阅读说明：本技术 一种里氏木霉rut-c30原生质体遗传转化方法 (Genetic transformation method for trichoderma reesei RUT-C30 protoplast ) 是由 相金悦 康丽芳 陶程程 于 2021-09-17 设计创作，主要内容包括：本发明提供一种里氏木霉RUT-C30遗传转化方法,包括如下步骤：a)将外源质粒与里氏木霉RUT-C30原生质体在聚乙二醇水溶液中混合并进行冰浴10-20min；b)向混合体系中继续加入8-10倍体积的聚乙二醇水溶液,室温下转化培养5-6min,加入STC试剂沉淀转化后的原生质体再悬浮；c)将转化后的原生质体移至木霉筛选培养基中培养5-7天后挑取转化株,再移至木霉琼脂培养基中扩繁,获得转化株孢子悬浮液,将孢子悬浮液接种至木霉种子培养基过夜培养获得转化株菌丝。本发明将pAN7-1潮霉素表达质粒成功转化RUT-C30菌株,转化株阳性率23-41％,成功建立里氏木霉RUT-C30的遗传转化系统。(The invention provides a Trichoderma reesei RUT-C30 genetic transformation method, which comprises the following steps: a) mixing the exogenous plasmid and Trichoderma reesei RUT-C30 protoplast in a polyethylene glycol aqueous solution, and carrying out ice bath for 10-20 min; b) continuously adding 8-10 times of polyethylene glycol aqueous solution into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent, precipitating and transforming the protoplast, and suspending again; c) transferring the transformed protoplast to a trichoderma screening culture medium, culturing for 5-7 days, then selecting a transformed strain, transferring the transformed strain to a trichoderma agar culture medium, carrying out propagation expansion to obtain a transformed strain spore suspension, and inoculating the spore suspension to a trichoderma seed culture medium for overnight culture to obtain transformed strain hypha. The invention successfully transforms the pAN7-1 hygromycin expression plasmid into the RUT-C30 strain, the positive rate of the transformant is 23-41%, and the genetic transformation system of Trichoderma reesei RUT-C30 is successfully established.)

1. A method for genetic transformation of Trichoderma reesei RUT-C30, comprising the following steps:

a) mixing the exogenous plasmid and Trichoderma reesei RUT-C30 protoplast in a polyethylene glycol aqueous solution, and carrying out ice bath for 10-20 min;

b) continuously adding 8-10 times of polyethylene glycol aqueous solution into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent, precipitating and transforming the protoplast, and suspending again;

c) transferring the transformed protoplast to a trichoderma screening culture medium, culturing for 5-7 days, then selecting a transformed strain, transferring the transformed strain to a trichoderma agar culture medium, carrying out propagation expansion to obtain a transformed strain spore suspension, and inoculating the spore suspension to a trichoderma seed culture medium for overnight culture to obtain transformed strain hypha.

2. The genetic transformation method of claim 1, wherein the foreign plasmid in step (a) comprises any plasmid containing DNA that can be transformed into protoplasts; the polyethylene glycol aqueous solution comprises the following components: 250-300g/L linear and/or multi-arm polyethylene glycol, 40-60mL of 1M CaCl₂5-15mL of 1M Tris.

3. The genetic transformation method as claimed in claim 2, wherein the molecular weight of the linear and/or multi-armed polyethylene glycol is 4000-6000, the multi-armed polyethylene glycol comprises one of two-armed polyethylene glycol, three-armed polyethylene glycol and four-armed polyethylene glycol,

the structural formula of the linear polyethylene glycol is as follows:

m is an integer between 90 and 136;

the double-arm polyethylene glycol has the structural formula:

n is an integer between 42 and 66;

the structural formula of the three-arm polyethylene glycol is as follows:

p is an integer between 29 and 44;

the structural formula of the four-arm polyethylene glycol is as follows:

q is an integer between 22 and 34.

4. The genetic transformation method according to claim 2, wherein the aqueous polyethylene glycol solution comprises: 250g/L multiarm polyethylene glycol, 50mL 1M CaCl₂10mL of 1M Tris (pH 7.5).

5. The genetic transformation method according to claim 1, wherein the selection medium in step (c) is an enhanced selection medium prepared by adding 0.5-1.5g/L betaine and 10-15mL/L dandelion leachate to the components of the selection medium.

6. The genetic transformation method according to claim 5, wherein the screening-enhancing medium is prepared by adding 1.5g/L betaine and 15mL/L dandelion leachate to the components of the screening medium.

7. The genetic transformation method according to claim 1, wherein the extraction method of trichoderma reesei RUT-C30 protoplast is:

1) inoculating the Trichoderma reesei RUT-C30 spore suspension to a Trichoderma complete culture medium, and culturing for 13-15 hours to obtain hyphae;

2) performing enzymolysis on the hyphae in an enzymolysis solution containing lyase for 1-3 hours, wherein the concentration of the lyase in the enzymolysis solution is 2.5-20 mg/mL;

3) trichoderma reesei RUT-C30 protoplasts were collected by filtration through a G2 glass frit funnel.

8. The genetic transformation method according to claim 7, wherein the Trichoderma reesei RUT-C30 hyphae in step (1) are Trichoderma reesei RUT-C30 hyphae obtained by culturing for 15 hours.

9. The genetic transformation method according to claim 7, wherein the lyase in step (2) is a lyase derived from Trichoderma harzianum (Trichoderma harzianum), the concentration of the lyase in the enzymatic hydrolysate is 5-15mg/mL, the enzymatic hydrolysis is performed at 25-30 ℃ and the enzymatic hydrolysis time is 2-3 hours.

10. The genetic transformation method according to claim 7, wherein the step (3) further comprises washing and centrifuging the Trichoderma reesei RUT-C30 protoplast collected by filtration through a G2 glass sand core funnel, wherein the G2 glass sand core funnel has a pore size of 30-50 μm.

Technical Field

The invention relates to the technical field of biology, in particular to an extraction method of trichoderma reesei RUT-C30 protoplast and a genetic transformation method of the protoplast.

Background

The lignocellulose biomass is the most abundant renewable resource on the earth, can be degraded into fermentable sugar under the action of cellulase, and can be fermented to produce green, clean and renewable biomass energy and new materials, so that the pollution-free and non-renewable fossil energy is supplemented and replaced, and the method is an inevitable direction for realizing economic transformation by human beings.

However, the production cost of cellulases is very high, which greatly limits the conversion utilization of lignocellulosic biomass. In order to reduce the cost of cellulase and improve the activity of cellulase, the development of efficient engineering strains for producing cellulase is an important research and development direction. At present, the most commonly used strain for producing cellulase is trichoderma reesei RUT-C30, the strain is a mutant strain obtained by screening an original strain QM6a of trichoderma reesei through mutagenesis methods such as ultraviolet mutagenesis, chemical reagent mutagenesis and the like, compared with QM6a, the cellulase production capacity is greatly improved, the industrial enzyme production requirement cannot be met, and further targeted modification is urgently needed.

In fact, as early as 1978, researchers used polyethylene glycol (PEG) mediated protoplast transformation to achieve genetic transformation of Saccharomyces cerevisiae (Saccharomyces cerevisiae). The method is the most widely used genetic transformation method in fungi at present, and the principle is that some commercial enzymes are utilized to remove the complex cell wall components of the fungi to obtain protoplast, and the protoplast is subjected to PEG and Ca²⁺Under the action of divalent cations, molecular bridges are formed between protoplast cells or between protoplasts and exogenous DNA to promote the adhesion of the protoplast cells or between the protoplasts and the exogenous DNA to form precipitates, and simultaneously, protoplast fine particles are formedThe surface charge of the cell membrane is disturbed, thereby changing the permeability of the cell membrane and promoting the protoplast to absorb the exogenous DNA. In the method, the yield and the state of the protoplast are critical, but the preparation process of the protoplast is complex, complicated and delicate, the cell wall components of different strains are different, and the regeneration capacity of the protoplast is also different, so that the standard unification of the preparation and transformation processes of different strains is difficult to realize. In addition, because the regeneration capacity of the cell wall of the Trichoderma reesei RUT-C30 is poor and the like, the research on genetic transformation of the Trichoderma reesei RUT-C3578 is less, and at present, a genetic transformation system of the Trichoderma reesei RUT-C30 strain is not established for a moment, which causes great inconvenience for realizing the strain transformation.

Disclosure of Invention

In view of the defects in the prior art, the invention preferably selects the optimal extraction method of the trichoderma reesei RUT-C30 protoplast aiming at the preparation conditions of the protoplast of the trichoderma reesei RUT-C30 strain, such as hypha culture time, enzymolysis time, enzyme concentration in enzymolysis liquid, enzymolysis conditions, collection mode and the like. In addition, through screening and optimizing PEG type and trichoderma screening culture medium, a trichoderma reesei RUT-C30 genetic transformation method with high positive transformation rate is finally established.

In a first aspect, the present invention provides a method for genetic transformation of trichoderma reesei RUT-C30, comprising the steps of:

a) mixing the exogenous plasmid and Trichoderma reesei RUT-C30 protoplast in polyethylene glycol (PEG) water solution, and performing ice bath for 10-20 min;

b) continuously adding 8-10 times of polyethylene glycol aqueous solution into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent, precipitating and transforming the protoplast, and suspending again;

c) transferring the transformed protoplast to a trichoderma screening culture medium, culturing for 5-7 days, then selecting a transformed strain, transferring the transformed strain to a trichoderma agar culture medium, carrying out propagation expansion to obtain a transformed strain spore suspension, and inoculating the spore suspension to a trichoderma seed culture medium for overnight culture to obtain transformed strain hypha.

The foreign plasmid described in step (a) includes any plasmid containing DNA that can be transformed into protoplasts. In a specific embodiment of the invention, the plasmid is the pAN7-1 hygromycin expression plasmid.

The polyethylene glycol aqueous solution comprises the following components: 250-300g/L linear and/or multi-arm polyethylene glycol, 40-60mL of 1M CaCl₂5-15mL of 1M Tris; preferably 250-300g/L linear and/or multi-arm polyethylene glycol, 50mL of 1M CaCl₂10mL of 1M Tris (pH 7.5).

Preferably, the molecular weight of the linear and/or multi-arm polyethylene glycol is 4000-6000, and the multi-arm polyethylene glycol comprises one of double-arm polyethylene glycol, three-arm polyethylene glycol and four-arm polyethylene glycol.

In a specific embodiment of the present invention, the linear polyethylene glycol has a structural formula of:

m is an integer between 90 and 136.

The double-arm polyethylene glycol has the structural formula:

n is an integer between 42 and 66.

The structural formula of the three-arm polyethylene glycol is as follows:

p is an integer between 29 and 44.

The structural formula of the four-arm polyethylene glycol is as follows:

q is an integer between 22 and 34.

Preferably, the polyethylene glycol aqueous solution comprises the following components: 250g/L multiarm polyethylene glycol, 50mL 1M CaCl₂10mL of 1M Tris (pH 7.5).

The components of the trichoderma screening culture medium in the step (c) are as follows: 1M sorbitol, 20g/L glucose, 15g/L KH₂PO₄，5g/L(NH₄)₂SO₄，0.6g/L MgSO₄·7H₂O，0.6g/L CaCl₂，0.005g/L FeSO₄·7H₂O，0.0016g/L MnSO₄·H₂O，0.0014g/L ZnSO₄·7H₂O，0.002g/L CoCl₂20g/L agar powder. After the preparation is finished, sterilizing in an autoclave (121 ℃, 25min), cooling to 45-50 ℃, adding hygromycin B (the final concentration is 175 mu g/mL) and ampicillin (the final concentration is 100 mu g/mL) into a super clean bench, and cooling for later use.

In a preferred embodiment of the present invention, the screening medium in step (c) is an enhanced screening medium, wherein the enhanced screening medium is prepared by adding 0.5-1.5g/L betaine and 10-15mL/L dandelion leachate to the components of the screening medium.

Specifically, the components of the enhanced screening medium are as follows: 1M sorbitol, 20g/L glucose, 15g/L KH₂PO₄，5g/L(NH₄)₂SO₄，0.6g/L MgSO₄·7H₂O，0.6g/L CaCl₂，0.005g/L FeSO₄·7H₂O，0.0016g/L MnSO₄·H₂O，0.0014g/L ZnSO₄·7H₂O，0.002g/L CoCl₂20g/L agar powder, 0.5-1.5g/L betaine and 10-15mL/L dandelion extract; sterilizing in a high pressure steam sterilizing pot (121 deg.C, 25 min); after the temperature is cooled to 45-50 deg.C (preferably without scalding hands), hygromycin B (final concentration 175. mu.g/mL) and ampicillin (final concentration 100. mu.g/mL) are added into the clean bench, and the mixture is poured into a plastic petri dish and cooled for later use.

In the most preferred embodiment of the present invention, the screening medium is supplemented with 1.5g/L betaine and 15mL/L dandelion extract.

Preferably, the preparation method of the dandelion leachate comprises the following steps: extracting 100g dry whole plant of herba Taraxaci with 1L boiling water for 3-4 hr, cooling, centrifuging, and collecting supernatant to obtain herba Taraxaci lixivium.

In an embodiment of the invention, the extraction method of the trichoderma reesei RUT-C30 protoplast comprises the following steps:

1) inoculating the Trichoderma reesei RUT-C30 spore suspension to a Trichoderma complete culture medium, and culturing for 13-15 hours to obtain hyphae;

2) performing enzymolysis on the hyphae in an enzymolysis solution containing lyase for 1-3 hours, wherein the concentration of the lyase in the enzymolysis solution is 2.5-20 mg/mL;

3) trichoderma reesei RUT-C30 protoplasts were collected by filtration through a G2 glass frit funnel.

In the above production method, preferably, the Trichoderma reesei RUT-C30 mycelium in the step (1) is Trichoderma reesei RUT-C30 mycelium obtained by culturing for 15 hours.

Preferably, the lyase in the step (2) is from Trichoderma harzianum (Trichoderma harzianum), the concentration of the lyase in the enzymolysis liquid is 5-15mg/mL, the enzymolysis is carried out at 25-30 ℃, and the enzymolysis time is 2-3 hours; preferably, the concentration of the lyase in the enzymatic hydrolysate is 10mg/mL, the enzymatic hydrolysis is carried out at 30 ℃, and the enzymatic hydrolysis time is 2.5 hours.

Preferably, the step (3) further comprises washing and centrifuging the Trichoderma reesei RUT-C30 protoplast collected by filtration through a G2 glass sand core funnel; more preferably, the aperture of the G2 glass sand core funnel is 30-50 μm.

The technical scheme of the invention has the advantages that:

1, the optimal conditions for protoplast generation are proposed as follows: the optimal culture time of hypha is 15h, the optimal enzyme concentration in the enzymolysis liquid is 10mg/mL, the optimal enzymolysis time is 2.5h, and the optimal enzymolysis condition is standing enzymolysis at 30 ℃; the best mode for collecting protoplast is G2 glass sand core funnel filtration, washing and centrifugation, and finally the concentration of extracted protoplast is multiplied by 10^{^8}one/mL, and good state of protoplasts.

2, successfully transforming the pAN7-1 hygromycin expression plasmid into the RUT-C30 strain, wherein the positive rate of the transformant is 23-41%, and successfully establishing a genetic transformation system of Trichoderma reesei RUT-C30.

3, the inventors have unexpectedly found that addition of betaine and dandelion leachate to a trichoderma reesei screening medium increased the conversion of trichoderma reesei RUT-C30 positive.

4, the inventor finds that the positive transformation rate of the Trichoderma reesei RUT-C30 can be influenced by changing the structure of PEG, and particularly, the number of positive transformants of the transformant is increased along with the increase of the number of PEG arms.

Drawings

FIG. 1 map of plasmid PAN 7-1;

FIG. 2 protoplasts collected by G2 funnel filtration;

FIG. 3 PCR identification of Trichoderma reesei protoplasts transformed with PAN7-1 plasmid;

FIG. 4 sequencing results of plasmid-transformed Trichoderma reesei protoplasts PAN 7-1;

FIG. 5 influence of hypha cultivation time and enzymolysis time on protoplast yield, wherein (A) protoplast yield was obtained by enzymolysis of 13h hypha, and (B) protoplast yield was obtained by enzymolysis of 15h hypha;

FIG. 6 effect of enzyme concentration on protoplast yield;

FIG. 7 effect of rotation speed on protoplast yield;

FIG. 8 protoplasts obtained by buffer extraction.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The components and the preparation method of the culture medium are as follows:

the components of the trichoderma complete culture medium are as follows: 20mL/L of trichoderma salt solution, 10mL/L of vitamin solution, 1.5g/L of casamino acid, 10g/L of glucose, 3g/L of YEP and the balance of 970mL ddH₂And O. The trichoderma salt solution comprises the following components: 26g/L KCl, 26g/L MgSO₄·7H₂O，76g/L KH₂PO₄50mL/L of trichoderma trace element solution; the components of the trichoderma trace element solution are as follows: 40mg/L Na₂B₄O₇·10H₂O，400mg/L CuSO₄·5H₂O，800mg/L Fe₂(SO₄)₃·2H₂O，800mg/L MnSO₄·2H₂O，8mg/L ZnSO₄·7H₂And O. The vitamin solution (100mL) was weighed in order: vitamin B15 mg, biotin 1mg, nicotinic acid 10mg, D-calcium pantothenate 20mg, pyridoxine HCl 5mg, riboflavin 10mg, p-aminobenzoic acid 50mg, and the balance water.

The components of the trichoderma agar culture medium are as follows: 10g/L glucose, 2g/L corn steep liquor, 2.8g/L KH₂PO₄，3.92g/L(NH₄)₂SO₄，0.84g/L MgSO₄·7H₂O, 0.84g/L urea, 2g/L Tween-80, 0.014g/L FeSO₄·7H₂O，0.00436g/L MnSO₄·H₂O，0.00392g/L ZnSO₄·7H₂O，0.0056g/L CoCl₂20g/L agar; after compounding, the pH was adjusted to 4.8 with 2M NaOH solution. Sterilizing in high pressure steam sterilizer (121 deg.C, 25 min); cooling to 45-50 deg.C (preferably not scalding hands), adding ampicillin (final concentration of 100 μ g/mL) into the clean bench, pouring into plastic culture dish, and cooling.

The components of the trichoderma seed culture medium are as follows: 12g/L glucose, 0.7g/L corn steep liquor, 1.96g/L KH₂PO₄，1.372g/L(NH₄)₂SO₄，0.294g/L MgSO₄·7H₂O, 0.294g/L urea, 0.0049g/L FeSO₄·7H₂O，0.0015g/L MnSO₄·H₂O，0.0014g/L ZnSO₄·7H₂O，0.002g/L CoCl₂. After preparation, the mixture was sterilized in an autoclave (121 ℃ C., 25 min).

The components and the preparation method of the reagent related by the invention are as follows:

the MW reagent comprises the following components: 147.9g/L MgSO₄·7H₂O。

The OM reagent comprises the following components: 295.8g/L MgSO₄·7H₂O, 10mL of 1M phosphate buffer (pH 6.5), adjusting the pH to 5.8; wherein the 1M phosphate buffer (pH 6.5) has the following components: 268.1g/L Na₂HPO₄·7H₂O，120g/L NaH₂PO₄The pH was adjusted to 6.5.

The STC reagent comprises the following components: 218.64g/L sorbitol, 10mL of 1M CaCl₂10ml of 1M Tris (pH 7.5), pH adjusted to 7.5; wherein 1M of CaCl₂Comprises the following components: 111g/L anhydrous CaCl₂(ii) a The composition of 1M Tris (pH 7.5) was: 121g/L Tris-base, pH adjusted to 7.5.

The linear PEG and the multi-arm PEG used by the invention are purchased from Xiamen Sainungge Biotech Co., Ltd, and the preparation method comprises the following steps:

the 25% linear PEG4000 solution had the composition: 250g/L PEG4000, 50mL of 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

The 25% linear PEG6000 solution had the composition: 250g/L PEG6000, 50mL of 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

The 25% two-arm PEG4000 solution had the following composition: 250g/L double arm PEG4000, 50mL 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

The 25% two-arm PEG6000 solution had the following composition: 250g/L double arm PEG6000, 50mL 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

The 25% three-arm PEG4000 solution had the following composition: 250g/L three-arm PEG4000, 50mL of 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

The 25% three-arm PEG6000 solution comprises the following components: 250g/L three-arm PEG6000, 50mL of 1M CaCl₂10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.

Example 1 genetic transformation of Trichoderma reesei RUT-C30 protoplasts

(1) Trichoderma reesei RUT-C30 protoplast extraction

S1, inoculating spore suspension into 250mL of sterilized complete trichoderma culture medium, and culturing overnight for 15h to obtain hyphae;

s2: filtering out the culture medium from the hyphae cultured in S1, washing the hyphae with 25mL MW reagent, weighing 3g of hyphae, placing the hyphae in a 50mL centrifuge tube, adding OM reagent into the centrifuge tube filled with the hyphae to 15mL scale marks, and shaking the centrifuge tube to uniformly disperse the hyphae;

s3: weighing 0.225g of lyase (Sigma L1412) and dissolving in 5mL of OM reagent, filtering and sterilizing with a 0.22 μm filter membrane, transferring to a centrifuge tube filled with mycelium and OM reagent in the step S2, continuously metering to 22.5mL of scale mark with OM reagent, and mixing uniformly; transferring the mixture to a 250mL glass beaker, wrapping the beaker with tin foil paper, and standing the beaker in an incubator at 30 ℃ for enzymolysis for 2.5 hours; taking 1 μ L of enzymolysis solution for enzymolysis for 2.5h, diluting 10 times, and counting under microscope to obtain the concentration of 8.9 × 10⁶Protoplasts per mL;

s4: adding an equivalent STC reagent into the enzymolysis liquid which is subjected to enzymolysis for 2.5h, slightly shaking and uniformly mixing, filtering the mixture through a G2 sand core glass funnel to a 50mL round-bottom centrifuge tube, centrifuging the mixture for 8min to precipitate the protoplast, pouring the supernatant, placing the centrifuge tube on ice, adding 10mL STC reagent, rotating the centrifuge tube, and dissolving the protoplast; centrifuging for 8min, precipitating protoplast, and collecting the protoplast precipitate aggregate as shown in FIG. 2A;

s5: pouring the supernatant, placing the centrifugal tube on ice, adding 500 mu L of STC reagent, rotating the centrifugal tube, and dissolving the protoplast; taking 1 μ L of the above protoplast, diluting 10 times, and counting under microscope to obtain final concentration of 2.56 × 10⁸Protoplasts per mL, microscopic protoplast status is shown in FIG. 2B.

(2) Trichoderma reesei RUT-C30 protoplast transformation

S6: subpackaging the extracted protoplast into 50mL centrifuge tubes, placing 200 μ L protoplast in each centrifuge tube on ice, adding 5 μ g exogenous plasmid (PAN7-1 plasmid, map is shown in figure 1) and 50 μ L25% linear PEG6000 solution into each centrifuge tube, mixing, placing the centrifuge tubes on ice for reaction for 20 min;

s7: inclining the centrifuge tube, slowly adding 2mL of 25% linear PEG6000 solution to the centrifuge tube, culturing at room temperature for 5min, adding 8mL of STC reagent, centrifuging for 8min, precipitating protoplast, pouring out supernatant, adding 100 μ L of STC reagent, rotating the centrifuge tube, and dissolving the protoplast;

s8: transferring the transformed protoplast (transformant) to a trichoderma screening culture medium by using a sterile pipette, uniformly coating, culturing in a mould incubator at 30 ℃ for about 5-7 days, then selecting 22 transformants, transferring to a trichoderma agar culture medium for propagation, scraping spores to obtain a spore suspension, and inoculating the spore suspension to a trichoderma seed culture medium for overnight culture to obtain hyphae.

The trichoderma screening medium described in this example comprises the following components: 1M sorbitol, 20g/L glucose, 15g/L KH₂PO₄，5g/L(NH₄)₂SO₄，0.6g/L MgSO₄·7H₂O，0.6g/L CaCl₂，0.005g/L FeSO₄·7H₂O，0.0016g/L MnSO₄·H₂O，0.0014g/L ZnSO₄·7H₂O，0.002g/L CoCl₂20g/L agar powder. After the preparation is finished, sterilizing in an autoclave (121 ℃, 25min), cooling to 45-50 ℃, adding hygromycin B (the final concentration is 175 mu g/mL) and ampicillin (the final concentration is 100 mu g/mL) into a super clean bench, and cooling for later use.

(3) Positive transformant detection

22 mycelia of overnight-cultured transformants and RUT-C30 strain were extracted with the use of the rapid plant genomic DNA extraction system (DP-321) of Tiangen Biochemical technology Ltd, and the procedures and reagent components were as described in the description of DP-321. The extracted DNA was used as a template DNA, and PCR amplification and agarose gel electrophoresis were performed using pAN7-F, pAN7-R primers to verify whether the transformant was successfully transformed.

Wherein, the PCR amplification and agarose gel electrophoresis program and the flow are as follows:

(1) PCR amplification

The PCR reaction (20. mu.L) was as follows:

the PCR amplification procedure was as follows:

(2) agarose gel electrophoresis

A1% agarose gel was prepared with electrophoresis buffer TAE (1X) and heated to complete dissolution in a microwave oven. Taking out, shaking, cooling to a temperature not to scald hands, adding nucleic acid dye (Genegreen, Tiangen Biochemical technology Co., Ltd.), and pouring onto the horizontal plate of the electrophoresis tank with the sample comb. After the agarose gel solidified, carefully pull out the comb, place the gel-loaded horizontal plate of the electrophoresis tank into the electrophoresis tank, and add the electrophoresis buffer TAE (1X).

Appropriate size Marker and PCR samples were spotted in the sample well in an amount of 5-10. mu.L, and the order of spotting the samples was recorded. The voltage is adjusted to be 100-150V, electrophoresis is carried out for 20-30min, then the gel is taken out, placed in a G-box and irradiated by blue light, electrophoresis bands are checked and analyzed by GeneSys software, wherein 5 products have the target bands, and the bands are shown in figure 3. The 5 PCR products were sequenced, and the sequencing results were completely aligned with the target fragment on the plasmid, as shown in FIG. 4, and all of the 5 transformants were positive (the positive rate was 23%).

Example 2 Trichoderma reesei RUT-C30 protoplast extraction condition optimization

(1) Screening of hypha culture time and enzymolysis time

The cultivation time of hyphae directly affects the yield and status of protoplasts because hyphae at different age stages have different sensitivities to enzymes due to differences in cell wall thickness, composition, etc. The tender hyphae have thinner cell walls and simpler components, so the hyphae are easier to be subjected to enzymolysis compared with old hyphae, but the hyphae which are too tender cannot normally produce protoplasts with large quantity and good quality. Therefore, it is necessary to search for the culture time of the mycelia in order to obtain protoplasts of high quality and large quantity. Secondly, the enzymolysis time of the hyphae also has great influence on the yield and the quality of the protoplast. The enzymolysis time is too short, hypha can not be fully enzymolyzed, and protoplast can not be obtained; the enzymolysis time is too long, the enzyme solution also damages the protoplast, and the yield and the activity of the protoplast are also reduced, thereby influencing the transformation and regeneration effects.

In order to determine the optimal hypha culture time and enzymolysis time, the culture time (13h, 15h) and the enzymolysis time (1h, 1.5h, 2h, 2.5h, 3h) were combined as variables under the enzymolysis condition of 5mg/mL enzyme concentration at 30 ℃ and 100rpm, respectively. The invention makes ten-group comparison experiments, each group of experiments is set to be repeated for three times, and the yield of each group of experimental protoplasts under a specific time is averaged. As shown in FIG. 5, the yield of protoplasts obtained after culturing mycelia for 13h and performing enzymolysis for 1.5h was 6.1X 10, which was the highest under the conditions of 5mg/mL enzyme concentration, 30 ℃ and 100rpm enzymolysis⁴Per mL; culturing mycelium for 15h, and performing enzymolysis for 2.5h to obtain protoplast with highest yield of 3.1 × 10⁵one/mL. In addition, microscopic examination shows that mycelia cultured for 15h are full and mellow and uniform in size of protoplasts obtained after enzymolysis for 2.5 h.

Therefore, it was found that the optimal culture time for mycelia was 15 hours, and the optimal enzymatic hydrolysis time was 2.5 hours. Under the parameters, the yield of the obtained protoplast is highest and the state is good.

(2) Screening of enzyme concentration

The filamentous fungal cell wall components are mainly chitin, proteins, lipids, glucan, and the like. The lyase used in the present invention was Sigma L1412 (lyase from Trichoderma harzianum), having mainly β -glucanase, cellulase, protease and chitinase activities. The protoplast cell membrane is mainly composed of lipids, proteins, and dextran. Therefore, the enzymolysis liquid not only can cause the cracking damage to the cell wall, but also can play a certain role in the enzymolysis to the cell membrane of the protoplast. Therefore, the enzyme concentration in the enzymatic hydrolysate should be maintained at a suitable level to ensure cell wall removal and to avoid excessive destruction of protoplasts.

In order to determine the optimum enzyme concentration, the enzyme was incubated at 30 ℃ and 100rpmHyphae were incubated for 15h for 2.5h and four enzyme concentration gradients of 2.5mg/mL, 5mg/mL, 10mg/mL, 15mg/mL and 20mg/mL were set with the enzyme concentration as a single variable. Four sets of comparative experiments were performed, each set of experiments was repeated three times, and the yield of protoplasts from each set of experiments was averaged for a particular enzyme concentration. As shown in FIG. 6, the enzyme concentrations of 0.81X 10 at 2.5, 5, 10, 15 and 20mg/mL, respectively, were obtained⁵、3.1×10⁵、9.5×10⁵、11×10⁵、9.82×10⁵Protoplast per mL.

Although the highest yield of protoplasts was obtained at 15mg/mL enzyme concentration, microscopic examination showed that the protoplast morphology began to deform and deteriorate at 15 and 20mg/mL enzyme concentrations; although the yield of the protoplast obtained under the enzyme concentration of 10mg/mL is slightly lower than that obtained under the enzyme concentration of 15mg/mL, the yield difference is not large, and meanwhile, the protoplast is mellow and full and is more suitable for transformation and regeneration.

Therefore, it was found that the optimal enzyme concentration for hyphal enzymolysis was 10mg/mL, and the yield of protoplasts obtained was the highest and the state was good under these parameters.

(3) Screening of conditions for enzymatic hydrolysis

The enzymolysis condition is also the key point which can influence the generation of the protoplast, and the enzymolysis temperature is determined to be 30 ℃ in the invention considering that the optimal temperature for the growth of the trichoderma is 30 ℃. In order to determine the optimal enzymolysis conditions of the mycelia, mycelia cultured for 15h were subjected to enzymolysis for 2.5h at an enzyme concentration of 10mg/mL, and the enzymolysis conditions were set as single variables at 30 ℃ and 0rpm, 30 ℃ and 50rpm, 30 ℃ and 100rpm, and each of the enzymolysis solutions was observed and counted under a microscope. Three groups of comparison experiments are carried out, each group of experiments is set to be repeated for three times, and the yield of each group of experimental protoplasts under a specific enzymolysis condition is averaged. As a result, as shown in FIG. 7, the yield of protoplasts counted under a microscope under enzymatic conditions of 30 ℃ and 0rpm was 8.9X 10⁶Per mL; the protoplast yield was 2.92X 10 at 30 ℃ and 50rpm for enzymatic digestion⁶Per mL; the protoplast yield was 9.5X 10 at 30 ℃ and 100rpm for enzymatic hydrolysis⁵one/mL.

Therefore, it was found that the conditions for the optimal enzymatic hydrolysis of mycelia were 30 ℃ and 0rpm, and the yield of protoplasts was the highest and the state was good.

(4) Screening of methods for protoplast Collection

To determine the best method for collecting protoplasts, mycelia cultured for 15 hours were subjected to enzymolysis for 2.5 hours under enzymolysis conditions of 30 ℃ and 0rpm at an enzyme concentration of 10mg/mL, and the protoplast collection method was set as a single variable, including buffer (0.6M sorbitol, 0.1M Tris-HCl, pH 7.0) extraction, centrifugation after filtration with G2 sand-core funnel (pore size 30-50 μ M, deer head brand), centrifugation after filtration with 400 mesh cell filtration sieve (pore size 37.5 μ M, Solarbio/Solibao), centrifugation after filtration with Miracloth (pore size 22-25 μ M, Merck Millipore/Merck Millipore), and protoplasts after enzymolysis were collected in four ways.

As a result, as shown in FIG. 8, the protoplasts collected by the extraction method had a very low yield and a poor condition, and a large amount of mycelia were mixed; the yield of protoplasts collected by filtration through a 400-mesh cell filter screen and a Miracloth is very low, and the existence of protoplast precipitates can not be seen almost by naked eyes after centrifugation.

And after filtration through a G2 sand core funnel, washing and centrifugation, a distinct macroscopic aggregation precipitate of protoplasts can be collected (FIG. 2A). The precipitate was gently dissolved with 500. mu.L of STC reagent and collected to a concentration of 2.56X 10⁸one/mL and good protoplast (FIG. 2B) (20 mL in the previous enzymolysis system, 8.9X 10 protoplast concentration in the original enzymolysis solution)⁶one/mL, protoplast recovery 72%).

Therefore, it was found that the best way to collect protoplasts was G2 sand-core funnel filtration, washing, and centrifugation, and the yield of the obtained protoplasts was the highest and the state was good.

Example 3 Trichoderma reesei RUT-C30 protoplast transformation conditions optimization

Polyethylene glycol (PEG) solution is added in the protoplast transformation process, and the principle is as follows: PEG can form a molecular bridge between the protoplast and the exogenous DNA to promote the mutual adhesion of the protoplast and the exogenous DNA to form a precipitate, and simultaneously cause charge disorder on the surface of a cell membrane of the protoplast, thereby changing the permeability of the cell membrane and promoting the protoplast to absorb the exogenous DNA. Based on this, the inventors tried to promote the fusion of exogenous DNA and protoplast by adding PEG solution with different molecular structures during the transformation process, and the positive transformation rate of the protoplast was improved.

A: the extraction of the Trichoderma reesei RUT-C30 protoplasts was performed in the same manner as in example 1, except that PEG6000 was replaced with PEG4000 and the concentration was changed in the transformation of the protoplasts, and the procedure was as in example 1.

B: the extraction of the protoplasts of Trichoderma reesei RUT-C30 was performed in the same manner as in example 1, except that PEG6000 was replaced with double-arm PEG (molecular weight: 4000) in the transformation of the protoplasts, and the procedure was as in example 1.

C: the extraction of the protoplasts of Trichoderma reesei RUT-C30 was performed in the same manner as in example 1, except that PEG6000 was replaced with double-arm PEG (molecular weight: 6000) in the transformation of the protoplasts, and the procedure was as in example 1.

D: the extraction of the Trichoderma reesei RUT-C30 protoplasts was performed in the same manner as in example 1, except that PEG6000 was replaced with three-arm PEG (molecular weight: 4000) in the protoplast transformation, and the procedure was as in example 1.

E: the extraction of the Trichoderma reesei RUT-C30 protoplasts was performed in the same manner as in example 1, except that PEG6000 was replaced with three-arm PEG (molecular weight: 6000) in the protoplast transformation, and the rest of the procedure was performed in the same manner as in example 1.

22 transformants were picked up and propagated on a screening medium for Trichoderma as described in example 1, and 22 mycelia of the transformants were finally obtained. PCR amplification and agarose gel electrophoresis were performed using the hyphal DNA as a template DNA and pAN7-F, pAN7-R primers to verify whether the transformants were successfully transformed. The results are shown in the following table:

TABLE 1

According to the data given in the table above, it can be seen that the PEG as a bridge between the protoplast and the exogenous DNA has an important influence on whether the exogenous DNA is fused into the protoplast. Meanwhile, the inventor also finds that the molecular weight of PEG has no influence on the positive conversion rate of the transformant, the arm number of PEG has substantial influence on the positive conversion rate, and the number of positive transformants of the transformant is increased along with the increase of the arm number of PEG. This is because when PEG is double-armed or triple-armed, it has a better effect of linking the protoplast to the foreign DNA than linear PEG, and the foreign DNA is better incorporated into the protoplast.

Example 4 Trichoderma selection Medium optimization

The technicians of the invention unexpectedly discover in tests that 0.5-1.5g/L betaine and 10-15mL/L dandelion leachate are added on the basis of the conventional trichoderma screening culture medium, the culture medium is named as enhanced screening culture medium, and the positive rate of the trichoderma reesei transformant obtained by final screening is higher. In order to optimize the addition ratio, the present invention sets the following 3 concentrations of enhanced screening media.

The trichoderma screening culture medium comprises the following components: 1M sorbitol, 20g/L glucose, 15g/L KH₂PO₄，5g/L(NH₄)₂SO₄，0.6g/L MgSO₄·7H₂O，0.6g/L CaCl₂，0.005g/L FeSO₄·7H₂O，0.0016g/L MnSO₄·H₂O，0.0014g/L ZnSO₄·7H₂O，0.002g/L CoCl₂20g/L agar powder. After the preparation is finished, sterilizing in an autoclave (121 ℃, 25min), cooling to 45-50 ℃, adding hygromycin B (the final concentration is 175 mu g/mL) and ampicillin (the final concentration is 100 mu g/mL) into a super clean bench, and cooling for later use.

A: the extraction mode and transformation method of trichoderma reesei RUT-C30 protoplast are the same as example 1, the transformed protoplast (transformant) is transferred to an enhanced screening culture medium I, the protoplast is coated evenly, 22 transformants are picked after being cultured in a mould incubator at 30 ℃ for about 5-7 days, then transferred to a trichoderma agar culture medium for propagation, then spores are scraped to obtain a spore suspension, and the spore suspension is inoculated to a trichoderma seed culture medium for overnight culture to obtain hypha.

B: the extraction mode and transformation method of the trichoderma reesei RUT-C30 protoplast are the same as example 1, the transformed protoplast (transformant) is transferred to an enhanced screening culture medium II, the protoplast is evenly coated, 22 transformants are picked after being cultured in a mould incubator at 30 ℃ for about 5 to 7 days, then the transformants are transferred to a trichoderma agar culture medium for propagation, then spores are scraped to obtain a spore suspension, and the spore suspension is inoculated to a trichoderma seed culture medium for overnight culture to obtain hypha.

C: the extraction mode and transformation method of trichoderma reesei RUT-C30 protoplast are the same as example 1, the transformed protoplast (transformant) is transferred to an enhanced screening culture medium III, the protoplast is evenly coated, 22 transformants are picked after being cultured in a mould incubator at 30 ℃ for about 5-7 days, then the transformants are transferred to a trichoderma agar culture medium for propagation, then spores are scraped to obtain a spore suspension, and the spore suspension is inoculated to a trichoderma seed culture medium for overnight culture to obtain hypha.

The obtained hyphal DNA was used as a template DNA, the positive transformation rate was determined in the same manner as in example 1, a desired band was found by PCR amplification and agarose gel electrophoresis, and PCR products on the band were sequenced, and as a result, the sequence of the detected product was completely aligned with that of the desired fragment on the plasmid, and all of the transformants corresponding to the desired band were positive transformants, and the number thereof was as shown in the following table.

TABLE 2

As can be seen from the data in the table above, compared with the original screening medium, the positive conversion rate is increased when the leaching solutions of betaine and dandelion with different concentrations are added into the screening medium, mainly because with the increase of the addition of betaine and dandelion, the bactericidal effect of betaine and dandelion can eliminate a part of false positive hygromycin resistant strains, so that more strains containing hygromycin resistant genes are contained in the picked transformant, and the positive conversion rate is higher. Wherein, when the concentration of the betaine is 1.5g/L and the concentration of the dandelion leaching solution is 15mL/L, the positive conversion rate of the trichoderma reesei protoplast is the highest.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

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