阅读说明：本技术 一种长柄类大型真菌的超大原生质体及其制备方法 (Oversized protoplast of macrofungi with long petioles and preparation method thereof ) 是由 严俊杰 韩星 甘炳成 苗人云 冯仁才 赵金 林俊彬 甘颖 于 2021-09-08 设计创作，主要内容包括：本发明公开了一种长柄类大型真菌的超大原生质体及其制备方法,属于原生质体制备技术领域,该制备方法主要包括以下步骤：首先将长柄类大型真菌子实体样品横切取下菌柄区段,然后将菌柄区段纵切得到若干细条型的样品组织块；再将所得的样品组织块置于装有酶解液的装置中涡旋振荡1～3分钟后,然后将装置置于恒温摇床中,于25～30℃温度下酶解2～4小时；最后将样品进行过滤得到滤液,并将所得的滤液进行离心去除酶解液,得到长柄类大型真菌的超大原生质体。与现有常规方法相比,本发明获得的原生质体直径可达20～50μm,是常规方法制备的原生质体的4～10倍,有效解决了现有技术中存在原生质体直径小和显微操作困难的问题。(The invention discloses a super large protoplast of a macrofungi with long handle and a preparation method thereof, belonging to the technical field of protoplast preparation, wherein the preparation method mainly comprises the following steps: firstly, transversely cutting a stipe section of a fruiting body sample of a long-stalked large fungus, and then longitudinally cutting the stipe section to obtain a plurality of strip-shaped sample tissue blocks; placing the obtained sample tissue block in a device filled with enzymolysis liquid, performing vortex oscillation for 1-3 minutes, then placing the device in a constant-temperature shaking table, and performing enzymolysis for 2-4 hours at the temperature of 25-30 ℃; and finally, filtering the sample to obtain filtrate, and centrifuging the obtained filtrate to remove enzymolysis liquid to obtain the oversized protoplast of the macrofungi belonging to the long stalk class. Compared with the conventional method, the diameter of the obtained protoplast can reach 20-50 μm, which is 4-10 times of the diameter of the protoplast prepared by the conventional method, and the problems of small diameter of the protoplast and difficult micromanipulation in the prior art are effectively solved.)

1. A preparation method of an oversized protoplast of a macrofungi with long handle is characterized by comprising the following steps:

step 1-pretreatment of the sample:

transversely cutting a stipe large-scale fungus fruiting body sample to take down a stipe section, and then longitudinally cutting the stipe section to obtain a plurality of strip-shaped sample tissue blocks;

step 2-enzymolysis of the sample:

placing the sample tissue block obtained in the step 1 in a device filled with enzymolysis liquid, performing vortex oscillation for 1-3 minutes, then placing the device in a constant-temperature shaking table, and performing enzymolysis for 2-4 hours at the temperature of 25-30 ℃;

step 3-filtration and centrifugation of the sample:

and (3) filtering the sample obtained after the enzymolysis in the step (2) to obtain a filtrate, and centrifuging the obtained filtrate to remove enzymolysis liquid to obtain the oversized protoplast of the macrofungi of the pedunculate class.

2. The method for producing oversized protoplasts of a macrophyte belonging to claim 1, wherein the macrophyte belonging to the genus macrophyte is a macrophyte having a long stalk structure, and the long stalk structure is a mature fruiting body of the macrophyte, and the length of the stalk is 6cm or more.

3. The method for preparing oversized protoplasts of a macrofungi of the longstalck type according to claim 1, wherein the diameter of the oversized protoplast is 20-50 μm.

4. The method for preparing oversized protoplasts of macrofungi belonging to the class consisting of longstalck, according to claim 1, wherein the sample of fruiting body of macrofungi belonging to the class consisting of longstalck in step 1 is a fresh fruiting body of macrofungi belonging to the class consisting of robust longstalck, with a length of 6-10 cm.

5. The method for preparing oversized protoplasts of a macrophyte belonging to claim 1, wherein the position traversed in step 1 is 2 to 4cm from the pileus of a fruiting body of a macrophyte belonging to the genus longissimus.

6. The method for preparing oversized protoplasts of a macrofungi of the long-stalk type according to claim 1, wherein the length of the stalk segment in step 1 is 0.15-0.35 cm.

7. The method for preparing oversized protoplasts of macrofungi belonging to claim 1, wherein the number of the sample tissue blocks added to 2% by mass/volume of 1mL of the enzymolysis solution is 4-7 during the enzymolysis of the sample in the step 2.

8. The method for preparing oversized protoplasts of macrofungi belonging to claim 1, wherein the specific operation of filtration in step 3 is as follows: and (3) filtering the sample obtained after enzymolysis in the step (2) through a funnel with sterile lens wiping paper, and washing the lens wiping paper for 2-3 times by using 1-2 mL of sterile 0.6-0.8 mol/L mannitol to obtain filtrate.

9. The method for preparing the oversized protoplast of the macrofungi of the petiolus type according to claim 1, further comprising the specific processes of re-suspending and microscopic examination: and (3) adding 200 mu L of 0.6mol/L mannitol into the oversized protoplast of the macrofungi with the long handle obtained in the step (3) for re-suspending, sucking 10-15 mu L of suspension, and observing the size of the protoplast under an optical microscope.

10. A protoplast produced by the method for producing an oversized protoplast of a macrofungi of the species Flavobacterium as defined in any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of protoplast preparation, in particular to an oversized protoplast of a macrofungi of longstalck species and a preparation method thereof.

Background

The large-scale fungus protoplast is widely applied to the aspects of crossbreeding, gene editing breeding, genetic transformation and the like, and is an important material for the research process of cell biology, molecular biology and genetics. For example, in the micro-operation processes of CRISPR-Cas9 RNP (ribonucleoprotein complex) gene editing technology, nuclear transplantation technology and the like, the size of protoplast is directly related to feasibility and difficulty of experimental development. Fungus protoplasts prepared by the conventional method are usually very small (taking needle mushroom as an example, the diameter of the protoplast is only about 5 microns), and are far smaller than the protoplast of animal cells (the diameter is about 30 microns) and most plants (the diameter is about 50 microns), so that the micromanipulation technology of large fungi such as needle mushroom is difficult to break through.

Disclosure of Invention

Aiming at the defects, the invention aims to provide the oversized protoplast of the macrofungi with long petioles and the preparation method thereof, which can effectively solve the problems of small diameter of the protoplast and difficult micromanipulation in the prior art.

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

the invention provides a preparation method of a super large protoplast of a macrofungi, which comprises the following steps:

step 1-pretreatment of the sample:

transversely cutting a stipe large-scale fungus fruiting body sample to take down a stipe section, and then longitudinally cutting the stipe section to obtain a plurality of strip-shaped sample tissue blocks;

step 2-enzymolysis of the sample:

placing the sample tissue block obtained in the step 1 in a device filled with enzymolysis liquid, performing vortex oscillation for 1-3 minutes, then placing the device in a constant-temperature shaking table, and performing enzymolysis for 2-4 hours at the temperature of 25-30 ℃;

step 3-filtration and centrifugation of the sample:

and (3) filtering the sample obtained after the enzymolysis in the step (2) to obtain a filtrate, and centrifuging the obtained filtrate to remove enzymolysis liquid to obtain the oversized protoplast of the macrofungi of the pedunculate class.

Further, the large fungi of the long stalk type in the present invention is large fungi having a long stalk structure, wherein the long stalk structure means that the length of the stalk of the mature fruiting body of the large fungi is more than 6cm, and the large fungi of the long stalk type specifically includes, but is not limited to, flammulina velutipes, pleurotus eryngii, oudemansiella radicata, collybia albuminosa, coprinus cinereus, hypsizigus marmoreus, pholiota nameko, agrocybe aegerita, and the like.

Furthermore, the diameter of the super large protoplast prepared by the method is 20-50 μm.

Further, the fruiting body sample of the macrofungi of the brachypodium in the step 1 is a fresh and robust fruiting body of the macrofungi of the brachypodium in the elongation stage, and the total length is 6-10 cm, preferably 8 cm.

Furthermore, the transverse position in the step 1 is 2-4 cm away from the pileus of the fruiting body of the macrofungi of the long stalk type, and preferably 3cm away from the pileus of the fruiting body of the macrofungi of the long stalk type.

Further, the length of the stipe segment in the step 1 is 0.15-0.35 cm, preferably 0.20 cm.

Further, the width of the sample tissue block in the step 1 is 1-2 mm, and preferably 1 mm.

Further, the preparation process of the enzymatic hydrolysate is as follows: adding 0.6mol/L mannitol into muramidase for full dissolution to prepare an enzymatic hydrolysate with the mass volume ratio of 1-3%, and filtering and sterilizing the enzymatic hydrolysate to obtain the muramidase; the mass volume ratio of the enzymatic hydrolysate is preferably 2%.

The above-mentioned lywallzyme of the present invention is a reagent commonly used in the art, and can be purchased as it is without any particular limitation.

Further, in the step 2, during the enzymolysis of the sample, 4-7 sample tissue blocks, preferably 5 sample tissue blocks, are added into 2% of enzymolysis liquid per 1 mL.

Further, during the enzymolysis of the sample in the step 2, the rotating speed of the constant temperature shaking table is 140-170 rpm, preferably 150 rpm.

Further, the specific operation process of filtering in step 3 is as follows: and (3) filtering the sample obtained after enzymolysis in the step (2) through a funnel with sterile lens wiping paper, and washing the lens wiping paper for 2-3 times by using 1-2 mL of sterile 0.6mol/L mannitol to obtain filtrate.

Further, the specific operation process of removing the enzymolysis liquid by centrifugation in the step 3 is as follows: and transferring the obtained filtrate into a centrifuge tube, placing the centrifuge tube into a centrifuge after precooling at 10 ℃, setting the centrifugal force to be 3000g, centrifuging for 10-20 minutes, and slowly removing the supernatant by using a pipette gun.

Further, the preparation method of the oversized protoplast suitable for the macrofungi with long petioles further comprises the following steps of resuspension and microscopic examination, and the specific process is as follows: and (3) adding 200 mu L of 0.6mol/L mannitol into the oversized protoplast of the macrofungi with the long handle obtained in the step (3) for re-suspending, sucking 10-15 mu L of suspension, and observing the size of the protoplast under an optical microscope.

The invention also provides a protoplast prepared by the preparation method of the oversized protoplast of the macrophyte of the long-petiole.

In summary, the invention has the following advantages:

1. the diameter of the protoplast of the large fungus with long stalks prepared by the conventional method is small, for example, the diameter of the protoplast of the needle mushroom is about 5 mu m, and the application of the technologies such as micromanipulation and the like is difficult to realize, the diameter of the protoplast obtained by the invention can reach 20-50 mu m, which is 4-10 times of that of the protoplast prepared by the conventional method, the smooth development of the micromanipulation technology can be fully ensured, the application of the modern cell biology technology on edible fungi with long stalks and the like of the needle mushroom is realized, and the basic biology research field of the edible fungi is expanded;

2. in the conventional method, mycelium preparation needs to be carried out in advance, and the time is long (generally, the mycelium needs to be cultured for 2-3 times, and the culture time for each propagation is 5-10 days); the method is especially inexperienced for wild edible fungi which cannot be artificially cultured; the invention directly adopts the stipe part of the sporocarp to prepare the protoplast, saves the time of hypha culture, and can directly carry out the protoplast preparation on edible fungi purchased in the market or collected in the wild and be used for subsequent researches such as cytology and the like.

Drawings

FIG. 1 is a microscopic observation result of Flammulina velutipes super large protoplast prepared by the present invention, wherein the magnification is 400x, and the spherical object shown by the arrow is the protoplast;

FIG. 2 is a microscopic observation result of the oversized protoplast of Pleurotus eryngii prepared by the present invention, wherein the magnification is 400x, and the spherical object shown by the arrow is the protoplast;

FIG. 3 is a microscopic observation result of Flammulina velutipes protoplast prepared by a conventional method, wherein the magnification is 400x, and the spherical objects shown by arrows are protoplasts;

FIG. 4 is a microscopic observation result of a conventional method for preparing Pleurotus eryngii protoplasts, wherein the magnification is 400x, and the spherical objects shown by arrows are protoplasts.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

The embodiment provides a preparation method of an oversized protoplast of a large epiphyte of the long stalk type, taking flammulina velutipes as an example, and the preparation method specifically comprises the following steps:

1. preparation of enzymolysis liquid: 0.02g of lywallzyme (produced by Guangdong institute of microorganisms) is fully dissolved by 1mL of sterile 0.6mol/L mannitol to prepare an enzymolysis liquid with the mass-volume ratio of 2 percent;

2. filtering and sterilizing enzymolysis liquid: sucking the enzymolysis liquid prepared in the step 1 by using a sterile syringe, filtering by using a disposable sterile needle type filter head with the aperture of 0.45nm, and filtering by using a disposable sterile needle type filter head with the aperture of 0.22nm for later use;

3. selection and pretreatment of samples: taking a fresh and strong needle mushroom fruiting body (the total length is 8cm) in an elongation stage, transversely cutting a 0.2cm long stipe section at a position 3cm away from a pileus by using a sterile scalpel, and longitudinally cutting the taken section into a plurality of strip-shaped sample tissue blocks;

4. enzymolysis of the sample: placing the sample tissue blocks taken out in the step 3 into sterile 5mL centrifuge tubes (each centrifuge tube is provided with 5 sections of tissue blocks), and adding 1mL of enzymolysis liquid with the mass volume ratio of 2% obtained in the step 2; vortex and shake for 1 minute to make the sample tissue block fully suspend in 2% enzymolysis liquid; fixing the centrifugal tube on a floating plate, placing the floating plate in a constant-temperature shaking table, and carrying out enzymolysis for 3 hours at 28 ℃ and 150 rpm;

5. and (3) filtering: filtering the sample subjected to enzymolysis in the step 4 into a new 5mL sterile centrifuge tube by using a funnel with sterile lens wiping paper, and flushing the lens wiping paper for 2 times by using 1mL sterile 0.6mol/L mannitol to enable the protoplast remaining on the lens wiping paper to fully flow into the new centrifuge tube;

6. centrifuging to remove enzymolysis liquid: placing the new centrifuge tube filled with the filtrate in a centrifuge after precooling at 10 ℃, setting the centrifugal force to be 3000g, and centrifuging for 10 minutes; slowly removing the supernatant by using a pipette to obtain a protoplast precipitate;

7. resuspending and microscopic examination: 200 μ L of 0.6mol/L mannitol was added to the protoplast pellet obtained in step 6 for resuspension, and 10 μ L of the suspension was aspirated to observe the size of the protoplast under an optical microscope (10 Xobjective and 40 Xobjective), and the results of microscopic examination of the protoplast obtained are shown in FIG. 1.

The diameter of the protoplasts prepared in this example was 42 μm.

Example 2

This example provides a method for preparing an oversized protoplast of a macrofungi belonging to the genus Pectinatus, which is different from example 1 in that: taking Pleurotus eryngii as an example, the other steps and parameters are the same.

The diameter of the protoplasts prepared in this example was 24 μm, and the microscopic examination results are shown in FIG. 2.

Comparative example 1

This example provides a method for preparing protoplasts of edible fungi (i.e., conventional method for preparing protoplasts by hypha), specifically, the method is as described in example 2 of patent No. CN107083337A, and the obtained protoplasts of Flammulina velutipes are observed under a microscope, and the diameter of the obtained protoplasts is about 5 μm (same magnification as example 1, 400 ×) as shown in FIG. 3.

Comparative example 2

This example provides a method for preparing protoplasts of edible fungi (i.e., a conventional method for preparing protoplasts by hypha), specifically, the method is as described in example 2 of patent No. CN107083337A, and the obtained protoplasts of Pleurotus eryngii are observed under a microscope, and the diameter of the obtained protoplasts is about 5 μm (same magnification as example 2, 400X) as shown in FIG. 4.

In conclusion, the invention provides the oversized protoplast of the macrofungi with long stalks and the preparation method thereof, the diameter of the prepared protoplast can reach 20-50 μm, which is 4-10 times of that of the protoplast prepared by the conventional method, the problems of small diameter of the protoplast and difficult micromanipulation in the prior art are effectively solved, the smooth development of the micromanipulation technology can be fully ensured, the application of the modern cell biology technology on the edible fungi with stalks such as flammulina velutipes, pleurotus eryngii and the like is realized, and the basic biology research field of the edible fungi is expanded.

The foregoing is merely exemplary and illustrative of the present invention and it is within the purview of one skilled in the art to modify or supplement the embodiments described or to substitute similar ones without the exercise of inventive faculty, and still fall within the scope of the claims.