Filamentous fungal mutant strains highly expressing superoxide dismutase and their applications

Abstract

The present application discloses a filamentous fungal mutant strain with high-efficient expression of superoxide dismutase and its application. The filamentous fungal mutant strain of the present application is formed by gene knockout of phytochrome genes PaPhy1 and / or PaPhy2 in the wild-type filamentous fungus Podospora anserina. The filamentous fungal mutant strain of the present application is a mutant strain with gene knockout of phytochrome gene PaPhy1, a mutant strain with gene knockout of phytochrome gene PaPhy2, or a mutant strain with gene knockout of phytochrome genes PaPhy1 and PaPhy2. The present application first discovers that the filamentous fungus Podospora anserina with gene knockout of phytochrome genes PaPhy1 and / or PaPhy2 can express superoxide dismutase and peroxidase more efficiently, which is of great significance for the high-efficient expression and preparation of superoxide dismutase and peroxidase.

Description

Technical Field

[0001] This application relates to the technical field of microbial expression of superoxide dismutase, and particularly to a filamentous fungal mutant strain that highly expresses superoxide dismutase and its application. Background Art

[0002] As an important environmental factor, light can trigger and coordinate various physiological processes of organisms. Fungi use light signals to regulate different life processes, such as circadian rhythm, vegetative growth, reproductive development, secondary metabolism, stress response, and pathogenicity. Fungi sense light through a complex light-sensing system composed of photoreceptors that can distinguish specific wavelengths and intensities of light. It has been reported that there are three types of photoreceptors in fungi, namely WC, VIVID, and cryptochrome that sense blue and violet light, rhodopsin that senses green light, and phytochrome that senses red and far-red light. So far, most studies on fungal photobiology have focused on blue light receptors, which have photorepair functions, as well as functions of regulating the biological clock and secondary metabolism. There are few research reports on the phytochrome, a red light receptor, in fungi.

[0003] Phytochromes have been reported in the ascomycete fungi Aspergillus nidulans, Neurospora crassa, Aspergillus fumigatus, Botrytis cinerea, Fusarium graminearum, and the basidiomycete Ustilago maydis. After the deletion of the phytochrome fphA in Aspergillus nidulans, the induction of sexual and asexual development of ΔfphA by red light becomes weaker. Neurospora crassa contains two phytochrome genes, phy1 and phy2, but phy1 and phy2 are not regulated by light, and the mRNA accumulation of phy1 is only regulated by the circadian rhythm. In the growth, development, and pathogenicity of Botrytis cinerea, the phytochrome Bcphy2 plays an important role. In addition, Bcphy2 also plays a histidine kinase-related function, but the functions of most fungal phytochromes in the light-sensing process are still unknown. It is thus speculated that different fungal phytochromes may be conserved in structure, but their biological roles are different.

[0004] The filamentous fungus Podospora anserina (P. anserina) is a model organism for studying aging. P. anserina has a short life cycle and only reproduces sexually, without asexual reproduction. According to research reports, light is essential for the sexual reproduction of P. anserina, which is completely opposite to the fact that light can inhibit the sexual development of Aspergillus oryzae and Aspergillus nidulans. Red light can regulate the balance between asexual development and sexual reproduction of Aspergillus nidulans by affecting the conformation of phytochrome proteins. Genomic analysis of P. anserina shows that it contains 9 photoreceptor genes, which may enable P. anserina to perceive light and respond to light. So far, only one blue light receptor, VVD, has been studied in P. anserina. After the deletion of the vvd gene, the development of male and female gametes of this species is abnormal, resulting in a decline in sexual reproductive ability. However, how light affects the physiological processes of the filamentous fungus Podospora anserina through phytochrome remains to be further clarified. Summary of the Invention

[0005] The purpose of this application is to provide a new filamentous fungal mutant strain that highly expresses superoxide dismutase and its application.

[0006] This application specifically adopts the following technical solutions:

[0007] The first aspect of this application discloses a filamentous fungal mutant strain that highly expresses superoxide dismutase, which is formed by gene knockout of phytochrome genes PaPhy1 and / or PaPhy2 in the wild-type filamentous fungus Podospora anserina.

[0008] It should be noted that in this application, the wild-type filamentous fungus Podospora anserina refers to a strain in which the phytochrome genes PaPhy1 and PaPhy2 are normally expressed; the filamentous fungal mutant strain refers to a strain in which the phytochrome genes PaPhy1 and / or PaPhy2 of the filamentous fungus Podospora anserina cannot be normally expressed, and more specifically, a strain in which the phytochrome genes PaPhy1 and / or PaPhy2 are not expressed or weakly expressed. In this application, highly expressing superoxide dismutase means that, compared with the wild-type filamentous fungus Podospora anserina, the filamentous fungal mutant strain can express superoxide dismutase more efficiently; similarly, highly expressing peroxidase means that, compared with the wild-type filamentous fungus Podospora anserina, the filamentous fungal mutant strain can express peroxidase more efficiently.

[0009] It should also be noted that the research of this application has found that the phytochrome genes PaPhy1 and PaPhy2 of the filamentous fungus Podospora anserina have a negative regulatory effect on the activities of superoxide dismutase (SOD enzyme) and peroxidase (POD enzyme). The research of this application has confirmed that the mutant strain with single-gene knockout of PaPhy1 (labeled as ΔPaPhy1), the mutant strain with single-gene knockout of PaPhy2 (labeled as ΔPaPhy2), and the strain with double-gene knockout of PaPhy1 and PaPhy2 (labeled as ΔPaPhy1ΔPaPhy2) have higher SOD enzyme activity than the wild-type filamentous fungus Podospora anserina. In one implementation manner of this application, the SOD enzyme activity of the mutant strain can reach up to 2.79 times that of the wild-type filamentous fungus Podospora anserina. It can be understood that the key of this application lies in the research and confirmation that the phytochrome genes PaPhy1 and PaPhy2 have a negative regulatory effect on the activities of SOD enzyme and POD enzyme; gene knockout is only a way to make the phytochrome genes PaPhy1 and PaPhy2 not expressed in one implementation manner of this application, and it does not exclude that other methods can also be used to make these two genes not expressed or weakly expressed to achieve the effect of highly expressing SOD enzyme or POD enzyme.

[0010] In one implementation manner of this application, the mutant strain is formed by gene knockout of the phytochrome genes PaPhy1 and PaPhy2 in the wild-type filamentous fungus Podospora anserina.

[0011] In one implementation manner of this application, the preservation number of the mutant strain is CCTCC M 20231657.

[0012] It should be noted that the research of this application has found that both single-gene mutations and double-gene mutations of the phytochrome genes PaPhy1 and PaPhy2 of the wild-type filamentous fungus Podospora anserina can more efficiently express SOD enzyme and POD enzyme; among them, the effect of the double-gene mutant strain ΔPaPhy1ΔPaPhy2 is better. Therefore, only the double-gene mutant strain ΔPaPhy1ΔPaPhy2 of this application has been preserved.

[0013] The second aspect of this application discloses the application of the mutant strain of this application in the preparation of superoxide dismutase or peroxidase.

[0014] It should be noted that the mutant strain of this application can efficiently express SOD enzyme and POD enzyme; therefore, it can be used to more efficiently prepare superoxide dismutase and peroxidase, thereby improving production efficiency and reducing production costs.

[0015] The third aspect of the present application discloses a method for highly expressing superoxide dismutase, which includes culturing the mutant strain of the present application.

[0016] In one implementation of the present application, the culture conditions for highly expressing superoxide dismutase are to culture for at least 3 days under white light, red light or dark conditions.

[0017] Preferably, culture for at least 3 days under white light or dark conditions.

[0018] Preferably, the culture temperature for highly expressing superoxide dismutase is constant temperature culture at 27°C.

[0019] It should be noted that the mutant strain of the present application can highly express superoxide dismutase. As for the culture conditions, the culture conditions of the existing wild-type filamentous fungus Podospora anserina can be referred to. Culturing for at least 3 days under white light, red light or dark conditions is only the specific culture method adopted in one implementation of the present application, and it does not exclude that there are more optimal culture methods; similarly, constant temperature culture at 27°C is also only the specific culture problem adopted in one implementation of the present application. However, the research of the present application finds that culturing the mutant strain of the present application under white light or dark conditions can obtain relatively more efficient expression of superoxide dismutase; especially under dark conditions, that is, under the condition of no light stimulation, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 are significantly higher than those of the wild type, and there are significant differences. In one implementation of the present application, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 under dark conditions are 1.94 times, 2.63 times and 2.79 times respectively of the SOD enzyme activity of the wild-type filamentous fungus Podospora anserina.

[0020] The fourth aspect of the present application discloses a method for highly expressing peroxidase, which includes culturing the mutant strain of the present application.

[0021] In one implementation of the present application, the culture conditions for highly expressing peroxidase are to culture for at least 5 days under white light, red light or dark conditions.

[0022] Preferably, culture for at least 5 days under white light or red light conditions.

[0023] Preferably, the culture temperature for highly expressing peroxidase is constant temperature culture at 27°C.

[0024] It should be noted that the culture conditions for highly expressing peroxidase in this application can also refer to the culture conditions of the existing wild-type filamentous fungus Podospora anserina. However, it was found in the research of this application that the culture conditions for highly expressing peroxidase vary among different mutant strains. For example, the single-gene mutant strains ΔPaPhy1 and ΔPaPhy2 both have higher expression efficiency under red light conditions, with the highest being 1.40 times and 1.78 times that of the wild-type filamentous fungus Podospora anserina respectively. Another example is that the double-gene mutant strain ΔPaPhy1ΔPaPhy2 has higher expression efficiency under white light conditions, with the highest being 1.29 times that of the wild-type filamentous fungus Podospora anserina.

[0025] The fifth aspect of this application discloses a method for enhancing the superoxide dismutase activity of the filamentous fungus Podospora anserina, which includes using gene knockout technology or gene silencing technology to make the phytochrome genes PaPhy1 and / or PaPhy2 of the filamentous fungus Podospora anserina not express or weakly express.

[0026] The sixth aspect of this application discloses a method for enhancing the peroxidase activity of the filamentous fungus Podospora anserina, which includes using gene knockout technology or gene silencing technology to make the phytochrome genes PaPhy1 and / or PaPhy2 of the filamentous fungus Podospora anserina not express or weakly express.

[0027] It should be noted that the key of this application lies in the research finding that the phytochrome genes PaPhy1 and PaPhy2 of the filamentous fungus Podospora anserina have a negative regulatory effect on the activities of SOD enzyme and POD enzyme. Therefore, in principle, as long as the phytochrome genes PaPhy1 and PaPhy2 do not express or weakly express, the expression of SOD enzyme and POD enzyme can be promoted, thereby enhancing the superoxide dismutase activity and peroxidase activity of the filamentous fungus Podospora anserina. As for the specific methods for making the genes not express or weakly express, reference can be made to the prior art, including but not limited to gene knockout and gene silencing.

[0028] The beneficial effects of this application are as follows:

[0029] The filamentous fungal mutant strains of the present application that highly express superoxide dismutase, namely the mutant strains with the phytochrome gene PaPhy1 knocked out, the mutant strains with the phytochrome gene PaPhy2 knocked out, and the mutant strains with both the phytochrome genes PaPhy1 and PaPhy2 knocked out. The present application first discovered that the filamentous fungus Podospora anserina with the phytochrome gene PaPhy1 and / or PaPhy2 knocked out can more efficiently express superoxide dismutase and peroxidase, which is of great significance for the high-efficiency expression and preparation of superoxide dismutase and peroxidase. Description of the Drawings

[0030] Figure 1 is the phylogenetic tree of P. anserina in the examples of the present application;

[0031] Figure 2 is the schematic structural diagram of the phytochrome genes PaPhy1 and PaPhy2 in the examples of the present application;

[0032] Figure 3 is the amplification electrophoresis result of the upstream and downstream fragments of the PaPhy gene and the screening resistance marker gene in the examples of the present application. Lanes 1, 2, and 3 are PaPhy1-5’, Nour, and PaPhy1-3’ in sequence;

[0033] Figure 4 is the amplification electrophoresis result of the upstream and downstream fragments of the PaPhy gene and the screening resistance marker gene in the examples of the present application. Lanes 1, 2, and 3 are PaPhy2-5’, Gene, and PaPhy2-3’ in sequence;

[0034] Figure 5 is the amplification electrophoresis result of the PaPhy gene fusion fragment in the examples of the present application. Lanes 1 and 2 are PaPhy1-5’-Nour and Nour-PaPhy1-3’ in sequence;

[0035] Figure 6 is the amplification electrophoresis result of the PaPhy gene fusion fragment in the examples of the present application. Lanes 1 and 2 are PaPhy2-5’-Gene and Gene-PaPhy2-3’ in sequence;

[0036] Figure 7 is the upstream and downstream verification result of the ΔPaPhy mutant gene knockout expression cassette in the examples of the present application. Lanes 1 and 3 are PaPhy1-vF / valid5’, and lanes 2 and 4 are PaPhy1-valid3’ / vR;

[0037] Figure 8These are the verification results of the upstream and downstream of the ΔPaPhy mutant gene knockout expression cassette in the embodiments of the present application. Lanes 1 and 3 are PaPhy2-vF / valid5’, and lanes 2 and 4 are PaPhy2-valid3’ / vR;

[0038] Figure 9 These are the verification results of the upstream and downstream of the ΔPaPhy mutant gene knockout expression cassette in the embodiments of the present application. Lane 1 is PaPhy2-vF / valid5’, lane 2 is PaPhy2-valid3’ / vR, lane 3 is PaPhy1-vF / valid5’, and lane 4 is PaPhy1-valid3’ / vR;

[0039] Figure 10 These are the electrophoresis results of the amplified fragments for the verification of the ΔPaPhy1 complemented strain in the embodiments of the present application;

[0040] Figure 11 These are the electrophoresis results of the amplified fragments for the verification of the ΔPaPhy2 complemented strain in the embodiments of the present application;

[0041] Figure 12 This is the result graph of observing the senescence of the complemented strain and the wild-type strain under dark conditions in the embodiments of the present application;

[0042] Figure 13 This is the result graph of comparing the colony growth rates of the wild-type and mutant strains under different light conditions in the embodiments of the present application;

[0043] Figure 14 This is the result graph of observing the colony morphology of the wild-type and mutant strains under different light conditions in the embodiments of the present application. The photos were taken after 7 days of culture;

[0044] Figure 15 These are the statistical results of the number of fruiting bodies of the wild-type and mutant strains under different light conditions in the embodiments of the present application;

[0045] Figure 16 These are the observation results of the senescence time of the wild-type and mutant strains under different light conditions in the embodiments of the present application;

[0046] Figure 17 This is the result graph of observing the vacuoles of the wild-type and mutant strains under dark conditions by laser confocal microscopy in the embodiments of the present application;

[0047] Figure 18 These are the statistical results of three types of vacuoles of the wild-type and mutant strains under dark conditions in the embodiments of the present application;

[0048] Figure 19 These are the detection results of the hyphal ROS content of the wild-type and mutant strains in the embodiments of the present application, with n = 3 for each strain;

[0049] Figure 20 These are the detection results of SOD enzyme activity of wild-type strains and mutant strains under different light conditions in M2 liquid medium in the embodiments of this application;

[0050] Figure 21 These are the detection results of POD enzyme activity of wild-type strains and mutant strains under different light conditions in M2 liquid medium in the embodiments of this application.

[0051] The mutant strain ΔPaPhy1ΔPaPhy2 of this application, with the Latin name: Podospora anserina, is preserved in the China Center for Type Culture Collection (CCTCC), address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation date: September 8, 2023, preservation number: CCTCC M 20231657. Detailed implementation manners

[0052] Phytochromes play a key role in bacterial and plant development, but their biological functions in fungi are not fully understood. To explore the roles and regulatory mechanisms of phytochrome genes PaPhy1 and PaPhy2 in the sexual reproduction and asexual development of P. anserina, this application uses homologous recombination methods to perform site-directed knockout of the two phytochrome genes PaPhy1 and PaPhy2 in P. anserina, obtaining phytochrome gene deletion strains ΔPaPhy1 and ΔPaPhy2, and constructing a double mutant ΔPaPhy1ΔPaPhy2 through genetic hybridization; analyze the differences between mutant strains and wild-type strains in various aspects under different lights, and clarify the main functions of phytochrome genes in P. anserina.

[0053] The results show that there are differences between mutant strains and wild-type strains in sexual reproduction, asexual development, senescence rate, and reactive oxygen metabolism, etc.; in particular, mutant strains can highly express SOD enzyme and POD enzyme. This application's research clarifies the functions of phytochrome genes in P. anserina, providing new ideas for further exploring the regulation mechanism of light on the reproduction of filamentous fungi and anti-aging research.

[0054] The following further elaborates on this application through specific embodiments. The following embodiments only further illustrate this application and should not be construed as limiting this application.

[0055] Embodiment

[0056] I. Materials and methods

[0057] 1.1 Materials

[0058] 1.1.1 Plasmids and strains: Wild-type filamentous fungus Podospora anserina strain (WT), Δmus51::phleoR strain (Δmus51::phleoR is a strain with the mus51 gene knocked out in the wild-type strain, and the homologous recombination efficiency is greatly improved after knocking out this gene). pBC-Nourseothricin and pBC-Geneticin are used for the construction of ΔPaPhy1 and ΔPaPhy2 mutants respectively.

[0059] 1.1.2 Main media: The formulations and preparation methods of media such as M2 minimal medium (M2), germination medium (G), and inoculation medium (Agar) refer to Podospora anserina Genome Project (http: / / podospora.i2bc.paris-saclay.fr / index.php).

[0060] 1.1.3 Main reagents and instruments

[0061] The M5 Fungal Genomic DNA Kit was purchased from Beijing Juhemei Biotechnology Co., Ltd.; Go DNAPolymerase, Ex DNA Polymerase and Max are all produced by Takara; The BCA Protein Quantification Kit was purchased from Novizan Biotech Co., Ltd.; Nourseothricin and Geneticin were purchased from Nanjing Dulai Biotechnology Co., Ltd.; Hygromycin and other reagents were purchased from Sangon Biotech Co., Ltd.; Lywallzyme was purchased from Sigma; NBT, DAB color development kits, and superoxide dismutase (SOD) activity detection kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; The light incubator (S / N: 21004 - 12938) was purchased from HaiboTe Co., Ltd. in Taiwan, China; The FM4-64 dye was purchased from Beijing Coolaber Technology Co., Ltd.; The laser confocal microscope LSM710 was purchased from Carl Zeiss in Germany.

[0062] 1.2 Bioinformatics analysis of the phytochrome gene PaPhy

[0063] Two putative phytochrome genes, Pa_4_59200 and Pa_4_890, were obtained from the P. anserina genome database and named PaPhy1 and PaPhy2, respectively. The corresponding amino acid sequences were PaPhy1 (XP_001905870.1) and PaPhy2 (XP_001903686.1). The amino acid sequences of another seven photoreceptor genes and 13 amino acid sequences of phytochrome proteins from Ascomycetes and Basidiomycetes were obtained from the NCBI genome database, namely: Podospora anserina (CDP28551.1), Podospora anserina (CDP29591.1), Podospora anserina (CDP22503.1), Podospora anserina (CDP23894.1), Podospora anserina (CDP22389.1), Podospora anserina (CDP29135.1), Podospora anserina (CDP22636.1), Ustilago bromivora (SYW76598.1), Neurospora crassa (AAZ57421.1), Sordaria macrospora (KAA8624047.1), Neurospora crassa (AAZ57422.1), Sordaria macrospora (KAA8633010.1), Aspergillus nidulans (CAI30283.1), Trichoderma reesei (XP_006965210.1), Alternaria alternata (QBZ93283.1), Fusarium fujikuroi (KLP13785.1), Lachnellula hyalina (XP_031001258.1), Beauveria bassiana (KAF1729566.1), Aspergillus oryzae (OOO09401.1). The amino acid sequences of the 21 proteins were aligned using the CLUSTALW algorithm in MEGA11.0 software, and a phylogenetic tree of the 21 protein sequences was constructed using the maximum likelihood method in MEGA11.0.

[0064] 1.3 Construction of PaPhy gene knockout mutants

[0065] Primers were designed using the NCBI online software. The primer sequences are shown in Table 1. The 5'-ends (PaPhy1-5' and PaPhy2-5'), 3'-ends (PaPhy1-3' and PaPhy2-3') of the PaPhy1 and PaPhy2 genes were amplified by PCR respectively, and the Nour and Gene gene fragments were amplified from the pBC-Nourseothricin and pBC-Geneticin plasmids respectively. The PCR products were purified and recovered. In this example, a purification and recovery kit purchased from OMEGA Company was specifically used to purify and recover the PCR products. The specific steps of purification and recovery refer to the kit instruction manual.

[0066] The PCR amplification primers are shown in Table 1. The PCR amplification used Go DNA polymerase. The reaction system was: Green Buffer 10 μL, dNTPs 2 μL, MgCl2 4 μL, upstream primer 2 μL, downstream primer 2 μL, genomic DNA 2 μL, Polymerase 0.5 μL, ddH2O 28.5 μL. The PCR reaction conditions were: pre-denaturation at 95 °C for 5 min, then entering 35 cycles: denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 3 min. After the cycle ended, extension at 72 °C for 7 min, standby at 4 °C.

[0067] The two flanking fragments of PaPhy1 and PaPhy2 purified in the first round and the resistance screening marker gene were fused by fusion PCR respectively. The fusion PCR used Prime MAX DNA polymerase. The reaction system was: PaPhy-5' / PaPhy-3' 2 μL, NourseothricinR / GeneticinR 2 μL, PaPhy-1F / PaPhy-MkF 2 μL, PaPhy-MkR / PaPhy-4R 2 μL, Prime MAX 25 μL, ddH2O 17 μL. The reaction conditions were: pre-denaturation at 98 °C for 2 min, then entering 35 cycles: denaturation at 98 °C for 10 s, annealing at 58 °C for 5 s, extension at 72 °C for 25 s. After the cycle ended, extension at 72 °C for 7 min, standby at 4 °C.

[0068] The fusion fragments were transferred into the protoplasts of the ΔPaMus51 strain, and transformants with stable resistance were screened using the resistance medium containing Nourseothricin or Geneticin respectively.

[0069] The specific transformation method included:

[0070] Preparation of P. anserina protoplasts

[0071] After thawing the ΔPaMus51::nourseoR(+) strain on ice for 20 min, pick a piece and inoculate it on M2 medium, and activate and culture it at 27 °C for 2 d.

[0072] (1) Take a piece of mycelium with an area of about 1 cm 2 and add it to an EP tube containing 500 μL of ddH2O. Place the EP tube in a cell disruptor and oscillate it at 5.0 m / s for 20 s, with an interval of 10 s, and oscillate 2 times to obtain a bacterial solution.

[0073] (2) Add tetracycline to 1 L of fungal liquid medium (final concentration 10 μg / mL). Each cell culture flask contains 200 mL. Prepare 5 washed and sterilized cell culture flasks in advance, and add 200 μL of the bacterial solution to each cell culture flask.

[0074] (3) After statically culturing in the dark at 27 °C for 2 - 3 d, filter the mycelium with two layers of sterilized medical gauze. Wash the filtered mycelium with TPS1 solution multiple times to thoroughly wash away the residual fungal liquid medium.

[0075] (4) Transfer the washed mycelium to an Erlenmeyer flask and weigh the mycelium. Add 20 mg / mL of cell wall degrading enzyme to the Erlenmeyer flask. The cell wall degrading enzyme is dissolved with TPS1, and the volume (mL) of the added TPS1 solution is equal to the numerical value of the mycelium mass (g), and note that the liquid needs to cover the mycelium.

[0076] (5) Incubate the Erlenmeyer flask at 37 °C on a shaker at a rotation speed of 70 rpm for 4 h, and observe under an optical microscope whether the mycelium is enzymatically hydrolyzed into spherical protoplasts.

[0077] (6) Fix four layers of sterilized gauze on a sterilized funnel, insert the lower end of the funnel into a 50 mL centrifuge tube, and wash the mycelium with TPS1 multiple times to collect protoplasts.

[0078] (7) Place the 50 mL centrifuge tube in a centrifuge and centrifuge at 20 °C for 10 min at a rotation speed of 3,200 rpm.

[0079] (8) Discard the supernatant, add 5 mL of TPS1 solution to resuspend the precipitate, gently pipette it with a pipette gun, then add TPS1 to 30 mL, and centrifuge at 20 °C for 10 min at a rotation speed of 3,200 rpm.

[0080] (9) Pour out the supernatant, add 5 mL of TPC solution to resuspend the precipitate, gently pipette it with a pipette gun, and then add TPC solution to 30 mL, and centrifuge at 20 °C for 10 min at a rotation speed of 3,200 rpm.

[0081] (10) After centrifugation, pour out the TPC supernatant and leave about 1 mL of the supernatant. Mix the supernatant and the precipitate, and then count using a hemocytometer to ensure that the final concentration of protoplasts is approximately 2×10 8 cells / mL.

[0082] (11) Aliquot the protoplasts into 1.5 mL EP tubes, with 5 tubes, approximately 200 μL per tube. After aliquoting, they can be stored in a -80 °C refrigerator or used directly.

[0083] Fungal transformation

[0084] (1) Thaw the protoplasts on ice or use them directly. Incubate at 48 °C for 5 min, immediately transfer to ice and place for 30 s, and then let stand at room temperature for 5 min.

[0085] (2) Add the amplified DNA fragment to the tube containing the protoplasts and let stand at room temperature for 15 min to ensure that the mass of each fragment is above 5 μg.

[0086] (3) Add 2 mL of TamponD solution to a 50 mL centrifuge tube in advance. Transfer the mixture in step (2) to the 50 mL centrifuge tube, mix well, and let stand at room temperature for 15 min.

[0087] (4) Centrifuge at 20 °C for 10 min at a speed of 3,200 rpm. Slowly aspirate and discard all the supernatant, and retain the precipitate.

[0088] (5) Add 5 mL of RG liquide to the 50 mL centrifuge tube. Gently mix the precipitate and the liquid, and culture in the dark at 27 °C for 12 h.

[0089] (6) Mix sorbitol and RG Top in a ratio of 3:1 in advance and place in a 60 °C oven to prevent the mixture from solidifying. Take 30 mL of the mixture and mix it with the protoplasts in step (5), with a final volume of 35 mL. Then add different antibiotics to the mixture. The final concentration of nourseothricin is 40 μg / mL (i.e., add 14 μL of nourseoticin with a concentration of 100 mg / mL), and the final concentration of geneticin is 100 μg / mL (i.e., add 35 μL of geneticin with a concentration of 100 mg / mL).

[0090] (7) Turn on the water bath in advance and adjust the temperature to 47 °C. Quickly transfer the above mixture to the water bath and water bath for 7 min. Pour 7 mL of the mixture into M2 culture dishes containing the corresponding antibiotics to prepare 5 resistant media. Culture at 27 °C for 2 - 5 d and wait for the transformants to grow. Re-inoculate the transformants onto the resistant plates for secondary verification.

[0091] It is understandable that after the transformants grow on the resistant plate, the transformant colonies are inoculated onto the resistant plate again for secondary resistance verification. The colonies that can grow on both resistant plates are the transformants with stable resistance.

[0092] The wild-type strain is hybridized with the transformant, and microspores with resistance to the resistance marker gene but without the resistance of Δmus51::phleoR protoplasts are screened to obtain a stably inheritable mutant strain. Specifically, the transformants verified for resistance twice are inoculated onto the same M2 medium together with the wild-type strain of the opposite mating type and co-cultured at 27°C. The hyphae of the two colonies will cross after 3 days of growth, and the hybrid hyphae can be fertilized to form mature fruiting bodies and eject spores after 7 days of growth.

[0093] The single mutants ΔPaPhy1 and ΔPaPhy2 are verified by PCR and gene sequencing. The PCR amplification products are sequenced by BGI, specifically including:

[0094] (1) The transformants verified on the secondary resistant plate are inoculated onto the M2 medium and cultured under white light at 27°C for 2 d.

[0095] (2) Add 20 μL of 0.5 mol / L NaOH and a small amount of fine glass beads into a 1.5 mL EP tube, collect the hyphae into this EP tube, put this tube into a cell disruptor, and oscillate at 5.0 m / s for 20 s, oscillating 2 times.

[0096] (3) Centrifuge at room temperature for 30 s at a speed of 12,000 rpm to make the glass beads and hyphae settle to the bottom, and then boil in boiling water for 1 min.

[0097] (4) Add 100 μL of 1 mol / L Tris-HCl and 1×TE mixture into the EP tube, mix well and cool in ice water. The cooled mixture is used as the reaction template.

[0098] (5) Verify the primers PaPhy-5T, 5-Test and 3-Test, PaPhy-3T, as shown in Table 1, and perform amplification according to the PCR reaction system of Ex DNA polymerase. The extension speed of Ex DNA polymerase is 1,000 bp / min, and the extension time is adjusted according to the length of the target fragment. PaPhy-5T and 5-Test are used to amplify and verify the sequence of about 1,000 bp at the 5' end of PaPhy; 3-Test and PaPhy-3T are used to verify the sequence of about 1,000 bp at the 3' end.

[0099] Ex The PCR reaction system for DNA polymerase is as follows: Template 1, 1 μL of PaPhy-5T / PaPhy-3T, 1 μL of 5-Test / 3-Test, Ex DNA Polymerase 12.5 μL, ddH2O 9.5 μL.

[0100] The reaction conditions are: pre-denaturation at 94°C for 10 min, and then enter 30 cycles: denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min. After the cycle ends, extend at 72°C for 5 min and standby at 4°C.

[0101] (6) After performing agarose gel electrophoresis on the PCR products, conduct comparative analysis.

[0102] (7) Purify the transformants: After obtaining the transformants with the target gene knockout, hybridize at least 3 transformants with the wild-type strain of the opposite mating type. After culturing for 7 days, collect the ejected spores and pick microspores under the microscope. Inoculate the microspores onto GY medium and culture for 2 days, and then verify the mating type category and resistance of the germinated microspores. Inoculate the hyphae of the germinated microspores onto the resistance plates containing protoplast resistance and resistance screening marker resistance respectively, and screen the strains that can grow on the screening resistance marker but cannot grow on the resistance plate containing protoplast resistance; inoculate the strains that can only grow on the resistance plate containing resistance marker resistance between the strains of the opposite mating type and culture for 7 days. Judge the resistance of the strains according to whether fruiting bodies can be formed between adjacent colonies, and then store the strains of the opposite mating type at -80°C to obtain the mutant with the target gene deletion.

[0103] After obtaining two single mutants, ΔPaPhy1 and ΔPaPhy2, perform genetic hybridization on the single mutants ΔPaPhy1 and ΔPaPhy2, screen the microspores with both Nourseothricin and Geneticin resistance, and verify the double mutant ΔPaPhy1ΔPaPhy2 by PCR.

[0104] The genetic hybridization of ΔPaPhy1 and ΔPaPhy2 includes: Activate two strains of ΔPaPhy1 and ΔPaPhy2 with opposite mating types. After activation for two days, take 1 cm 2 of the hyphae and dilute the hyphae by crushing with 1 mL of ddH2O. Take 10 μL of the crushed bacterial liquid and inoculate it onto the same M2 solid medium, and culture at 27°C. After 3 days of growth of the two strains, the hyphae will cross, and after 7 days of culture, the hyphae of the two opposite mating types will be fertilized to form mature fruiting bodies.

[0105] The screening of the double mutant ΔPaPhy1ΔPaPhy2 includes:

[0106] (1) Take a small piece of the single variant ΔPaPhy1 (+) and a small piece of the single mutant ΔPaPhy2 (-), inoculate them on an M2 petri dish, and culture them under white light at 27 °C for 3 days;

[0107] (2) After culturing for 3 days, add 1.5 mL of ddH2O to the petri dish to submerge the mycelium. After gently shaking the plate, continue to culture under white light at 27 °C until the fruiting bodies mature;

[0108] (3) After observing that the fruiting bodies are mature, remove the lid of the M2 medium, replace it with an agar medium covering the M2 medium, and collect ascospores for 6 h;

[0109] (4) Remove the inoculated spore medium that has collected ascospores, pick out microspores under a microscope, and inoculate the microspores onto a germination medium, and culture them in the dark at 27 °C for about 2 days;

[0110] (5) After the microspores germinate, inoculate the microspores onto resistance plates containing nourseothricin and geneticin respectively; the microspores that can grow on both resistance media are the double-target gene deletion mutants.

[0111] PCR verification of the double mutant strain ΔPaPhy1ΔPaPhy2 includes:

[0112] (1) Add 20 μL of 0.5 mol / L NaOH and fine glass beads to a 1.5 mL EP tube, collect the mycelium of the double-gene deletion strain into this EP tube, put this tube into a cell disruptor, and oscillate at 5.0 m / s for 20 s, oscillating 2 times.

[0113] (2) Centrifuge at room temperature for 30 s at a speed of 12,000 rpm to sediment the glass beads and mycelium to the bottom, and place it in boiling water for 1 min.

[0114] (3) Add 100 μL of a 1 mol / L Tris-HCl and 1×TE mixture to the EP tube, mix well and cool it in ice water. The cooled mixture is used as a reaction template.

[0115] (4) Verify the primers PaPhy1-5’Test, PaPhy1-3’Test, 5Test, 3Test and PaPhy2-5’Test, PaPhy2-3’Test, 5Test, 3Test, as shown in Table 1, and perform amplification according to the PCR reaction system of Ex DNA polymerase. The extension speed of Ex DNA polymerase is 1,000 bp / min, and the extension time is adjusted according to the length of the target fragment.

[0116] (5) After performing agarose gel electrophoresis on the PCR products, conduct comparative analysis.

[0117] Ex The PCR reaction system for DNA polymerase is as follows: Template 1, 1 μL of PaPhy-5T / PaPhy-3T, 1 μL of 5-Test / 3-Test, Ex 12.5 μL of DNA Polymerase, 9.5 μL of ddH2O.

[0118] The reaction conditions are as follows: Pre-denaturation at 94°C for 10 min, and then enter 30 cycles: Denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min. After the cycles are completed, extend at 72°C for 5 min and standby at 4°C.

[0119] 1.4 Construction of complementation strains of ΔPaPhy1 and ΔPaPhy2

[0120] PCR amplify the complete coding regions of PaPhy1 and PaPhy2 genes of the wild-type strain of P. anserina and 500 bp upstream and downstream of the target genes. Specifically, using the WT genomic DNA as a template, design primers at 500 bp upstream and downstream of the two phytochrome gene fragments, named PaPhy1-1 and PaPhy1-2, PaPhy2-1 and PaPhy2-2, as shown in Table 1.

[0121] Transfer the target gene PaPhy1 and PaPhy2 gene fragments and the digested pBC-Hygromycin plasmid vector into the protoplasts of ΔPaPhy1 and ΔPaPhy2, and then perform resistance verification of the transformants to obtain the complementation strains ΔPaPhy1com and ΔPaPhy2com, and observe whether the phenotypes of the complementation strains ΔPaPhy1com and ΔPaPhy2com are restored.

[0122] Specifically in this experiment, the gene fragments of PaPhy1 and PaPhy2 and the pBC-Geneticin and pBC-Nourseothricin plasmid vectors are digested with a single enzyme overnight at 37°C. Analyze by agarose gel electrophoresis to confirm the band sizes.

[0123] The enzyme digestion reaction system is as follows: 2 μL of Buffer, 0.2 μL of BSA, template ≥ 1 μg, 0.5 μL of EcoRV, and supplement ddH2O to 20 μL.

[0124] Transfer the pBC-Hygromycin plasmid vector into the protoplasts of ΔPaPhy1 and ΔPaPhy2, specifically including:

[0125] (1) The protoplasts are thawed on ice or used directly, incubated at 48 °C for 5 min, immediately transferred to ice for 30 s, and then left standing at room temperature for 5 min.

[0126] (2) Add the digested pBC-Hygromycin plasmid vector and the DNA fragments of PaPhy1 and PaPhy2 to the tube containing the protoplasts.

[0127] (3) Add 2 mL of TamponD solution to a 50 mL centrifuge tube in advance, transfer the mixture in step (2) to the 50 mL centrifuge tube, mix well, and leave standing at room temperature for 15 min.

[0128] (4) Centrifuge at 20 °C for 10 min at a speed of 3,200 rpm, slowly aspirate and discard all the supernatant, and retain the precipitate.

[0129] (5) Add 5 mL of RG liquide to a 50 mL centrifuge tube, mix the precipitate and the liquid well, and culture in the dark at 27 °C for 12 h.

[0130] (6) Sorbitol and RG Top are mixed in advance at a ratio of 3:1 and placed in an oven at 60 °C to prevent the mixture from solidifying. Take 30 mL of the mixture and mix it with the protoplasts in step (5), and the final volume is 35 mL. Then add the antibiotic Hygromycin to the mixture, and the final concentration is 75 μg / mL.

[0131] (7) Turn on the water bath in advance and adjust the temperature to 47 °C. Quickly transfer the above mixture to the water bath and water bath for 7 min. Pour 7 mL of the mixture into an M2 petri dish containing Hygromycin antibiotic, and prepare 5 resistant media. Culture at 27 °C for 2 - 5 d and wait for the transformants to grow.

[0132] The resistance verification of the transformants includes: taking 1 cm of the mycelium of the transformants growing on the M2 medium containing Hygromycin antibiotic 2 and inoculating it again onto the M2 medium containing Hygromycin antibiotic, which is the resistance verification of the transformants.

[0133] Table 1 Primer sequences

[0134]

[0135]

[0136] 1.5 Comparison of growth rates and colony morphologies under different light conditions

[0137] Cut 0.25 cm from the colonies of WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 that have been activated for 2 d2 The mycelium was placed at the edge of the M2 medium and incubated at a constant temperature of 27°C under white light, red light, and darkness. The colony diameter was measured daily, the average growth rate over 7 days was calculated, and the colony morphology was photographed and recorded.

[0138] Activation method: The strain stored in an -80°C refrigerator was thawed on ice for 10 min. A portion of the mycelium was taken out and inoculated onto the M2 medium without complete thawing, and cultured at 27°C under white light for 2 days to obtain the activated strain.

[0139] 1.6 Determination of sexual reproductive ability under different light conditions

[0140] The activated WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains of different mating types were mixed and inoculated onto the M2 medium, and incubated at a constant temperature of 27°C under white light, blue-red light, blue light, red light, and darkness for 7 days. The fruiting body maturation time and quantity were recorded.

[0141] 1.7 Determination of senescence time under different light conditions

[0142] The activated WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains of the same mating type were inoculated onto a medium with a diameter of 15 cm and cultured under white light, red light, and darkness. The time when obvious melanin accumulation appeared at the hyphal tip was the time when the colony began to senesce, and the time from hyphal inoculation to hyphal growth cessation was the life cycle. The senescence time and life cycle were observed and recorded, with n = 3 for each strain.

[0143] 1.8 Vacuole FM4-64 staining

[0144] Under darkness, the WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 were cultured to 10 days and 18 days respectively. A small amount of mycelium was scraped and transferred to a 4 μg / mL FM4-64 fluorescent dye, stained for 10 min in the dark, and then washed 3 times with 10 mmol / L PBS. The culture condition was 27°C. The size and morphology of the vacuoles were observed under a laser confocal microscope at a 100× oil immersion objective, and the size and distribution of the vacuoles were manually measured using ZEN 3.0. According to the size of the vacuoles, they were divided into three categories: small vacuoles with a size less than 2.5 μm 2 were small vacuoles, 2.5 μm 2 to 8 μm 2 were medium-sized vacuoles, and those larger than 8 μm 2 were large vacuoles.

[0145] 1.9 ROS content determination

[0146] The activated 2d WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains were inoculated onto M2 medium and incubated at 27°C in the dark for 3 days. The mycelia were stained with a DAB color development kit and NBT reagent respectively, incubated in the dark for 2 h, the staining reagent was removed, and the mycelia were washed with sterile water. The staining of the mycelia was observed.

[0147] 1.10 Determination of antioxidant enzyme activities under different light conditions

[0148] Peroxidase (POD enzyme) and superoxide dismutase (SOD enzyme) are important antioxidant enzymes in organisms, also known as free radical scavenging enzymes. They can scavenge free radicals in cells, protect cells from free radical damage, and delay cell aging. WT, ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 were inoculated into M2 liquid medium and incubated at 27°C under white light, red light, and in the dark for 5 days. Total protein was extracted using a filamentous fungus protein extraction kit (product number BB-3136) according to the kit instructions, and the protein concentration was determined using a BCA protein concentration assay kit (product number E112) according to the instructions.

[0149] The activity of POD enzyme was determined by the guaiacol method. The product number of the kit used was BC0090. In the presence of H2O2, POD enzyme can catalyze the oxidation of guaiacol to form a brown-red substance, tetraguaiacol, which has a characteristic absorption peak at 470 nm, and the color intensity is proportional to the concentration of the product within a certain range. The activity of SOD enzyme was determined using a SOD enzyme activity detection kit produced by Beijing Solarbio Science & Technology Co., Ltd. The specific operation steps are shown in the kit instructions.

[0150] II. Results and analysis

[0151] 2.1 Bioinformatics analysis of PaPhy genes

[0152] 2.1.1 Phylogenetic analysis of phytochrome proteins in P. anserina: MEGA11.0 was used to construct a phylogenetic tree of phytochrome proteins from 13 ascomycetes, 1 basidiomycete, and 7 other predicted photoreceptors in P. anserina. The results are as Figure 1 shown. Phylogenetic analysis showed that the blue light receptor and the green light receptor have a higher genetic relationship. Among the red light receptors, PaPhy1 and PHY 1 of the ascomycete Neurospora crassa have a higher genetic relationship; Neurospora crassa PHY2 lacks 360 amino acids compared to PHY1 and is located on the outer side of this phylogenetic tree. PaPhy2 and Neurospora crassa PHY2 have a higher genetic relationship. The schematic structures of the proteins encoded by PaPhy1 and PaPhy2 genes are as Figure 2As shown, compared with the amino acid sequence of PaPhy1, the amino acid sequence of PaPhy2 is slightly shorter. PaPhy1 has a 160 - amino - acid extension at the C - terminus and 200 amino acids at the N - terminus separating the HKRD and RR domains, but shows extremely high similarity in the core conserved region. Both PaPhy1 and PaPhy2 contain the domains typical of phytochromes. Analysis of the conserved domains of PaPhy1 and PaPhy2 shows that PAS has a chromophore - binding site cysteine (C) that can bind linear tetrapyrrole. The cysteine in GAF of PaPhy1 and PaPhy2 is replaced by isoleucine (I) and valine (V) respectively. The histidine kinase domain (HKRD) has a conserved histidine site, and the response regulator domain (RR) has a conserved aspartic acid site. Therefore, it is considered that the two phytochrome proteins of P. anserina are light - regulated histidine kinases that play dual functions as red - light receptors and histidine kinases.

[0153] 2.2 Construction and identification of PaPhy mutants

[0154] 2.2.1 Construction of PaPhy1 and PaPhy2 gene knockout expression cassettes: Using the wild - type genomic DNA of P. anserina as a template, the upstream fragment PaPhy - 5’ and downstream fragment PaPhy - 3’ of the PaPhy gene, about 1000 bp, were amplified by PCR. The resistance screening marker Gene and Nour gene fragments were amplified from the pBC - Nourseothricin and pBC - Geneticin plasmid DNAs respectively. The results are as Figure 3 and Figure 4 shown. The knockout expression cassette PaPhy1 - 5’ - Nour and Nour - PaPhy1 - 3’ of PaPhy1 was constructed by fusion PCR, as Figure 5 shown, and the knockout expression cassette PaPhy2 - 5’ - Gene and Gene - PaPhy2 - 3’ of PaPhy2, as Figure 6 shown.

[0155] 2.2.2 Verification of ΔPaPhy1 and ΔPaPhy2 mutants: After transferring the knockout expression cassette into the protoplasts of the Δmus51::phleoR strain, transformants containing the Nourseothricin or Geneticin resistance markers were screened. The homologous recombination fragments were amplified by verification primers, as Figures 7 to 9 shown, and the verification primer sequences are shown in Table 1. The purified amplification products were sequenced, and the sequencing results were consistent with the target sequences, indicating that the screened resistance marker genes were successfully integrated into the PaPhy gene locus. Figure 7Electrophoretogram of PaPhy1-vF / valid5’ and PaPhy1-valid3’ / vR, where lanes 1 and 3 are PaPhy1-vF / valid5’, and lanes 2 and 4 are PaPhy1-valid3’ / vR. Figure 8 Electrophoretogram of PaPhy2-vF / valid5’ and PaPhy2-valid3’ / vR, where lanes 1 and 3 are PaPhy2-vF / valid5’, and lanes 2 and 4 are PaPhy2-valid3’ / vR. Figure 9 Electrophoretogram of PaPhy1-vF / valid5’, PaPhy1-valid3’ / vR, PaPhy2-vF / valid5’ and PaPhy2-valid3’ / vR, where lane 3 is PaPhy1-vF / valid5’, lane 4 is PaPhy1-valid3’ / vR, lane 1 is PaPhy2-vF / valid5’, and lane 2 is PaPhy2-valid3’ / vR.

[0156] Based on the above results, it is indicated that the PaPhy gene in the target transformant has been replaced by the resistance screening marker fragment, that is, the target transformant is a successfully constructed PaPhy gene deletion mutant strain.

[0157] 2.3 Construction and identification of ΔPaPhy complemented strains

[0158] Using the genomic DNA of P. anserina wild type as a template, two gene fragments to be complemented were amplified by PCR. The complemented gene fragments were separately transferred into the ΔPaPhy1 and ΔPaPhy2 mutants. After transformation, transformants with corresponding resistance were selected for PCR verification, and the target gene fragments were amplified. The results are as Figure 10 and Figure 11 shown. Under dark conditions, the senescence time of the complemented strains was the same as that of the wild type strain, as shown in Figure 12 , indicating successful complementation of the PaPhy1 and PaPhy2 genes.

[0159] 2.4 Effects of phytochrome on the growth rate and colony morphology of P. anserina

[0160] After successfully constructing the mutants, it was observed whether the growth and development of the strains were affected by different light conditions. WT, ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were inoculated on M2 minimal medium and cultured at 27 °C for 7 d. The growth rates were compared. The results are as Figure 13 and Figure 14 shown. From Figure 13 and Figure 14, it can be seen that the growth rate of all strains is the fastest under red light, followed by darkness, and the slowest under white light. This indicates that red light alone can promote the growth of P. anserina hyphae, and white light composite light, as a light stress, can inhibit the growth of hyphae. Under the same light, there is no significant difference in the growth rate between mutant strains and wild-type strains. Observing the colony morphology of P. anserina, it can be seen that under the same light conditions, there is no difference between wild-type strains and mutant strains, and the colony morphology is the same under red light and darkness; whether under white light, blue / red light or blue light alone, the colony morphology of all strains is the same, showing more obvious melanin accumulation. This indicates that P. anserina is similar to another filamentous fungus Neurospora crassa, and the photomorphogenesis of P. anserina is mainly regulated by blue light. Figure 13 In this study, n = 3 for each strain, T-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0161] 2.5 Effects of phytochrome genes on the sexual reproductive ability of P. anserina

[0162] The fruiting body is a reproductive body that can produce spores formed by higher filamentous fungi during the sexual reproductive stage and is an important organ for fungi to carry out sexual reproduction. The sexual development level of P. anserina is usually measured by the number of fruiting bodies. P. anserina cannot carry out sexual reproduction after 7 days of hybrid culture in the dark. Under different light conditions, the sexual reproductive abilities of wild-type strains and mutant strains were compared, and the results are as Figure 15 shown. Figure 15 The results show that under white light, compared with WT, the fruiting body maturation time and ascospore ejection time of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 are delayed. All strains can produce fruiting bodies under blue / red light or blue light alone, but the number of fruiting bodies produced is less than that under white light. Under blue light, the number of fruiting bodies of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 decreased by 2.4%, 15.2%, and 24.3% respectively, indicating that PaPhy1 and PaPhy2 genes have a certain promoting effect on the sexual reproduction of the strains. Under red light, all strains cannot carry out sexual reproduction, but under blue / red light composite light, the number of fruiting bodies produced by all strains is more than that under blue light alone, indicating that under the combined action of blue light and red light, the sexual reproductive ability of the strains is enhanced, which is more conducive to the strains to carry out sexual reproduction compared with only blue light irradiation. Figure 15 In this study, n = 3 for each strain, T-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0163] 2.6 Phytochrome genes are involved in the regulation of P. anserina senescence

[0164] In the exploration of the effect of light on the sexual reproduction of P. anserina, it was found that the fruiting body maturation times of the wild-type strain and mutant strains were different. The fruiting body of ΔPaPhy1 matured earliest, followed by that of ΔPaPhy2, and the fruiting body of ΔPaPhy1ΔPaPhy2 matured latest. Therefore, it was speculated that the life cycle of the phytochrome-deficient strains was affected and the senescence time might change. As Figure 16 shown, WT began to senesce on the 10th day of cultivation in the dark, and the mycelium stopped growing on the 28th day of cultivation; ΔPaPhy1 began to senesce on the 14th day of cultivation, ΔPaPhy2 began to senesce on the 16th day of cultivation; ΔPaPhy1ΔPaPhy2 began to senesce on the 18th day of cultivation, and the mycelium stopped growing on the 45th day of cultivation. The senescence times of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 were delayed by 40%, 60%, and 70% respectively. This indicates that the two phytochrome genes play an important role in regulating the senescence of P. anserina mycelium. The senescence delay of the PaPhy2-deficient strain was more obvious, and the senescence delay time of the double-deletion mutant strain was the longest. It may be that the regulatory effects of the two genes on the senescence of the strain do not completely overlap, and they have a synergistic effect in regulating cell senescence. WT began to senesce on the 9th day and the 12th day under white light and red light respectively, while the senescence times of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 under white light and red light were on the 14th, 16th, and 18th d respectively. This shows that white light can promote the senescence of WT, and red light can delay the senescence of WT.

[0165] 2.7 Effects of phytochrome gene deletion on the vacuoles of P. anserina

[0166] Fungal vacuoles have many functions in cells, such as storing substances, maintaining cell pressure balance, decomposing substances, etc. Changes in their size and number will affect cell metabolism and function, thus affecting the growth and senescence of fungi. When the strain enters the senescent state, the size and morphology of the vacuoles will change. This may be because as the strain ages, the substances in the vacuoles will gradually decompose, resulting in changes in the size and number of vacuoles. Autophagy regulates senescence by degrading damaged proteins and organelles and other structures. Fungal vacuoles are the main organs for the degradation and utilization of intracellular substances. Therefore, this experiment further studied the relationship between PaPhy deletion, vacuole function changes, and the degree of strain senescence. First, the sizes of the PaPhy-deleted strains and wild-type strains at 10 d and 18 d of cultivation were analyzed, as Figure 17 and Figure 18 shown. Figure 17Results of staining the vesicle membranes of 10-day-old ΔPaPhy and wild-type strains with 4 μg / mL FM4-64 for 10 minutes and analyzing the vesicle size and morphology by laser confocal microscopy. Figure 18 Statistical results of three types of vacuoles. In M2 culture, after 9 days and 17 days of strain culture, nitrogen starvation induction culture was carried out for 1 day to promote the formation of vacuoles. Next, FM4-64 staining was used to measure the vacuole size, less than 2.5 μm 2 for small vacuoles, 2.5 μm 2 to 8 μm 2 for medium-sized vacuoles, greater than 8 μm 2 for large vacuoles. At the 10th day of culture, the vacuoles in the wild-type strain were round, as shown in Figure 17 and Figure 18 . Among these vacuoles, 39% were small vacuoles, 61% were medium-sized vacuoles, and 5% were large vacuoles. In contrast, the proportion of different types of vacuoles in the ΔPaPhy1 strain changed, with 32% being small vacuoles, 55% being medium-sized vacuoles, and 13% being large vacuoles; in the ΔPaPhy2 strain, 26% were small vacuoles, 38% were medium-sized vacuoles, and 36% were large vacuoles; in the ΔPaPhy1ΔPaPhy2 strain, 23% were small vacuoles, 13% were medium-sized vacuoles, and 64% were large vacuoles. The proportion of large vacuoles in the ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains was higher than that in the wild-type. At the 18th day of culture, all strains entered the senescent state, and there were no large vacuoles. The proportion of medium-sized vacuoles increased, and the senescence degree of the wild-type strain further increased. The proportion of small vacuoles increased to 78%. At the same culture time, compared with the ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains, the WT hyphae had the greatest senescence degree. Figure 18 In, n = 3 for each strain, T-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

[0167] 2.8 Effects of phytochrome on ROS

[0168] Nitroblue tetrazolium (NBT) staining method and 3,3'-diaminobenzidine (DAB) staining method are often used to detect the accumulation of superoxide anion radicals and hydrogen peroxide in hyphal tissues. The accumulation of superoxide anion radicals and hydrogen peroxide is an important component of ROS. ROS will attack cellular molecules such as DNA, proteins, and lipids, leading to their oxidative damage and loss of function. These damages may cause cells to enter the aging state and accelerate their senescence process. The depth of hyphal staining is proportional to the ROS content. The lighter the color, the lower the ROS content, and vice versa. The staining results are as shown in Figure 19As shown, NBT staining revealed that the blue color on WT hyphae was deeper, followed by that on ΔPaPhy1 hyphae, the blue on ΔPaPhy2 hyphae was lighter than that on ΔPaPhy1, and the blue on ΔPaPhy1ΔPaPhy2 hyphae was the lightest; DAB staining showed that the brown color at the center of WT hyphae was the darkest, followed by that on ΔPaPhy1 hyphae, the brown on ΔPaPhy2 hyphae was lighter than that on ΔPaPhy1, and the brown on ΔPaPhy1ΔPaPhy2 hyphae was the lightest. The above results indicated that the ROS content in WT hyphae was the highest; the color of ΔPaPhy1 hyphae was lighter than that of WT, and the ROS content was relatively lower; the color of ΔPaPhy2 hyphae was lighter than that of ΔPaPhy1, and the ROS content was further reduced; the color of ΔPaPhy1ΔPaPhy2 hyphae was the lightest, and the ROS content was the lowest. Therefore, it was speculated that the deletion of the phytochrome gene would reduce ROS production, avoid the generation of excessive oxidative stress, and thus delay the aging process of the mutants.

[0169] 2.9 Effects of phytochrome gene deletion on the antioxidant enzyme activities of P. anserina

[0170] 2.9.1 Analysis of superoxide dismutase (SOD enzyme) activity: The SOD enzyme is an important enzyme in the antioxidant enzyme system, which can scavenge superoxide anion free radicals in cells, protect cells from free radical damage, delay cell aging, and affect the aging rate of organisms.

[0171] In this study, 100 μL of liquid was inoculated into 50 mL of liquid medium, and after culturing for 5 days, the total protein of the mycelium was extracted to determine the SOD enzyme activity of the strains. For each strain, n = 3, T test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and the results are as Figure 20 shown. Figure 20The results showed that, under the condition of no light stimulation, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were significantly higher than those of the wild type, and there were significant differences, which were 1.94 times, 2.63 times and 2.79 times the SOD enzyme activity of the wild type, respectively. This indicated that the PaPhy1 and PaPhy2 genes had a negative regulatory effect on the SOD enzyme activity and there was a complementary effect. When cultured under white light for 5 days, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were 1.4 times, 2.25 times and 2.05 times the SOD enzyme activity of the wild type, respectively; when cultured under red light for 5 days, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were 0.95 times, 2.39 times and 2.10 times the SOD enzyme activity of the wild type, respectively. This indicated that when there was light stimulation, the strains had higher SOD enzyme activities to better cope with the oxidative stress response caused by light. Thus, it can be seen that the phytochrome genes played an important regulatory role in the antioxidant enzyme system of P. anserina. The SOD enzyme activity of the mutant strains was higher than that of the wild type strains, improving the ability to scavenge ROS, which was consistent with the phenomenon of decreased ROS content observed in the ROS content determination experiment.

[0172] 2.9.2 Analysis of peroxidase (POD enzyme) activity: The POD enzyme can effectively scavenge hydrogen peroxide and reduce cell damage caused by hydrogen peroxide. To measure the POD enzyme activities of the wild type strains and mutant strains, in this study, 100 μL of the liquid was inoculated into 50 mL of the liquid medium. After culturing for 5 days, the total protein of the mycelium was extracted to measure the POD enzyme activity of the strains, with n = 3 for each strain. T-test: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. The results are as Figure 21 shown. Figure 21The results showed that when there was no light stimulation, the POD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were higher than those of the wild type, which were 1.29, 1.12 and 1.06 times the POD enzyme activity of the wild type, respectively. This indicated that the expression of POD enzyme in the mutant strains was increased, which could enhance their antioxidant capacity, and the regulation of POD enzyme activity was independent of each other. The deletion of two phytochrome genes could have a synergistic effect. This result was consistent with the decrease in ROS content measured, indicating that the mutant strains had stronger antioxidant capacity. When cultured under white light for 5 days, the POD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were 1.09 times, 1.36 times and 1.29 times the SOD enzyme activity of the wild type, respectively; when cultured under red light for 5 days, the SOD enzyme activities of ΔPaPhy1, ΔPaPhy2 and ΔPaPhy1ΔPaPhy2 were 1.40, 1.78 and 1.10 times the POD enzyme activity of the wild type, respectively. This indicated that the deletion of phytochrome genes would have different degrees of influence on POD enzyme activity, and different wavelengths of light had different regulatory effects on this. The increase in POD enzyme activity in the mutant strains could reduce the cell damage caused by ROS. Thus, it could be seen that the PaPhy1 and PaPhy2 genes played an important regulatory role in the antioxidant enzyme system of P. anserina.

[0173] Based on the results of the determination of the activities of the two antioxidant enzymes, it could be seen that the mutant strains had higher antioxidant enzyme activities, could scavenge more free radicals, reduce the oxidative damage of free radicals to cells, and thus delayed the senescence of the mutant strains.

[0174] III. Discussion and Conclusions

[0175] In ascomycetes, the functions of photoreceptors have been relatively well studied in Neurospora crassa and Aspergillus nidulans. Through bioinformatics analysis in this study, it was found that P. anserina contained two phytochrome homologous proteins, PaPhy1 and PaPhy2. These two proteins included the photosensory domain that binds chromophore at the N-terminus and the output regulatory domain at the C-terminus, which are possessed by typical phytochrome proteins. The photosensory domain consists of PAS-GAF-PHY, and the C-terminus consists of HisKA and HATPase-C, belonging to two-component histidine kinases. Two-component histidine kinases play an important role in biological sensing and environmental adaptation. Therefore, it was analyzed that the phytochromes of P. anserina played the function of red light receptors.

[0176] In fungi, research on red light and phytochromes has mainly been conducted in Neurospora crassa, Aspergillus fumigatus, and Aspergillus nidulans. Red light controls the balance between asexual and sexual development as well as the germination process. In Neurospora crassa, blue light activates many light responses, including the biosynthesis of mycelial carotenoids and the formation of vegetative spores (macroconidia); while red light has no obvious effect on its growth and development. Strains lacking the phy1 and phy2 genes are not affected in the known light responses. Aspergillus nidulans mainly produces sexual reproductive structures - cleistothecia in the dark and mainly produces asexual reproductive structures - conidia in the light. Only red light can control the switch between asexual and sexual development in Aspergillus nidulans. In this study, it was found that blue light can activate the light responses of P. anserina, such as the formation of black pigments and the occurrence of sexual reproduction. As Figure 14 and Figure 15 shown, there is no difference in the colony morphology of WT under darkness and red light. This fungus cannot carry out sexual reproduction and can only undergo asexual development, indicating that P. anserina is similar to Neurospora crassa and the photomorphogenesis is mainly regulated by blue light. The level of sexual development in P. anserina is usually measured by the number of fruiting bodies. White light and blue light can effectively induce the formation of sexual reproductive structures - fruiting bodies in P. anserina, while red light has no such effect. As Figure 14 and Figure 15 shown, but red light can act as a cofactor for the occurrence of sexual reproduction in the presence of blue light. At the same light duration, the strains lacking phytochrome produce fewer fruiting bodies compared to the wild-type strain. As Figure 15 shown, for example, under blue light, the number of fruiting bodies of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 decreased by 2.4%, 15.2%, and 24.3% respectively. This indicates that phytochrome, as a red light receptor, can also respond to blue light. The inductive effect of blue light on the sexual development of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 weakens in turn. PaPhy2 responds more strongly to blue light. The two phytochrome genes may coordinate with each other. When one gene is deleted, the other gene can supplement its function to a certain extent. The phytochrome genes have a positive regulatory effect on the process of fruiting body formation in P. anserina.

[0177] Sexual reproduction and asexual reproduction in fungi alternate. During asexual reproduction, the sexual reproductive process is initiated under the influence of environmental inductive factors. Compared with WT, the number of fruiting bodies of ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 is less, the senescence is delayed in the dark, and the life cycle is longer. As Figure 16As shown, the delay of ΔPaPhy1ΔPaPhy2 was 61.7%. This may be because after the deletion of the phytochrome gene, light, as an important environmental factor, affects the antioxidant enzyme system and vacuolar metabolic capacity of P. anserina, thus changing the aging rate of the fungus and altering the entire life cycle of the strain. SOD and POD enzymes are important antioxidant enzymes in organisms, also known as free radical scavenging enzymes, which respectively scavenge superoxide anion radicals and hydrogen peroxide, reduce the intracellular ROS content, reduce cell damage caused by ROS, and delay the aging of organisms. The determination of the ROS content and the activities of two oxygen free radical scavenging enzymes in the phytochrome gene deletion strains found that the ROS produced by ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 after 3 days of dark culture was lower than that of the wild type, and the SOD enzyme activity was significantly higher than that of the wild type strain. Thus, it can be seen that the mutant strains have a stronger ability to scavenge free radicals. The decrease in the ROS content and the increase in the free radical scavenging enzyme activity of the mutant strains can both weaken the oxidative damage of free radicals to cells, resulting in a delay in the aging of the strains. Autophagy is an important cell metabolic process that is widespread and evolutionarily conserved in eukaryotes. This process can turnover the substances inside the cells. Some damaged proteins or organelles will be wrapped in autophagosomes with a double-membrane structure and sent to vacuoles (yeast and plants) or lysosomes (animals) for degradation and reuse. At the same culture time, compared with the ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 strains, the proportion of small vacuoles in the WT hyphae is the largest, as Figure 18 shown, the degree of aging is the greatest. After the deletion of the PaPhy1 and PaPhy2 genes, the proportion of the large vacuoles in the hyphae increases, which may accelerate the metabolic process of intracellular substances and lead to a delay in the aging time of the strain. P. anserina, as a model organism for studying aging, explores the influence of photoreceptors on the aging of P. anserina, providing a research basis for the study of the aging of complex multicellular organisms. In addition, the ROS content of the mutant strains changes, the aging is delayed, the life cycle is extended, and more cellulase can be produced. Therefore, it can be used as an engineering strain for cellulose degradation.

[0178] In summary, P. anserina has a strong response to white light and blue light, but is insensitive to red light. White light and blue light can effectively induce sexual reproduction; the two phytochrome genes have a positive regulatory effect on the sexual reproduction process of P. anserina and a negative regulatory effect on the POD enzyme activity and SOD enzyme activity. Therefore, the mutant strains ΔPaPhy1, ΔPaPhy2, and ΔPaPhy1ΔPaPhy2 can all highly express SOD and POD enzymes.

[0179] The above content is a further detailed description of the present application in combination with specific implementation manners, and it cannot be determined that the specific implementation of the present application is only limited to these descriptions. For those of ordinary skill in the technical field to which the present application pertains, without departing from the concept of the present application, several simple deductions or substitutions can also be made.

Claims

1. A filamentous fungal mutant strain with high - level expression of superoxide dismutase, characterized in that: The mutant strain is formed by gene knockout of the phytochrome genes PaPhy1 and / or PaPhy2 in the wild-type filamentous fungus Podospora anserina; the amino acid sequence corresponding to PaPhy1 is XP_001905870.1, and the amino acid sequence corresponding to PaPhy2 is XP_001903686.

1.

2. The mutant strain according to claim 1, wherein: The mutant strain is formed by gene knockout of the phytochrome genes PaPhy1 and PaPhy2 in the wild-type filamentous fungus Podospora anserina.

3. The mutant strain according to claim 2, wherein: The preservation number of the mutant strain is CCTCC M 20231657.

4. Use of the mutant strain according to any one of claims 1-3 in the preparation of superoxide dismutase or peroxidase.

5. A method for efficiently expressing superoxide dismutase, characterized in that: It includes culturing the mutant strain according to any one of claims 1-3.

6. The method according to claim 5, wherein: The conditions of the culturing are culturing for at least 3 days under white light, red light or dark conditions.

7. The method according to claim 6, wherein: The temperature of the culturing is constant temperature culturing at 27°C.

8. A method for efficiently expressing peroxidase, characterized in that: It includes culturing the mutant strain according to any one of claims 1-3.

9. The method according to claim 8, characterized in that: The conditions of the culturing are culturing for at least 5 days under white light, red light or dark conditions.

10. The method according to claim 9, wherein: The temperature of the culturing is constant temperature culturing at 27°C.

11. A method for enhancing the superoxide dismutase activity of filamentous fungus Podospora anserina, characterized in that: It includes using gene knockout technology or gene silencing technology to make the phytochrome genes PaPhy1 and / or PaPhy2 of the filamentous fungus Podospora anserina not express or weakly express; the amino acid sequence corresponding to PaPhy1 is XP_001905870.1, and the amino acid sequence corresponding to PaPhy2 is XP_001903686.

1.

12. A method for improving the peroxidase activity of the filamentous fungus Podospora anserina, characterized in that: It includes using gene knockout technology or gene silencing technology to make the phytochrome genes PaPhy1 and / or PaPhy2 of the filamentous fungus Podospora anserina not express or weakly express; the amino acid sequence corresponding to PaPhy1 is XP_001905870.1, and the amino acid sequence corresponding to PaPhy2 is XP_001903686.1.

Images