Culture medium and method for rapidly preparing verticillium dahliae protoplast

Abstract

The invention discloses a culture medium and a method for rapidly preparing verticillium dahliae protoplast. Relates to the field of agricultural biotechnology. The invention provides specific components of the culture medium, a method for preparing verticillium dahliae protoplast by using the culture medium, and a culture medium combined with the verticillium dahliae protoplast for use. The protoplast preparation system provided by the invention can shorten the successful preparation time of verticillium dahliae protoplast, can obtain a large amount of protoplast, which reaches 5.9 * 10 < 7 > / mL, and can efficiently realize transformation of exogenous genes at low cost. The practicability is high, and the popularization and application value is high.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural biotechnology, and more particularly to a culture medium and a method for rapidly preparing protoplasts of Verticillium dahliae. Background Art

[0002] Verticillium dahlia belongs to the genus Verticillium of the subphylum Ascomycota. Verticillium dahlia has a wide host range and can harm a variety of field crops such as tomatoes, eggplants, potatoes, melons, watermelons, cucumbers, peanuts, kidney beans, mung beans, soybeans, cotton, sesame, etc. Among them, cotton Verticillium wilt caused by Verticillium dahliae has brought serious economic losses to the cotton industry and is one of the important factors restricting the sustainable development of cotton production. Therefore, in-depth research on the pathogenesis of cotton Verticillium wilt and the development of effective prevention and control measures are of great significance to ensure the healthy development of the cotton industry.

[0003] At present, a lot of research work is focused on the functional study of the key genes of virulence and pathogenicity of Verticillium dahliae. Although Agrobacterium-mediated genetic transformation is commonly used for the transformation of exogenous genes, a faster transformation method is still needed. Most laboratories engaged in fungal gene function research use protoplasts for genetic transformation because the protoplast-based transformation procedure is simple to operate and requires less time and equipment. There are also some studies on the genetic transformation of Verticillium dahliae using protoplasts, but it takes a long time from the cultivation of Verticillium dahliae to the successful preparation of protoplasts.

[0004] Therefore, whether a culture medium and method for rapidly preparing protoplasts of Verticillium dahliae can be provided is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a culture medium and method for rapidly preparing protoplasts of Verticillium dahliae, aiming to establish a method for rapidly preparing protoplasts, and to lay a foundation for genetic transformation and functional research of Verticillium dahliae.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A culture medium for rapidly preparing protoplasts of Verticillium dahliae comprises: 10 g / L D-Glucose, 2 g / L Peptone, 1 g / L Yeast Extract, 1 g / L Casamino Acids, 50 mL / L 20×Nitrate Salts, 1 mL / L Vitamin Solution, 1 mL / L Trace Elements, and the balance is water.

[0008] Preferably: when preparing solid culture medium, add 15 g / L agar powder.

[0009] Preferred: 50mL / L of 20×Nitrate Salts including: 120g / L of NaNO 3 , 10.4 g / L KCl, 10.4 g / L MgSO 4. 7H 2 O and 30.4 g / L KH 2 PO 4 , the balance is water.

[0010] Preferably, 1 mL / L Vitamin Solution includes: 0.1 g / L Biotin, 0.1 g / L Pyridoxin, 0.1 g / L Thiamine, 0.1 g / L Riboflavin, 0.1 g / L P-aminobenzoic acid and 0.1 g / L Nicotinic Acid, and the balance is water.

[0011] Preferred: 1mL / L Trace Elements includes: 22g / L ZnSO 4. 7H 2 O, 11g / L H 3 BO 3 , 5g / L MnCl 2. 4H 2 O, 5g / L FeSO 4. 7H 2 O, 1.7 g / L CoCl 2. 6H 2 O, 1.6 g / L CuSO 4. 5H 2 O, 50 g / L Na 4 EDTA and 1.5 g / L Na 2 MnO 4. 2H 2 O, the balance is water.

[0012] The present invention also provides a TB3 medium for use in combination with any of the above-mentioned mediums, the TB3 medium comprising: 3 g of Yeast Extract, 3 g of Casamino Acids, 200 g of Sucrose, and the balance being water;

[0013] If preparing as upper layer TB3 solid medium, add 5g / L agar powder;

[0014] If preparing as the lower layer TB3 solid culture medium, add 7.5 g / L agar powder.

[0015] The present invention also provides a method for preparing protoplasts of Verticillium dahliae using any of the above culture media, comprising the following steps:

[0016] Step 1, inoculating the Verticillium dahliae strain onto the solid culture medium of claim 2, and culturing;

[0017] Step 2, cutting hyphae from the edge of the cultured Verticillium dahliae colony and inoculating them onto the culture medium of claim 1 for further cultivation;

[0018] Step 3, fold the sterile magic filter cloth into a funnel shape, filter the mycelium cultured in step 2 into the magic filter cloth, rinse the mycelium, absorb the water, and squeeze out the excess culture medium to obtain the mycelium;

[0019] Step 4: placing the hyphae obtained in step 3 in a decomposition enzyme solution and an osmotic pressure stabilizer, and performing enzymolysis to obtain the protoplasts of Verticillium dahliae.

[0020] Preferably: the culture in step 1 is carried out at 25° C. for 4 days;

[0021] Continue culturing as described in step 2: 25°C, 180 rpm for 36 h;

[0022] Step three is as follows: fold the sterile magic filter cloth into a funnel shape, filter the hyphae cultured in step two into the magic filter cloth, rinse the hyphae with sterile water to wash away excess conidia, then dry the water with a paper towel, and squeeze out the excess culture medium to obtain the hyphae;

[0023] In step 4, the concentration of the decomposition enzyme solution is 15 mg / mL; the osmotic pressure stabilizer is 0.7 mol / L NaCl; the enzymatic hydrolysis is carried out at 28° C. and 70 rpm for 3 h.

[0024] The present invention also provides the application of the Verticillium dahliae protoplasts prepared by the method in genetic transformation.

[0025] The present invention also provides application of the Verticillium dahliae protoplasts prepared by the method in preventing and controlling cotton diseases.

[0026] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a culture medium and method for rapidly preparing protoplasts of Verticillium dahliae. The technical effect achieved is that the protoplast preparation system provided by the present invention can shorten the time for successfully preparing protoplasts of Verticillium dahliae, and a large number of protoplasts can be obtained, reaching 5.9×10 7 / mL, and can achieve the transformation of exogenous genes at low cost and high efficiency. It is highly practical and has strong promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0028] Figure 1 The attached figure is a colony morphology diagram of Verticillium dahliae after 36 hours of cultivation according to the method provided by the present invention.

[0029] Figure 2 The accompanying drawings are observation diagrams of GFP transfer into the protoplasts of Verticillium dahliae provided by the present invention, wherein A: observation of hyphae into which GFP is transferred into the protoplasts of Verticillium dahliae; B: observation of spores into which GFP is transferred into the protoplasts of Verticillium dahliae. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] The embodiment of the invention discloses a culture medium and a method for rapidly preparing protoplasts of Verticillium dahliae.

[0032] The raw materials not mentioned in the examples are all commercially available, and the steps and processes not mentioned are all conventional processes, for example:

[0033] Verticillium dahliae WX-1 was isolated, purified and preserved by the Plant Disease Biological Control Laboratory of the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. See the literature (Wang Jun. Preliminary study on the interaction between Bacillus subtilis NCD-2 and Verticillium dahliae WX-1 [D]. Hebei Agricultural University)

[0034] Miracloth, used to filter the mycelium of Verticillium dahliae after shaking, was purchased from Sigma-Aldrich.

[0035] Driselase, used to enzymatically degrade the cell wall of Verticillium dahliae to release protoplasts, was purchased from Sigma-Aldrich.

[0036] STC solution: 200 g of Sucrose, 7.88 g of TrisCl, 5.55 g of CaCl 2, add water to fully dissolve it, adjust the pH to 8.0, make up to 1L, place at 121℃ and sterilize for 20min.

[0037] PTC solution: 600 g of PEG4000 was dissolved in 1 L of STC solution, placed at 121°C and sterilized for 20 min.

[0038] Example 1

[0039] A culture medium (CM liquid culture medium) for rapidly preparing protoplasts of Verticillium dahliae comprises: 10 g / L D-Glucose, 2 g / L Peptone, 1 g / L Yeast Extract (Shenggong), 1 g / L Casamino Acids (Sigma-Aldrich), 50 mL / L 20×Nitrate Salts, 1 mL / L Vitamin Solution, 1 mL / L TraceElements, and the balance is water;

[0040] To further optimize the embodiment, if a solid culture medium (CM solid culture medium) is prepared, 15 g / L agar powder (Agar) is added.

[0041] To further optimize the example, 50 mL / L of 20× Nitrate Salts includes: 120 g / L of NaNO 3 , 10.4 g / L KCl, 10.4 g / L MgSO 4. 7H 2 O and 30.4 g / L KH 2 PO 4 , the rest is water. (No need for high pressure sterilization, can be stored in a sealed glass bottle at room temperature for a long time)

[0042] To further optimize the example, 1mL / L Vitamin Solution includes: 0.1g / L Biotin, 0.1g / L Pyridoxin, 0.1g / L Thiamine, 0.1g / L Riboflavin, 0.1g / L P aminobenzoic acid and 0.1g / L Nicotinic Acid, and the balance is water. (No need for high pressure sterilization, can be stored at 4°C for a long time)

[0043] To further optimize the example, 1 mL / L of Trace Elements includes: 22 g / L of ZnSO 4. 7H 2 O, 11g / L H 3 BO 3 , 5g / L MnCl 2. 4H2 O, 5g / L FeSO 4. 7H 2 O, 1.7 g / L CoCl 2. 6H 2 O, 1.6 g / L CuSO 4. 5H 2 O, 50 g / L Na 4 EDTA and 1.5 g / L Na 2 MnO 4. 2H 2 O, the rest is water. (No need for high pressure sterilization, can be stored at 4°C for a long time, keep away from light)

[0044] TB3 liquid culture medium includes: 3g Yeast Extract, 3g Casamino Acids, 200g Sucrose, and the balance is water (sterile water is made up to 1L), and stirred to completely dissolve;

[0045] If the upper layer of TB3 solid medium (upper layer of regeneration medium) is prepared, 5 g / L agar powder (the concentration of agar is 0.5%) is added;

[0046] If the lower layer TB3 solid culture medium (lower layer of regeneration culture medium) is prepared, 7.5 g / L agar powder (the concentration of agar is 0.75%) is added; place at 121° C. and sterilize for 20 min.

[0047] Example 2

[0048] The cultivation of Verticillium dahliae comprises the following steps:

[0049] Step 1: inoculate the Verticillium dahliae strain onto a CM solid culture medium plate and culture at 25°C for 4 days.

[0050] Step 2: Use a sterile scalpel to cut about 50 pieces of 2mm×2mm mycelium from the edge of the 4-day-old Verticillium dahliae colony, inoculate them into 80mL CM liquid culture medium (be careful to prevent contamination) and continue to culture at 25℃ and 180rpm for 36h. The state of the cultured colony is as follows: Figure 1 shown.

[0051] Example 3

[0052] A method for rapidly obtaining mycelium of Verticillium dahliae comprises the following steps:

[0053] Steps 1 and 2 are the same as in Example 2;

[0054] Step 3: Fold the sterile magic filter cloth into a funnel shape on the clean bench, filter the mycelium cultured in step 2 onto the magic filter cloth, rinse the mycelium with sterile water to wash away excess conidia, then dry the water with a paper towel, squeeze out excess culture medium to obtain mycelium for preparing protoplasts.

[0055] Example 4

[0056] The preparation of the protoplasts of Verticillium dahliae comprises the following steps:

[0057] Steps 1, 2 and 3 are the same as those in Example 3;

[0058] Step 4: Place the obtained mycelium in a 50mL sterile centrifuge tube containing 20mL enzyme solution (containing 30mg of lyase and osmotic pressure stabilizer, the osmotic pressure stabilizer is 20mL of 0.7mol / L NaCl solution, note that it should be prepared and used immediately, and sterilized by filtering with a 0.45μm sterile filter), and place it (lying flat) on a 28℃ shaker, 70rpm for 3h of enzymolysis. (A large number of protoplasts can be obtained, reaching 5.9×10 7 pcs / mL)

[0059] Technical effect verification:

[0060] PEG-mediated genetic transformation of protoplasts and screening and identification of transformants

[0061] Steps 1, 2, 3 and 4 are the same as those in Example 4;

[0062] Step 5. Also in the clean bench, fold the sterile magic filter cloth into a funnel shape, gently shake the enzymatic hydrolysate, pour the enzymatic hydrolysate onto the magic filter cloth to leave the mycelium on the magic filter cloth, collect the filtrate in a new 50mL sterile centrifuge tube, and centrifuge at 4°C, 3500rpm for 8min.

[0063] Step 6. Carefully discard the supernatant in the centrifuge tube after centrifugation in an ultra-clean workbench, add 10 mL of STC solution, and use a 1 mL pipette tip with the tip cut off to mix the protoplasts of Verticillium dahliae to fully suspend them, and centrifuge at 4°C and 3500 rpm for 8 minutes.

[0064] Step 7: Add an appropriate amount of STC solution to fully suspend the protoplasts of Verticillium dahliae and examine them under a microscope. Adjust the concentration to 1.0×10 5 / mL, dispense 150μL of protoplasts into 10mL sterile centrifuge tubes, and transform immediately or freeze at -70℃.

[0065] Add 5 μg of GFP plasmid to 150 μL of protoplasts obtained in step 7, mix gently, and let stand at room temperature for 25 min.

[0066] Add 1 mL of PTC, mix gently, and let stand at room temperature for 25 min.

[0067] Add 9 mL of TB3 liquid culture medium (as recovery medium) to the above-mentioned static protoplasts, and place them flat on a shaking incubator at 28°C and 70 rpm for recovery for 2 hours.

[0068] The revived protoplasts and the lower layer TB3 solid culture medium with selected resistance and suitable temperature were mixed to make up to 50 mL, and 10 mL was poured into 9 cm culture dishes respectively. After solidification, 10 mL of the upper layer TB3 solid culture medium with selected resistance and suitable temperature was poured into each culture dish (note that the selection resistance in the upper culture medium is generally twice that of the lower culture medium), blown on the clean bench for 30 minutes, sealed the plate, and cultured at 25°C for 10 days.

[0069] The transformants that broke through the upper TB3 solid medium were picked up with a sterile toothpick to a new medium (CM solid medium) for rapid preparation of protoplasts of Verticillium dahliae. After growing for 3 days, the vegetative mycelium was placed under an upright fluorescence microscope to observe whether it had green fluorescence. A 5mm punch was used to punch a bacterial cake and placed in 1 mL of sterile water. The spore liquid of the transformant was prepared and placed under an upright fluorescence microscope to observe whether it had green fluorescence. The untransformed plasmid was used as a blank control. The observation results of the GFP transformation were as follows: Figure 2 shown.

[0070] The experimental results showed that compared with the control without GFP, green fluorescence was observed in the mycelium and spores of the transformants with GFP plasmid, indicating that GFP can be transiently expressed in protoplasts. This shows that the rapid preparation of protoplasts established in this study can be used for the genetic transformation of Verticillium dahliae, laying a foundation for further research on the functional genes of Verticillium dahliae.

[0071] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0072] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for rapidly preparing protoplasts of Verticillium dahliae, characterized in that: include: 10g / L D-Glucose, 2g / L Peptone, 1g / L Yeast Extract, 1g / L Casamino Acids, 50mL / L 20×Nitrate Salts, 1mL / L Vitamin Solution, 1mL / L Trace Elements, and the balance is water.

2. The culture medium according to claim 1, characterized in that When preparing solid culture medium, add 15 g / L agar powder.

3. The culture medium according to claim 1, characterized in that The 50 mL / L 20×Nitrate Salts includes: 120 g / L NaNO3, 10.4 g / L KCl, 10.4 g / L MgSO 4. 7H2O and 30.4g / L KH2PO4, the balance is water.

4. The culture medium according to claim 1, characterized in that The 1 mL / L Vitamin Solution includes: 0.1 g / L Biotin, 0.1 g / L Pyridoxin, 0.1 g / L Thiamine, 0.1 g / L Riboflavin, 0.1 g / L P-aminobenzoic acid and 0.1 g / L Nicotinic Acid, and the balance is water.

5. The culture medium according to claim 1, characterized in that The 1 mL / L Trace Elements include: 22 g / L ZnSO 4. 7H2O, 11g / L H3BO3, 5g / L MnCl 2. 4H2O, 5g / L FeSO 4. 7H2O, 1.7g / L CoCl 2. 6H2O, 1.6g / L CuSO 4. 5H2O, 50g / L Na4EDTA and 1.5g / L Na2MnO 4. 2H2O, the balance is water.

6. A TB3 medium for use in combination with any one of the mediums of claims 1 to 5, characterized in that: The TB3 culture medium includes: 3g Yeast Extract, 3g Casamino Acids, 200g Sucrose, and the balance is water; If preparing as upper layer TB3 solid medium, add 5g / L agar powder; If preparing as the lower layer TB3 solid culture medium, add 7.5 g / L agar powder.

7. A method for preparing protoplasts of Verticillium dahliae using the culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: Step 1, inoculating the Verticillium dahliae strain onto the solid culture medium of claim 2, and culturing; Step 2, cutting hyphae from the edge of the cultured Verticillium dahliae colony and inoculating them onto the culture medium of claim 1 for further cultivation; Step 3, fold the sterile magic filter cloth into a funnel shape, filter the mycelium cultured in step 2 into the magic filter cloth, rinse the mycelium, absorb the water, and squeeze out the excess culture medium to obtain the mycelium; Step 4: placing the hyphae obtained in step 3 in a decomposition enzyme solution and an osmotic pressure stabilizer, and performing enzymolysis to obtain the protoplasts of Verticillium dahliae.

8. The method according to claim 7, characterized in that Cultivation in step 1: 25°C for 4 days; continued cultivation in step 2: 25°C, 180 rpm for 36 hours; Step three is as follows: fold the sterile magic filter cloth into a funnel shape, filter the hyphae cultured in step two into the magic filter cloth, rinse the hyphae with sterile water to wash away excess conidia, then dry the water with a paper towel, and squeeze out the excess culture medium to obtain the hyphae; In step 4, the concentration of the enzyme solution is 15 mg / mL; the osmotic pressure stabilizer is 0.7 mol / L NaCl; the enzymatic hydrolysis is carried out at 28° C. and 70 rpm for 3 h.

9. Use of the Verticillium dahliae protoplasts prepared by the method of claim 7 or 8 in genetic transformation.

10. Use of the protoplasts of Verticillium dahliae prepared by the method of claim 7 or 8 in preventing and controlling cotton diseases.

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