CN113789342B - Genetic transformation method for Trichoderma reesei RUT-C30 protoplast - Google Patents
Genetic transformation method for Trichoderma reesei RUT-C30 protoplast Download PDFInfo
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Abstract
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 polyethylene glycol aqueous solution, and ice-bathing for 10-20min; b) Continuously adding polyethylene glycol aqueous solution with the volume of 8-10 times into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent to precipitate transformed protoplast, and suspending again; c) Transferring the transformed protoplast into a trichoderma screening culture medium for 5-7 days, picking up a transformant, transferring into a trichoderma agar culture medium for propagation to obtain a transformant spore suspension, and inoculating the spore suspension into a trichoderma seed culture medium for overnight culture to obtain transformant hyphae. The invention successfully converts pAN7-1 hygromycin expression plasmid into RUT-C30 strain, the positive rate of the converted strain is 23-41%, and a genetic transformation system of Trichoderma reesei RUT-C30 is successfully established.
Description
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
Lignocellulose biomass is the most abundant renewable resource on 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 as to supplement and replace polluted and non-renewable fossil energy, which is a necessary direction for realizing economic transformation of human beings.
However, the cost of cellulase production is very high, which greatly limits the conversion utilization of lignocellulosic biomass. In order to reduce the cost of cellulase and improve the cellulase activity, the development of efficient engineering strains for producing cellulase is an important development direction. At present, the most commonly used strain for producing cellulase is Trichoderma reesei RUT-C30, which is a mutant strain obtained by screening an original strain QM6a of Trichoderma reesei by a mutagenesis method such as ultraviolet mutagenesis, chemical reagent mutagenesis and the like, and compared with the mutant strain QM6a, the cellulase production capability of the mutant strain is greatly improved, but the industrial enzyme production requirement is still not met, and further targeted modification is needed.
Indeed, as early as 1978, researchers have achieved genetic transformation of Saccharomyces cerevisiae (Saccharomyces cerevisiae) using polyethylene glycol (polyethylene glycol, PEG) -mediated protoplast transformation methods. 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 complex cell wall components of the fungi to obtain protoplasts, and PEG and Ca are used for the transformation 2+ Under the action of the divalent cations, molecular bridges are formed between protoplast cells or between protoplasts and exogenous DNA, so that the protoplast cells or the protoplasts and the exogenous DNA are promoted to adhere to each other to form a precipitate, and meanwhile, the surface charge of the cell membrane of the protoplast is disturbed, so that the permeability of the cell membrane is changed, and the protoplast is promoted 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, tedious and carefulThe cell wall components of different strains are different, and the regeneration capacity of protoplasts is also different, so that the standard unification is difficult to realize for the preparation and transformation processes of different strains. Moreover, because of poor cell wall regeneration capability of Trichoderma reesei RUT-C30 and the like, the research on genetic modification of the Trichoderma reesei RUT-C30 is less, and at present, a genetic transformation system of the Trichoderma reesei RUT-C30 strain is not established, which causes great inconvenience for realizing the strain modification.
Disclosure of Invention
In view of the defects existing in the prior art, the invention aims at the preparation conditions of the protoplast of the Trichoderma reesei RUT-C30 strain, such as mycelium culture time, enzymolysis time, enzyme concentration in enzymolysis liquid, enzymolysis conditions, collection mode and the like, and preferably provides the optimal Trichoderma reesei RUT-C30 protoplast extraction method. In addition, through screening and optimizing the screening culture medium such as PEG type and Trichoderma, a Trichoderma reesei RUT-C30 genetic transformation method with higher 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 ice-bathing for 10-20min;
b) Continuously adding polyethylene glycol aqueous solution with the volume of 8-10 times into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent to precipitate transformed protoplast, and suspending again;
c) Transferring the transformed protoplast into a trichoderma screening culture medium for 5-7 days, picking up a transformant, transferring into a trichoderma agar culture medium for propagation to obtain a transformant spore suspension, and inoculating the spore suspension into a trichoderma seed culture medium for overnight culture to obtain transformant hyphae.
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 a 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 1M CaCl 2 5-15mL of 1M Tris; preferably 250-300g/L linear and/or multi-arm polyethylene glycol, 50mL 1M CaCl 2 10mL of 1M Tris (pH 7.5).
Preferably, the linear and/or multi-arm polyethylene glycol has a molecular weight of 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 the structural formula:
m is an integer between 90 and 136.
The structural formula of the double-arm polyethylene glycol is as follows:
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 multi-arm polyethylene glycol, 50mL 1M CaCl 2 10mL of 1M Tris (pH 7.5).
The trichoderma screened culture medium in the step (c) comprises the following components: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L agar powder. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min), cooling to 45-50deg.C, adding hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) into the ultra-clean bench, and cooling.
In a preferred embodiment of the present invention, the screening medium in the step (c) is an enhanced screening medium, and the enhanced screening medium is prepared by adding 0.5-1.5g/L betaine and 10-15mL/L dandelion extract to the components of the screening medium.
Specifically, the components of the enhanced screening culture medium are as follows: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L of agar powder, 0.5-1.5g/L of betaine and 10-15mL/L of dandelion extract; sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min); after cooling to 45-50deg.C (preferably without scalding hands), hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) are added into the ultra-clean bench, and the mixture is poured into a plastic culture dish and cooled for use.
In a most preferred embodiment of the present invention, the enhanced screening medium is prepared by adding 1.5g/L betaine and 15mL/L dandelion extract to the components of the screening medium.
Preferably, the preparation method of the dandelion extract comprises the following steps: 100g of dandelion dried whole herb is added into 1L of boiling water to be extracted for 3-4 hours, cooled and centrifuged to obtain supernatant, thus obtaining dandelion leaching liquid.
In an embodiment of the invention, the trichoderma reesei RUT-C30 protoplast extraction method comprises the steps of:
1) Inoculating Trichoderma reesei RUT-C30 spore suspension into Trichoderma complete culture medium, and culturing for 13-15 hr to obtain mycelium;
2) Carrying out enzymolysis on hyphae in an enzymolysis liquid containing lyase for 1-3 hours, wherein the concentration of the lyase in the enzymolysis liquid is 2.5-20mg/mL;
3) Trichoderma reesei RUT-C30 protoplasts were collected by filtration through a G2 glass sand core funnel.
In the above preparation method, preferably, the Trichoderma reesei RUT-C30 hypha in the step (1) is Trichoderma reesei RUT-C30 hypha obtained by culturing for 15 hours.
Preferably, the lyase in the step (2) is a lyase 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, and the enzymatic hydrolysis is performed at 30 ℃ for 2.5 hours.
Preferably, step (3) further comprises washing and centrifuging said trichoderma reesei RUT-C30 protoplasts 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 formation are: the optimal culture time of mycelium 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 30 ℃ for standing enzymolysis; the best mode of protoplast collection is G2 glass sand core funnel filtration, washing and centrifugation, and the concentration is finally extracted to be multiplied by 10 ^8 Protoplast with good status and per mL.
2, successfully transforming the pAN7-1 hygromycin expression plasmid into the RUT-C30 strain, wherein the positive rate of the transformed strain is 23-41%, and successfully establishing a genetic transformation system of the Trichoderma reesei RUT-C30.
3, the inventors have unexpectedly found that the addition of betaine and dandelion extract to trichoderma reesei screening media increases the positive conversion rate of trichoderma reesei RUT-C30.
4, the inventors found that the positive conversion rate of Trichoderma reesei RUT-C30 can be affected by changing the structure of PEG, and in particular, as the number of PEG arms increases, the number of positive transformants of the transformant increases.
Drawings
FIG. 1 is a map of plasmid PAN 7-1;
FIG. 2 protoplasts collected by filtration through a G2 funnel;
FIG. 3 PCR identification of PAN7-1 plasmid transformed Trichoderma reesei protoplasts;
FIG. 4 sequencing results of PAN7-1 plasmid transformed Trichoderma reesei protoplasts;
FIG. 5 influence of mycelium culture time and enzymolysis time on protoplast yield, wherein (A) protoplast yield of 13h mycelium subjected to enzymolysis, and (B) protoplast yield of 15h mycelium subjected to enzymolysis;
FIG. 6 effect of enzyme concentration on protoplast production;
FIG. 7 effect of rotational speed on protoplast production;
FIG. 8 protoplasts obtained by buffer extraction.
Detailed Description
The technical solutions of 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, but not all. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The composition and preparation method of the culture medium related by the invention are as follows:
the complete culture medium of trichoderma comprises the following components: 20mL/L of trichoderma salt solution, 10mL/L of vitamin solution, 1.5g/L of casein amino acid, 10g/L of glucose, 3g/L of YEP and the balance of 970mL of ddH 2 O. The trichoderma salt solution comprises the following components: 26g/L KCl,26g/L MgSO 4 ·7H 2 O，76g/L KH 2 PO 4 50mL/L of trichoderma trace element solution; the trichoderma trace element solution comprises the following components: 40mg/L Na 2 B 4 O 7 ·10H 2 O，400mg/L CuSO 4 ·5H 2 O，800mg/L Fe 2 (SO 4 ) 3 ·2H 2 O，800mg/L MnSO 4 ·2H 2 O，8mg/L ZnSO 4 ·7H 2 O. The vitamin solution (100 mL) was weighed in sequence for the following reagents: vitamins15mg of biotin, 1mg of biotin, 10mg of nicotinic acid, 20mg of D-calcium pantothenate, 5mg of pyridoxine HCl, 10mg of riboflavin, 50mg of para-aminobenzoic acid and the balance of water.
The trichoderma agar medium comprises the following components: 10g/L glucose, 2g/L corn steep liquor, 2.8g/L KH 2 PO 4 ，3.92g/L(NH 4 ) 2 SO 4 ，0.84g/L MgSO 4 ·7H 2 O,0.84g/L urea, 2g/L Tween-80, 0.014g/L FeSO 4 ·7H 2 O，0.00436g/L MnSO 4 ·H 2 O，0.00392g/L ZnSO 4 ·7H 2 O，0.0056g/L CoCl 2 20g/L agar; after formulation, the pH was adjusted to 4.8 with 2M NaOH solution. Sterilizing in a high-pressure steam sterilizing pot (121 ℃ for 25 min); after cooling to 45-50deg.C (preferably without scalding hands), ampicillin (final concentration 100 μg/mL) is added into the ultra-clean bench, and the mixture is poured into a plastic culture dish and cooled for use.
The trichoderma seed culture medium comprises the following components: 12g/L glucose, 0.7g/L corn steep liquor, 1.96g/L KH 2 PO 4 ，1.372g/L(NH 4 ) 2 SO 4 ，0.294g/L MgSO 4 ·7H 2 O,0.294g/L urea, 0.0049g/L FeSO 4 ·7H 2 O，0.0015g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 . After preparation, the mixture was sterilized in an autoclave (121 ℃ C., 25 min).
The composition and the preparation method of the reagent components related by the invention are as follows:
the MW reagent comprises the following components: 147.9g/L MgSO 4 ·7H 2 O。
The components of the OM reagent are: 295.8g/L MgSO 4 ·7H 2 O,10mL of 1M phosphate buffer (pH 6.5), pH was adjusted to 5.8; wherein the 1M phosphate buffer (pH 6.5) comprises the following components: 268.1g/L Na 2 HPO 4 ·7H 2 O，120g/L NaH 2 PO 4 The pH was adjusted to 6.5.
The STC reagent comprises the following components: 218.64g/L sorbitol, 10mL 1M CaCl 2 10ml of 1M Tris (pH 7.5), fine-tuning the pH to 7.5; wherein 1M CaCl 2 The components of (1) are as follows: 111g/L anhydrous CaCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The composition of 1M Tris (pH 7.5) was: 121g/L Tris-base, pH was adjusted to 7.5.
The linear PEG and the multi-arm PEG used in the invention are purchased from Xiaomen Saunobang biotechnology Co., ltd, and the specific preparation method is as follows:
the components of the 25% linear PEG4000 solution were: 250g/L PEG4000, 50mL 1M CaCl 2 10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.
The 25% linear PEG6000 solution had the following composition: 250g/L PEG6000, 50mL 1M CaCl 2 10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.
The 25% double arm PEG4000 solution had the following composition: 250g/L double arm PEG4000, 50mL 1M CaCl 2 10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.
The 25% double arm PEG6000 solution comprises the following components: 250g/L double arm PEG6000, 50mL 1M CaCl 2 10mL 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 1M CaCl 2 10mL 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 1M CaCl 2 10mL of 1M Tris (pH 7.5), the pH was adjusted to 7.5.
EXAMPLE 1 genetic transformation method of Trichoderma reesei RUT-C30 protoplasts
(1) Trichoderma reesei RUT-C30 protoplast extraction
S1, inoculating spore suspension into 250mL of sterilized trichoderma complete culture medium, and culturing overnight for 15h to obtain hypha;
s2: filtering the mycelium cultured in the step S1, washing the mycelium by using 25mL MW reagent, weighing 3g mycelium, placing the mycelium in a 50mL centrifuge tube, adding OM reagent to 15mL scale marks into the centrifuge tube filled with mycelium, and shaking the centrifuge tube to uniformly disperse the mycelium;
s3: 0.225g of lyase (Sigma L1412) was weighed and dissolved in 5mL of OM reagent, and after filtration and sterilization with a 0.22 μm filter, transferred to a centrifuge tube containing mycelium and OM reagent in step S2, and continued with OMThe volume of the reagent is fixed to 22.5mL of scale mark, and the mixture is uniformly mixed; transferring the mixture into a 250mL glass beaker, wrapping with tinfoil paper, and standing in a 30 ℃ incubator for enzymolysis for 2.5h; taking 1 μl of enzymolysis solution for 2.5h, diluting for 10 times, and counting under microscope to obtain 8.9X10 6 Protoplasts of individual/mL;
s4: adding an equivalent amount of STC reagent into the enzymolysis liquid for enzymolysis for 2.5 hours, slightly shaking and uniformly mixing, filtering into a 50mL round bottom centrifuge tube through a G2 sand core glass funnel, centrifuging for 8 minutes to precipitate protoplasts, pouring out supernatant, placing the centrifuge tube on ice, adding 10mL of STC reagent, rotating the centrifuge tube, and dissolving the protoplasts; centrifuging for 8min, precipitating protoplast, and collecting the protoplast precipitate in figure 2A;
s5: pouring out the supernatant, placing the centrifuge tube on ice, adding 500 mu L of STC reagent, rotating the centrifuge tube, and dissolving protoplast; 1. Mu.L of the protoplast was diluted 10-fold and counted under a microscope to give a final concentration of 2.56X10 8 The protoplast status of each/mL of protoplast under the microscope is shown in FIG. 2B.
(2) Trichoderma reesei RUT-C30 protoplast transformation
S6: subpackaging the extracted protoplast into 50mL centrifuge tubes, placing 200 mu L protoplast in each centrifuge tube on ice, adding 5 mu g exogenous plasmid (PAN 7-1 plasmid, map is shown in figure 1) into each centrifuge tube, mixing evenly 50 mu L25% linear PEG6000 solution, and placing the centrifuge tubes on ice for reaction for 20min;
s7: tilting a centrifuge tube, slowly attaching 2mL of 25% linear PEG6000 solution to the centrifuge tube, culturing for 5min at room temperature, adding 8mL of STC reagent to the centrifuge tube, centrifuging for 8min, precipitating protoplast, pouring out supernatant, adding 100 mu L of STC reagent, rotating the centrifuge tube, and dissolving protoplast;
s8: transferring the transformed protoplast (transformant) to a trichoderma screening culture medium by using a sterile straw, uniformly coating, culturing in a mold incubator at 30 ℃ for about 5-7 days, picking up 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 KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L agar powder. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min), cooling to 45-50deg.C, adding hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) into the ultra-clean bench, and cooling.
(3) Positive transformant detection
22 overnight cultured transformant mycelia and RUT-C30 strain were used to extract DNA using the rapid plant genomic DNA extraction system (DP-321) from Tiangen Biotechnology Co., ltd, and the procedure and reagent composition were as described in DP-321. The extracted DNA was used as a template DNA, and the PCR amplification and agarose gel electrophoresis were performed using pAN7-F, pAN7-R primer to confirm whether the transformation of the above transformant was successful.
The PCR amplification and agarose gel electrophoresis program and flow are as follows:
(1) PCR amplification
The PCR reaction system (20. Mu.L) was as follows:
the PCR amplification procedure was as follows:
(2) Agarose gel electrophoresis
A1% agarose gel was prepared with running buffer TAE (1X) and heated in a microwave oven until completely dissolved. Taking out, shaking, adding nucleic acid dye (Genegreen, tiangen Biochemical technology Co., ltd.) when cooling to avoid scalding hands, and lightly pouring onto electrophoresis tank horizontal plate with sample comb inserted. After the agarose gel had solidified, the comb was carefully pulled out, the horizontal plate of the electrophoresis tank with the gel was placed in the electrophoresis tank, and electrophoresis buffer TAE (1×) was added.
Marker and PCR samples of appropriate size were spotted in the sample well at a spot dose of 5-10. Mu.L and the sample spot order was recorded. The voltage was adjusted to 100-150V, electrophoresis was performed for 20-30min, after which the gel was removed, placed in a G-box, and the electrophoresis bands were checked and analyzed by the GeneSys software, wherein 5 products were present as bands of interest, as shown in FIG. 3. The 5 PCR products were sequenced and the sequencing result was fully aligned with the fragment of interest on the plasmid, as shown in FIG. 4, demonstrating that all 5 transformants were positive transformants (23% positive rate).
EXAMPLE 2 optimization of Trichoderma reesei RUT-C30 protoplast extraction conditions
(1) Screening of mycelium culture time and enzymolysis time
The culture time of mycelium directly affects the yield and status of protoplasts, because mycelium at different age stages has different sensitivity to enzymes due to differences in cell wall thickness, composition, etc. Tender hyphae have thinner cell walls and simpler components, so the hyphae are easier to be hydrolyzed compared with older hyphae, but too tender hyphae cannot normally produce protoplasts with large quantity and good quality. Therefore, it is necessary to search for the culture time of mycelium in order to obtain a large amount of protoplasts with excellent quality. Secondly, the enzymolysis time of hyphae has great influence on the yield and quality of protoplasts. The enzymolysis time is too short, hyphae cannot be fully hydrolyzed, and protoplasts cannot be obtained; the enzymolysis time is too long, the enzyme liquid also damages protoplast, the yield and activity of the protoplast are reduced, and the transformation and regeneration effects are affected.
In order to determine the optimal mycelium culture time and enzymolysis time, the culture time (13 h, 15 h) and the enzymolysis time (1 h,1.5h, 2h, 2.5h, 3 h) were combined as variables under the enzymolysis conditions of 5mg/mL enzyme concentration, 30 ℃ and 100rpm, respectively. Ten groups of comparative experiments were performed, three replicates were set for each group, and the yield of protoplasts at a given time was averaged for each group. As a result, as shown in FIG. 5, mycelia were cultured for 13 hours under the conditions of an enzyme concentration of 5mg/mL, a temperature of 30℃and an enzyme hydrolysis speed of 100rpm, and the yield of protoplasts obtained by the enzyme hydrolysis for 1.5 hours was the highest and found to be 6.1X10 4 individual/mL; culturing mycelium for 15 hr and performing enzymolysis for 2.5 hr to obtain protoplast with highest yield of 3.1X10 5 And each mL. In addition, the mycelia cultured for 15h are found through microscopic examination, and the whole state of the protoplast obtained after enzymolysis for 2.5h is full and round and uniform in size.
Therefore, the optimal cultivation time of the mycelium is 15 hours, and the optimal enzymolysis time is 2.5 hours. Under this parameter, the yield of protoplasts obtained was highest and the situation was good.
(2) Screening of enzyme concentration
The filamentous fungal cell wall components are mainly chitin, protein, lipids, dextran, and the like. The lyase used in the present invention is Sigma L1412 (lyase from Trichoderma harzianum (Trichoderma harzianum)), which has mainly beta-glucanase, cellulase, protease and chitinase activities. Protoplast cell membranes are mainly composed of lipids, proteins, glucans, etc. Therefore, the enzymolysis liquid can cause cracking damage to cell walls and plays a certain role in enzymolysis to cell membranes of protoplasts. Therefore, the enzyme concentration in the enzymatic hydrolysate should be maintained at a suitable level, both to ensure removal of the cell wall and to avoid excessive damage to the protoplasts.
To determine the optimal enzyme concentration, mycelia were subjected to enzymatic hydrolysis at 30℃and 100rpm for 15 hours, 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 groups of comparison experiments are carried out, each group of experiments is repeated three times, and each group of experiment is performedThe yield of the biomass at a specific enzyme concentration was averaged. As a result, as shown in FIG. 6, at enzyme concentrations of 2.5, 5, 10, 15 and 20mg/mL, the concentrations were 0.81X 10, respectively 5 、3.1×10 5 、9.5×10 5 、11×10 5 、9.82×10 5 Protoplasts per mL.
Although the yield of the obtained protoplasts was highest at an enzyme concentration of 15mg/mL, microscopic examination showed that the morphology of the protoplasts began to deform and deteriorate at enzyme concentrations of 15 and 20mg/mL; the yield of protoplast obtained at the enzyme concentration of 10mg/mL is slightly lower than that obtained at the enzyme concentration of 15mg/mL, but the yield difference is not large, and the protoplast state is round and full, so that the protoplast is more suitable for transformation and regeneration.
Therefore, it was found that the optimal enzyme concentration for mycelium enzymolysis was 10mg/mL, and that the yield of the obtained protoplasts was highest and the state was good under this parameter.
(3) Screening of enzymolysis conditions
The enzymolysis condition is also a key point affecting the generation of protoplast, and the enzymolysis temperature is set to be 30 ℃ in consideration of the optimal temperature of trichoderma growth of 30 ℃. To determine the optimal enzymatic hydrolysis conditions of the mycelia, mycelia cultured for 15 hours were enzymatically hydrolyzed for 2.5 hours at an enzyme concentration of 10mg/mL, and the enzymatic hydrolysis conditions were set as single variables at 30℃and 0rpm, 30℃and 50rpm, 30℃and 100rpm, and the enzymatic solutions were counted under a microscope. Three groups of comparison experiments are carried out, each group of experiments is repeated three times, and the yield of protoplasts under specific enzymolysis conditions in each group of experiments is averaged. As a result, as shown in FIG. 7, under the enzymatic hydrolysis conditions of 30℃and 0rpm, the yield of protoplasts obtained by counting under a microscope was 8.9X10 6 individual/mL; under enzymolysis conditions of 30 ℃ and 50rpm, the yield of protoplast is 2.92 multiplied by 10 6 individual/mL; at 30℃and 100rpm, the protoplast yield was 9.5X10 @ 5 And each mL.
Therefore, it was found that the optimal conditions for the enzymatic hydrolysis of mycelium were 30℃and 0rpm, and that the yield of the obtained protoplasts was highest and the conditions were good under these parameters.
(4) Screening of protoplast collection methods
To determine the optimal protoplast collection method, mycelia cultured for 15h were subjected to enzymolysis for 2.5h under an enzymolysis condition of 30℃and 0rpm at an enzyme concentration of 10mg/mL, and the protoplast collection mode was set as a single variable, and protoplasts after enzymolysis were collected by a buffer (0.6M sorbitol, 0.1M Tris-HCl, pH 7.0) extraction method, a G2 sand core funnel (pore size: 30-50 μm, deer head plate) filtration followed by centrifugation, a 400 mesh cell filtration sieve (pore size: 37.5 μm, solarbio/Soliebao) filtration followed by centrifugation, and Miracloth magic filter cloth (pore size: 22-25 μm, merck Millipore/Merck Miybag) filtration followed by centrifugation.
As a result, as shown in FIG. 8, the protoplasts collected by the extraction method were very low in yield and poor in state, and mixed with a large amount of mycelia; the yield of protoplasts collected by filtration using a 400 mesh cell filter screen and Miracloth magic filter cloth was also very low, and the presence of protoplast sediment was hardly seen by the naked eye after centrifugation.
And the clear macroscopic protoplast aggregate pellet could be collected after filtration through G2 sand funnel, washing and centrifugation (fig. 2A). The precipitate was gently dissolved with 500. Mu.L of STC reagent and collected at a concentration of 2.56X10 8 Protoplast (FIG. 2B) with good condition at a volume of per mL (20 mL of the pre-enzymolysis system, 8.9X10% of the protoplast concentration in the raw enzymolysis solution) 6 Protoplast recovery was 72% per mL.
Therefore, the best collection mode of protoplast is G2 sand core funnel filtration, washing and centrifugation, under which the yield of the obtained protoplast is highest and the state is good.
EXAMPLE 3 optimization of Trichoderma reesei RUT-C30 protoplast transformation conditions
Polyethylene glycol (PEG) solution is added in the protoplast transformation process, and the principle is as follows: PEG can form a molecular bridge between protoplast and exogenous DNA, promote the mutual adhesion of the protoplast and the exogenous DNA to form a precipitate, and simultaneously cause the disturbance of the surface charge of the cell membrane of the protoplast, thereby changing the permeability of the cell membrane and promoting the absorption of the exogenous DNA by the protoplast. Based on this, the inventors tried to promote fusion of exogenous DNA with protoplasts by adding PEG solutions of different molecular structures during transformation, increasing the positive transformation rate of protoplasts.
A: trichoderma reesei RUT-C30 protoplast was extracted in the same manner as in example 1, and in protoplast transformation, PEG6000 was replaced with PEG4000 at a constant concentration, and the rest was performed in the same manner as in example 1.
B: trichoderma reesei RUT-C30 protoplast was extracted in the same manner as in example 1, and in protoplast transformation, PEG6000 was replaced with double arm PEG (molecular weight 4000), and the other steps were performed in the same manner as in example 1.
C: trichoderma reesei RUT-C30 protoplast was extracted in the same manner as in example 1, and in protoplast transformation, PEG6000 was replaced with double arm PEG (molecular weight 6000), and the other steps were performed in the same manner as in example 1.
D: trichoderma reesei RUT-C30 protoplast was extracted in the same manner as in example 1, and in protoplast transformation, PEG6000 was replaced with three-arm PEG (molecular weight 4000), and the other steps were performed in the same manner as in example 1.
E: trichoderma reesei RUT-C30 protoplast was extracted in the same manner as in example 1, and in protoplast transformation, PEG6000 was replaced with three-arm PEG (molecular weight 6000), and the other steps were performed in the same manner as in example 1.
22 transformants were picked on a Trichoderma screening medium and propagated as described in example 1 to obtain 22 transformant mycelia. PCR amplification and agarose gel electrophoresis were performed using pAN7-F, pAN7-R primers with hypha DNA as template DNA, and the success of transformation of the above transformants was verified. The test results are shown in the following table:
TABLE 1
From the data presented in the above table, it can be seen that PEG as a bridge between protoplasts and exogenous DNA has an important influence on whether exogenous DNA is incorporated into the protoplasts. Meanwhile, the inventors also found that the molecular weight of PEG had no effect on the positive conversion rate of the transformant, while the number of arms of PEG had a substantial effect on the positive conversion rate, and as the number of arms of PEG increased, the number of positive transformants of the transformant also increased. This is because when PEG is double-armed or triple-armed, the ligation of protoplasts to foreign DNA is better and the foreign DNA is better incorporated into the protoplasts than linear PEG.
EXAMPLE 4 Trichoderma selection Medium optimization
The unexpected discovery of the technical staff in the experiment is that 0.5-1.5g/L betaine and 10-15mL/L dandelion extract are added on the basis of the conventional trichoderma reesei screening culture medium, the method is named as an enhanced screening culture medium, and the positive rate of the trichoderma reesei transformant obtained by final screening is higher. In order to optimize the optimal addition ratio, the present invention sets the following 3 concentrations of the enhancement screening medium.
The trichoderma screening culture medium comprises the following components: 1M sorbitol, 20g/L glucose, 15g/L KH 2 PO 4 ，5g/L(NH 4 ) 2 SO 4 ，0.6g/L MgSO 4 ·7H 2 O，0.6g/L CaCl 2 ，0.005g/L FeSO 4 ·7H 2 O，0.0016g/L MnSO 4 ·H 2 O，0.0014g/L ZnSO 4 ·7H 2 O，0.002g/L CoCl 2 20g/L agar powder. Sterilizing in high pressure steam sterilizing pot (121deg.C, 25 min), cooling to 45-50deg.C, adding hygromycin B (final concentration 175 μg/mL) and ampicillin (final concentration 100 μg/mL) into the ultra-clean bench, and cooling.
A: the method for extracting and transforming Trichoderma reesei RUT-C30 protoplast is the same as in example 1, transferring the transformed protoplast (transformant) to an enhanced screening culture medium I, uniformly coating, culturing in a mold incubator at 30 ℃ for about 5-7 days, picking up 22 transformants, transferring to a Trichoderma agar medium for propagation, scraping spores to obtain spore suspension, and inoculating the spore suspension to a Trichoderma seed medium for overnight culture to obtain hyphae.
B: the method for extracting and transforming Trichoderma reesei RUT-C30 protoplast is the same as in example 1, transferring the transformed protoplast (transformant) to an enhanced screening medium II, uniformly coating, culturing in a mold incubator at 30 ℃ for about 5-7 days, picking up 22 transformants, transferring to a Trichoderma agar medium for propagation, scraping spores to obtain spore suspension, and inoculating the spore suspension to a Trichoderma seed medium for overnight culture to obtain mycelia.
C: the method for extracting and transforming Trichoderma reesei RUT-C30 protoplast is the same as in example 1, transferring the transformed protoplast (transformant) to an enhanced screening medium III, uniformly coating, culturing in a mold incubator at 30 ℃ for about 5-7 days, picking up 22 transformants, transferring to a Trichoderma agar medium for propagation, scraping spores to obtain spore suspension, and inoculating the spore suspension to a Trichoderma seed medium for overnight culture to obtain hyphae.
The positive conversion rate was detected by using the obtained hypha DNA as a template DNA in the same manner as in example 1, a target band was found by PCR amplification and agarose gel electrophoresis, the PCR product on the band was sequenced, and the result was that the sequence of the product to be examined was completely aligned with the target fragment on the plasmid, and the transformants corresponding to the target band were positive transformants, the numbers of which are shown in the following table.
TABLE 2
As can be seen from the above table data, compared with the original screening medium, positive conversion rate is improved when betaine and dandelion leaching solutions with different concentrations are added into the screening medium, mainly because the sterilization effect of the betaine and dandelion can eliminate part of false positive hygromycin resistant strains along with the increase of the addition amount of the betaine and the dandelion, so that more strains containing hygromycin resistant genes are selected from the selected transformants, and the detected positive conversion rate is higher. Wherein, when the concentration of betaine is 1.5g/L and the concentration of dandelion extract is 15mL/L, the positive conversion rate of trichoderma reesei protoplast is highest.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> institute of plant Material at national academy of sciences
SHANGHAI HANHE BIOLOGICAL NEW MATERIAL TECHNOLOGY Co.,Ltd.
<120> a method for genetic transformation of Trichoderma reesei RUT-C30 protoplast
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Claims (6)
1. A Trichoderma reesei RUT-C30 genetic transformation method comprises the following steps:
a) Mixing the exogenous plasmid and Trichoderma reesei RUT-C30 protoplast in polyethylene glycol aqueous solution, and ice-bathing for 10-20min;
b) Continuously adding polyethylene glycol aqueous solution with the volume of 8-10 times into the mixed system, performing transformation culture at room temperature for 5-6min, adding STC reagent to precipitate transformed protoplast, and suspending again;
c) Transferring the transformed protoplast into a trichoderma screening culture medium for 5-7 days, picking up a transformant, transferring into a trichoderma agar culture medium for propagation to obtain a transformant spore suspension, inoculating the spore suspension into a trichoderma seed culture medium, and culturing overnight to obtain transformant hyphae;
wherein the PEG in the step a) is multi-arm polyethylene glycol with the molecular weight of 4000-6000, the multi-arm polyethylene glycol comprises one of double-arm polyethylene glycol, three-arm polyethylene glycol and four-arm polyethylene glycol, and the polyethylene glycol aqueous solution comprises the following components: 250-300g/L multi-arm polyethylene glycol, 40-60mL 1M CaCl 2 5-15mL of 1M Tris;
the structural formula of the double-arm polyethylene glycol is as follows:
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;
wherein the screening culture medium in the step c) contains 1.5g/L betaine and 15mL/L dandelion extract.
2. The genetic transformation method according to claim 1, wherein the aqueous polyethylene glycol solution comprises the following components: 250g/L multi-arm polyethylene glycol, 50mL 1M CaCl 2 10mL of 1M Tris.
3. The genetic transformation method according to claim 1, wherein the trichoderma reesei RUT-C30 protoplast extraction method is as follows:
1) Inoculating Trichoderma reesei RUT-C30 spore suspension into Trichoderma complete culture medium, and culturing for 13-15 hr to obtain mycelium;
2) Carrying out enzymolysis on hyphae in an enzymolysis liquid containing lyase for 1-3 hours, wherein the concentration of the lyase in the enzymolysis liquid is 2.5-20mg/mL;
3) Trichoderma reesei RUT-C30 protoplasts were collected by filtration through a G2 glass sand core funnel.
4. The genetic transformation method according to claim 3, wherein the Trichoderma reesei RUT-C30 hypha obtained by culturing the Trichoderma reesei RUT-C30 hypha in the step (1) for 15 hours.
5. The genetic transformation method according to claim 3, wherein the lyase in the 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.
6. The genetic transformation method according to claim 3, wherein step (3) further comprises washing and centrifuging the trichoderma reesei RUT-C30 protoplasts collected by filtration through a G2 glass sand core funnel, the G2 glass sand core funnel having a pore size of 30 to 50 μm.
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| CN1385519A (en) * | 2002-04-19 | 2002-12-18 | 浙江大学 | Li's Trichoderma strains and use thereof |
| CN112175842A (en) * | 2020-11-13 | 2021-01-05 | 上海汉禾生物新材料科技有限公司 | Method for producing cellulase with high yield by fermenting trichoderma reesei |
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| Title |
|---|
| Gaston E. Ortiz et al..Characterization, optimization, and scale-up of cellulases production by trichoderma reesei cbs 836.91 in solid-state fermentation using agro-industrial products.《Bioprocess and Biosystems Engineering》.2015,第第38卷卷2117-2128. * |
| 刘士旺 ; 尤玉如 ; 黄俊 ; 吴元锋 ; 毛建卫 ; .聚乙二醇介导木霉的原生质体转化.中国酿造.(第01期),第36-37页. * |
| 顾斌涛 ; 熊大维 ; .农杆菌介导转化木霉原生质体的研究.生物化工.(第02期),全文. * |
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