Application of nicotinamide in improving growth performance of pleurotus ostreatus

Abstract

The application belongs to the technical field of edible fungi, and relates to cultivation of pleurotus ostreatus, in particular to application of nicotinamide (NAM) in improving growth performance of pleurotus ostreatus. The specific operation is as follows: (1) different concentrations of nicotinamide are added in the culture medium of pleurotus ostreatus; (2) the influence of adding different concentrations of nicotinamide on the high-temperature stress resistance of pleurotus ostreatus is analyzed. The research results show that adding nicotinamide in the culture medium of pleurotus ostreatus can obviously improve the high-temperature stress recovery growth rate of mycelium of pleurotus ostreatus.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of edible fungi, and relates to cultivation of Pleurotus ostreatus, in particular to application of nicotinamide (NAM) in improving growth performance of Pleurotus ostreatus. BACKGROUND

[0002] The scientific name of Pleurotus ostreatus is (Jacq.) P.Kumm. Pleurotus ostreatus (Jacq.) P.Kumm. belongs to Basidiomycota, Agaricomycetes, Agaricales, Pleurotaceae and Pleurotus in taxonomy, is one of the most important edible fungi, and is also a fungus for both medicine and food, and has the effects of treating waist and leg pain, numbness of hands and feet, and relieving tumor.

[0003] High temperature is one of the main abiotic stress factors that limit the growth and development of edible fungi. High temperature stress can inhibit the growth of edible fungi, cause a large burst of reactive oxygen species, lead to lipid peroxidation, damage cell structure, and reduce the activity of enzymes such as laccase and cellulase. High temperature stress can also cause the mycelium to produce pigment and cause wood rot, resulting in huge losses in cultivation production. Improving the ability of Pleurotus ostreatus to resist high temperature stress helps to improve the yield of Pleurotus ostreatus. Patent CN112126632A discloses a method for improving the laccase activity of Pleurotus ostreatus, which uses an induction medium containing mixed metal ions to culture and obtain Pleurotus ostreatus with high laccase activity. Patent CN103449914A discloses the application of trehalose in prolonging the life of Pleurotus ostreatus. However, there is no relevant research on the growth of edible fungi under high temperature conditions. In order to improve the performance of Pleurotus ostreatus under high temperature stress, the present application has conducted in-depth research. SUMMARY

[0004] In view of the above technical problems, the present application provides application of nicotinamide in improving growth performance of Pleurotus ostreatus, which solves the problem of growth of Pleurotus ostreatus under high temperature stress.

[0005] The technical scheme of the present application is as follows:

[0006] One of the purposes of the present application is to protect the application of nicotinamide in improving heat stress resistance of Pleurotus ostreatus.

[0007] The second purpose of the present application is to protect the application of nicotinamide in improving the recovery ability of Pleurotus ostreatus under heat stress.

[0008] The third purpose of the present application is to protect the application of nicotinamide in improving the growth rate of Pleurotus ostreatus.

[0009] The fourth purpose of the present application is to protect the application of nicotinamide in promoting the growth of mycelium of Pleurotus ostreatus.

[0010] The fifth object of the present application is to protect the use of nicotinamide in improving the laccase activity in the mycelium of P. ostreatus.

[0011] In the above use, the laccase activity in the mycelium of P. ostreatus is increased to 6.28 times of the original by adding nicotinamide in the culture medium / substrate of P. ostreatus under heat stress.

[0012] In the above use, the use of P. ostreatus is realized by regulating the acetylation modification level through the nicotinamide.

[0013] The nicotinamide regulates the acetylation modification level through Sirt2.

[0014] The operation steps for realizing the above use are as follows: adding nicotinamide in the culture medium / substrate, and then inoculating P. ostreatus liquid to culture.

[0015] Further, the adding concentration of the nicotinamide is 1-10 mM.

[0016] The formula of the culture medium is as follows: yeast extract powder 5 g / L, glucose 20 g / L, VB1 0.01 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, and agar 2%.

[0017] The formula of the substrate is as follows: dry material and water in a mass ratio of 1:1.3, wherein the dry material includes 88% cottonseed hulls, 10% wheat bran, and 2% lime.

[0018] The present application has the following beneficial effects:

[0019] (1) The present application first studies the change of the acetylation level in P. ostreatus, and the role of acetylation modification in large fungi has not been reported yet. The response mechanism of edible fungi to heat stress is analyzed, which can find a path for the innovation of heat-resistant cultivation technology.

[0020] (2) The present application first adds nicotinamide in the plate substrate and cultivation substrate of P. ostreatus to improve the ability of P. ostreatus to resist high-temperature stress.

[0021] (3) The present application can obviously improve the high-temperature stress recovery growth rate of P. ostreatus mycelium by adding different concentrations of nicotinamide in the culture medium.

[0022] (4) The present application can obviously improve the time of P. ostreatus mycelium present primordium by adding nicotinamide in the culture medium.

[0023] (5) The present application first finds that deacetylase SIRT2 can deacetylate GST to improve the enzyme activity of GST, which can remove active oxygen to protect the structure of plant cell membrane and protein activity, and plays an important role in the response mechanism of stress resistance. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 A graph showing the changes in total protein acetylation levels at different times during heat stress in the rough-skinned lateral ear.

[0026] Figure 2 Key regulatory proteins (SIRT2 and GST) were screened for acetylated proteomes; A represents GO enrichment; B represents protein domain enrichment; and C represents the key regulatory proteins identified in this study. Black dots indicate the number and location of lysine acetylation sites in the target proteins.

[0027] Figure 3 The effects of exogenous addition of SIRT substrate NAM on mycelial growth and recovery growth after heat stress in Pleurotus ostreatus agar plates under normal culture and heat stress conditions were investigated. A represents the mycelial growth rate in plates cultured under normal conditions with low concentrations of SIRT substrate NAM; B represents the mycelial growth rate after heat stress at 40℃ with low concentrations of SIRT substrate NAM; C represents the mycelial growth rate in plates cultured under normal conditions with high concentrations of SIRT substrate NAM; and D represents the mycelial growth rate after heat stress at 40℃ with high concentrations of SIRT substrate NAM.

[0028] Figure 4 The study investigated the effect of adding the optimal concentration of NAM on the growth of bag-cultured Pleurotus ostreatus mycelium and its recovery growth after heat stress. In this study, A represents the growth rate of bag-cultured mycelium under normal culture conditions, and B represents the recovery growth rate of bag-cultured mycelium under 40℃ heat stress.

[0029] Figure 5 The effects of exogenous NAM addition on the primordia of Pleurotus ostreatus under normal culture and heat stress conditions.

[0030] Figure 6 The effects of exogenous NAM addition on the activity of matrix-degrading enzymes of Pleurotus ostreatus; where A represents the effect of exogenous NAM addition on laccase activity; B represents the effect of exogenous NAM addition on filter paper cellulase activity; and C represents the effect of exogenous NAM addition on carboxymethyl cellulase activity.

[0031] Figure 7A is the H2O2 content of the mycelium of the exogenous NAM added strain; B is the NADPH content of the mycelium of the exogenous NAM added strain; C is the GSH content of the mycelium of the exogenous NAM added strain.

[0032] Figure 8 A is the influence of SIRT2 deacetylation of GST1 and GST1 mutants on the enzyme activity; B is the influence of SIRT2 deacetylation of GST2 and GST2 mutants on the enzyme activity. DETAILED DESCRIPTION

[0033] The technical solutions of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0034] The test strain of the present application is Pleurotus ostreatus strain Xin 831 Pleurotus ostreatus The strain is currently a widely cultivated variety in China.

[0035] The solid GYE culture medium has the following components: yeast extract powder 5 g / L, glucose 20 g / L, VB1 0.01 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, and agar 2%.

[0036] The liquid GYE culture medium has the following components: yeast extract powder 5 g / L, glucose 20 g / L, VB1 0.01 g / L, MgSO4·7H2O 0.5 g / L, and KH2PO4 1 g / L.

[0037] The solid SA culture medium has the following components: sucrose 20 g / L, asparagine 0.88 g / L, ammonium dihydrogen phosphate 2.0 g / L, L-valine 1.0 g / L, dipotassium hydrogen phosphate trihydrate 0.224 g / L, potassium dihydrogen phosphate 0.803 g / L, magnesium sulfate heptahydrate 0.99 g / L, calcium chloride 0.02 g / L, trace element solution 5 mL / L, vitamin nucleotide solution 5 mL / L, and agar powder 2%.

[0038] Example 1

[0039] In this embodiment, the following steps are used to detect the changes in the acetylation level of total proteins of Pleurotus ostreatus under heat stress at different times.

[0040] (1) Preparation of solid strain of P. ostreatus. In the clean bench, the edge mycelium block of P. ostreatus was punched with a sterile puncher with a diameter of 5 mm, inoculated on a GYE solid plate, and cultured at 25°C in the dark until the mycelium grew over the plate.

[0041] (2) The full plate was placed in a 40°C incubator for high-temperature stress for 0 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h, and then the mycelium was collected, with 3 plates for each treatment and 3 repetitions.

[0042] (3) The collected mycelium was quickly frozen in liquid nitrogen and ground, and total protein was extracted by adding a protein extraction solution.

[0043] (4) Western blot was performed using the extracted total protein, and acetylated antibodies were used to detect the change in the acetylation level of the total protein.

[0044] The results are shown in Figure 1 , and it can be seen from Figure 1 that with the increase of the heat stress time, the acetylation level of the mycelium of P. ostreatus showed a trend of first decreasing and then increasing. The acetylation level decreased at 0-0.5 h, and gradually increased after 0.5 h. The acetylation level increased significantly at 24 h.

[0045] Example 2

[0046] In this example, the key regulatory proteins of acetylation were screened from proteomics:

[0047] (1) Preparation of solid strain of P. ostreatus. In the clean bench, the edge mycelium block of P. ostreatus was punched with a sterile puncher with a diameter of 5 mm, inoculated on a GYE solid plate, and cultured at 25°C in the dark until the mycelium grew over the plate.

[0048] (2) The full plate was placed in a 40°C incubator for high-temperature stress for 0 h and 24 h, and then the mycelium was collected, with 3 plates for each treatment and 3 repetitions.

[0049] (3) The collected mycelium was quickly frozen in liquid nitrogen and sent to a company for acetylation modification quantitative proteomics sequencing.

[0050] (4) The results of proteomics were analyzed to screen the key regulatory proteins.

[0051] The analysis results are shown in Figure 2 , GO enrichment ( Figure 2 A) and protein domain enrichment ( Figure 2 B) analysis showed that glutathione transferase (GST) and some proteins related to oxidation and reduction were acetylated. Therefore, the focus was placed on the proteins related to oxidation and reduction, and from the acetylation proteomics database, proteins such as Figure 2C shows several redox-related proteins, as well as acetyltransferases and deacetylases. This demonstrates that acetylation modification plays an important role in the thermal stress experienced by *Pleurotus ostreatus*.

[0052] Example 3

[0053] This example investigated the effects of nicotinamide addition on the mycelial growth of *Pleurotus ostreatus* agar plates under normal and heat stress cultures, as well as the recovery growth after heat stress. The steps are as follows:

[0054] (1) Preparation of solid culture of Pleurotus ostreatus. In a clean bench, use a 5 mm diameter sterile punch to take mycelial blocks from the edge of Pleurotus ostreatus and inoculate them onto GYE solid plates. Incubate in the dark at 25°C until the mycelium has fully colonized the plate.

[0055] (2) Preparation of culture plates containing different concentrations of nicotinamide. In a clean bench, nicotinamide stock solution (2M) prepared with ultrapure water was added to melted solid GYE medium to make the exogenous nicotinamide concentrations in the medium 0mM, 0.25mM, 0.5mM, 1mM, 2mM, 4mM, 10mM, 20mM, 30mM, 40mM and 50mM respectively. After shaking well, the plates were poured. Hyphae blocks of Pleurotus ostreatus on the edge were punched with a 5mm diameter sterile punch and inoculated into GYE medium plates containing different concentrations of nicotinamide. The plates were then incubated in the dark at 25°C.

[0056] (3) Determination of mycelial growth rate and recovery growth rate after high-temperature stress in plate experiments. Inoculated plates were incubated in the dark at 25℃ until one-third of the plate was filled. A line was drawn around the edge of the mycelium on the bottom of the plate, and the date was recorded. After recording, the plates were placed in a 40℃ incubator for 24 hours of high-temperature stress, and then returned to a 25℃ incubator to continue incubation until one group of mycelium filled the plate. A line was drawn again, and the date was recorded. The growth length of the mycelium in each group was measured, and the mycelial growth rate and recovery growth rate were calculated (growth rate (cm / d) = mycelial growth length (cm) / mycelial growth days (days); recovery growth rate (cm / d) = mycelial recovery growth length (cm) / mycelial recovery growth days (days)). Five plates were used for each treatment, with three replicates.

[0057] The results are as follows Figure 3 As shown, by Figure 3 As shown in A and B, the recovery growth rate of *Pleurotus ostreatus* hyphae in the experimental groups at low concentrations was higher than that in the control group. Except for the experimental group with 10 mM nicotinamide, which showed no significant difference in recovery growth rate between the two groups (P>0.05), the recovery growth rates of all other experimental groups at low concentrations were significantly higher than those in the control group (P<0.05). Figure 3As shown in C and D, the recovery growth rate of *Pleurotus ostreatus* mycelia in the experimental groups with low concentrations was higher than that in the control group. Except for the experimental groups with nicotinamide concentrations of 2 mM and 4 mM, which showed no significant difference in recovery growth rate compared to the control group (P>0.05), the recovery growth rates of the 0.25, 0.5, 1, and 2 mM experimental groups were significantly higher than those in the control group (P<0.05). Therefore, adding a certain amount of nicotinamide to the culture medium can improve the growth rate of *Pleurotus ostreatus* mycelia and enhance their resistance to high-temperature stress.

[0058] Example 4

[0059] This example studies the effect of adding the optimal concentration of NAM on the mycelial growth of bag-cultured Pleurotus ostreatus and its recovery growth after heat stress. The steps are as follows:

[0060] (1) Preparation of liquid spawn for oyster mushrooms. In a clean bench, use a 5mm diameter sterile punch to collect mycelial blocks from the edge of the oyster mushroom and inoculate them into 200mL of GYE liquid medium (containing 8-10 glass beads). Incubate at 25℃ and 160rpm in a shake flask until the spawn becomes a thick, porridge-like consistency (approximately 5-7 days). Expand the prepared liquid spawn to a scale-up level of 5% (V / V).

[0061] (2) Preparation of cultivation bags with different concentrations of nicotinamide. The nicotinamide concentration was set to 0 mM and 1 mM. The culture medium formula was: medium:water = 1:1.3, of which the dry medium formula was: cottonseed hulls 88%, wheat bran 10%, lime 2%. The mixture was thoroughly stirred until it could be shaped by hand without releasing water, and left at room temperature for half an hour. The culture medium was packed into 14cm×28cm×0.004cm polypropylene folded corner bags at a rate of 500g wet medium / bag, sealed with plastic rings, and sterilized by high-pressure steam at 126℃ for 3 hours. After sterilization, the bags were removed and cooled to room temperature. The corresponding mass of nicotinamide powder was added to the shaken liquid inoculum, and then inoculated into the cooled bags.

[0062] (3) Determination of the recovery growth rate of oyster mushroom mycelium after high temperature stress in bagged cultivation. When the mycelium grew to near the middle of the bag, the bag was placed in a 40℃ incubator for 48 hours of high temperature stress treatment. Then the bag was returned to a 25℃ incubator and cultured until a certain group of mycelium filled the bag. The length of mycelium recovery growth in each group was measured, and the mycelium recovery growth rate was further calculated (recovery growth rate (cm / d) = recovery growth length (cm) / recovery growth days (days)). Each treatment consisted of 10 bags, with 3 replicates.

[0063] The results are as follows Figure 4 As shown, by Figure 4 As shown in Figure A, the mycelial growth rate of *Pleurotus ostreatus* in the experimental group with added 1 mM nicotinamide was significantly higher than that in the control group (P < 0.05). Figure 4B As can be seen from the table, the growth rate of the experimental group with 1 mM nicotinamide added was significantly higher than that of the control group (P<0.05). It can be seen that adding a certain amount of nicotinamide in the culture medium can promote the growth of the mycelium and improve the ability of the mycelium of Pleurotus ostreatus to resist high temperature stress.

[0064] Example 5

[0065] This example adds nicotinamide to the effect of high temperature stress on the growth of the mycelium of Pleurotus ostreatus, using the following steps:

[0066] (1) Preparation of solid spawn of Pleurotus ostreatus. In the clean bench, use a sterile puncher with a diameter of 5 mm to punch the edge mycelium block of Pleurotus ostreatus, inoculate it into GYE solid plates, and cultivate at 25°C in the dark until the mycelium covers the plate.

[0067] (2) Preparation of plates with different concentrations of nicotinamide in the present primordium medium. In the clean bench, add the nicotinamide stock solution (2M) prepared with ultrapure water to the melted solid SA medium to make the exogenous nicotinamide concentration in the medium 0 mM and 1 mM, respectively. Shake well and pour the plates. Use a sterile puncher with a diameter of 5 mm to punch the edge mycelium block of Pleurotus ostreatus, inoculate it into the SA medium plates containing different concentrations of nicotinamide, and cultivate at 25°C in the dark.

[0068] (3) Effect of adding nicotinamide on the present primordium of Pleurotus ostreatus in plate test.

[0069] After inoculation, the plates were incubated at 25°C in the dark until they were almost full. The date was recorded. After recording, some plates were placed in a 40°C incubator for 24 hours of high temperature stress. Then all the plates were placed in a 15°C incubator to observe, take pictures and record the different development stages, and record the date. The time of each group entering different stages is shown in Table 1, with 5 plates for each treatment and 3 repeats.

[0070] Table 1 Time of Pleurotus ostreatus entering different stages

[0071]

[0072] The results of the photographs are shown in Figure 5 As can be seen from Figure 5 , the mycelium grows white and dense after adding NAM under normal culture. As can be seen from Figure 5 , the time of strobilus stage and fruiting body stage is advanced after adding NAM under normal culture; the time of primordium stage, strobilus stage and fruiting body stage is advanced in the heat stress group after adding NAM. It is shown that after adding NAM, the strobilus stage and fruiting body stage of Pleurotus ostreatus are promoted, the growth cycle is shortened, which is beneficial to the growth of Pleurotus ostreatus. It can be seen that adding a certain amount of nicotinamide in the culture medium can improve the growth ability of the mycelium of Pleurotus ostreatus

[0073] Example 6

[0074] The present embodiment studies the effect of exogenous NAM on substrate degradation enzyme activity of P. ostreatus, using the following steps:

[0075] (1) P. ostreatus solid strain preparation. In the clean bench, use a 5mm diameter sterile punch to take the edge mycelium block of P. ostreatus, inoculate it into GYE solid plate, and cultivate at 25°C in the dark until the mycelium covers the plate.

[0076] (2) Preparation of medium plates containing different concentrations of nicotinamide. In the clean bench, add the nicotinamide stock solution (2M) prepared with ultrapure water to the melted solid GYE medium to make the exogenous nicotinamide concentration in the medium 0mM and 1mM, respectively. Shake well and pour the plate, then soak the sterilized glass paper in sterile water, and use sterile forceps to spread it on the cooled plate. Use a 5mm diameter sterile punch to take the edge mycelium block of P. ostreatus, inoculate it into the GYE medium plate containing different concentrations of nicotinamide, and cultivate at 25°C in the dark.

[0077] (3) Determination of P. ostreatus mycelium substrate degradation enzyme activity after adding nicotinamide in plate test

[0078] The inoculated plate was cultured at 25°C in the dark until the plate was full, then the mycelium liquid was collected, frozen in liquid nitrogen, and stored at -80°C. The laccase, filter paper cellulase, and carboxymethyl cellulase enzyme activities were determined according to the corresponding enzyme activity determination method.

[0079] The results are shown in Figure 6, which shows that Figure 6 A, the laccase activity of P. ostreatus mycelium decreased by 69.31% under heat stress, and 1mM NAM increased the laccase activity to 1.68 times the original under normal conditions, and the effect was more obvious under heat stress, and the laccase activity increased to 3.25 times the original after adding NAM. As shown in Figure 6 B, the filter paper cellulase activity of P. ostreatus mycelium decreased by 86.48% under heat stress, and 1mM NAM increased the filter paper cellulase activity to 1.23 times the original under normal conditions, and the effect was more obvious under heat stress, and the filter paper cellulase activity increased to 6.28 times the original after adding NAM. As can be seen, the addition of a certain amount of nicotinamide in the culture medium can improve the ability of P. ostreatus mycelium to resist high temperature stress. As shown in Figure 6 C, the carboxymethyl cellulase activity of P. ostreatus mycelium decreased by 95.15% under heat stress, and 1mM NAM increased the carboxymethyl cellulase activity to 1.15 times the original under normal conditions, and the effect was more obvious under heat stress, and the carboxymethyl cellulase activity increased to 4.86 times the original after adding NAM. As can be seen, the addition of a certain amount of nicotinamide in the culture medium can improve the ability of P. ostreatus mycelium to resist high temperature stress.

[0080] Example 7

[0081] The present embodiment studies the effect of exogenous NAM on the oxidation-reduction homeostasis of P. ostreatus, using the following steps:

[0082] (1) Preparation of P. ostreatus solid strain. In a clean bench, a 5mm-diameter sterile puncher was used to punch the edge mycelium block of P. ostreatus, which was inoculated on a GYE solid plate and cultured at 25°C in the dark until the mycelium grew over the plate.

[0083] (2) Preparation of medium plates containing different concentrations of nicotinamide. In a clean bench, 2M NAM stock solution prepared with ultrapure water was added to the melted solid GYE medium to obtain a medium containing 0mM and 1mM exogenous NAM, respectively. After shaking, the medium was poured into plates, and sterile glass paper soaked in sterile water was used to cover the plates. A 5mm-diameter sterile puncher was used to punch the edge mycelium block of P. ostreatus, which was inoculated on the GYE medium plates containing different concentrations of NAM and cultured at 25°C in the dark.

[0084] (3) Determination of oxidation-reduction related indicators of P. ostreatus with exogenous NAM. The inoculated plates were cultured at 25°C in the dark until the plates were full, then the mycelium was collected, frozen in liquid nitrogen, and stored at -80°C. The contents of H2O2, NADPH, and GSH were determined according to the corresponding determination methods.

[0085] The results are shown in Figure 7 As shown in Figure 7 A, the content of H2O2 in the mycelium of P. ostreatus increased under heat stress, and decreased after adding 1mM NAM under normal conditions, which was more obvious under heat stress. The content of H2O2 decreased more after adding NAM. As shown in Figure 7 B and C, the contents of NADPH and GSH in the mycelium of P. ostreatus decreased under heat stress, and increased after adding 1mM NAM, which was more obvious under heat stress. Thus, adding a certain amount of NAM in the culture medium can improve the ability of P. ostreatus mycelium to resist high temperature stress, reduce the content of H2O2, increase the contents of NADPH and GSH, and play an important role in maintaining the oxidation-reduction homeostasis of P. ostreatus.

[0086] Example 8

[0087] The present embodiment studies the effect of SIRT2 on the activity of GST through deacetylation, using the following steps:

[0088] Prokaryotic expression and protein purification of deacetylase SIRT2 and target proteins glutathione transferase GST1 and GST2. The deacetylase SIRT2 and key target proteins screened from acetylation data were cloned, and the pET-28a empty plasmid and CDS fragments of the deacetylase SIRT2 and key target proteins were subjected to double enzyme digestion reaction, and the enzyme digestion products were recovered by OMEGA gel recovery kit. The recovered fragments and the vector were ligated by T4 ligase, and then the ligation products were transformed into E. coli DH5a competent cells, and positive clone screening and plasmid extraction were performed by colony PCR. Referring to the E. coli transformation method, 1 μL of plasmid was transformed into E. coli BL21 expression strain, and positive transformants were screened and identified.

[0089] The correct transformants were picked and verified in fresh LB liquid medium containing 100 μg / mL Kana, activated at 37 °C and 220 rpm for about 2 h, and stopped when the OD600 of the bacterial solution was between 0.4 and 0.6; in the experimental group, 1 mM IPTG solution was added, and induced at 37 °C and 220 rpm for 4 h; after induction, the bacterial cells were centrifuged at 4 °C and 6000 rpm, and the bacterial cells were collected into a centrifuge tube, the supernatant was discarded, and the bacterial cells were resuspended with appropriate PBS buffer, the residual culture medium was removed, and the bacterial cells were centrifuged again under the above conditions, the supernatant was discarded, and the bacterial cells were resuspended with appropriate PBS buffer; the bacterial solution was broken by ultrasonic cell disrupter until the bacterial solution was clear, and then centrifuged at 4 °C and 6000 rpm, and the supernatant and precipitate were collected respectively, and the precipitate was resuspended with appropriate PBS buffer. Stored in a 4 °C refrigerator for standby, and then verified by SDS-PAGE for protein size.

[0090] Take the cultured bacteria liquid 2 mL into 200 mL liquid LB containing 200 μL Kana, 37°C shaking table, 220 rpm, culture to bacteria liquid OD600=0.4;Add 1mM of IPTG, put into 37°C shaking table 220pm, induce 4h;The 200mL bacteria liquid after induction 4℃, 8000rpm, 3min, collect into 50 mL centrifuge tube, add 20mL PBS Buffer resuspend, 4℃, 8000rpm, 2min;Pour off the supernatant, add 20mL PBS Buffer resuspend, put on ice for standby;The collected bacteria liquid is broken by ultrasonic disrupter; After breaking, 4℃, 8000rpm, centrifuge for 30 min, the supernatant is filtered twice with a filter head with a pore size of 0.22 μm, and placed on ice for standby;Assemble HisTrapTMHP preloaded nickel column, install a 0.22 μm filter head above the nickel column to prevent impurities from blocking the nickel column, then use a 10 mL medical syringe to draw 5 mL sterile water to flush the nickel column, and then add 5 mL Binding Buffer to balance the nickel column;The prepared cell breakage supernatant is loaded onto the column and repeated 5 times;Respectively, 20mM, 50mM, 100mM, 150mM, 200mM, 250mM, 300mM, 500mM are used to wash the column protein, and 2mL centrifuge tube is used to collect the eluate;After elution, the nickel column is washed with 5mL sterile water, then 5mL Binding Buffer is used to balance the nickel column, finally 2mL prepared 20% ethanol is used to column seal, the nickel column is placed in 4℃ refrigerator, and the protein eluate is verified by SDS-PAGE.

[0091] Prokaryotic expression and protein purification of mutants of glutathione transferases GST1 and GST2. The acetylation modification sites detected by omics data of GST1 and GST2 were subjected to site-directed mutagenesis using a kit, and then a prokaryotic expression vector was constructed for protein purification, the method steps being the same as above.

[0092] In vitro deacetylation reaction of deacetylase SIRT2 and target proteins glutathione transferases GST1 and GST2 and their mutants. The purified proteins were determined for concentration by BCA protein concentration determination kit, and the deacetylation reaction was 10 ug GST protein, 10 ug SIRT2 protein, 60uM NAD+, 25uM Tris-HCl (pH8.0), 137mM NaCl, 2.7mM KCl, 1mM MgCl2, 1mM DTT, total volume 50ul, mixed, 30 degrees reaction for 4 hours, WB detection of acetylation level was performed on the reacted sample, and GST and GST mutant enzyme activity was determined according to the kit instructions.

[0093] The results are as follows:Figure 8 As shown in Fig. 2A, Figure 8 As shown in Fig. 2B, SIRT2 can reduce the acetylation level of GST1 and GST1K66R, and significantly increase the enzyme activity, but SIRT2 cannot reduce the acetylation level of the mutant GST1K66R, and cannot increase the enzyme activity. It is indicated that SIRT2 acts on the K66 site of GST1 to reduce the acetylation level and then increase the enzyme activity. Figure 8 As shown in Fig. 2B, SIRT2 can reduce the acetylation level of GST1 and GST1K66R, and significantly increase the enzyme activity, but SIRT2 cannot reduce the acetylation level of the mutant GST1K66R, and cannot increase the enzyme activity. It is indicated that SIRT2 acts on the K66 site of GST1 to reduce the acetylation level and then increase the enzyme activity.

[0094] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of nicotinamide for increasing the heat stress resistance of Pleurotus ostreatus, characterized in that: The nicotinamide regulates acetylation modification level mediated by Sirt2; Under heat stress conditions, nicotinamide is added in the culture medium / substrate of Pleurotus ostreatus, and the added concentration of the nicotinamide is 1-10 mM.

2. Use of nicotinamide for improving the heat stress recovery ability of P. ostreatus, characterized in that: The nicotinamide regulates acetylation modification level mediated by Sirt2; Under heat stress conditions, nicotinamide is added in the culture medium / substrate of Pleurotus ostreatus, and the added concentration of the nicotinamide is 1-10 mM.

3. Use of nicotinamide for increasing laccase activity in mycelium of P. ostreatus, characterized in that: The nicotinamide regulates acetylation modification level mediated by Sirt2; Under heat stress conditions, nicotinamide is added in the culture medium / substrate of Pleurotus ostreatus, and the added concentration of the nicotinamide is 1-10 mM.

4. Use according to claim 3, characterized in that: The laccase activity in the mycelium of Pleurotus ostreatus is increased to 6.28 times of the original.

Images