CN116286593A

CN116286593A - Preparation method and kit of aquatic plant water vegetable flower leaf protoplast

Info

Publication number: CN116286593A
Application number: CN202310194089.9A
Authority: CN
Inventors: 杨星宇; 张思思; 梁玉婷; 刘淑超; 李娜; 章晓琴; 李苗; 史红文; 于静亚; 康凯丽; 曾宪沂; 王宁迪
Original assignee: Wuhan Institute of Landscape Architectural Design Co Ltd
Current assignee: Wuhan Institute of Landscape Architectural Design Co Ltd
Priority date: 2023-02-28
Filing date: 2023-02-28
Publication date: 2023-06-23

Abstract

The invention provides a preparation method and a kit of a water lily leaf protoplast. The invention uses the leaves of the water hyacinth as the material, and establishes a water hyacinth protoplast preparation system by adjusting an enzymolysis system, a washing system and a purification method. The enzymolysis effect is good when 1% of cellulase, 0.5% of pectase and 0.5% of educing enzyme are adopted in the enzymolysis liquid system, the compounding of MES, sorbitol and KCl can ensure that the osmotic potential of an external medium is the same as that of a cell when the protoplast is dissociated, the activity of the protoplast is maintained, and the osmotic pressure of the protoplast can be well maintained by adopting 26% of sucrose solution; the method can rapidly and efficiently dissociate the protoplast cells of the leaf tissue of the cauliflower, and the number of the separated protoplasts is large, the activity is high, and the background is pure. The result can be directly put on a machine to carry out single-cell sequencing, and provides an important basis for exploring molecular mechanisms of molecular cell biological research and development of the dysmorphism leaves of the broccoli.

Description

Preparation method and kit of aquatic plant water vegetable flower leaf protoplast

Technical Field

The invention belongs to the technical field of plant molecular cell biology, and particularly relates to a preparation method and a kit of aquatic plant water vegetable flower leaf protoplast.

Background

The watercress (Ottelia cordiata) is an annual or perennial herb of the genus psyllium of the family Eupolyphaga, which is grown in a running water channel and has a camptotheca habit. The water hyacinth flower has two special-shaped leaves, namely a submerged leaf and a floating leaf, wherein the submerged leaf is usually in a belt shape and a needle shape, and the floating leaf is usually in a long oval shape. In addition to morphological differences, leaves in two forms have larger differences in terms of cell structure, photosynthetic capacity and the like, and the research on how the same tissues of plants grow into different phenotypes under the same genetic background has important ecological significance to adapt to different environments.

In the past, research on plant biology has generally used traditional sequencing technology, taking whole tissues as samples, and can not reveal the heterogeneity of expression among different types of cells, even if a specific tissue part is obtained by using a micro-cutting technology, other tissue parts can be polluted, and the obtained cell number is highly limited. In order to determine the functional characteristics of specific genes at the cellular level and to mine key factors potentially regulating plant vital activities, single-cell sequencing technology is becoming a new generation of research means, and by dissociating plant tissues into single cells and then sequencing, histology information can be obtained from the single cell level.

However, the first step in performing single cell sequencing requires that single cells be obtained. At present, a mechanical method or an enzymolysis method is generally used for obtaining the protoplast single-cell suspension. The mechanical method has complex operation steps and low efficiency, but the enzymolysis method is used, the cell wall components of different plants are greatly different, each plant has a specific enzymolysis liquid proportion, and the existing method has no universality and is basically blank in application in aquatic plants such as water flowers and the like. Moreover, the single-cell sequencing instrument has higher requirements on the quality and purity of single-cell suspension, and the existing protoplast preparation method is mainly oriented to somatic cell fusion or transgenic technology and has low requirements on the purity of protoplasts. The method for extracting the leaf protoplast of the cauliflower by adopting the prior art has the advantages of less quantity and low activity of leaf protoplast of the cauliflower, and can influence the follow-up experiment to a certain extent. Leaf protoplasts are extracted by a double enzymolysis method, and protoplasts are purified by the sedimentation method, so that plant tissues or cell fragments in protoplast suspension have more impurities, and the protoplasts with low purity can influence the sequencing on an upper machine.

Therefore, a rapid and efficient preparation method of the water lettuce peanut body is urgently needed at present, and the prepared protoplast can be directly used for single-cell sequencing research.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art, and provides a preparation method and a kit of the water plant leaf protoplast, so as to fill the blank of the preparation of the water plant leaf protoplast and provide a powerful tool for the molecular cell biology research of the water plant.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first object of the invention is to provide a preparation method of a water lily leaf protoplast, which comprises the following specific steps:

step S1, taking water rape flowers to sink leaves or float leaves to obtain water rape flower leaves; the water rape flower leaf is a first leaf newly grown from a water rape flower plant;

s2, cutting the water vegetable flower leaves into strips of 0.5-1 mm by using a double-sided blade, completely soaking the water vegetable flower leaves in enzymolysis liquid, and reacting in dark condition to obtain an enzymolysis primary product;

the enzymolysis liquid consists of deionized water and substances with the following concentrations:

step S3, adding a W5 solution into the enzymolysis primary product obtained in the step S2 to terminate the reaction, centrifuging at a low speed, and discarding the supernatant to obtain a precipitate;

the W5 solution consists of deionized water and the following substances in concentration:

s4, adding 26% sucrose solution into the precipitate obtained in the step S3 for purification treatment, centrifuging at bottom speed, and collecting protoplast bands floating on the solution interface;

and S5, adding the protoplast band obtained in the step S4 into a cleaning solution for resuspension to obtain the water cauliflower leaf protoplast for single-cell sequencing.

Further, in step S2, the conditions for the enzymolysis reaction are: the temperature of the enzymolysis reaction is 28-29 ℃, and the enzymolysis reaction is cultured for 4.5-5.5 h on a horizontal shaking table of 45-50 r/min under the dark condition.

Further, in step S3, the pH of the W5 solution is 5.7 to 5.8.

Further, the specific process of the step S3 is that the mixed solution of the enzymolysis primary product obtained in the step S2 and the W5 solution is filtered by a sterile 40m cell sieve to obtain plant tissue fragments, and the plant tissue fragments are centrifuged for 3min at a rotating speed of 100g, and the filtrate is collected.

Further, in step S4, the centrifugation condition is centrifugation at 100g of rotation speed for 2min.

Further, in step S5, the cleaning solution is a solution containing 20mmol MES and 0.4mol sorbitol in each liter of sterilized deionized water.

Further, in step S1, the step of obtaining the water lily leaves includes: selecting submerged leaf blades only growing submerged leaf water vegetable flower plants or floating leaf blades only growing floating leaf water vegetable flower plants, removing leaf stalks, and performing aseptic water cleaning treatment.

The second object of the invention is to provide a kit for preparing kohlrabi leaf protoplast, which comprises the enzymolysis liquid and the W5 solution.

Compared with the prior art, the technical scheme provided by the invention has the beneficial effects that:

(1) The invention provides a preparation method of a water lily leaf protoplast, which uses a newly grown first leaf of a water lily plant as a material, and establishes a water lily leaf protoplast preparation system by adjusting an enzymolysis system, a washing system and a purification method. The observation result shows that the enzymolysis effect is better when the enzymolysis liquid system contains 1 percent of cellulase, 0.5 percent of pectase and 0.5 percent of educing enzyme; the compounding of MES, sorbitol and KCl can ensure that the osmotic potential of an external medium and a cell is the same when the protoplast is released, and maintain the activity of the protoplast; caCl2 and BSA are compounded, so that the integrity of a protoplast membrane can be maintained, the protoplast is prevented from being damaged by proteolytic enzyme in enzymolysis liquid, the osmotic pressure of the protoplast can be well maintained to be stable by adopting 26% sucrose solution, the number of the protoplast is large, and the number of fragment impurities is small. The method can quickly and efficiently dissociate the protoplast cells of the leaf tissue of the cauliflower, and the number of the separated protoplasts is large, the activity is high, and the background is pure. The result can be directly put on a machine to carry out single-cell sequencing, and provides an important basis for exploring molecular mechanisms of molecular cell biological research and development of the dysmorphism leaves of the broccoli.

Drawings

FIG. 1 is a view of a submerged leaf protoplast of the broccoli observed at a 10-fold magnification;

FIG. 2 shows the water leaf protoplasts of the water hyacinth flower observed under a 10-fold field of view;

FIG. 3 shows the submerged leaf protoplasts of the broccoli observed in 40-fold field of view;

FIG. 4 shows the water leaf protoplasts of the water hyacinth flower observed under 40-fold field of view;

FIG. 5 is a floating purified leaf protoplast;

FIG. 6 shows an interface-purified leaf protoplast;

FIG. 7 is a sedimentation purified leaf protoplast;

FIG. 8 is a graph showing the results of protoplast activity assay in example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The compound drugs used in the practice of the present invention were purchased from the division of biological engineering (Shanghai) and the enzymes used were all from Yault brand in Japan.

The enzyme activity of the cellulase used in the invention is more than 10000U/g, the enzyme activity of the educing enzyme is more than 3000U/g, the enzyme activity of the pectic enzyme is more than 1000U/g, the cellulase is cellulase R-10, the educing enzyme is educing enzyme R-10, and the pectic enzyme is pectic enzyme Y-23.

MES:2- (N-morpholino) ethanesulfonic acid; BSA: serum proteins.

Protoplast counting was performed as follows: 10. Mu.L of the protoplast heavy suspension was aspirated, and the resulting suspension was dropped onto a hemocytometer, and the number of protoplasts was measured by the hemocytometer, whereby the total number of protoplasts (number/g) = (total number of protoplasts in 5 squares/80X 400X 10) ⁴ X dilution factor)/total mass of leaf (g).

Protoplast activity assay: detecting the activity of cells by using Fluorescein Diacetate (FDA), if the cell membrane is complete, enabling protoplast to emit green fluorescence, if the protoplast is broken, not emitting green fluorescence, uniformly mixing the purified protoplast heavy suspension with 0.01% FDA working solution, standing for 5min at room temperature, then dripping the mixture onto a blood cell counting plate, and observing the staining condition of the cells by using a fluorescence confocal microscope, wherein the activity counting formula is as follows: (fluorogenic protoplasts/total number of protoplasts). Times.100%.

The preparation method of the water rape leaf cell protoplast comprises the following steps:

step S1, obtaining submerged leaves and floating leaves of the water lettuce flowers;

s2, cutting the leaves of the water lettuce flowers into strips with the diameter of about 1mm by using a double-sided blade, and completely soaking the strips in enzymolysis liquid to react in dark condition;

step S3, adding a W5 solution to terminate the reaction, sieving the mixed solution of the primary enzymolysis product obtained in the step S2 and the W5 solution by using a 40 mu m sterile cell filter, collecting a new test tube, centrifuging at a low speed, and discarding the supernatant;

s4, adding the precipitate obtained in the step S3 into 26% sucrose solution for purification treatment, centrifuging at a low speed, and collecting protoplast bands floating on the solution interface;

and S5, adding the protoplast purified in the step S4 into a cleaning solution for resuspension, and observing the cell concentration and activity by using a microscope.

Example 1

The preparation method of cell protoplast in submerged leaf blade of submerged leaf water vegetable flower plant comprises the following steps:

(1) Preparation of materials:

selecting submerged leaf blades which only grow submerged leaf rape flower plants, wherein the blades are the first blades for plant new growth, removing leaf stalks, and performing aseptic water cleaning treatment.

(2) Preparation of consumable:

prepared sterile double-sided blade, culture dish, centrifuge tube (1.5 mL), pasteur pipette, 40m sterile cell sieve, and different sizes of transferLiquid gun, ddH ₂ O, etc.

(3) And (3) preparing an enzymolysis liquid:

the enzymolysis liquid system is: 1% (w/v) cellulase, 0.5% (w/v) educt enzyme, 0.5% (w/v) pectinase, 0.4mol/L sorbitol, 20mmol/L MES, 20mmol/L KCl, 10mmol/L CaCl ₂ 0.1% (w/v) BSA, sterilized ddH as solvent ₂ O。

Mixing cellulase, educing enzyme, pectase, MES, sorbitol and KCl, and water-bathing at 55deg.C for 10min to obtain a mixed solution; cooling the mixed solution to room temperature, and mixing the mixed solution with CaCl ₂ Mixing with BSA to obtain enzymolysis solution. The enzymolysis liquid should be brown and clear, and the enzyme liquid should be prepared at present.

(4) Dissociation of protoplasts:

the prepared leaf was cleaned with sterile water, rapidly cut into approximately 1mm wide strips with a double-sided blade, and fully immersed in a petri dish containing 10mL of enzymatic hydrolysate. The protoplasts were incubated for 5h in the dark at 28℃with shaking at 45r/min, during which time release was observed under a microscope.

(5) Filtering, purifying and collecting protoplast:

firstly adding 5mL of precooled W5 solution to terminate the reaction, gently shaking a culture dish to promote the release of protoplast, filtering plant tissue fragments from the mixed solution of the enzymolysis primary product obtained in the previous step and the W5 solution through a sterile 40 mu m cell sieve, centrifuging for 3min at a rotating speed of 100g, and collecting filtrate into a 1.5mL centrifuge tube; gently removing the supernatant by using a Pasteur pipette and a pipette, adding a proper amount of precooled 26% sucrose solution to resuspend protoplasts, centrifuging for 2min at a rotating speed of 100g, and collecting protoplast bands floating on the solution interface; finally, re-suspending the suspension by a proper amount of pre-cooled cleaning liquid to prepare the protoplast suspension.

The formula of the W5 solution is as follows: 2mmol/L MES, 154mmol/L NaCl, 125mmol/L CaCl ₂ And 5mmol/L KCl, finally adding sterilized ddH ₂ Mixing O uniformly; the pH of the W5 solution was 5.8.

The formula of the cleaning liquid is as follows: 20mmol/L MES, 0.4mol/L sorbitol, and finally sterilized ddH were added ₂ And mixing the O evenly.

(6) Microscopic examination of protoplasts:

the protoplast suspension was taken and placed on a glass slide and examined under a normal optical microscope.

As a result, as shown in FIG. 1, the number of the water lily protoplasts in the visual field was observed under a 10-fold mirror, and the water lily protoplasts were excellent in morphology and complete in structure and without breakage. Counting of submerged leaves by hemocytometer the first leaf protoplast yield was approximately 5.53×10 ⁶ The activity per gram was found to be 88.6%.

As shown in FIG. 5, it can be seen that the floating purified protoplasts were the purest background, the number of protoplasts was large and the debris impurities were small.

As shown in FIG. 8, it was found that the number of fluorescence-emitting protoplasts was objective and the activity quality was high.

Example 2

The preparation method of the cell protoplast in the submerged leaf blade of the floating water vegetable flower plant comprises the following steps:

substantially the same as in example 1, except that: the material is floating leaf blades for only growing floating leaf water vegetable flower plants, and the blades are first blades for new growth of the plants; the temperature of the enzymolysis reaction is 29 ℃, and the enzymolysis reaction is cultured for 5 hours on a horizontal shaking table of 50r/min under the dark condition; the pH of the W5 solution was 5.7.

As a result, as shown in FIG. 1, the number of the water lily protoplasts in the visual field was observed under a 10-fold mirror, and the water lily protoplasts were excellent in morphology and complete in structure and without breakage. Counting of submerged leaves by hemocytometer the first leaf protoplast yield was approximately 5.92 x 10 ⁶ The activity per gram was 89.1% as measured by activity.

Example 3

substantially the same as in example 1, except that: the enzymolysis time is 4.5 hours. Counting of submerged leaves by hemocytometer the first leaf protoplast yield was approximately 5.46×10 ⁶ The activity per gram was 86.7% as measured by activity.

Example 4

substantially the same as in example 1, except that: the enzymolysis time is 5.5 hours. Counting of submerged leaves by hemocytometer the first leaf protoplast yield was approximately 5.46×10 ⁶ The activity per gram was found to be 88.2%.

Comparative example 1

substantially the same as in example 1, except that: the submerged leaf blade of the submerged leaf water vegetable flower plant is a newly grown second leaf. As a result, as shown in FIG. 3, the number of the thin portions in the visual field was found to be sparse under a magnification of 40. Counting of the second leaf protoplast yield of submerged leaves by means of a hemocytometer approximately 2.10X10 ⁶ The activity of the cell wall per gram is 48.1 percent according to activity detection, the number of the cell wall per gram is obviously reduced, and part of protoplast still has cell walls, so that the fragment rate is higher.

Comparative example 2

substantially the same as in example 2, except that: the submerged leaf blade of the submerged leaf water vegetable flower plant is a newly grown second leaf. As a result, as shown in FIG. 4, the number of the thin portions in the visual field was found to be sparse under a magnification of 40. Counting of the second leaf protoplast yield of submerged leaves by means of a hemocytometer approximately 2.56X10 ⁶ The activity of the cell wall per gram is 44.6 percent according to activity detection, the number of the cell wall per gram is obviously reduced, and part of protoplasts still exist on the cell wall, so that the fragment rate is higher.

Comparative example 3

substantially the same as in example 1, except that: the enzymolysis liquid contains 1.5% of cellulase and 0.4% of educing enzyme. Protoplast yield was counted as 3.56X10 by means of a hemocytometer ⁶ The activity of the protoplast per gram is 72.5 percent, and the cell is found to exist in part of the protoplast after the activity detectionA wall.

Comparative example 4

substantially the same as in example 1, except that: firstly, enzymatic hydrolysis is carried out for 3 hours by using enzymatic hydrolysate containing 1% of cellulase, 0.5% of eductive enzyme and 0.5% of pectase; and then the enzymolysis liquid containing 1.2 percent of cellulase and 0.4 percent of eductive enzyme is used for enzymolysis for 2 hours. Protoplast yield was counted as 4.83×10 by means of a hemocytometer ⁶ The activity per gram was found to be 60.9% by activity.

Comparative example 5

substantially the same as in example 1, except that: and the enzymolysis time is 6 hours. Protoplast yield was counted as 4.78X10 by means of a hemocytometer ⁶ The activity per gram was measured to be 74.5%.

Comparative example 6

substantially the same as in example 1, except that: the enzymolysis time is 4 hours. Protoplast yield was counted as 3.56X10 by means of a hemocytometer ⁶ The activity per gram was detected to be 62.7%.

Comparative example 7

substantially the same as in example 1, except that: the primary enzymatic hydrolysate obtained by enzymatic hydrolysis for 5 hours using an enzymatic hydrolysate containing 1% cellulase, 0.5% pectinase and 0.5% educt was purified by an interfacial method using a 13% mannitol solution and a 26% sucrose solution, and the purification was observed by an optical microscope.

As a result, as shown in FIG. 6, the interface method was used to purify protoplasts with a relatively pure background, but with a relatively small number of protoplasts.

Comparative example 8

substantially the same as in example 1, except that: the primary enzymatic hydrolysate obtained by enzymatic hydrolysis of an enzymatic hydrolysate containing 1% cellulase, 0.5% pectinase and 0.5% educt enzyme for 5 hours was purified by sedimentation using a 0.4mol/L sorbitol solution, and the purification was observed by an optical microscope.

As a result, as shown in FIG. 7, the number of protoplasts purified by the sedimentation method was large, but the background impurities were large.

Comparative example 9

substantially the same as in example 1, except that: 25% sucrose solution. Protoplast yield was 5.06X10 by means of a hemocytometer ⁶ The activity of the extract per gram is 69.4 percent according to the activity detection

Comparative example 10

substantially the same as in example 1, except that: 27% sucrose solution. Protoplast yield was counted as 4.97X10 by means of a hemocytometer ⁶ The activity of the extract per gram is 73.8 percent according to activity detection

The preparation method of the water lily leaf protoplast can quickly and efficiently dissociate the protoplast cells of the water lily leaf tissue, and the number of the separated protoplasts is large, the activity is high, and the background is pure. The result can be directly put on a machine to carry out single-cell sequencing, and provides an important basis for exploring molecular mechanisms of molecular cell biological research and development of the dysmorphism leaves of the broccoli.

The embodiments described above and features of the embodiments herein may be combined with each other without conflict.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. The preparation method of the water lettuce leaf protoplast is characterized by comprising the following specific steps:

s1, obtaining a water-taking cauliflower leaf blade by using a water-taking cauliflower submerged leaf blade or a water-floating leaf blade; the water rape flower leaf is a first leaf newly grown from a water rape flower plant;

s3, adding a W5 solution into the enzymolysis primary product obtained in the step S2 to terminate the reaction, centrifuging at a low speed, and discarding the supernatant to obtain a precipitate;

s4, adding 26% sucrose solution into the precipitate obtained in the step S3 for purification treatment, centrifuging at a low speed, and collecting protoplast bands floating on the solution interface;

s5, adding the protoplast band obtained in the step S4 into a cleaning solution for resuspension to obtain the water lily protoplast for single cell sequencing.

2. The preparation method of the enzyme hydrolysis solution according to claim 1, wherein in the step S2, the preparation method comprises the steps of mixing cellulase, eductive enzyme, pectase, MES, sorbitol and KCl, and carrying out water bath at 55 ℃ for 10min to obtain a mixed solution; cooling the mixed solution to room temperature, and mixing the mixed solution with CaCl ₂ Mixing with BSA to obtain the final product.

3. The method according to claim 2, wherein in step S2, the conditions for the enzymolysis reaction are: the temperature of the enzymolysis reaction is 28-29 ℃, and the enzymolysis reaction is cultured for 4.5-5.5 h on a horizontal shaking table of 45-50 r/min under the dark condition.

4. The method according to claim 3, wherein in step S3, the pH of the W5 solution is 5.7 to 5.8.

5. The method according to claim 4, wherein the step S3 is performed by filtering the mixture of the primary product of enzymolysis obtained in the step S2 and the W5 solution through a sterile 40 μm cell sieve, and centrifuging at 100g for 3min.

6. The method according to claim 5, wherein in step S4, the centrifugation is performed at 100g for 2min.

7. The method of claim 6, wherein in step S5, the cleaning solution is a solution containing 20mmol MES and 0.4mol sorbitol per liter of sterilized deionized water.

8. The method of claim 7, wherein the step of obtaining the water lettuce leaves in step S1 comprises: selecting submerged leaf blades only growing submerged leaf water vegetable flower plants or floating leaf blades only growing floating leaf water vegetable flower plants, removing leaf stalks, and performing aseptic water cleaning treatment.

9. A kit for preparing a kohlrabi leaf protoplast, comprising the enzymatic hydrolysate of claim 1 and a W5 solution.