CN115354013B - Tobacco protoplast preparation method - Google Patents
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- 241001002356 Valeriana edulis Species 0.000 claims abstract description 17
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 210000001519 tissue Anatomy 0.000 claims description 25
- 229940088598 enzyme Drugs 0.000 claims description 19
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- 230000002255 enzymatic effect Effects 0.000 claims description 14
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 10
- 229930195725 Mannitol Natural products 0.000 claims description 10
- 239000000594 mannitol Substances 0.000 claims description 10
- 235000010355 mannitol Nutrition 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 239000007987 MES buffer Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 6
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- 238000005520 cutting process Methods 0.000 claims description 5
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- 108010029182 Pectin lyase Proteins 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 125000000567 diterpene group Chemical group 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- 239000001814 pectin Substances 0.000 description 1
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- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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Abstract
The application belongs to the technical field of tobacco tissue organ dissection, and particularly relates to a preparation method of tobacco protoplast. The method aims at the preparation and separation application of tobacco root cells or protoplast of tobacco leaf cells, and comprises the following steps: preparing enzymolysis liquid, preprocessing materials, extracting by enzymolysis, separating protoplast and the like. Based on the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco cells, combines the research requirement of metabonomics generated by secondary metabolism, carries out further optimization design on the type and the dosage of biological enzyme required in the process of preparing protoplast by an enzymolysis method, and constructs the protoplast preparation method suitable for tobacco root tip sources or tobacco glandular hair sources. Preliminary experiment results show that the protoplast obtained by the preparation method has a large quantity and high activity, and meanwhile, the related extraction method is simple, quick and short in time consumption, so that the application has good scientific research practical value.
Description
Technical Field
The application belongs to the technical field of tobacco tissue organ dissection, and particularly relates to a preparation method of tobacco protoplast.
Background
Tobacco is used as a special cash crop, is highly relevant to national economy development, and is relatively simple in cultivation management due to relatively short growth cycle, so that the tobacco is a common mode plant in botanic research.
In the plant study, when the study is carried out on various metabolites or cell levels, the preparation and the acquisition of protoplasts are the basis of relevant experiments. Plant protoplasts refer to naked cells surrounded by only the plasma membrane after removal of the cell wall.
In the prior art, the commonly used protoplast preparation and separation methods comprise a mechanical method, a chemical method, an enzymolysis method and the like, wherein the enzymolysis method is a mainstream plant protoplast preparation method at present because of the advantages of safety, effectiveness, relative controllability, rapidity and the like. However, in objective terms, since the differences in the structural composition of different plant cells are large and the research purposes are different, there is no general preparation method for preparing plant protoplasts, and only the optimal protoplast isolation preparation method can be respectively explored according to the differences among different plants, different plant tissues and organs and different cells.
Disclosure of Invention
The application aims at providing a protoplast preparation and separation method suitable for research of tobacco secondary metabolites, thereby laying a certain technical foundation for research of tobacco cell metabonomics.
The technical scheme adopted by the application is described in detail below.
The preparation method of the tobacco protoplast is suitable for the protoplast preparation and separation application aiming at root cells or tobacco leaf (glandular hair) cells in the study of tobacco metabonomics, and specifically comprises the following steps:
firstly, preparing enzymolysis liquid
Taking tobacco root tips or tobacco leaves (glandular hairs) as a protoplast preparation material (raw material) source;
when aiming at tobacco root tip materials, the enzymolysis liquid prepared by the protoplasm is as follows: MES buffer containing cellulase celllast, pectolyase (or Macerozyme); the specific formula comprises the following components:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.0-1.5% pectase (or educase Macerozyme) +0.1% BSA;
when aiming at tobacco leaf (glandular wool) materials, the enzymolysis liquid prepared by the protoplasm is as follows: CPW buffer containing cellulase cellust and Macerozyme; the specific formula comprises the following components:
buffer CPW (pH 5.8) +mannitol Manmitol (pH 5.7), 0.4 M+1% cellulase celllast+0.5% educase Macerozyme;
the buffer CPW (pH 5.8): KH (KH) 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 is, for example, cellulase Onozuka R-10 with the enzyme activity of 10000U/g;
the pectase is, for example, pectase Y-23 with the enzyme activity of 1000U/g;
the isolated enzyme macerozyme is, for example, macerozyme R-10 with an enzyme activity of 3000U/g;
(2) Pretreatment of materials
Taking sterile cultured tobacco or fresh tobacco (glandular hair) as a protoplast preparation material (raw material) source; the pretreatment comprises the following steps:
taking root tip tissue of tobacco which germinates and grows for 7-10 days aiming at root tissue, cleaning with sterile water (not less than 3 times), sucking water by filter paper, and cutting with a blade along the growth direction of the root tip to obtain 0.5cm;
selecting tobacco leaves which are fully stretched in 4-6 leaf periods after tobacco sprouting and growing according to tobacco leaf tissues, placing the tobacco leaves on ice, and rapidly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
such as in particular K326, burley or yellow tobacco;
(3) Enzymatic 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 performing vibration treatment for 1-5 h at 26-30 ℃ (for example, 80rpm vibration treatment for 3 h);
the specific dosage aspect is as follows:
every 100 root tips, the consumption of the enzymolysis liquid is 5mL; the consumption of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the root tip filtrate, retaining the precipitate, and re-suspending the precipitate with MES buffer solution (without lyase) to obtain tobacco root tip protoplast extractive solution;
and centrifuging the filtrate of the tobacco leaves at 500rpm for 5-10 min, and reserving supernatant to obtain tobacco gland hair cell protoplast extract with higher activity and purity.
Based on the research summary of the existing plant cell protoplast extraction method, the inventor combines the structural characteristics of tobacco root tissues and tobacco leaf cells, combines the research requirement of secondary metabolism generation metabonomics (the root is the synthesis part of tobacco alkaloid, and the tobacco glandular hair is the main synthesis part of terpenes, especially diterpenes), carries out further optimization design on the type and the dosage of biological enzyme required in the protoplast preparation process by an enzymolysis method, and constructs the protoplast preparation method suitable for tobacco root tip sources or tobacco glandular hair sources.
After the related extraction process parameters are optimized, the preliminary experimental result shows that the number of the prepared protoplast is large, the activity is high, wherein the number of the protoplast in the tobacco root extract can reach 1.0x10 6 The number of the protoplast in the tobacco gland hair cell leaf extract per ml can reach 5.3 multiplied by 10 5 The activity of the protoplast is higher per ml>85%). Meanwhile, the related extraction method is simple, quick and short in time consumption, so that the application has good scientific research practical value, and a good technical foundation can be laid for related cytology research and metabonomics research.
Drawings
FIG. 1 shows root tip protoplasts obtained from combinations of different enzymes of K326;
FIG. 2 shows root tip protoplasts obtained with different enzyme concentrations of K326;
FIG. 3 shows root tip protoplasts obtained by treatment at different treatment times of K326;
FIG. 4 shows root tip protoplasts obtained after treatment of root tips of different growth times of K326;
FIG. 5 shows the results of activity assays of root protoplasts of different tobacco varieties; in the figure: a: benshiyan; b: k326; under the color view, the color is bright yellow: living cells; blue (blue-green): dead cells or cell debris;
FIG. 6 shows tobacco leaf (glandular hair) protoplasts obtained by treatment at different times of K326;
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 for K326;
FIG. 9 shows tobacco leaf (glandular hair) protoplasts obtained after cleavage of K326 and yellow flower tobacco leaves.
Detailed Description
The present application is further illustrated below with reference to examples. Before describing the specific embodiments, the following description will briefly explain some experimental contexts in the following embodiments.
Experimental materials:
cellulase cellurast: cellulase Onozuka R-10 (enzyme activity 10000U/g), educt enzyme macerozyme: macerozyme R-10 (enzyme activity 3000U/g), pectase pectolyase: pectolylase Y-23 (enzyme activity 1000U/g), all of the products of Yakult Japan company;
tobacco material:
culturing tobacco K326 and yellow flower tobacco in a greenhouse, picking leaves which are fully stretched in 4-6 leaf periods, and preparing gland Mao Yuansheng plastids;
root tissue materials adopt root tissues which germinate (K326, nicotiana benthamiana) under aseptic conditions and grow for 7-10 days;
the experimental method comprises the following steps:
when the number of protoplasts is read by cytometry: firstly, taking 20 mu L of protoplast suspension, and adding 20 mu L of trypan blue for uniformly mixing; then 10. Mu.L of the suspension was added to a cell counting plate, allowed to stand for 1 minute, and counted by an insertion cell counter; cell concentration N (number/mL) was read and cell number was calculated:
cell number = N x cell volume (mL).
Example 1
In this example, tobacco root tissue is taken as an example, and the procedure for preparing the relevant protoplasts is briefly described below.
Firstly, preparing enzymolysis liquid
In combination with the summary of the prior art and the analysis of the tissue structure of the tobacco root (compared with the tissue of the tobacco flake, the degree of fibrosis of the tobacco root is high, and the cellulose content is high), the inventor considers that the enzymolysis of cellulase and pectase is mainly dependent when preparing the root tissue protoplast, but in order to determine the proper biological enzyme dosage proportion, and in combination with the related pre-test result, the inventor performs further screening on a plurality of better biological enzyme liquid combinations, in particular:
enzymatic hydrolysate 1:
MES、5 mM + CaC1 2 10 mM +nacl, 10 mM +mannitol Manmitol (pH=5.7), 0.35M +1.5% cellulase celllast+1.5% educase Macerozyme+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+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+0.1% BSA;
(2) Pretreatment of materials
Taking tobacco root tip tissue, cleaning with sterile water (3 times), sucking water by filter paper, and cutting root tip with length of 0.5cm along the growth direction of root tip;
(3) Enzymatic extraction
Placing the root tip pretreated in the step (2) into a container, respectively adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment at 28 ℃;
the specific dosage aspect is as follows:
every 100 root tips, the consumption of the enzymolysis liquid is 5mL;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the filtrate at 700rpm for 10min, retaining the precipitate, and re-suspending the precipitate with 500 μl of buffer;
the buffer solution is the solution without biological enzyme in the step (I) (namely, the enzymolysis solution in the step (I) does not contain the rest components of cellulase, pectase and educing enzyme; when in resuspension, the corresponding buffer solution is adopted for resuspension, namely, protoplast obtained by the treatment group of the enzymolysis solution 1 adopts the buffer solution without the corresponding biological enzyme in the enzymolysis solution 1);
protoplasts obtained from different treatment groups were counted using a cytometer and activity was detected using a fluorescence microscope. The detection results of protoplasts obtained from different material sources and different enzymolysis parameters are shown in fig. 1-4. Specifically:
when different types of enzymolysis liquid are adopted for treatment (the materials are root tissues growing for 7 days, and the enzymolysis oscillation treatment is carried out for 3 hours), the fact that the enzymolysis liquid 2 obtains the largest number of protoplasts (the treatment group result of the enzymolysis liquid 2 in the figure) can be seen, and the best preparation effect is shown; the reason for this analysis is that: for the tender tobacco root tip tissue, the pectin content is relatively high, so that the combination of cellulase and pectase is more suitable.
In terms of the concentration of biological enzymes in the enzymatic hydrolysate (materials all grow into root tissue for 7 days, and the enzymatic shaking treatment is carried out for 3 hours), it can be seen (fig. 2): the use amount of pectase is properly increased, which is more favorable for dissociating cell walls, thereby obtaining more protoplasts.
As regards the enzymatic treatment time (materials all growing in root tissue for 7 days, treated with enzymatic hydrolysate 2), it can be seen (fig. 3): the enzymolysis treatment time is properly improved, 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, enzymatic hydrolysate 2 is treated for 3 h) it can be seen (fig. 4): root tip tissue grown for 7 days is more favorable for obtaining higher number of protoplasts, and analysis considers that the root tip tissue grown for 10 days has higher fibrosis degree, which is unfavorable for preparing and obtaining protoplasts (or, the relevant enzymolysis dosage proportion needs to be explored and adjusted again).
In addition, under the same lysis conditions (7 tissues are grown, 2h treatment with enzymatic hydrolysate) for different tobacco varieties, the results indicate that: the number of protoplasts was comparable (10 5 Orders of magnitude), but the root protoplasts of nicotiana benthamiana were more active and less fragmented (fig. 5A), while the root protoplasts of K326 were less active and more fragmented (fig. 5B).
Based on the optimized conditions of the experimental results, the inventor further prepares protoplast cells by taking root tip parts of root tissues (K326) growing for 7 days as materials, treats the protoplast cells for 3 hours by adopting enzymolysis liquid 2, and further separates and resuspents the protoplast cells, and the cell count result shows that the number of the protoplasts can reach 1.0x10 6 The cell activity is higher, and the preparation effect is better.
Example 2
In this example, protoplasts were prepared from tobacco leaf material, and the detailed procedure is outlined below.
Firstly, preparing enzymolysis liquid
The enzymolysis liquid is as follows: CPW buffer containing cellust and an educase Macerozyme; the specific formula comprises the following components:
enzymatic hydrolysate 1:
buffer CPW (pH 5.8) +Manmitol (pH 5.7), 0. M +1% celllast+0.5% Macerozyme;
enzymolysis liquid 2
Buffer CPW (pH 5.8) +Manmitol (pH 5.7), 0. M +1% celllast+0.5% Macerozyme;
the buffer CPW (pH 5.8): KH (KH) 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 fully stretched in 4-6 leaf periods after the tobacco germinates and grows, placing the tobacco leaves on ice, and rapidly cutting the tobacco leaves into filaments with the width of about 1mm for later use;
(3) Enzymatic 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 vibrating at 30 ℃ and 50 rpm;
the specific dosage aspect is as follows: the consumption of the enzymolysis liquid is 10mL for each fresh tobacco leaf;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, the mixed solution is filtered by a 250-mesh cell sieve;
centrifuging the filtrate at 500rpm for 10min, and keeping supernatant to obtain tobacco leaf (glandular hair) cell protoplast extractive solution with high activity and purity.
Protoplasts obtained from different treatment groups were counted using a cytometer and activity was detected using a fluorescence microscope. The detection results of protoplasts obtained from different material sources and different enzymolysis parameters are shown in fig. 5-8. Specifically:
as for the enzymolysis treatment time (K326 tobacco leaf, enzymolysis liquid 1 treatment), it can be seen (fig. 6): the enzymolysis treatment time is properly prolonged, so that more protoplasts can be effectively obtained;
regarding the protoplast isolation method (K326 tobacco leaf, enzymolysis solution 1 treatment for 3 h), it can be seen (FIG. 7): different from the separation mode of root tip source protoplast, the number of protoplast in supernatant after centrifugation is more and more complete; analysis suggests that the main cause is: the glandular hair cells on the surface of the tobacco leaves contain a large amount of metabolites such as terpenoid compounds and 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 as the main glandular hair cell protoplasts of the tobacco leaves; in the precipitation, although a certain amount of protoplasts exist, the impurity cells and mesophyll cells are more, so that the influence of factors such as purity and the like is considered, and the method is not suitable for the subsequent metabonomics research and study;
as for mannitol concentration (K326 tobacco leaves, different enzymatic solutions were treated for 3 h), it can be seen (fig. 8): the protoplast prepared by 0.4M mannitol has more complete structure and more quantity, and the result shows that the osmotic pressure under the condition of 0.4M mannitol is more suitable for preparing the protoplast of the tobacco leaves;
as for the tobacco variety distinction (enzymatic hydrolysate 1 treatment for 3 h), it can be seen (fig. 9): the difference between the two is not great, which shows that the method has better universality, but compared with the method, when the K326 tobacco leaves are treated, the prepared protoplast has better dispersity and is more suitable.
Based on the optimized conditions of the experimental results, the inventor further uses K326 tobacco leaves as materials to prepare protoplast cells, adopts enzymolysis liquid 1 to treat for 3 hours, and further separates and resuspents, and the cell count result shows that the number of protoplasts in the supernatant can reach 5.3X10 5 The cell activity is higher, and the preparation effect is better.
Claims (2)
1. The preparation method of the tobacco protoplast is characterized by comprising the following steps:
(1) Preparing enzymolysis liquid
Taking tobacco root tips as a protoplast preparation material source; the tobacco is K326 or Benshi tobacco;
aiming at tobacco root tip materials, the enzymolysis liquid prepared by the protoplasm is as follows: MES buffer solution containing cellulase celllast and pectase; the formula comprises the following components:
MES、5 mM + CaC1 2 10 mM +NaCl, 10 mM +mannitol Manmitol, 0.35M +1.5% cellulase celllast+1.5% pectase+0.1% BSA;
the cellulase is cellulase Onozuka R-10 with the enzyme activity of 10000U/g;
the pectase is pectolyase Y-23 with the enzyme activity of 1000U/g;
(2) Pretreatment of materials
Taking root tip tissue of tobacco which germinates and grows for 7 days aiming at root tissue, and cutting the root tip tissue along the growth direction of the root tip after cleaning with sterile water;
(3) Enzymatic extraction
Placing the root tip pretreated in the step (2) into a container, adding the corresponding enzymolysis liquid prepared in the step (1), and carrying out oscillation treatment for 3 hours at the temperature of 28 ℃;
every 100 root tips, the consumption of the enzymolysis liquid is 5mL;
(4) Protoplast isolation
After the vibration treatment in the step (3) is finished, filtering the mixed solution;
and (3) centrifuging the filtrate of the root tip, reserving the precipitate, and re-suspending the precipitate with a buffer solution to obtain the tobacco root tip protoplast extract.
2. The method for preparing tobacco protoplasts according to claim 1, wherein in the step (4), the centrifugation is: and centrifuging at 500-700 rpm for 5-15 min.
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