Filamentous fungus mutant strain with sporocarp development defect and application thereof

Abstract

The invention discloses a filamentous fungus mutant strain with sporocarp development defects and application of the filamentous fungus mutant strain. The invention discloses a filamentous fungus mutant strain with sporocarp development defects. The filamentous fungus mutant strain is formed by carrying out gene knockout on a mitochondrial complex I intermediate assembly factor CIA30 gene on a wild type filamentous fungus Podospora anserina. According to the application, the gene knockout mitochondrial complex I intermediate assembly factor CIA30 gene is found for the first time, the extension capacity of hyphae can be improved, the growth life of the hyphae can be prolonged, and the application has important value and significance for breeding industrial strains for efficiently growing the hyphae.

Description

Technical Field

[0001] The present application relates to the technical field of filamentous fungus mutation, and in particular to a filamentous fungus mutant strain with fruiting body development defects and application thereof. Background Art

[0002] Fruiting bodies are important reproductive organs of filamentous fungi, which are formed during sexual reproduction and used to protect spores produced by meiosis. Fungal fruiting bodies have various morphologies, including umbrella-shaped, pen-shaped, spherical, bell-shaped, etc., and are complex multicellular structures in fungi. The morphological development of fruiting bodies and which genes mediate or regulate them have always been the research topics of some filamentous fungi. For example, in Neurospora crassa, MAP kinase and transcription factor ADA-6 were found to regulate fruiting body development, and in Sordaria macrospora, glyoxylate protease LON2 was found to be involved in fruiting body development and ascospore formation.

[0003] Since the 1830s, the filamentous fungus Podospora anserina has been studied as a model organism for more than 100 years, including life processes such as fungal nutritional incompatibility, fruiting body development, sexual reproduction, aging, prions, cell differentiation, respiratory metabolism, etc. The fruiting body formation of P. anserina begins with the fertilization development of male and female gametes differentiated by hyphae. After a series of morphological changes, it differentiates into a body with a distinct neck and asci. Then, ascogenous hyphae extend from the fertilized female gametophyte (ascogonium) to undergo nuclear division to form hooks (croziers). These hooks undergo nuclear fusion and meiosis in the asci to form ascospores, and finally eject ascospores under neck pressure. The entire life cycle can be completed in 7 days.

[0004] The P. anserina mitochondrial i-AAA protease PaIAP-deficient strain was severely impaired in fruiting body formation and spore germination under high temperature culture at 37°C, and aging was accelerated. Sun Yue's research found that knocking out the P. anserina mitochondrial NADH dehydrogenase encoding gene also blocked fruiting body development, but aging was delayed, accompanied by reduced ATP synthesis and other characteristics.

[0005] The development of P. anserina fruiting bodies is complex, involving multiple biological processes such as cell recognition, meiosis, cell differentiation, and various vesicle transport. Which genes mediate or regulate the development of P. anserina fruiting bodies remains a difficult problem that needs to be solved urgently in the field of P. anserina research. Summary of the invention

[0006] The purpose of the present application is to provide a new mutant strain of filamentous fungi with fruiting body development defects and its application.

[0007] This application specifically adopts the following technical solutions:

[0008] The first aspect of the present application discloses a mutant strain of filamentous fungi with defective fruiting body development, which is formed by knocking out the mitochondrial complex I intermediate assembly factor CIA30 gene from the wild-type filamentous fungus Podospora anserina.

[0009] It should be noted that in this application, the wild-type filamentous fungus Podospora anserina refers to a strain in which the mitochondrial complex I intermediate assembly factor CIA30 gene is normally expressed, and the filamentous fungus mutant strain refers to a strain in which the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina cannot be normally expressed. This application study found that the mutant strain (labeled as ΔPaCIA30) obtained by knocking out the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina showed excellent hyphae extension ability and significantly prolonged growth lifespan, which is of great value and significance for the breeding of industrial strains with efficient hyphae growth.

[0010] In one implementation of the present application, the deposit number of the mutant strain is CCTCC M 20242756.

[0011] It should be noted that the present application found that knocking out the mitochondrial complex I intermediate assembly factor CIA30 gene in the wild-type filamentous fungus Podospora anserina can improve the mycelial extension ability and prolong the mycelial growth lifespan; therefore, the present application has preserved the mutant strain ΔPaCIA30 obtained by gene knockout.

[0012] The second aspect of the present application discloses the use of the mutant strain of the present application in preparing efficient growing mycelium.

[0013] In the present application, efficient mycelium growth means that, relative to the wild-type filamentous fungus Podospora anserina, the mutant strain has a longer mycelium lifespan and / or a stronger mycelium extension ability.

[0014] It should be noted that the mutant strain of the present application can improve the mycelium extension ability and prolong the mycelium growth life; therefore, it can be used as an industrial strain with long mycelium life and / or strong mycelium extension ability to obtain a larger mycelium yield.

[0015] The third aspect of the present application discloses a method for increasing the mycelium yield of the filamentous fungus Podospora anserina, comprising culturing the mutant strain of the present application for at least 15 to 21 days.

[0016] It should be noted that the mutant strain of the present application can continue to grow hyphae after 15 days of culture, and the hyphae can grow for 21 days without aging; compared with the wild-type strain, the hyphae almost no longer extend and grow for 7 to 9 days, and the aging characteristics appear at 11 days. The mutant strain of the present application has significantly improved hyphae extension ability and hyphae growth life; therefore, the mutant strain of the present application is cultured for at least 15 to 21 days, and more hyphae can be obtained, thereby increasing the hyphae yield. As for the culture conditions of the filamentous fungus Podospora anserina, reference can be made to the prior art, such as constant temperature culture at 27°C in M2 medium under light conditions.

[0017] In one implementation of the present application, the culture condition is constant temperature culture at 27° C. under light conditions.

[0018] In one implementation of the present application, the culture medium used for culture is M2 culture medium.

[0019] The fourth aspect of the present application discloses a method for extending the life span of mycelium of the filamentous fungus Podospora anserina, comprising using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

[0020] The fifth aspect of the present application discloses a method for improving the hyphae extension ability of the filamentous fungus Podospora anserina, comprising using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

[0021] The sixth aspect of the present application discloses a method for regulating the fruiting body development of the filamentous fungus Podospora anserina, comprising using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

[0022] It should be noted that the key to this application is that the research found that the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina is negatively correlated with the hyphae extension ability and hyphae growth lifespan; therefore, in principle, as long as the mitochondrial complex I intermediate assembly factor CIA30 gene is not expressed or the expression is weakened, the hyphae extension ability and hyphae growth lifespan can be improved. As for the specific method of not expressing or weakly expressing the gene, reference can be made to the existing technology, including but not limited to gene knockout and gene silencing.

[0023] The beneficial effects of this application are:

[0024] The present application discloses a mutant strain of filamentous fungi with defects in fruiting body development, namely, a mutant strain with gene knockout of the mitochondrial complex I intermediate assembly factor CIA30 gene. The present application is the first to discover that gene knockout of the mitochondrial complex I intermediate assembly factor CIA30 gene can improve the mycelium extension ability and prolong the mycelium growth life, which is of great value and significance for the breeding of industrial strains with efficient mycelium growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a comparison diagram of multiple sequences of PaCIA30 in the examples of this application;

[0026] Figure 2 is the PaCIA30 developmental evolution tree in the examples of this application;

[0027] Figure 3 It is the construction and verification result of the ΔPaCIA30 mutant in the examples of this application;

[0028] Figure 4 It is the construction and verification result of the ΔPaCIA30com complementation strain in the embodiment of the present application;

[0029] Figure 5 This is a graph of hyphae growth of the WT strain and the ΔPaCIA30 mutant within 7 days of culture in the examples of the present application;

[0030] Figure 6 This is a graph showing the growth life span of the WT strain and the ΔPaCIA30 mutant in the examples of the present application;

[0031] Figure 7 This is a graph showing the growth diameter measurement of the WT strain and the ΔPaCIA30 mutant in the examples of the present application;

[0032] Figure 8 It is a microscopic observation picture of the fruiting body morphology and ascospores of WT and ΔPaCIA30 in the examples of the present application;

[0033] Fig. 9This is the result of Elisa immunocrosslinking experiment between WT strain and ΔPaCIA30 mitochondrial complex I in the example of this application;

[0034] Fig.10 This is a graph showing the NADH content in mycelium of the WT strain and the ΔPaCIA30 mutant in the examples of the present application;

[0035] Fig.11 This is a graph showing the growth diameter of the WT strain and ΔPaCIA30 in 10 μM rotenone medium in the examples of the present application;

[0036] Fig.12 This is a graph showing the detection of ATP content in hyphae of the WT strain and the ΔPaCIA30 mutant in the examples of the present application;

[0037] Fig.13 This is the ROS tissue staining result of the WT strain and the ΔPaCIA30 mutant in the examples of this application;

[0038] Fig.14 It is a volcano plot of differential protein analysis between the WT strain and the ΔPaCIA30 mutant in the example of this application;

[0039] Fig.15 This is a bar graph of differential protein screening between the WT strain and the ΔPaCIA30 mutant in the examples of this application;

[0040] Fig.16 It is a heat map of hierarchical clustering analysis of WT strain and ΔPaCIA30 mutant in the examples of this application.

[0041] The mutant strain ΔPaCIA30 of the present application, whose Latin name is: Podospora anserina, is deposited in the China Center for Type Culture Collection (CCTCC), address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: December 9, 2024, deposit number: CCTCC M 20242756. DETAILED DESCRIPTION

[0042] The fruiting body development of P. anserina is complex, involving multiple biological processes such as cell recognition, meiosis, cell differentiation and various vesicle transport, and these processes are inseparable from the support of cellular energy supply. Mitochondria are the center of cell metabolism and regulation, and are an important guarantee for a series of physiological and biochemical processes such as energy production and metabolism, biosynthesis, oxidative stress, calcium homeostasis, cell aging and death. CIA30 protein is an assembly factor of mitochondrial complex I intermediate (MCIA). It was first identified in N. crassa. Mutations in the gene encoding this protein in corn can cause abnormal seed development. At the same time, this gene is also related to many human diseases. However, no relevant studies have yet specifically characterized the specific molecular functions of CIA30 protein.

[0043] Based on the split-marker homologous recombination technology, this study constructed a knockout mutant ΔPaCIA30 of the putative coding gene PaCIA30 (Pa-1-20440) of the MCIA assembly factor CIA30. It was found that the fruiting body development of this mutant was severely impaired and ascospores could not be formed. However, the mycelium could grow for 21 days without aging and had a strong mycelium extension ability, which is of great value and significance for the breeding of industrial strains with efficient mycelium growth.

[0044] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be construed as limiting the present application.

[0045] Example

[0046] 1. Materials and Methods

[0047] 1. Construction and cultivation of experimental strains

[0048] The experimental strains in this study include the wild-type filamentous fungus Podospora anserina strain (WT) and the P. anserina protoplast Δmus51::nourseoR (mat+ / -) that effectively improves the efficiency of homologous recombination. The strain culture conditions and methods refer to the Podospora anserina Genome Project.

[0049] Visit address: http: / / podospora.i2bc.Paris-saclay.fr / index.php.

[0050] In this study, the gene knockout mutant ΔPaCIA30 was constructed based on the principle of homologous recombination. Hygromycin B was selected as the screening marker, and the primers MKF / MKR were used to amplify the resistance fragment with PBC-Hygro as the gene template; the primers 1F / 2R and 3F / 4R were used to amplify the upstream 5' fragment and the downstream 3' fragment of the target gene PaCIA30, respectively. The two fragments were connected to the resistance gene by fusion PCR to obtain two fusion fragments, and the primers were 1F / MKR and MKF / 4R. The two fusion fragments were transferred into the protoplast Δmus51::nourseoR(mat+ / -), and the transformants were screened using primers YzF / YzR. At least three identified transformants were selected for genetic hybridization with WT(mat-), and microspores that only retained the resistance to Hygromycin B were screened and identified by PCR sequencing.

[0051] In this study, the complementing strain ΔPaCIA30 was constructed based on the co-transfection principle. comGeneticin was selected as a screening marker, and the complete coding region of PaCIA30 was amplified using primers HbF / HbR. The complemented fragment and PBC-geneticin were co-transfected into ΔPaCIA30 protoplasts (mat+). The transformants with hygromycin and geneticin resistance were verified using primers HbF / HbR and identified by phenotypic analysis. The specific method for constructing mutant strains is referenced to patent application 202311166400.5. The primers used in this study are shown in Table 1.

[0052] Table 1 Primer sequences

[0053]

[0054] The main reagents and instruments used in this case include: M5 Fungal Genomic DNA Kit purchased from Beijing Juhemei Biotechnology Co., Ltd.; Go DNA Polymerase, Ex DNA Polymerase and Max were all produced by Takara; BCA Protein Quantification Kit was purchased from Novozymes Biotech Co., Ltd.; Hygromycin and other reagents were purchased from Sangon Biotech Co., Ltd.; lytic enzyme was purchased from Sigma; mitochondrial complex I content detection kit (ELISA, MM-92768702); NADH content determination kit (Biyuntian, S0175); ATP content determination kit (Biyuntian, S0027); NBT, DAB colorimetric kit and superoxide dismutase (SOD) activity detection kit were purchased from Beijing Solebow Technology Co., Ltd.; light incubator (S / N: 21004-12938) was purchased from Taiwan Hibot Co., Ltd.; upright microscope (Primo Star) was purchased from ZEISS; inverted microscope (12VDC) was purchased from Motic.

[0055] 2. Bioinformatics Analysis

[0056] The gene encoding the putative CIA30 protein was searched from the P. anserina genome library and named PaCIA30 (Pa_1_20440). The species whose CIA30 protein had been identified or had high credibility were searched from the Uniprot website, including Neurosporacrassa (Uniprot accession number: O42636), Schizosaccharomyces pombe (Uniprot accession number: O14297), Mus musculus (Uniprot accession number: Q9CWX2), Gorilla gorilla (Uniprot accession number: Q0MQ83), Drosophila melanogaster (Uniprot accession number: Q9VAI1), Homo sapiens (Uniprot accession number: Q9Y375), Pan troglodytes (Uniprot accession number: Q0MQ84), Caenorhabditis briggsae (Uniprot accession number: Q61FQ3), and Arabidopsis pilosula (Uniprot accession number: Q61FQ5). thaliana (Uniprot accession number: Q9LQI7). Mega 11 automatically adjusted the multiple sequence alignment and constructed the evolutionary tree using the maximum likelihood method.

[0057] 3. Characterization Analysis

[0058] Growth morphology and life span: The WT strain and the ΔPaCIA30 mutant were grown in dextrin medium (M2) for 2 days, the bacterial blocks were cut, and the agar blocks were dispersed by fast-prep to prepare bacterial liquid. 10 μL of bacterial liquid was inoculated in the center of M2 medium (d = 9 cm), and cultured at 27°C constant light and temperature for one week. The mycelial growth diameter was recorded and the growth morphology of the strain was preserved by taking photos. 10 μL of bacterial liquid was inoculated at the edge of M2 medium (d = 18 cm), and cultured at 27°C constant light and temperature. The growth diameter was measured every day until the mycelium stopped growing. At this time, the mycelial growth time was defined as the growth life span of P. anserina. Reference: Stumpferl SW, Stephan OFau-Osiewacz HD, Osiewacz HD. Impact of a disruption of a pathway delivering copper to mitochondria on podospora anserina metabolism and life span[J].(1535-9778(Print)).

[0059] Gamete fertility analysis: Male and female gamete fertility of the ΔPaCIA30 mutant was determined as described previously. Male and female fertility of ΔPaCIA30 was determined by growing the strain on the same M2 medium plate with the WT strain of the opposite mating type. When the hyphae of the two cultures were conjugated, the culture was continued to observe the formation of fruiting bodies. References: LiY, Yan P, Lu X, et al. Involvement of pasnf1 in fungal development, sterigmatocystin biosynthesis, and lignocellulosic degradation in the filamentous fungus podospora anserina[J].(1664-302X(Print)); and Sellem CH, Lemaire C Fau-Lorin S, Lorin S Fau-Dujardin G, et al. Interaction between theoxa1 and rmp1 genes modulates respiratory complex assembly and life span inpodospora anserina[J].(0016-6731(Print)).

[0060] Observation of fruiting body morphology: 10 μL of different mating type bacterial liquid was inoculated in the center of the M2 culture dish, cultured at 27℃ for 7 days, and observed under an inverted microscope. The number of fruiting bodies formed was observed under a 4x objective lens, and then a single fruiting body was picked and placed on a slide, covered with a cover glass and lightly crushed, and the contents of the fruiting body, i.e. the eruption of ascospores, were observed under a 10x objective lens.

[0061] 4. Determination of mitochondrial complex I content

[0062] P. anserina mitochondria were isolated according to the study of Adam et al. (Lee YW, Adam C, Picard M, et al. Biological roles of the podospora anserina mitochondrial lon protease and the importance of its n-domain [J]. PLoS ONE, 2012, 7 (5)). The strain was grown on M2 medium covered with cellophane for 2-3 days at 27°C under constant light and temperature. Mycelia were collected and dissolved in 0.7 M sorbitol buffer (50 mM Tris-HCl, pH 7.5 and 0.2 mM EDTA) containing protein inhibitors. Mitochondria were purified by differential centrifugation. The protein was resuspended in 0.7 M sorbitol buffer again. The mitochondrial protein concentration was determined by Brad-ford method and used for subsequent mitochondrial complex I content Elisa analysis. The detection steps were referred to the manufacturer's instructions for the kit (ELISA, MM-92768702).

[0063] 5. NADH content determination

[0064] P. anserina was cultured in M2 medium covered with cellophane at 27°C for 3 days. The lysis solution was added at a ratio of about 400 μL of lysis solution per 10-30 mg of mycelium, mixed, and placed on ice for 5-8 minutes to allow lysis to fully release NADH. After lysis, the supernatant was taken and heated in a water bath at 60°C for 30 minutes. The NAD + The solution will be decomposed and only NADH will be retained. The mixture is mixed and the protein concentration is determined by BCA. The solution is also used for the determination of NADH content in mycelium. The detection steps refer to the manufacturer's instructions for the kit (Biyuntian, S0175).

[0065] 6. ATP content determination

[0066] P. anserina was cultured in M2 medium covered with cellophane at 27°C for 2 days and 5 days, and the hyphae were collected respectively. The hyphae cell walls were destroyed by Fast-prep, and lysis solution was added at a ratio of about 100-200 μL of lysis solution per 20 mg of hyphae. The mixture was mixed and placed on ice for 3-5 min to lyse it and fully release ATP. After lysis, the mixture was centrifuged at 12000×g at 4°C for 5 min, and the supernatant was collected and the protein concentration was determined by BCA, and it was also used for the determination of hyphae ATP content. The detection steps were referred to the manufacturer's instructions for the kit (Biyuntian, S0027).

[0067] 7. ROS content analysis

[0068] The determination of ROS content in P. anserina was based on the study of Warnsmann et al. (Warnsmann V, Hainbuch S, Osiewacz H D. Quercetin-induced lifespan extension in podospora anserina requires methylation of the flavonoid by the o-methyltransferase pamth1 [J]. Frontiers in Genetics, 2018, 9). Take an appropriate amount of DAB colorimetric reagent or NBT reagent to infiltrate the mycelium, shake it at a low speed in a dark place for 3-5 hours, slowly pour out the staining reagent, wash the mycelium with sterile water to terminate the staining reaction, and take pictures to preserve the staining of the mycelium of the strain.

[0069] 8. Proteomic Analysis

[0070] P. anserina was cultured at 27°C on M2 medium covered with cellophane for 3 days, and the mycelium was collected for proteomic analysis based on the label-free principle. Proteins were extracted, and the protein quality was verified by SDS-PAGE and then digested by trypsin. Each sample was separated by nano-HPLC reverse phase chromatography and mass spectrometry, and protein identification and LFQ quantitative analysis were performed using PD / MaxQuant library search software. References: Plubell DL, Wilmarth PA, Zhao Y, et al. Extended multiplexing of tandem mass tags (tmt) labeling reveals age and high fat diet-specific proteome changes in mouse epididymal adipose tissue [J]. Molecular & Cellular Proteomics, 2017, 16 (5): 873-890.

[0071] 2. Results Analysis

[0072] 1. Evolution analysis of PaCIA30

[0073] The gene encoding the putative mitochondrial complex I intermediate assembly factor CIA30 was searched from the P. anserina genome sequence and named PaCIA30 (Pa-1-20440). Mega 11 was used to perform multiple sequence comparison analysis on PaCIA30 and construct a phylogenetic evolutionary tree. The comparison species included animals, plants, and fungi. Among them, CIA30 of N. crassa has been identified as a mitochondrial complex I intermediate assembly factor. Figure 1 and Figure 2 As shown, CIA30 protein is somewhat conserved among different species. PaCIA30 and N.crassa are paralogs with high similarity. The full length of the amino acid multiple sequence alignment of PaCIA30 and N.crassa is 295 (including redundant deleted sequences), 111 amino acid sequences are inconsistent, and 184 amino acid sequences are similar, indicating that PaCIA30 has more than 50% sequence similarity with N.crassa, and also suggests that PaCIA30 has similar protein functions to N.crassa.

[0074] Figure 1 This is a comparison chart of multiple sequences of PaCIA30. Figure 2 This is the developmental tree of PaCIA30.

[0075] 2. ΔPaCIA30 mutant and ΔPaCIA30 com Construction of complementation strains

[0076] Using the WT (mat+) genome as a template, PCR amplification was performed to obtain the flanking fragments within about 2000 bp of PaCIA30, and the PBC-Hygro plasmid DNA was used as a template to amplify the resistance gene Hygro screening fragment. Fusion PCR was used to construct the gene expression knockout cassette PaCIA30-5'-Hygro and Hygro-PaCIA30-3' two connecting fragments. The construction and transformant verification results are shown in Figure 3 However, we were unable to purify transformants of the opposite mating type by genetic hybridization. The model fungus P. anserina has a non-recombining region of about 0.8 Mb on chromosome 1, which is located near the mating type gene (mating type), showing recombination inhibition. PaCIA30 is located exactly in this non-recombining region and is close to the centromere. Therefore, unlike the previous method of obtaining mutant strains of different mating types through genetic hybridization, this study directly constructed two mating type ΔPaCIA30 mutants (mat+ and mat-).

[0077] Figure 3The construction and verification results of the ΔPaCIA30 mutant, where (A) shows the amplification electrophoresis results of the upstream and downstream fragments of the PaCIA30 gene and the resistance marker gene; (B) shows the amplification electrophoresis results of the PaCIA30 gene fusion fragment; (C) shows the upstream and downstream verification electrophoresis results of the ΔPaCIA30 (mat+) mutant transformant; (D) shows the upstream and downstream verification electrophoresis results of the ΔPaCIA30 (mat-) mutant transformant.

[0078] Using WT (mat+) genomic DNA as a template, PCR amplification was performed to obtain the complete coding region of PaCIA30. The complemented fragment and PBC-geneticin were co-transfected into ΔPaCIA30 (mat+) protoplasts. Transformants with dual resistance to hygromycin and geneticin were screened for PCR verification. Phenotypic analysis showed that the growth characteristics of the complemented strain were restored to the WT level, indicating that the complemented strain ΔPaCIA30com was successfully constructed. Figure 4 shown.

[0079] Figure 4 The construction and verification results of the ΔPaCIA30com complementing strain, where (A) shows the electrophoresis results of PaCIA30 and geneticine resistance genes; (B) shows the electrophoresis results of the ΔPaCIA30com transformant verification; (C) shows the phenotype of the ΔPaCIA30com complementing strain.

[0080] 3. The growth lifespan of ΔPaCIA30 was significantly prolonged, and the fruiting body development was hindered

[0081] The WT strain and the ΔPaCIA30 mutant were inoculated on M2 medium to analyze the mycelial growth rate and lifespan. Figures 5 to 7 As shown in the figure, after the WT strain entered the sexual reproduction process, that is, 7 to 9 days of culture, its hyphae almost stopped extending and growing, and the outer circle of the hyphae showed obvious brown-green pigments, showing aging characteristics, that is, cultured to 11 days. In comparison, the hyphae growth rate of the ΔPaCIA30 mutant was significantly slower than that of the WT before 7-9 days, but it showed excellent hyphae extension characteristics during the subsequent growth period. After 15 days, the hyphae can still continue to grow and can extend to the end of the culture dish (d = 19 cm).

[0082] Figure 5 This is the hyphal growth diagram of the WT strain and the ΔPaCIA30 mutant within 7 days of culture. Figure 6 Figure 2 shows the growth lifespan of the WT strain and the ΔPaCIA30 mutant. Figure 7 Diameter measurements of the WT strain and the ΔPaCIA30 mutant. t-test: *P<0.05, **P<0.01, ***P<0.001.

[0083] The ΔPaCIA30 mutant and the WT strain of the opposite mating type were inoculated on M2 medium to analyze the sexual reproduction of ΔPaCIA30, including gamete fertility, fruiting body morphology, and ascospore eruption. Figure 8 As shown in the figure, ΔPaCIA30 (mat+ / mat-) and WT (mat- / mat+) were genetically hybridized, and both formed fruiting bodies and sprayed ascospores after 7-9 days of culture, indicating that both male and female gametes of the ΔPaCIA30 mutant can maintain fertility. However, when the opposite mating type of ΔPaCIA30 was hybridized, the sexual reproduction process of the mutant was abnormal after 7-9 days. The mature fruiting bodies produced by the WT strain usually show plump asci at the bottom and hooks with spray holes on the neck. The morphological development of the fruiting bodies of the ΔPaCIA30 mutant is abnormal. The fruiting bodies are very small and spherical, the neck structure is missing, the asci development is blocked, and ascospores cannot be produced.

[0084] 4. Mitochondrial complex I is affected in the ΔPaCIA30 mutant

[0085] In N.crassa and humans, CIA30 protein has been shown to be involved in the assembly of mitochondrial complex I intermediate (MCIA). The loss of this protein will lead to the accumulation of MCIA membrane arms, while affecting the assembly of other modules of mitochondrial complex I (CI), and ultimately affecting the function and activity of the whole enzyme. In order to explore whether the loss of PaCIA30 has a similar effect on P.anserina mitochondrial complex I, this study analyzed the content of mitochondrial complex I and NADH. The content of mitochondrial complex I in the ΔPaCIA30 mutant was significantly increased (3d, Fig. 9 ), NADH content decreased significantly (3d, Fig.10 ). Rotenone is one of the respiratory inhibitors that can compete with ubiquinone to close the mitochondrial complex I respiratory channel. When 10 μM rotenone was added to M2 medium, the mycelial growth rate of WT and ΔPaCIA30 mutant decreased significantly, and there was no significant difference in the degree of inhibition between the two ( Fig.11 ).

[0086] Fig. 9 The results of Elisa immunocrosslinking experiments of mitochondrial complex I between WT strain and ΔPaCIA30 mutant. Fig.10 This is the detection diagram of NADH content in the hyphae of WT strain and ΔPaCIA30 mutant. Fig.10 Diameter measurement of WT strain and ΔPaCIA30 mutant in 10 μM rotenone medium. Fig.12 The graph shows the detection of ATP content in mycelium of WT strain and ΔPaCIA30 mutant. t test: *P<0.05, **P<0.01, ***P<0.001.

[0087] 5. ATP synthesis is affected in the ΔPaCIA30 mutant

[0088] ATP is an important indicator of cell energy status. After 2 days of growth, the ATP content of the ΔPaCIA30 mutant did not show a significant downward trend compared with the WT; as time went on, the ATP content of the ΔPaCIA30 mutant decreased significantly after 5 days of growth ( Fig.12 ), indicating that PaCIA30 may be related to the ATP synthesis process.

[0089] 6. Altered ROS production in the ΔPaCIA30 mutant

[0090] In P. anserina, strains with extended lifespan or delayed aging often show the characteristics of reduced content of reactive oxygen free radicals (ROS). Mitochondria are the main sites of ROS generation, namely mitochondrial complex III and mitochondrial complex I. In order to explore whether the extended lifespan of the ΔPaCIA30 mutant is related to changes in ROS content, this study used hyphal tissue staining to determine whether 3,3'-diaminobenzidine (DAB) and nitro blue tetrazolium chloride (NBT) staining were used to visualize the relative levels of reactive oxygen free radicals in the strains. DAB can react with hydrogen peroxide to produce a reddish-brown precipitate, while yellow NBT can be reduced by superoxide radicals to blue water-insoluble formazan. The staining results are shown in the figure. Fig.13 As shown, the hydrogen peroxide content of the ΔPaCIA30 mutant was significantly reduced compared with the WT strain ( Fig.13 A). However, after 7 days of culture, NBT staining was deeper than that of the WT strain ( Fig.13 B), but overall, the ROS content of the ΔPaCIA30 mutant was reduced, indicating that the ΔPaCIA30 mutant produced superoxide radicals in addition to hydrogen peroxide in the late growth stage.

[0091] Fig.13 The results of ROS tissue staining of the WT strain and the ΔPaCIA30 mutant are shown in Figure 1, where Figure A is the DAB staining result and Figure B is the NBT staining result.

[0092] 7. Proteomic Analysis of ΔPaCIA30 Mutant

[0093] In the proteomics analysis, the screening criteria were P-adjust < 0.05, 0.83 ≤ |log2FC| ≤ 1.2, there were 861 up-regulated proteins, 667 down-regulated proteins, and a total of 1528 differentially expressed proteins were screened, such as Fig.14 and 15There are 43 differentially expressed proteins related to cell cycle, cell differentiation and chromatin remodeling, as shown in Table 2. A series of changes in the abundance of respiratory chain-related proteins were also found in the ΔPaCIA30 mutant, such as Fig.16 shown.

[0094] The expression levels of mitochondrial complex I subunit encoding genes Pa_4_7160, Pa_5_6010, Pa_1_1162, Pa_3_4100, Pa_1_11820, Pa_2_5150, Pa_7_11270 and ND4 were significantly upregulated, while the expression levels of genes such as ND2, Pa_2_12930, Pa_2_7140, Pa_5_5380, Pa_1_22280, Pa_2_12660, Pa_4_7950 and Pa_1_12970 were significantly downregulated, which may reflect a compensatory response to the complex I assembly defect of the ΔPaCIA30 mutant and indicate the important role of PaCIA30 in maintaining the composition and function of mitochondrial complex I.

[0095] Mitochondrial complex II subunit encoding genes Pa_1_3300 and Pa_3_10550, cytochrome c oxidase encoding genes PaCOX1, PaCOX2 and PaCOX3, and ATP synthase subunit ATP6 all showed up-regulated expression levels to varying degrees. In particular, in the differential protein screening, the expression of alternative NADH dehydrogenase encoding genes increased, namely PaNDE2 (external, Pa_7_5390), while PaNDⅠ1 (internal, Pa_7_1820) also showed an upward trend, although not significantly, as shown in Table 2.

[0096] Table 2 Some differentially expressed proteins in proteomic analysis

[0097]

[0098]

[0099]

[0100] Fig.14This is a volcano plot of differential protein analysis between the WT strain and the ΔPaCIA30 mutant. Each point represents a detected protein, and the horizontal axis represents the fold change of each protein in the group (taking the logarithm with base 2). The greater the difference, the whiter the distribution is at both ends of the X-axis. The positive and negative values ​​of the LOG_FOLDCHANGE value can be used to determine the up- and down-regulation of the protein. The vertical axis represents Q-VALUE (taking the negative number of the logarithm with base 10). The smaller the Q value, the larger the value after taking the logarithm, and the higher it goes in the vertical axis direction, the more significant the difference is. The significantly up-regulated differentially expressed proteins are represented in red, the significantly down-regulated differentially expressed proteins are represented in blue, and the proteins with non-significant differences are represented in gray. Fig.15 This is a bar graph of differential protein screening between WT strain and ΔPaCIA30 mutant. Red represents significantly upregulated differential proteins, and blue represents significantly downregulated differential proteins. In the figure, A represents WT strain and B represents ΔPaCIA30 mutant. Fig.16 Heat map of hierarchical clustering analysis of WT strain VSΔPaCIA30 mutant, red represents high expression level and blue represents low expression level.

[0101] Discussion

[0102] In this study, the putative coding gene PaCIA30 of the MCIA assembly factor CIA30 in P. anserina was successfully knocked out by homologous recombination technology to construct the ΔPaCIA30 mutant. This mutant showed abnormalities in fruiting body development, morphology and function, and exhibited excellent mycelial growth ability in the late growth stage.

[0103] 1. PaCIA30 is involved in regulating the development of P. anserina fruiting bodies

[0104] The ΔPaCIA30 mutant showed obvious defects in fruiting body development and ascospore formation, and the sexual reproduction process was severely blocked. Compared with WT, the fruiting bodies of the ΔPaCIA30 mutant could not form neck structures normally, ascus development was blocked, and ascospores could not be produced. These changes indicate that PaCIA30 may play a role in the sexual reproduction of P. anserina, especially in fruiting body development and ascospore formation. Even though PaCIA30 is located in the mating type non-recombination region (Pa_1_18610 to Pa_1_20880), the male and female gametes of the ΔPaCIA30 mutant can still maintain normal fertility. In the study of Grognet et al., knocking out the Pa_1_20880 gene caused P. anserina to be unable to erupt ascospores, although the fruiting body formation process remained normal, while knocking out the Pa_1_19950 gene did not observe obvious phenotypic changes, indicating that the abnormal morphological development of the fruiting body of the ΔPaCIA30 mutant is due to the loss of PaCIA30.

[0105] In P. anserina, studies on the regulation or influence of fruiting body development on NAD / NADP redox-related enzymes, MAP kinases, phosphorylation status of inositol triphosphate messengers, nucleotide metabolism-related enzymes or cofactors, peroxisomes, and DNA methylation have been reported one after another. In the ΔPaCIA30 mutant, 43 differentially expressed proteins related to cell cycle, cell differentiation, and chromatin remodeling were found, as shown in Table 2. PODANS_3_8080 encodes a putative mob1 protein, which is directly involved in N. crassa meiosis and ascospore formation, and the growth rate of the Δmob1 mutant is reduced to 40% of WT, the aerial hyphae are reduced, and the female reproductive structure cannot be produced. In budding yeast, PP2A-Cdc55 phosphatase regulates mitosis and can offset Cdk1- and Cdc5-dependent phosphorylation. PP2A-related differential expression was also found in mutants, such as PODANS_6_4830 and PODANS_7_9720, indicating that the abnormal fruiting body development of ΔPaCIA30 may be related to changes in cell cycle and differentiation.

[0106] 2. Abnormal fruiting body development in ΔPaCIA30 may be related to reduced ATP synthesis

[0107] Mitochondrial complex I (CI), also known as NADH dehydrogenase, has an L-shaped structure, with one side extending into the hydrophobic matrix (matrix arm) and one side extending into the hydrophilic membrane arm (membrane arm). It is the starting protein on the mitochondrial respiratory chain. CI couples the two-electron transfer reaction from NADH to ubiquinone with the vectorial translocation of four protons, providing the main part of the proton motive force that drives ATP synthesis, and is a key link in energy metabolism. Many mitochondrial dysfunctions are related to structural defects or assembly disorders of mitochondrial complexes, which can easily cause human diseases. It is reported that mitochondrial complex IV deficiency is usually caused by mutations in assembly factors, and similar situations also occur in complex I. The loss of any key assembly factor may affect CI assembly and function. Knockout of NDUFB10 leads to incomplete assembly of the N module of complex I, affecting the activity of complex I; impaired respiration of complex I was also found in NDUFS2 knockout cell lines.

[0108] The structure of CⅠ depends on the action of many assembly factors. PaCIA30 encodes the putative CⅠ intermediate assembly factor CIA30, and the loss of this protein leads to the accumulation of membrane arm intermediates. In the ΔPaCIA30 mutant, the number of mitochondrial complex I increased significantly (P=0.0017) and the content of hyphae NADH decreased significantly (P=0.0013) after 3 days of culture. At the same time, it caused dynamic changes in P. anserina mitochondrial proteins, which is similar to previous studies, indicating that the loss of PaCIA30 affects the structural stability of complex I. Marques et al. found that the loss of the 9.8KDa subunit of N. crassa mitochondrial complex I caused the inability to form spores. Andrea et al. found that the putative mitochondrial complex I assembly factor PRO34, which is homologous to N. crassa CIA84, is required for rapid vegetative growth, fruiting body and ascospore formation. Sun Yue found that knocking out the key gene encoding NADH dehydrogenase hindered the reproductive growth of the filamentous fungus P. anserina.

[0109] However, the ΔPaCIA30 mutant still retains sensitivity to the complex I inhibitor rotenone, which may be related to the parallel assembly mode of complex I. Previous studies have shown that the matrix arm and membrane arm of complex I are independent. In N.crassa, the loss of the 20.8KDa subunit in complex I prevents the complete assembly of complex I, but the assembly intermediates of the matrix arm and membrane arm of complex I can still be formed in the mutant. In yeast, the auxiliary subunit NUYM (NDUFS4) was knocked out, and the mutant could also maintain nearly 50% of the NADH-ubiquinone oxidoreductase activity of the wild type. Therefore, we speculate that the ΔPaCIA30 mutant can still form assembly intermediates of each module and perform partial enzyme functions.

[0110] On the other hand, the ATP6 subunit of the ΔPaCIA30 mutant was significantly upregulated, as shown in Table 2. The ATP6 subunit, together with other subunits, constitutes the F0 structure of the ATP synthase, which is embedded in the inner membrane of the mitochondria and participates in the transmembrane transport of protons. It interacts with the F1 structure and uses the electrochemical energy generated by the proton gradient to synthesize ATP, which is an essential step in the ATP synthesis process. Mutations in the ATP6 gene are associated with a variety of clinical phenotypes, including neurodegenerative diseases and many systemic diseases. At the same time, signs of possible impaired mitochondrial respiration were also found in the ΔPaCIA30 mutant, such as the significant upregulation of the external alternative NADH dehydrogenase PaNDE2, complex IV encoding subunits PaCOX1, PaCOX2 and PaCOX3, and respiratory supercomplex RCF1 factors, which are related to the triggering of respiratory compensatory pathways by fungi when mitochondrial respiration is blocked. The most notable feature of the alternative respiratory pathway is its low energy demand, which is consistent with the result of reduced ATP synthesis in the ΔPaCIA30 mutant.

[0111] Therefore, the abnormal fruiting body development of ΔPaCIA30 may be related to the reduction of ATP synthesis. The loss of PaCIA30 affects the structural homeostasis of complex I, resulting in the blockage of the mitochondrial ATP production pathway, and its energy supply cannot meet the needs of sexual reproduction, thus affecting the fruiting body development and ascospore formation.

[0112] 3. Mycelial growth of the ΔPaCIA30 mutant compensates for the fruiting body development defect

[0113] During the electron transfer process of the mitochondrial respiratory chain, electron leakage is prone to occur, forming free radicals, causing cell damage, and ultimately causing fungal aging. Mitochondrial complex I is also one of the main target sites for ROS generation. Complex I is the largest component of the respiratory chain. The mitochondrial-encoded core subunit is wrapped in the innermost layer of complex I and contains all enzyme catalytic sites. NADH oxidation in mitochondrial complex I begins at the top of the matrix arm. The release of protons in the matrix is ​​followed by the transfer of two electrons. The electrons will pass through the Fe-S cluster along the matrix arm to the junction with the membrane arm, where the electrons are transferred to ubiquinone.

[0114] Many studies have shown that the growth lifespan of P. anserina is related to the generation of mitochondrial ROS, including gene mutations of cytochrome c1, abnormalities of mitochondrial contact sites and cristae organization system (MICOS), and oleic acid metabolism, which can affect the mitochondrial respiratory chain, produce less ROS, and delay fungal aging. The absence of PaCIA30 also leads to a reduction in the ROS content of the strain. Although the fruiting body development and ascospore production are impaired, the mutant shows excellent hyphal extension ability in the late growth stage and can grow for more than 21 days without aging, which is of great significance for screening P. anserina strains with industrial application prospects. P. anserina can degrade and transform aromatic compounds and organic waste, and can be used in environmental protection fields such as degradation of lignocellulose and organic waste. However, P. anserina fruiting bodies and ascospores cannot effectively degrade lignocellulose and organic waste, and the various biological enzymes secreted by the hyphae are the effective components of biodegradation and transformation.

[0115] In addition, the loss of PaCIA30 caused significant expression of mitochondrial proteins such as Pa_1_740 (encoding a putative mitochondrial import inner membrane transporter subunit TIM50), Pa_7_5320 (encoding a putative mitochondrial import inner membrane transporter subunit TIM23), Pa_2_11710 (encoding a putative mitochondrial import inner membrane transporter subunit TIM22), OXA1 and rmp1, as shown in Table 2. The biosynthesis and function of respiratory chain complexes and F1F0-ATP synthase depend on the import of several mitochondrial precursor proteins produced on cytoplasmic ribosomes. Nuclear-encoded mitochondrial proteins form import channels through the outer membrane translocase (TOM) and interact with the mitochondrial membrane to enter various parts of mitochondria. The assembly and insertion of some subunits of complex I depend on the OXA1 gene and interact with the Rmp1 gene. The loss of the OXA1 gene in P. anserina leads to a phenotype of extended lifespan in mutants, and also shows defects in the activity of complex I and IV.

[0116] In summary, PaCIA30 deficiency reduced ROS generation, altered mitochondrial protein balance, led to impairment of the main source of energy ATP synthesis, and caused defective fruiting body development in the strain, which was replaced by vegetative growth of hyphae.

[0117] IV. Conclusion

[0118] This study revealed the key role of CIA30, an intermediate assembly factor of mitochondrial complex I, in the fruiting body development of P. anserina. The construction and analysis of the ΔPaCIA30 mutant showed that CIA30 is essential for the normal morphological establishment of fruiting bodies and the formation of ascospores. The fruiting body development of the mutant was blocked and ascospores could not be formed, which may be due to insufficient energy supply caused by defective assembly of mitochondrial complex I and reduced ATP synthesis. Nevertheless, the ΔPaCIA30 mutant showed excellent hyphal extension ability in the late growth stage and significantly extended growth lifespan, indicating that it compensated by enhancing hyphal growth in the case of fruiting body development defects, emphasizing the importance of CIA30 in regulating the development and aging of P. anserina fruiting bodies, laying a foundation for the breeding of industrial strains with efficient hyphal growth.

[0119] The above contents are further detailed descriptions of the present application in combination with specific implementation methods, and it cannot be determined that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A mutant strain of a filamentous fungus with a defect in fruiting body development, characterized in that: The mutant strain is formed by knocking out the mitochondrial complex I intermediate assembly factor CIA30 gene from the wild-type filamentous fungus Podospora anserina.

2. The mutant strain according to claim 1, characterized in that: The deposit number of the mutant strain is CCTCCM20242756.

3. Use of the mutant strain according to claim 1 or 2 in preparing efficient growing mycelium.

4. The use according to claim 3, characterized in that: The efficient mycelium growth means that the mutant strain has a longer mycelium lifespan and / or a stronger mycelium extension ability compared to the wild-type filamentous fungus Podospora anserina.

5. A method for increasing the mycelial yield of the filamentous fungus Podospora anserina, characterized in that: The method comprises culturing the mutant strain according to claim 1 or 2 for at least 15 to 21 days.

6. The method according to claim 5, characterized in that: The culture conditions are constant temperature culture at 27° C. under light conditions.

7. The method according to claim 5 or 6, characterized in that: The culture medium used in the culture is M2 culture medium.

8. A method for extending the life span of mycelium of filamentous fungus Podospora anserina, characterized in that: This includes using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

9. A method for improving the mycelial extension ability of the filamentous fungus Podospora anserina, characterized in that: This includes using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

10. A method for regulating the development of fruiting bodies of the filamentous fungus Podospora anserina, characterized in that: This includes using gene knockout technology or gene silencing technology to prevent or weaken the expression of the mitochondrial complex I intermediate assembly factor CIA30 gene of the filamentous fungus Podospora anserina.

Images