIGS. 1 to 4. Specifically, the method comprises the following steps:
when different types of enzymatic hydrolysate are adopted for treatment (the materials are root tissues growing for 7 days, and the enzymatic oscillation treatment is carried out for 3 hours), it can be seen (figure 1) that the number of protoplasts obtained by the enzymatic hydrolysate 2 is the largest (the result of the treatment group of the enzymatic hydrolysate 2 in the figure) and the best preparation effect is shown; the reason for this analysis is: aiming at tender tobacco root tip tissues, the pectin content is relatively high, so the combination of cellulase and pectinase is more suitable.
In terms of the concentration of the biological enzyme in the enzymatic hydrolysate (the materials are root tissues growing for 7 days, and the enzymatic oscillation treatment is carried out for 3 hours), it can be seen that (fig. 2): the use level of the pectinase is properly increased, so that the cell wall can be dissociated more conveniently, and a larger number of protoplasts can be obtained.
In terms of the enzymolysis treatment time (the materials were all root tissues grown for 7 days, treated with the enzymolysis solution 2), it can be seen (fig. 3): the enzymolysis treatment time is properly prolonged, which is beneficial to the separation of the prepared protoplast, thereby facilitating the subsequent research and application.
For the preparation of protoplast material (material is root tissue grown for 7 or 10 days, enzymolysis solution 2 treatment for 3 h), it can be seen (fig. 4): the root tip tissue growing for 7 days is more beneficial to obtaining a higher number of protoplasts, and the analysis shows that the root tip tissue growing for 10 days is not beneficial to preparing and obtaining the protoplasts because of higher fibrosis degree (or the related enzymolysis dosage ratio needs to be searched and adjusted).
In addition, for different tobacco varieties, under the same lysis conditions (7 tissues were grown and the enzymolysis solution was treated for 2 hours), the results show that: the number of protoplasts was comparable (10) 5 Order of magnitude), but the activity of nicotiana benthamiana protoplasts was higher and the number of fragments was less (fig. 5A), whereas K326 protoplasts were less active and the number of fragments was more (fig. 5B).
Combining the optimization conditions of the experimental results, the inventor further takes the root tip part of root tissue (K326) growing for 7 days as a material to prepare protoplast cells, adopts enzymolysis liquid 2 for treatment for 3 hours, and further separates and resuspends the protoplast cells, and the cell counting result shows that the number of the protoplasts can reach 1.0 multiplied by 10 6 The cell has high cell activity per ml, and shows good preparation effect.
Example 2
This example uses tobacco leaf material to prepare protoplasts, and the specific process is briefly described below.
Preparing enzymolysis liquid
The enzymolysis liquid is: CPW buffer containing celllast and the eductase Macerozyme; the specific formula comprises:
enzymatic hydrolysate 1:
buffer CPW (pH 5.8) + Manmitol (pH 5.7), 0.4M + 1% celllast + 0.5% Macerozyme;
enzymolysis liquid 2
Buffer CPW (pH 5.8) + Manmitol (pH 5.7), 0.5M + 1% celllast + 0.5% Macerozyme;
the buffer CPW (ph 5.8): KH (natural Kill) 2 PO 4 、27.2 mg/L + KNO 3 、101.0 mg/L + CaCl 2 •2H 2 O、 1480.0 mg/L + MgSO 4 •7H 2 O、246.0 mg/L + KI、0.16 mg/L + CuSO 4 •5H 2 O、0.025 mg/L。
(2) Pretreatment of materials
Selecting tobacco leaves which are completely stretched in the leaf stages of 4-6 after the tobacco sprouts and grows, placing the tobacco leaves on ice, and quickly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
(3) Enzymolysis extraction
Placing the root tips or tobacco filaments pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment at 30 ℃ and 50 rpm;
the specific dosage aspect is as follows: the dosage of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the oscillation treatment in the step (3) is finished, filtering the mixed solution by a 250-mesh cell sieve;
centrifuging the filtrate at 500rpm for 10min, and retaining the supernatant to obtain the tobacco leaf (glandular hair) cell protoplast extractive solution with high activity and purity.
Counting the protoplasts obtained from different treatment groups by using a cell counter, and performing activity detection by using a fluorescence microscope. The detection results of the protoplast obtained from different material sources and different enzymolysis processing parameters are shown in FIGS. 5 to 8. Specifically, the method comprises the following steps:
as regards the enzymatic treatment time (K326 tobacco leaves, enzymatic hydrolysate 1 treatment), it can be seen (fig. 6): the enzymolysis treatment time is properly prolonged, and a larger number of protoplasts can be effectively obtained;
in the case of the protoplast isolation protocol (K326 tobacco leaves, treatment with enzymatic hydrolysate 1 for 3 h), it can be seen (FIG. 7): different from the separation mode of root tip source protoplast, the number of the protoplast in the supernatant after centrifugation is more and more complete; the analysis considers that the main reason is that: glandular hair cells on the surface of the tobacco leaves contain a large amount of metabolic products such as terpenoids, acyl sugar and the like, and are relatively light, so that the protoplasts after centrifugation are mainly distributed in the supernatant, namely the protoplasts prepared by the supernatant can be considered to be mainly the glandular hair cell protoplasts of the tobacco leaves; in the precipitation, although a certain amount of protoplasts also exist, the impurity cells and mesophyll cells are more, so the influence of factors such as purity is considered, and the method is not suitable for the subsequent metabonomics research;
in terms of mannitol concentration (K326 tobacco leaves, different enzymatic hydrolysate treatment for 3 h), it can be seen (fig. 8): the protoplast prepared by the mannitol with the concentration of 0.4M has more complete structure and more quantity, and the result shows that the osmotic pressure under the mannitol with the concentration of 0.4M is more suitable for preparing the protoplast of the tobacco leaves;
for the tobacco variety differentiation (treatment with enzymatic hydrolysate 1 for 3 h), it can be seen (fig. 9): the difference between the two is not large, which shows that the method has better universality, but compared with the method for treating the K326 tobacco leaves, the prepared protoplast has better dispersity and is more suitable.
Combining the optimized conditions of the experimental results, the inventor further prepares the protoplast cells by taking K326 tobacco leaves as the material, treats the protoplast cells for 3 hours by using the enzymolysis solution, and further separates and resuspends the protoplast cells, and the cell counting result shows that the number of the protoplasts in the supernatant can reach 5.3 multiplied by 10 5 The cell has high activity per ml and shows good preparation effect.
Claims (5)
1. A preparation method of tobacco protoplasts is characterized in that the method is applied to the preparation and separation of protoplasts of tobacco root cells or tobacco leaf cells, and specifically comprises the following steps:
(I) preparing an enzymatic hydrolysate
Taking tobacco root tips or tobacco leaves as a raw material source for preparing protoplasts;
aiming at the tobacco root tip material, the enzymatic hydrolysate for the protoplasm preparation is as follows: MES buffer containing cellulase cellulast, pectinase pectolyase or eductase Macerozyme; the specific formula comprises:
MES、5 mM + CaC1 2 10 mM + NaCl, 10 mM + mannitol, 0.35M + 1.5% cellulase cellulose + 1.0 to 1.5% pectinase pectolyase or Macerozyme + 0.1% BSA;
aiming at the tobacco leaf material, the enzymatic hydrolysate for the original plasmid preparation is as follows: CPW buffer containing celllast and the eductase macrozyme; the specific formula comprises the following components:
buffer CPW + mannitol, 0.4M + 1% celllast + 0.5% macrozyme;
the buffer CPW: KH (Perkin Elmer) 2 PO 4 、27.2 mg/L + KNO 3 、101.0 mg/L + CaCl 2 •2H 2 O、 1480.0 mg/L + MgSO 4 •7H 2 O、246.0 mg/L + KI、0.16 mg/L + CuSO 4 •5H 2 O、0.025 mg/L；
(2) Pretreatment of materials
Taking aseptically cultured tobacco or fresh tobacco leaves as a material source for preparing protoplasts; during pretreatment:
taking root tip tissues of tobacco which germinate and grow for 7-10 days according to the root tissues, and cutting the root tip tissues by a blade along the growth direction of the root tip after the root tip tissues are cleaned by sterile water;
aiming at the tobacco leaf tissue, selecting the tobacco leaves which are completely stretched in the leaf period of 4-6 after the tobacco germination and growth, and cutting the tobacco leaves into filaments on ice for later use;
(3) Enzymolysis extraction
Placing the root tip or the tobacco leaf filament pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out vibration treatment for 1 to 5 hours at the temperature of 26 to 30 ℃;
(4) Protoplast isolation
After the oscillation treatment in the step (3) is finished, filtering the mixed liquid;
centrifuging the filtrate of the root tip, retaining the precipitate, and re-suspending the precipitate to obtain the tobacco root tip protoplast extracting solution;
and (4) centrifuging the filtrate of the tobacco leaves, and reserving the supernatant to obtain the tobacco leaf protoplast extracting solution.
2. The method for preparing tobacco protoplasts according to claim 1, wherein in the step (1), the cellulase cellulose is cellulose Onozuka R-10 having an enzyme activity of 10000U/g;
the pectase pectolyase is pectolyase Y-23 with the enzyme activity of 1000U/g;
the macerozyme R-1 with enzyme activity of 3000U/g.
3. The method for producing tobacco protoplasts according to claim 1, wherein the tobacco is K326 or daylily.
4. The method for preparing tobacco protoplasts according to claim 1, wherein in the step (3), the amount of the enzymolysis solution is 5mL per 100 root tips; the dosage of the enzymolysis liquid is 10mL per fresh tobacco leaf.
5. The method for preparing tobacco protoplasts according to claim 1, wherein in step (4), the centrifugation is performed by: centrifuging at 500-700rpm for 5-15min.
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