CN113717923A

CN113717923A - Preparation of hickory protoplast and establishment of transient transformation system

Info

Publication number: CN113717923A
Application number: CN202111124221.6A
Authority: CN
Inventors: 沈锦波; 易芳; 胡帅; 高燕丽; 张启香; 黄坚钦
Original assignee: Zhejiang A&F University ZAFU
Current assignee: Zhejiang A&F University ZAFU
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2021-11-30

Abstract

The invention discloses preparation of hickory protoplast and establishment of an instant transformation system. The method comprises the steps of carrying out enzymolysis on the Carya cathayensis Sarg callus by using a combination of 3.0-3.5% of cellulase R10, 0.7-1.0% of macerase R-10, 0.3-0.5% of Driselase and 40mM MES enzyme, carrying out separation, purification and pretreatment on the obtained protoplast, and carrying out efficient transfer of an exogenous gene into the Carya cathayensis Sarg protoplast by using PEG-Ca2+ for transient expression. The invention provides a method for separating, purifying and transiently transforming hickory protoplasts, and further solves the problem of heterologous expression of hickory genes in other plants.

Description

Preparation of hickory protoplast and establishment of transient transformation system

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to preparation of hickory protoplast and establishment of an instant transformation system

Background

Plant protoplasts are plant cells that are enzymatically or mechanically removed from their cell wall to render them viable cells that are only enveloped by a plasma membrane. Since protoplasts contain all genetic material, they are totipotent, and are the basis for molecular, biochemical and physiological research and production of transgenic plants, and promote genetic improvement of various plants. Klercker in 1982 first attempted to mechanically prepare protoplasts from algal plants, but this method was not widely used due to the low efficiency of protoplast production. Since the preparation method of plant protoplast was first published by Cooking in 1960, plant protoplast has been widely used in many fields, and it is usually used as receptor cell to perform transient transformation on target gene, and further perform subcellular localization analysis, cell signal transduction, protein interaction analysis and gene editing, etc., and is a fast and convenient technical means.

The protoplasts can be isolated by mechanical methods and enzymatic methods. Mechanical methods refer to the situation where cells are in a hypertonic solution environment, allowing plasmolysis of the cells, and after the protoplasts contract into spheres, the tissues are ground and the protoplasts are released from the wound. The method is suitable for protoplasts isolated from cells that are highly vacuolated, such as carrot, beetroot tissue, and the like. The obtained complete protoplast has less quantity, but the irreversible damage of the protoplast caused by the enzyme preparation can be avoided, thereby influencing the further experiment. Since the Cooking in 1960 firstly adopts cellulase in fungal culture to dissociate the root tips of tomato seedlings to obtain a large number of protoplasts, almost all the dissociation work of the protoplasts is carried out by an enzymatic hydrolysis method. Commonly used cell wall degrading enzymes are cellulase, hemicellulase, pectinase, etc. According to the method, a large amount of protoplasts can be obtained, but the enzyme preparation contains nuclease, protease, peroxidase and phenolic substances, so that the activity of the protoplasts is influenced to a certain extent. Protoplasts have been successfully isolated from the model plants Arabidopsis thaliana (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.), maize (Zea maysL.), rice (Oryza sativa L.), and other non-model plants switchgrass (Panicum virgatum L.), cabbage (Brassica oleracea L.), Citrus (Citrus reticulata Blanco), cucumber (Cucumis sativus L.).

Linan hickory (Carya cathayensis Sarg.) as a deciduous tree belongs to jugaceae hickory, is a unique woody oil tree species in China and is also one of important economic forests, and the fruit of the Linan hickory is rich in high nutritional value, is mainly produced in Tianmu mountain at the junction of Zhejiang and Wan, and is an important source of economic income of local farmers. Carya illinoinensis, also called thin-shelled hickory nut and pecan, is used as a tall tree and belongs to the family Juglandaceae. It is native to the United states, and has introduction applications in China, France, Mexico, Italy, and other countries. As a perennial woody plant, due to the long fruiting period, the male flower can consume a large amount of nutrition and other physiological characteristics of the tree body during the development, the development of the hickory industry is greatly limited, and the fast breeding, improvement and breeding of the hickory are research hotspots.

At present, a transient transformation system and a protein subcellular localization research system in the hickory are not mature, and a target gene can be expressed in a receptor cell in a short time through a transient transformation technology, so that a quick and effective method is provided for subsequent gene-protein research in the hickory.

Disclosure of Invention

The invention aims to solve the technical problem of heterologous expression of hickory gene in other plants, provides a method for separating, purifying and transiently transforming hickory protoplast, and provides a quick and effective method for subsequent gene-protein research in hickory.

A preparation method of Linan hickory protoplast comprises the following steps:

step (1), selecting yellow, fresh, crisp and tender Linan hickory nut callus with good growth state and grinding;

preferably, the Linan hickory callus is adopted in 7 days after subculture.

Placing the ground Linan hickory nut callus into an enzymolysis liquid, and carrying out enzymolysis for 4.5-5.5 h on a shaking table at 50-65 rpm/min under the condition of keeping out of the sun at 25 ℃;

the enzymolysis solution comprises the following components: cellulase R10 Cellulase R103-3.5% (wt/vol), Macerozyme R10 eductase R100.7-1% (wt/vol), Driselase crashase (From Basidiomycetes sp) 0.3-0.5% (wt/vol), D-mannitol 0.4M, KCl 40mM, MES (pH5.7) 40mM, CaCl ₂10 mM、BSA0.1％(wt/vol)；

Filtering the enzymolysis product, and centrifuging the filtrate at low speed to obtain a precipitate; adding a precooled W5 solution into the precipitate, gently washing the precipitate, stopping enzymolysis, centrifuging at a low speed, taking the precipitate, repeating for 2 times, and removing the enzymolysis liquid;

preferably, the volume ratio of pre-cooled W5 solution to precipitate is 2: 1;

step (4), adding the precooled W5 solution into the precipitate again, washing the precipitate gently, carrying out ice bath for 30min, and then centrifuging at low speed to obtain the precipitate; and (4) carrying out heavy suspension by using an MMG solution to obtain purified protoplasts.

Preferably, the volume ratio of pre-cooled W5 solution to precipitate is 1: 1;

the W5 solution comprises the following components: NaCl 154mM, CaCl₂125 mM、KCl 5mM、MES 2mM；

The MMG solution comprises the following components: 0.4mM of D-mannitol, 215 mM of MgCl and MES4 mM.

It is a second object of the present invention to provide PEG-Ca²⁺The mediated transient transformation method of the Linan hickory protoplast comprises the following steps:

step (1), mixing the exogenous gene DNA (the concentration is 3000ng/ul) to be transformed, 100ul of protoplast which is re-suspended by MMG solution and PEG-Ca2+ solution gently, and keeping the mixture at 25 ℃ in a dark condition for 20-25min to obtain a mixture after incubation and transformation; the mass-volume ratio of the exogenous gene DNA to the protoplast is 5-15 mug: 100 ul; the volume ratio of the PEG-Ca2+ solution to the total amount of the exogenous gene DNA and the protoplast is 1: 1;

the PEG-Ca²⁺The solution contains D-mannitol 0.5M, CaCl₂1 M、PEG4000 40％(wt/vol)；

Step (2), after incubation and transformation, adding an equal volume of W5 solution into the mixture, terminating transformation, and centrifuging at low speed to take a precipitate;

and (3) adding 1000ul of W5 solution into the precipitate, mixing the mixture gently, and culturing the mixture overnight at 25 ℃ in a dark condition.

The third purpose of the invention is to provide a preparation method of the hickory protoplast, which comprises the following steps:

selecting callus of the Carya cathayensis Sarg with good growth state, and cutting the callus with a clean scalpel;

placing the minced pecan callus into an enzymolysis solution, carrying out enzymolysis for 4-4.5 h on a table concentrator at the speed of 65rpm/min under the condition of keeping away from light at 25 ℃;

the enzymolysis solution comprises the following components: cellulase RS 1.0-1.5% (wt/vol), macerozyme R100.7-1.0% (wt/vol), pectinase 0.5-0.8% (wt/vol), D-mannitol 0.7M, KCl 40mM, MES (pH5.7) 40mM, CaCl ₂10 mM、BSA 0.1％(wt/vol)；

Filtering the enzymolysis product, and centrifuging the filtrate at low speed to obtain a precipitate; adding an equal volume of precooled W5 solution into the precipitate, gently washing the precipitate, stopping enzymolysis, and centrifuging at a low speed to obtain the precipitate;

step (4), adding the equal volume of precooled W5 solution into the precipitate again, washing the precipitate gently, carrying out ice bath for 30min, and then centrifuging at low speed to obtain the precipitate; and (4) carrying out heavy suspension by using an MMG solution to obtain purified protoplasts.

The MMG solution comprises the following components: d-mannitol 0.4mM, MgCl215 mM, MES4 mM.

It is a fourth object of the present invention to provide PEG-Ca²⁺The method for mediating the transient transformation of the hickory protoplast comprises the following steps:

step (1), mixing the protoplast and the PEG-Ca2+ solution which are re-suspended by the MMG solution in the exogenous gene plasmid DNA (with the concentration of 4000-; the mass-volume ratio of the exogenous gene DNA to the protoplast is 5-15 mug: 100 ul; the volume ratio of the PEG-Ca2+ solution to the total amount of the exogenous gene DNA and the protoplast is 1: 1;

the PEG-Ca²⁺The solution contains D-mannitol 0.5M, CaCl₂1 M、PEG4000 50％(wt/vol)；

Hydrolyzing the Carya cathayensis Sarg callus with a combination of 3-3.5% (wt/vol) Cellulase R10, 0.7-1.0% (wt/vol) Macerozyme R-10, 0.3-0.5% (wt/vol) Driselase (from Basidiomycetes sp) and 40mM MES (PH5.7) enzymes and using 1.0-1.5% (wt/vol) Cellulase RS, 0.7-1.0% (wt/vol) Macerozyme R-10 and 0.5-0.8% (wt/vol) Pectinase to obtain protoplasts, separating, purifying and pretreating the protoplasts, and hydrolyzing the protoplasts with PEG-Ca²⁺A method for transferring exogenous genes into Linan hickory protoplast with high efficiency for transient expression.

The invention provides a method for separating, purifying and instantaneously transforming hickory protoplast, further solves the problem of heterogenous expression of hickory gene in other plants, and provides a quick and effective method for the subsequent gene-protein research in hickory.

Drawings

FIG. 1 is a graph showing the results of the detection of the viability of the protoplasts of Linan hickory using FDA; the Loran hickory protoplast presents green fluorescence after A.FDA dyeing; B. the method comprises the following steps of (1) shooting Linan hickory protoplast under a laser confocal bright field condition; c, superposing the A image and the B image; a scale: 100 μm.

FIG. 2 shows the effect of PEG4000 content on transient expression efficiency of Linan hickory protoplast.

FIG. 3 shows the effect of different plasmid concentrations on transient expression efficiency of Linan hickory protoplast.

FIG. 4 is the subcellular localization of transient expression of unloaded GFP in Linan hickory protoplasts; wherein the GFP expression region in the Linan hickory protoplast observed under the A.488nm exciting light shows green fluorescence; B. the method comprises the following steps of (1) carrying out laser confocal shooting on a Linan hickory protoplast under a bright field condition; c, superposing the A image and the B image; a scale: 10 μm.

FIG. 5 is a graph showing the results of the detection of the viability of the protoplasts of Carya cathayensis Sarg using FDA; wherein the American hickory protoplast presents green fluorescence after being dyed by A.FDA; B. the method comprises the following steps of (1) shooting Linan hickory protoplast under a laser confocal bright field condition; c, superposing the A image and the B image; a scale: 100 μm.

FIG. 6 is the effect of PEG4000 content on the transient expression efficiency of Carya cathayensis protoplasts.

FIG. 7 is a graph of the effect of different plasmid concentrations on the transient expression efficiency of Carya cathayensis protoplasts.

FIG. 8 is the subcellular localization of transient expression of unloaded GFP in American hickory protoplasts; wherein the GFP expression region in the Carya cathayensis protoplast observed under the excitation light of A.488nm shows green fluorescence; B. the method comprises the following steps of (1) carrying out laser confocal shooting on an American hickory protoplast under a bright field condition; c, superposing the A image and the B image; a scale: 10 μm.

Detailed Description

The present invention is further analyzed with reference to the following specific examples.

Example 1: linan hickory instantaneous transformation system

1. The preparation method of the Linan hickory protoplast comprises the following steps:

1) selecting yellow fresh crisp and tender Lin' an hickory callus (preferably in 7d after subculture), and grinding lightly with a grinding pestle;

2) placing the ground Linan hickory nut callus into an enzymolysis solution prepared in advance, and carrying out enzymolysis for 4-4.5 h on a shaking table at 50-65 rpm/min under the condition of keeping out of the sun at 25 ℃;

wherein, the enzymolysis solution comprises the following components:

TABLE 1 enzymatic hydrolysate

Note: in the preparation process of the enzymolysis liquid, a mixed solution of 40mM MES (pH5.7), 3% (wt/vol) cellulose R10 (3-3.5% all), 0.7% (wt/vol) macrocezyme R10 (0.7-1% all), 0.3% driselase (from Basidiomycetes sp) (0.3-0.5% all), 0.4M mannitol and 40mM KCl is dissolved, and is placed in a water bath kettle at 55 ℃ for heating for 10min, so that the enzyme activity is favorably improved, and the DNase and the protease are inactivated. After cooling to room temperature, 10mM CaCl was added₂0.1% BSA, filtered through a 0.22 μm filter.

3) Filtering the enzymolysis product through a 300-mesh nylon net, collecting filtrate in a round-bottom centrifuge tube, and centrifuging at a low speed of 800rpm/2 min;

4) removing supernatant with a fine plastic pasteur pipette, adding precooled W5 solution with twice volume of precipitate, washing the precipitate gently, terminating enzymolysis, centrifuging at low speed of 800rpm/1min, repeating the step for 2 times, and removing the enzymolysis solution;

5) removing the supernatant with a fine plastic Pasteur pipette, adding precooled W5 solution with the same volume as the precipitate, gently washing the precipitate, carrying out ice bath for 30min, and then carrying out low-speed centrifugation at 800rpm/1 min;

6) the supernatant was removed with a fine plastic pasteur pipette and resuspended in MMG solution to obtain purified protoplasts.

Wherein, the W5 and MMG solution comprises the following components:

TABLE 2W 5 solution

NaCl	154mM
		CaCl₂	125mM
KCl	5mM
		MES	2mM

TABLE 3 MMG solution

D-Mannitol	0.4mM
		MgCl2	15mM
MES	4mM

2.PEG-Ca²⁺The mediated transient transformation expression of the Linan hickory protoplast comprises the following steps:

1) about 10ul plasmid (3000 ng/ul concentration) in 2ml round-bottom EP tube, add 100ul MMG solution heavy suspension protoplast;

2) adding equal volume of 110ul PEG solution, mixing gently, and keeping away from light at 25 deg.C for 20-25 min;

TABLE 4 PEG-Ca²⁺Solutions of

D-Mannitol	0.5M
		CaCl₂	1M
PEG4000	40％

Note: PEG-Ca²⁺The solution needs to be prepared for use.

3) Adding equal volume of 440ul W5 solution, terminating the conversion, mixing, and centrifuging at low speed at 750rpm/2 min;

4) the supernatant was discarded, 1000ul of W5 solution was added, mixed gently and mixed, and cultured overnight at 25 ℃ in the dark.

3. The method is used for obtaining the Linan hickory protoplast by enzymolysis and the PEG-Ca is utilized²⁺The method is a result of transferring GFP into the Linan hickory protoplast with high efficiency for transient expression.

1) The influence of different enzymolysis liquid combinations on the yield and the activity of the linan hickory protoplast is researched:

TABLE 5 influence of different ratios of resolvase on the protoplast activity and yield of Linan hickory

Note: the protoplast yield is calculated as: protoplast yield (per g) ((protoplast concentration): a predetermined amount)

Volume of protoplast suspension)/fresh weight of material used to prepare protoplasts.

Protoplast viability ═ (number of protoplasts unstained by FDA in field ÷ total number of protoplasts in field) × 100%

This result showed that group E7 exhibited higher yields and activity of dissociated Carya illinoensis protoplasts when combined with 3% (wt/vol) CellulaseR-10, 0.7% (wt/vol) Macerozyme R-10, 0.3% (wt/vol) Driselase (from Basidiomycetes sp), and 40mM MES (pH5.7) enzymes.

2) Since protoplasts do not have cell walls, the low external osmotic pressure can cause the rupture and death of the protoplasts, and a certain amount of osmotic pressure stabilizer is required to be added into the enzyme solution in order to ensure the activity of the separated protoplasts and the stability of plasma membranes, and the commonly used osmotic pressure stabilizer comprises Mannitol (D-Mannitol), Glucose (Glucose), Sorbitol (Sorbitol) and the like. The influence of different osmotic pressure (mannitol) concentrations on the activity and yield of the hickory protoplast is explored:

TABLE 6 Effect of osmotic concentration on the Activity and yield of Linan hickory protoplasts

The results show that in the experimental group 1, the yield and activity of the dissociated Lopa hickory protoplast are higher when the concentration of Mannitol (D-Mannitol) is 0.4M.

3) The proper enzymolysis time is the key for obtaining high-quality and high-activity protoplast. Table 7 explores the effect of different enzymatic hydrolysis times on the activity and yield of hickory protoplasts:

TABLE 7 Effect of enzymolysis time on the Activity and yield of Linan Carya cathayensis protoplasts

The result shows that in the experimental group 1, after enzymolysis is carried out for 4.5 hours, the yield and the activity of the juglans mandshurica protoplast are higher.

4) The influence of different PEG4000 contents on the transient expression efficiency of the Linan hickory protoplast is researched:

TABLE 8 influence of PEG4000 content on the transient expression efficiency of Linan hickory protoplasts (final concentration)

FIG. 1 is a diagram showing the results of the FDA detection of the viability of the Linan hickory protoplast. A.FDA dyed Lin' an hickory protoplast showing green fluorescence; B. the method comprises the following steps of (1) shooting Linan hickory protoplast under a laser confocal bright field condition; c.a and B image superposition result scale: 100 μm.

FIG. 2 shows the effect of PEG4000 content on transient expression efficiency of Linan hickory protoplast. The results show that the instantaneous transformation efficiency of the Linan hickory protoplast is the best when the final PEG4000 content is 20%.

5) The influence of different plasmid concentrations on the transient expression efficiency of the Linan hickory protoplast is explored:

TABLE 9 Effect of different plasmid concentrations on the transient expression efficiency of Linan Carya cathayensis protoplasts

FIG. 3 shows the effect of different plasmid concentrations on transient expression efficiency of Linan hickory protoplast. The result shows that the transient transformation efficiency of the Linan hickory protoplast is the highest when the concentration of the plasmid is 3000 ng/mu l.

FIG. 4 subcellular localization of transient expression of unloaded GFP in Linan hickory protoplasts. The GFP expression area in the Linan hickory protoplast observed under A.488nm exciting light shows green fluorescence; B. the method comprises the following steps of (1) carrying out laser confocal shooting on a Linan hickory protoplast under a bright field condition; c.a and B image superposition result scale: 10 μm.

Example 2: american hickory instantaneous transformation system

1. The preparation method of the hickory protoplast comprises the following steps:

1) selecting callus of Carya cathayensis Sarg with good growth state, and cutting with clean scalpel;

2) placing the minced pecan callus into an enzymolysis solution prepared in advance, carrying out enzymolysis for 4-4.5 h on a table concentrator at 65rpm/min under the condition of keeping away from light at 25 ℃;

wherein, the enzymolysis solution contains the following components:

TABLE 9 enzymatic hydrolysate

CellulaseRS	1％
		MacerozymeR10	0.7％
Pectinase	0.7％
		D-Mannitol	0.7M
KCl	40mM
		MES(PH5.7)	40mM
CaCl₂	10mM
		BSA	0.1％

Note: in the preparation process of the enzymolysis liquid, a mixed solution of 40mM MES (pH5.7), 1% (wt/vol) cellulose RS (1-1.5% all), 0.7% (wt/vol) macroczyme R10 (0.7-1.0% all), 0.7% (wt/vol) Pectinase (0.5-0.8% all), 0.7M mannitol and 40mM KCl is firstly dissolved and placed at 55 ℃ for bakingCulturing for 10min in a box, which is favorable for improving enzyme activity and inactivating DNA enzyme and protease. After cooling to room temperature, 10mM CaCl was added₂0.1% BSA, filtered through a 0.22 μm filter.

3) Filtering the enzymolysis product through a 300-mesh nylon net, collecting the filtrate in a round-bottom centrifuge tube, and centrifuging at a low speed of 100g/5 min;

4) removing supernatant with a fine plastic pasteur pipette, adding precooled W5 solution with the same volume as the precipitate, gently washing the precipitate, stopping enzymolysis, and centrifuging at low speed for 100g/2 min;

5) removing the supernatant with a fine plastic Pasteur pipette, adding precooled W5 solution with the same volume as the precipitate, gently washing the precipitate, carrying out ice bath for 30min, and carrying out centrifugation for 100g/2 min;

6) carefully aspirating the supernatant with a fine plastic pasteur pipette, discarding the supernatant, and resuspending with MMG solution to obtain purified protoplasts.

TABLE 10W 5 solution

NaCl	154mM
		CaCl₂	125mM
KCl	5mM
		MES	2mM

TABLE 11 MMG solution

D-Mannitol	0.4mM
		MgCl2	15mM
MES	4mM

1) about 10ul of GFP plasmid (concentration 4000-6000ng/ul) was placed in a 2ml round-bottom EP tube, and 100ul of protoplast resuspended in MMG solution was added;

2) adding equal volume of 110ul PEG solution, mixing gently, and keeping away from light at 25 deg.C for 15 min;

TABLE 12 PEG-Ca²⁺Solutions of

D-Mannitol	0.5M
		CaCl₂	1M
PEG4000	50％

3. The method is used for obtaining the Linan hickory protoplast through enzymolysis, and the PEG-Ca2+ method is used for transferring GFP into the Linan hickory protoplast efficiently for transient expression.

1) The influence of different enzymolysis liquid combinations on the yield and the activity of the protoplast of the Carya cathayensis Hance is researched:

TABLE 13 Effect of different ratios of resolvase on the Activity and yield of hickory protoplasts

Note: the protoplast yield is calculated as: protoplast yield (per g) — (protoplast concentration x volume of protoplast suspension)/fresh weight of material used to prepare the protoplasts.

This result gave group E1, which showed higher yields and activity of dissociated hickory protoplasts when 1% (wt/vol) CellulaseRS, 0.7% (wt/vol) Macerozyme R-10 and 0.7% (wt/vol) Pectinase enzyme combination were used.

2) Since protoplasts do not have cell walls, the low external osmotic pressure can cause the rupture and death of the protoplasts, and a certain amount of osmotic pressure stabilizer is required to be added into the enzyme solution in order to ensure the activity of the separated protoplasts and the stability of plasma membranes, and the commonly used osmotic pressure stabilizer comprises Mannitol (D-Mannitol), Glucose (Glucose), Sorbitol (Sorbitol) and the like. The influence of different osmotic pressure (mannitol) concentrations on the activity and yield of hickory protoplasts in america was explored:

TABLE 14 Effect of osmolality on the Activity and yield of Carya cathayensis protoplasts

The results show that in experimental group 2, the yield and activity of the dissociated hickory protoplast are higher when the concentration of Mannitol (D-Mannitol) is 0.7M.

3) The proper enzymolysis time is the key for obtaining high-quality and high-activity protoplast. The influence of different enzymolysis time on the activity and yield of the hickory protoplast is researched:

TABLE 15 Effect of enzymolysis time on the Activity and yield of hickory protoplasts

The result shows that in the experimental group 1, after enzymolysis is carried out for 4.5 hours, the yield and the activity of the protoplast of the Carya cathayensis nakai are high.

4) The influence of different PEG4000 contents on the transient expression efficiency of the hickory protoplast is researched:

TABLE 16 influence of PEG4000 content on the Activity of hickory protoplasts and the efficiency of transient expression (final concentration)

FIG. 5 is a graph showing the results of the FDA detection of the viability of the protoplasts of Carya cathayensis. A.FDA dyed hickory protoplast showing green fluorescence; B. the method comprises the following steps of (1) shooting Linan hickory protoplast under a laser confocal bright field condition; c.a and B image superposition result scale: 100 μm.

FIG. 6 the effect of PEG4000 content on the transient expression efficiency of Carya cathayensis protoplasts. The results show that the transient transformation efficiency of hickory protoplasts is best when the final concentration of PEG4000 is 25%.

4) The influence of different plasmid concentrations on the transient expression efficiency of the hickory protoplast is explored:

TABLE 17 Effect of different plasmid concentrations on the transient expression efficiency of Carya cathayensis protoplasts

FIG. 7 effect of different plasmid concentrations on transient expression efficiency of Carya cathayensis protoplast. The result shows that the transient transformation efficiency of the hickory protoplast is the highest when the concentration of the plasmid is 3000 ng/mul.

FIG. 8 subcellular localization of transient expression of unloaded GFP in American hickory protoplasts. The GFP-expressing region of the Carya cathayensis Roxb protoplast observed under A.488nm exciting light shows green fluorescence; B. the method comprises the following steps of (1) carrying out laser confocal shooting on an American hickory protoplast under a bright field condition; c.a and B image superposition result scale: 10 μm.

Claims

1. A preparation method of Linan hickory protoplast is characterized by comprising the following steps:

the enzymolysis solution comprises the following components: cellulase R103-3.5% (wt/vol), Macerozyme R100.7-1% (wt/vol), Driselase 0.3-0.5% (wt/vol), D-mannitol 0.4M, KCl 40mM, MES (PH5.7) 40mM, CaCl₂10mM、BSA0.1％(wt/vol)；

2. The method of claim 1, wherein the Linan Carya illinoensis callus is used within 7 days after subculture.

3. The method of claim 1, wherein the volume ratio of the pre-cooled W5 solution to the precipitate in step (3) is 2: 1; and (4) the volume ratio of the precooled W5 solution to the precipitate is 1: 1.

4. The method of claim 1, wherein the W5 solution comprises the following components: NaCl 154mM, CaCl₂125mM, KCl 5mM, MES 2 mM; the MMG solution comprises the following components: d-mannitol 0.4mM, MgCl215 mM, MES4 mM.

5.PEG-Ca²⁺The mediated transient transformation method of the Linan hickory protoplast is characterized by comprising the following steps:

step (1), mixing the exogenous gene DNA to be transformed, 100ul of the hickory protoplast prepared by the method of any one of claims 1 to 4 after being resuspended by MMG solution, and PEG-Ca2+ solution gently, and keeping away from light at 25 ℃ for 20-25min to obtain a mixture after incubation and transformation; the mass-volume ratio of the exogenous gene DNA to the protoplast is 5-15 mug: 100 ul; the volume ratio of the PEG-Ca2+ solution to the total amount of the exogenous gene DNA and the protoplast is 1: 1;

6. The method according to claim 5, wherein the concentration of the foreign gene DNA to be transformed is 3000 ng/ul.

7. The method of claim 5, wherein said PEG-Ca is²⁺The solution comprisesD-mannitol 0.5M, CaCl₂1M、PEG4000 40％(wt/vol)。

8. A preparation method of a Carya cathayensis protoplast is characterized by comprising the following steps:

the enzymolysis solution comprises the following components: cellulase RS 1.0-1.5% (wt/vol), macerozyme R100.7-1.0% (wt/vol), pectinase 0.5-0.8% (wt/vol), D-mannitol 0.7M, KCl 40mM, MES (pH5.7) 40mM, CaCl₂10mM、BSA 0.1％(wt/vol)；

9. The method of claim 8, wherein the W5 solution comprises the following components: NaCl 154mM, CaCl₂125mM, KCl 5mM, MES 2 mM; the MMG solution comprises the following components: d-mannitol 0.4mM, MgCl215 mM, MES4 mM.

10.PEG-Ca²⁺The method for mediating the transient transformation of the hickory protoplast comprises the following steps:

step (1), mixing the exogenous gene plasmid DNA to be transformed, the hickory protoplast prepared by the method of claim 8 or 9 after being resuspended by MMG solution, and PEG-Ca2+ solution gently, and keeping out of the sun at 25 ℃ for 15min to obtain a mixture after incubation and transformation; the mass-volume ratio of the exogenous gene DNA to the protoplast is 5-15 mug: 100 ul; the volume ratio of the PEG-Ca2+ solution to the total amount of the exogenous gene DNA and the protoplast is 1: 1;