CN108315292B

CN108315292B - Method for extracting lycoris plant protoplast

Info

Publication number: CN108315292B
Application number: CN201810378784.XA
Authority: CN
Inventors: 李青竹; 蔡友铭; 张永春; 杨柳燕; 许俊旭; 王桢; 周琳
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2018-04-25
Filing date: 2018-04-25
Publication date: 2021-02-26
Anticipated expiration: 2038-04-25
Also published as: CN108315292A

Abstract

The invention provides a method for extracting lycoris plant protoplast, which comprises the following steps: (1) dark treating Lycoris plant seedling for 10-12 hr, respectively cutting young leaf or bulb; (2) treating the fragment material obtained in the step (1) by using a reagent A, and slowly oscillating on a shaking table to release protoplasts; (3) filtering the protoplast solution obtained in the step (2) by using a 40-50 mu m filter screen, centrifuging the filtrate for 8-10min, washing the precipitate by using a reagent B, repeatedly centrifuging, and finally suspending the protoplast by using a reagent C. The method for extracting the lycoris plant protoplast has the advantages of large quantity of obtained protoplasts and good integrity, can be directly used for detecting the lycoris alkaloid galanthamine, and provides an important material for protoplast fusion and protoplast transformation.

Description

Method for extracting lycoris plant protoplast

Technical Field

The invention relates to the technical field of plant protoplasts, in particular to a method for extracting lycoris plant protoplasts.

Background

Lycoris spp is a bulbous herbaceous plant of Lycoris (Amaryll idaceae), and many Lycoris are ornamental and medicinal flowers specific to China, are rich in alkaloid components specific to various Lycoris plants, and have high medicinal value. Narciscine has antitumor activity, and lycorine can block cell cycle and induce apoptosis of cancer cell line HL-60. Galantamine and lycoramine as acetylcholinesterase inhibitor can affect brain nicotine receptor and inhibit acetylcholinesterase activity, and is one of the first choice drugs for clinical treatment of Alzheimer's disease (early symptoms of senile dementia). To date, wild lycoris resources are still the most important source of medicinal galanthamine, and the plant resources are reduced day by day due to the slow growth of the plant and the predatory development of the resources, so that development and protection of the existing resources by using biotechnology and creation of new germplasm resources by using a genetic transformation method are urgently needed.

The plant protoplast is a cell which is wrapped by a plasma membrane after the cell wall is removed, can obtain a large number of genetically homogeneous cells in a short time, and can directly take foreign plasmids, DNA and the like, thereby becoming a good receptor material for plant genetic transformation and having great significance for the research of gene function; the protoplast contains all genetic information of an individual, can generate important secondary metabolites, and can be separated, extracted and detected, so that a new idea can be provided for metabolite production; the protoplast is also widely applied to somatic cell distant hybridization, can overcome the hybridization obstacle, and is an effective way for germplasm innovation.

The method for separating and purifying the lycoris radiate protoplast and developing and utilizing the lycoris radiate protoplast lay a foundation for the follow-up germplasm utilization and improvement through bioengineering, has wide application prospect, and has no report on the separation and purification method and the application of the lycoris radiate different plant seedling protoplast in domestic and overseas research.

Disclosure of Invention

The invention aims to provide an extraction method of lycoris plant protoplasts, which comprises the following steps:

(1) raw material treatment: performing dark treatment on Lycoris plant seedlings with diameter of 0.5-1cm for 10-12h, cutting the seedlings into leaves and bulbs, and cutting into pieces with width of about 1 mm;

(2) and (3) enzymolysis release of protoplast: injecting a reagent A to treat the fragment material obtained in the step (1), performing enzymolysis in a dark place at the temperature of 22-28 ℃, vibrating for 1-8 hours, sucking and beating the mixed solution after the enzymolysis is finished, and fully releasing protoplasts;

(3) and (3) purifying protoplasts: filtering the protoplast solution obtained in the step (2) by using a 40-50 mu m filter screen, collecting filtrate, and centrifuging for 5-10min at the centrifugal temperature of 4 ℃; washing the precipitate with reagent B, centrifuging for 2-5 times, and suspending the precipitate with reagent C to obtain the protoplast.

The method for measuring the yield of the protoplast prepared in the method for extracting the lycoris plant protoplast comprises the following steps:

gently sucking and beating the protoplast suspended in the reagent C by using a pipette tip with a tip removed to ensure that the protoplast is uniformly distributed; a piece of clean blood counting plate is taken, 8-10 mu l of plasma body fluid is sucked by a pipette to fill the counting area, the counting is repeated for 5 times, and the average value is taken. Calculating the number of protoplast cells per gram (number/g) according to the weight of the lycoris radiate seedling pieces added in the step (1).

Specifically, the extraction method of the lycoris plant protoplast comprises the following steps:

(1) raw material treatment: selecting Lycoris radiata seedling with diameter of 0.5-1cm in leaf stage of Lycoris radiata plant, dark treating for 10-12 hr, cutting the seedling into leaves and bulb, and cutting into strips with width of about 1 mm;

(2) and (3) enzymolysis release of protoplast: injecting a reagent A to treat the fragment material obtained in the step (1), wherein the addition amount of the reagent A is proper to just submerge the fragment small section, performing enzymolysis in a dark place at the temperature of 22-28 ℃, slowly shaking the fragment material on a shaking table for 1-8h, and controlling the rotation speed of the shaking table to be 50-200rpm, and slightly sucking the mixed solution by using a tip-removed pipette tip after the enzymolysis is finished, so as to fully release the protoplast;

(3) and (3) purifying protoplasts: filtering the protoplast solution obtained in step (2) with a 40-50 μm filter screen, collecting the filtrate, centrifuging at 50-200g for 5-10min at 4 deg.C. The precipitate is repeatedly washed and centrifuged 2-5 times with reagent B, and finally the precipitate is centrifuged and the protoplast is suspended with reagent C.

Wherein the components of the reagent A are shown in a table 1, the components of the reagent B are shown in a table 2, and the components of the reagent C are shown in a table 3:

TABLE 1 reagent A Components

TABLE 2 reagent B Components

TABLE 3 reagent C Components

The extraction method of the lycoris plant protoplast provided by the invention uses lycoris plant bulbs and leaves as materials, obtains the protoplast with a large quantity and high purity through enzymolysis and purification, and lays a technical foundation for the analysis of lycoris resource secondary metabolites and germplasm innovation.

Specifically, the method for extracting the lycoris plant protoplast has the following advantages and beneficial effects:

1. the extraction method is simple and easy to operate, and has high repeatability.

2. The yield of the protoplast obtained by using the lycoris longituba leaves of the lycoris plant can reach 3.45 multiplied by 10⁷The content of a secondary metabolite galanthamine is 510 mg/g; the protoplast yield of the lycoris radiata bulb can reach 2.88 multiplied by 10⁷The content of the secondary metabolite galanthamine is 408mg/g, and good receptor materials can be provided for somatic cell hybridization, genetic transformation and secondary metabolite analysis of the lycoris plants.

Drawings

FIG. 1 is a microscopic observation (40-fold magnification) of the protoplast of the Lycoris radiata bulb in example 1.

FIG. 2 is a microscopic observation (40-fold magnification) of the protoplast of Lycoris longiradiata leaf in example 2.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.

Material sources are as follows:

and (3) short-tube lycoris seedling: lycoris radiata germplasm resource garden from Shanghai city academy of agricultural sciences, and is Lycoris radiata

Lycoris longituba seedlings: lycoris radiata germplasm resource garden from Shanghai city agricultural academy of sciences, and is prepared from Lycoris longiradiata (Lycoris longituba)

Cellulase R10 (Cellulase R10): BIOSHARP

Macrozyme R10 (eductase R10): BIOSHARP

Mannitol (Mannitol): BIOSHARP

MES (2-morpholinoethanesulfonic acid): BIOSHARP

2-Hydroxy-1-ethanethiol (thioethylene glycol): gucose (Glucose) by national chemical group chemical agents limited: BIOSHARP

Galantamine standard: chemical reagents of national drug group Co Ltd

The remaining reagents are conventional commercial products.

Example 1 isolation and application of Lycoris radiata bulb protoplast

(1) Selecting Lycoris radiata seedling (1 year old seedling produced by Lycoris radiata cutting propagation) with diameter of 0.5cm, dark treating for 10 hr, cutting off bulb of seedling, weighing 5g bulb, and cutting bulb into strips with width of about 1 mm;

(2) injecting 5ml of reagent A to treat the fragment material obtained in the step (1), carrying out dark enzymolysis, carrying out slow shaking treatment on a shaking table for 8 hours at the temperature of 22 ℃, wherein the rotating speed of the shaking table is 50rpm, and slightly sucking and beating the mixed solution by using a tip-removed pipette tip after the enzymolysis is finished, so as to fully release protoplasts;

the components of the reagent A are shown in Table 4

Table 4 example 1 reagent a ingredients

(3) Filtering the protoplast solution obtained in step (2) with a 50 μm filter screen, collecting the filtrate, centrifuging at 50g for 5min at 4 ℃. The pellet was centrifuged 2 times with 5ml of reagent B and finally the pellet was centrifuged to suspend the protoplasts with 1ml of reagent C.

The components of the reagent B and the reagent C in example 1 are shown in Table 5 and Table 6 respectively

TABLE 5 reagent B Components

TABLE 6 reagent C Components

And (4) gently sucking and beating the protoplast obtained in the step (3) by using a pipette tip with a tip removed to make the protoplast uniformly distributed. A piece of clean blood counting plate was taken, 10. mu.l of plasma fluid was aspirated by a pipette to fill the counting area, and the counting was repeated 5 times to take an average value. Calculating the number of protoplast cells obtained per gram of bulb based on the weight of 5g added in step (1) to be 2.88X 10⁷One per gram.

And (3) analyzing the content of galanthamine in the lycoris radiata bulbs:

taking 1mL of the bulb protoplast of the lycoris radiata prepared above as a sample to be detected, adding 2mL of 70% ethanol, carrying out ultrasonic extraction for 2 times, each time for 28min, then centrifuging at 12000rpm for 10min, taking the supernatant, carrying out nitrogen blowing concentration until the supernatant is nearly dry, redissolving by 1mL of methanol, and filtering by a 0.22 mu m filter membrane.

Accurately weighing appropriate amount of Galanthamine standard (Gal, Galanthamine, C) 0.010g with analytical balance₁₇H₂₁NO₃Molecular weight 287.359), dissolving with methanol and fixing the volume to 100mL to obtain standard stock solution, wherein the concentration of the stock solution is 100 mug/mL; accurately transferring 1mL of standard stock solution into a 100mL volumetric flask, and preparing a mixed standard intermediate solution with the constant volume of 1.0 mu g/mL by using methanol; the standard intermediate solution was removed as needed and diluted with methanol to standard working solutions of 2ng/mL, 6ng/mL, 20ng/mL, 60ng/mL and 100ng/mL, respectively.

Measuring the galanthamine standard solution by using a chromatograph connected with a secondary mass spectrometer in series, drawing a standard curve by using the measured peak area data and the concentration of the galanthamine standard solution to obtain a linear equation, wherein Y is 6.449e⁶X+6.584e⁶Coefficient of correlation R²0.9992. Substituting the mass spectrum peak area of the sample analyte into a linear equation to obtain the concentration C (in ng/ml) of the galanthamine solution, and calculating the galanthamine content M (in ng/g) in the sample according to the volume V (in ml) of the reconstituted methanol, the dilution multiple D and the mass M (in g) of the sample, wherein the calculation formula is as follows: and (3) M-CxV xD/M (wherein M-galantamine content in the sample is ng/g, C-galantamine concentration in the sample is ng/ml, V-reconstituted methanol volume is ml, D-dilution multiple, M-sample mass is g), and finally the galantamine content in the lycoris bulb protoplast is calculated to be 408 mg/g.

Example 2 Long tube lycoris leaf protoplast separation and application

(1) Selecting Lycoris longituba seedlings with diameter of 1.0cm (1 year old seedlings produced by cutting and propagating Lycoris longituba), dark treating for 12h, cutting off seedling leaves, weighing 10g of leaves, and cutting the leaves into pieces with width of about 1 mm;

(2) injecting 10ml of reagent A to treat the fragment material obtained in the step (1), carrying out dark enzymolysis, carrying out slow shaking treatment on a shaking table for 5 hours at the temperature of 28 ℃, wherein the rotating speed of the shaking table is 50rpm, and slightly sucking and beating the mixed solution by using a tip-removed pipette tip after the enzymolysis is finished, so as to fully release protoplasts;

example 2 the composition of reagent A is shown in Table 7

Table 7 example 1 reagent a ingredients

(3) Filtering the protoplast solution obtained in step (2) with a 40 μm filter screen, collecting the filtrate, centrifuging at 60g for 5min at 4 ℃. The pellet was centrifuged 4 times with 10ml of reagent B and finally the pellet was centrifuged to suspend the protoplasts with 1ml of reagent C.

The components of the reagent B and the reagent C in example 2 are shown in Table 8 and Table 9 respectively

TABLE 8 reagent B Components

TABLE 9 reagent C Components

And (4) gently sucking and beating the protoplast obtained in the step (3) by using a pipette tip with a tip removed to make the protoplast uniformly distributed. A piece of clean blood counting plate was taken, 10. mu.l of plasma fluid was aspirated by a pipette to fill the counting area, and the counting was repeated 5 times to take an average value. Calculating the number of protoplast cells per gram of leaf from the weight of 10g added in step (1) to be 3.45X 10⁷One per gram.

Analyzing the content of galanthamine in the longtube lycoris leaves:

taking 1mL leaf protoplast of Lycoris longituba as sample to be tested, adding 2mL 70% ethanol, ultrasonic extracting for 2 times, each time for 28min, then centrifuging at 12000rpm for 10min, taking supernatant, blowing nitrogen to concentrate to near dry, redissolving 1mL methanol, and filtering with 0.22 μm filter membrane.

Accurately weighing a proper amount of Galanthamine standard substance (national drug group chemical reagent, Inc.) 0.010g (Gal, Galanthamine, C17H21NO3, molecular weight 287.359) by using an analytical balance, dissolving by using methanol and fixing the volume to 100mL to obtain a standard stock solution, wherein the concentration of the stock solution is 100 mug/mL; accurately transferring 1mL of standard stock solution into a 100mL volumetric flask, and preparing a mixed standard intermediate solution with the constant volume of 1.0 mu g/mL by using methanol; the standard intermediate solution was removed as needed and diluted with methanol to give standard working solutions of concentrations 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, and 100ng/mL, respectively.

Measuring the galanthamine standard solution by using a chromatograph connected with a secondary mass spectrometer in series, drawing a standard curve by using the measured peak area data and the concentration of the galanthamine standard solution to obtain a linear equation, wherein Y is 7.9e⁶X+8.7e³Coefficient of correlation R²0.9999. Substituting the mass spectrum peak area of the sample analyte into the linear equation obtained in the step to obtain the concentration C (in ng/ml) of the galanthamine solution, and calculating the content M (in ng/g) of the galanthamine in the sample according to the volume V (in ml) of the reconstituted methanol, the dilution factor D and the mass M (in g) of the sample, wherein the calculation formula is as follows: and (3) M-CxV xD/M (wherein M-galantamine content in the sample is ng/g, C-galantamine concentration in the sample is ng/ml, V-reconstituted methanol volume is ml, D-dilution multiple, M-sample mass is g), and finally the galantamine content in the lycoris longituba leaf protoplast is calculated to be 510 mg/g.

Claims

1. A method for extracting lycoris plant protoplast comprises the following steps:

(1) raw material treatment: performing dark treatment on Lycoris plant seedlings with diameter of 0.5-1cm for 10-12h, cutting the seedlings into leaves and bulbs, and cutting into pieces;

(2) and (3) enzymolysis release of protoplast: injecting a reagent A to treat the fragments obtained in the step (1), performing enzymolysis in a dark place at the temperature of 22-28 ℃, performing vibration treatment for 5-8 hours, sucking and beating the mixed solution after the enzymolysis is finished, and fully releasing protoplasts;

(3) and (3) purifying protoplasts: filtering the protoplast solution obtained in the step (2) by using a 40-50 mu m filter screen, collecting filtrate, and centrifuging for 5-10min at the centrifugal temperature of 4 ℃; washing the precipitate with reagent B, centrifuging for 2-5 times, and finally suspending the precipitate with reagent C to obtain protoplast;

wherein the components of the reagent A, the reagent B and the reagent C are shown in the following tables 1, 2 and 3:

TABLE 1 reagent A Components

TABLE 2 reagent B Components

TABLE 3 reagent C Components