Trichoderma harzianum vacuole phosphate transporter gene TrVPT1 and application thereof

Abstract

The invention belongs to the technical field of genetic engineering and biology, and relates to application of trichoderma harzianum TrVPT1 in regulation and control of trichoderma phosphorus accumulation and gibberellic disease resistance. The nucleotide sequence of the trichoderma harzianum vacuole phosphate transporter gene TrVPT1 is as shown in SEQ ID NO: 1. The invention proves that the TrVPT1 gene has an important regulation and control effect in trichoderma phosphorus steady-state regulation and control and biological prevention and control functions.

Description

Technical Field

[0001] The invention belongs to the fields of genetic engineering and biotechnology, and in particular relates to the application of Trichoderma harzianum TrVPT1 in regulating Trichoderma phosphate accumulation and resistance to scab. Background Art

[0002] Phosphorus is an essential nutrient for plant growth and development and a key component of many macromolecular metabolites and compounds in plants, such as ATP, nucleic acids, and phospholipids. Plants primarily absorb phosphorus as orthophosphate. However, phosphorus in soil readily chelates with metal ions to form insoluble compounds. Furthermore, soil microorganisms convert some phosphorus into organic compounds, significantly reducing the availability of phosphorus for plant uptake and utilization. This is a key limiting factor in crop yield. To increase crop yields, large amounts of phosphorus fertilizer are often applied in agricultural production. However, long-term excessive application of phosphorus fertilizers leads to significant phosphorus accumulation in the soil, increasing production costs and contributing to a range of environmental problems, including eutrophication and harmful algal blooms. To cope with relatively scarce phosphorus environments, plants have evolved two strategies to improve phosphorus utilization efficiency: microbial-independent pathways (direct phosphorus uptake through roots) and microbial-dependent pathways (phosphorus uptake through interactions with microorganisms). However, research on soil phosphorus uptake and responses by soil microorganisms is relatively scarce. Therefore, in-depth understanding of the molecular regulatory networks underlying these pathways is crucial for improving phosphorus fertilizer use efficiency.

[0003] Trichoderma spp., a beneficial fungus widely found in soil, not only promotes plant growth but also exhibits significant biocontrol capabilities, particularly in inhibiting plant pathogens. Studies have shown that Trichoderma can secrete substances such as organic acids and phosphatases, converting insoluble phosphates in the soil into forms that can be absorbed and utilized by plants, thereby increasing the availability of soil phosphorus. However, how Trichoderma regulates the absorption, transport, and homeostasis of phosphorus, particularly its phosphorus metabolism mechanisms in response to pathogen infection, remains unclear.

[0004] As the primary storage organ within the cell, the vacuole plays a key role in maintaining intracellular phosphorus homeostasis. Vacuolar phosphate transporters are a class of membrane proteins responsible for transporting phosphate from the cytoplasm to the vacuole, and their function directly affects the distribution and utilization efficiency of intracellular phosphorus. In recent years, researchers have discovered multiple vacuolar phosphate transporter genes in plants and yeast and confirmed that they play an important role in regulating phosphorus homeostasis. However, in Trichoderma, the function of vacuolar phosphate transporters and their role in biocontrol have not been fully studied.

[0005] Wheat head blight, caused by the fungus Fusarium graminearum, is a serious disease that not only reduces wheat yields but also produces mycotoxins, posing a threat to human and livestock health. Currently, chemical control is the primary means of controlling wheat head blight. However, long-term use of chemical agents can lead to increased resistance in pathogens and environmental pollution. Therefore, the development of microbial-based biocontrol technologies is of great significance. As a potential biocontrol agent, the relationship between Trichoderma's disease resistance mechanism and phosphorus metabolism has not been thoroughly explored.

[0006] Based on this background, the present study cloned and identified a vacuolar phosphate transporter gene, TrVPT1, from Trichoderma harzianum for the first time, and found that this gene plays an important role in regulating Trichoderma phosphate homeostasis and resistance to wheat head blight. This discovery provides a new theoretical basis for a deeper understanding of Trichoderma phosphate metabolism and its application in biocontrol, and also provides a potential gene resource for the development of novel biocontrol technologies based on phosphorus regulation. Summary of the Invention

[0007] The present invention aims to provide a Trichoderma vacuolar phosphate transporter gene TrVPT1 and its application in regulating Trichoderma phosphate homeostasis and resistance to wheat fusarium head blight.

[0008] In order to achieve the above object, the present invention provides a Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1, the nucleotide sequence of the Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1 is shown in SEQ ID NO: 1.

[0009]

[0010] The sequence of the expressed protein is shown in SEQ ID NO: 2:

[0011] (SEQ ID NO: 2).

[0012] The second aspect of the present invention provides the use of the above-mentioned Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1 in regulating Trichoderma phosphate accumulation and / or resistance to ergot disease.

[0013] According to a specific embodiment of the present invention, the regulation of Trichoderma phosphate accumulation is to control the total phosphorus content of Trichoderma mycelium.

[0014] More specifically, the regulation of Trichoderma phosphatase accumulation is to transport phosphorus from the vacuole to the cytoplasm.

[0015] According to a specific embodiment of the present invention, the fusarium head blight is a plant disease caused by Fusarium graminearum. The plants include but are not limited to wheat.

[0016] This study demonstrates that the TrVPT1 gene plays a crucial regulatory role in Trichoderma's phosphorus homeostasis and biocontrol functions. Using homologous recombination, a TrVPT1 knockout mutant, Δvpt1, was successfully generated, providing important experimental material for in-depth study of the TrVPT1 gene's role in Trichoderma's phosphorus metabolism and biocontrol. The study further reveals that the absence of the TrVPT1 gene leads to phosphorus accumulation in the vacuole, which in turn affects Trichoderma's growth and metabolism. Furthermore, the TrVPT1 gene may indirectly affect Trichoderma's biocontrol functions by regulating phosphorus metabolism and affecting the synthesis or secretion of Trichoderma's secondary metabolites.

[0017] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0019] Figure 1 : Expression analysis of TrVPT1 under phosphorus-deficient conditions and in antagonistic Fusarium graminearum, where A: relative expression level of TrVPT1 gene under phosphorus-deficient and phosphorus-deficient conditions; B: relative expression level of TrVPT1 gene in T-aloe alone and in co-culture with Fusarium graminearum.

[0020] Figure 2 : TrVPT1 transformant identification results, wherein A: TrVPT1 transformant identification primer design; B: TrVPT1 identification electrophoresis diagram.

[0021] Figure 3 : Determination of growth rate and total phosphorus content of T-aloe and mutant Δvpt1, where A: Growth of T-aloe and mutant Δvpt1 on PDA plates; B: Growth rates of T-aloe and mutant Δvpt1 at different time points; C: Growth of T-aloe and mutant Δvpt1 in liquid PDA; D: Total phosphorus content of T-aloe and mutant Δvpt1.

[0022] Figure 4: The inhibitory ability of T-aloe and mutant Δvpt1 metabolites on Fusarium graminearum, wherein, A: the phenotype of Fusarium graminearum grown in the medium of T-aloe and mutant Δvpt1 metabolites for three days; B: the inhibition rate of T-aloe and mutant Δvpt1 metabolites on Fusarium graminearum for 3 days; C: the phenotype of Fusarium graminearum grown in the medium of T-aloe and mutant Δvpt1 metabolites for five days; D: the inhibition rate of T-aloe and mutant Δvpt1 metabolites on Fusarium graminearum for 5 days.

[0023] In each figure, different letters represent the level of significant difference among treatments (P-Value<0.05), * represents P-Value<0.05, ** represents P-Value<0.01, and *** represents P-Value<0.001. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0025] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0027] Materials and methods used in the examples:

[0028] Strain: Trichoderma harzianum T-aloe, Gene Bank registration number KC753766, deposit number GDMCC NO.66254, deposited in Guangdong Microbial Culture Collection Center (GDMCC) on April 30, 2025; the pathogen of ergot disease is Fusarium graminearum PH-1.

[0029] PDA medium: Cut 200 g of potatoes into pieces, add distilled water and boil for 20 min, filter. Add 20 g of glucose and 15 g of agar, dilute to 1 L, and sterilize at 121°C for 20 min.

[0030] PNM medium: 5 mM potassium nitrate, 2 mM calcium nitrate, 2 mM magnesium sulfate, 70 μM boric acid, 14 μM manganese chloride, 1 μM zinc sulfate, 0.2 μM sodium molybdate, 0.01 μM cobalt chloride, 0.5 μM copper sulfate, 0.01 μM ferrous sulfate, 20 g / L glucose. Dissolve each component in distilled water at the above concentrations, stir thoroughly until completely dissolved, then add 15 g / L agar and sterilize by autoclaving at 121°C for 20 min.

[0031] Phosphorus deficiency treatment: Phosphorus-deficient medium (PNM) was used, while phosphorus-containing medium (2 mM sodium dihydrogen phosphate) was added to phosphorus-deficient medium. T-aloe was inoculated onto both the phosphorus-deficient and phosphorus-containing mediums, respectively. After 5 days of incubation at 28°C, mycelia were harvested and placed into 1.5 mL sterile centrifuge tubes. These were quickly frozen in liquid nitrogen and stored at -80°C until further use.

[0032] Face-off experiment: Cover a petri dish containing PDA medium with sterile cellophane. Take a 5mm diameter mycelial disk from the growing edge of one-week-old Fusarium graminearum and one-week-old Trichoderma spp. and place it equidistantly on either end of the cellophane-covered PDA plate. The control group is divided into two groups: one containing only Trichoderma spp., and the other containing only Fusarium graminearum. Each group has five replicates. After incubation at 28°C in the dark for three days, hyphae from the control group (Fusarium graminearum), Trichoderma spp., and hyphae from the interface between Fusarium graminearum and Trichoderma spp. in the treated group are placed in 1.5mL sterile centrifuge tubes, quickly frozen in liquid nitrogen, and stored in a -80°C freezer until needed.

[0033] Inhibition rate: I = (CT) / C*100

[0034] Note: I represents the inhibition rate; C represents the growth radius of the pathogen in the control dish; T represents the growth radius of the pathogen in the confrontation dish.

[0035] Example 1 Analysis of the expression level of Trichoderma harzianum TrVPT1 gene in phosphorus-deficient and antagonistic Fusarium graminearum

[0036] To investigate the expression pattern of the TrVPT1 gene in Trichoderma harzianum under phosphorus deficiency and antagonism to Fusarium graminearum, Trichoderma samples were subjected to the following treatments: (1) Phosphorus deficiency culture: T-aloe was inoculated into phosphorus-supplemented and phosphorus-deficient culture media, respectively, and cultured at 28°C for 5 days; (2) Antagonistic treatment: Trichoderma was co-cultured with Fusarium graminearum and cultured at 28°C for 7 days. Total RNA from Trichoderma samples in each treatment group was extracted using a Fungal RNA Extraction Kit (OMEGA), and the RNA was reverse transcribed into first-strand cDNA using a reverse transcription kit. Using cDNA as a template, the expression level of the TrVPT1 gene was detected by real-time quantitative PCR (RT-qPCR).

[0037] RT-qPCR reaction system is as follows: SYBR Green Premix Ex TaqTM 5 μL, 0.2 μL each of upstream primer (5'-ACCTTTATCAGCCACACAGTAG-3', SEQ ID NO: 3) and downstream primer (5'-CCACCCATAACAAGGAGGTTAG-3', SEQ ID NO: 4), 0.2 μL of cDNA template, and sterile water were added to 10 μL. The reaction procedure was: 95°C pre-denaturation for 30 seconds, followed by 40 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 30 seconds. The internal reference gene Actin of Trichoderma harzianum (upstream primer: 5'-ATGGTATGGGTCAGAAGGA-3', SEQ ID NO: 5, downstream primer: 5'-ATGTCAACACGAGCAATGG-3', SEQ ID NO: 6) was used as a normalization control, and 2 -△△CT Methods The relative expression levels of target genes were calculated.

[0038] The results are as follows Figure 1 As shown in the results, the expression of the TrVPT1 gene was significantly upregulated under phosphorus deficiency compared with normal phosphorus supply (P < 0.05), indicating that TrVPT1 plays an important role in Trichoderma's response to phosphorus deficiency. Furthermore, during co-culture of Trichoderma with Fusarium graminearum, the expression of the TrVPT1 gene was suppressed (P < 0.05), suggesting that this gene may be involved in the physiological process of Trichoderma's antagonism to Fusarium graminearum. These results indicate that the TrVPT1 gene plays an important regulatory role in Trichoderma's phosphorus homeostasis and biocontrol function.

[0039] Example 2 Creation of TrVPT1 gene knockout mutant Δvpt1 strain

[0040] To study the function of the TrVPT1 gene, a TrVPT1 gene knockout mutant strain, Δvpt1, was constructed using homologous recombination technology. The specific experimental steps are as follows:

[0041] First, the genomic DNA of Trichoderma harzianum was extracted, and the upstream homologous arm of the TrVPT1 gene was amplified with the upstream primer being 5'-AACAGTTTGGACGTTTTGGG-3', SEQ ID NO: 7, and the downstream primer being 5'-CATTGATGTGTTGACCTCCCTCGTCCTGTGTCTCCAGCCA-3', SEQ ID NO: 8, and the amplified product I was recovered; the downstream homologous arm of the TrVPT1 gene was amplified with the upstream primer being 5'-AAACGTCCGCAATGTGTATGGTGAGACAGCTGCGTGA-3', SEQ ID NO: 9, and the downstream primer being 5'-TGTAAGGGTCTCAATGAGGTCGTA-3', SEQ ID NO: 10, and the amplified product II was recovered.

[0042] The HYG gene was amplified using a vector containing a hygromycin fragment as a template. The upstream primer was 5'-TGGCTGGAGACACAGGACGAGGGAGGTCAACACATCAATG-3', SEQ ID NO: 11, and the downstream primer was 5'-TCACGCAGCTGTCTCACCATACACATTGCGGACGTTT-3', SEQ ID NO: 12. Amplified product III was recovered. PCR products I, II, and III were then ligated by homologous recombination. The homologous recombinant sequence was then transformed into Trichoderma protoplasts by protoplast transformation. Transformants were screened on PDA plates containing hygromycin (50 mg / L).

[0043] The genomic DNA of the wild type and transformant Δvpt1 Trichoderma strains were extracted for PCR verification, and the identification primers were as follows: Figure 2 As shown in A. The target gene TrVPT1 of transformants Δvpt1-37 and Δvpt1-43 was replaced by the HYG gene, and the full-length amplified product had obvious band size differences with the wild-type Trichoderma ( Figure 2 B), indicating that the TrVPT1 gene was successfully knocked out and can be used for subsequent experiments.

[0044] In this example, the TrVPT1 gene knockout mutant Δvpt1 strain was successfully created by homologous recombination technology, which provides important experimental materials for in-depth study of the function of the TrVPT1 gene in Trichoderma phosphate metabolism and biological control.

[0045] Example 3 TrVPT1 gene knockout affects the stability of Trichoderma phosphatase

[0046] To investigate the function of the TrVPT1 gene in regulating phosphorus homeostasis in Trichoderma, the growth rate and total phosphorus content of wild-type Trichoderma strains and TrVPT1 gene knockout mutants Δvpt1 (Δvpt1-37 and Δvpt1-43) were measured. The specific experimental steps are as follows:

[0047] The wild type and transformant Δvpt1 Trichoderma strains were inoculated onto PDA plates and cultured at 28°C. The colony radius was measured at 18, 30, and 42 hours, and the growth curve was drawn. Figure 3 As shown in A and B, the growth rate of the transformant Δvpt1 was significantly reduced compared with the wild-type strain.

[0048] The wild-type Trichoderma strain and the Δvpt1 mutant strain were inoculated into PD liquid medium and cultured at 28°C and 180 rpm for 3 days. The mycelium was collected and rinsed with deionized water three times to remove the residual culture medium on the surface. The mycelium was dried at 60°C to constant weight and ground into powder. The total phosphorus content of the mycelium was determined by the molybdenum antimony colorimetric method. The results are as follows Figure 3 As shown in Figures C and D, compared to the wild-type strain, the TrVPT1 gene mutation caused more phosphorus to be retained in the vacuole. The total phosphorus content of the hyphae of the Δvpt1-37 and Δvpt1-43 mutants was significantly higher than that of the wild type, almost twice that of the wild type. This result indicates that the TrVPT1 gene knockout causes phosphorus to be retained in the vacuole and cannot be effectively transported to the cytoplasm to participate in metabolic processes, thereby affecting the phosphorus homeostasis and normal growth of Trichoderma.

[0049] This example demonstrates the key role of the TrVPT1 gene in regulating phosphorus homeostasis in Trichoderma by measuring growth rate and total phosphorus content in mycelia. Loss of the TrVPT1 gene leads to phosphorus accumulation in the vacuole, which in turn affects the growth and metabolism of Trichoderma, providing important evidence for further analysis of the TrVPT1 gene's function.

[0050] Example 4 TrVPT1 gene knockout weakens the inhibitory ability of Trichoderma to Fusarium graminearum

[0051] To investigate the function of the TrVPT1 gene in Trichoderma antagonism against Fusarium graminearum, the antibacterial activity of the metabolites of wild-type Trichoderma strains and TrVPT1 gene knockout mutants Δvpt1 (Δvpt1-37 and Δvpt1-43) was determined. The specific experimental steps are as follows:

[0052] The wild-type Trichoderma strain and the Δvpt1 mutant strain were inoculated into PD liquid culture medium and cultured with shaking at 28°C and 180 rpm for 6 days. After the incubation period, the mycelium was filtered through a sterile filter membrane and the metabolite solution was collected. The metabolite solution was autoclaved at 121°C for 20 minutes. The metabolite solution was mixed with PDA culture medium in a ratio of 3:7 to prepare a metabolite-containing culture medium. The control group consisted of a culture medium containing PDB and PDA in a ratio of 3:7. Fusarium graminearum was inoculated into the center of the metabolite-containing culture medium and the control culture medium and cultured in a constant temperature incubator at 28°C. The colony diameter of Fusarium graminearum was measured after 3 and 5 days of incubation, and the inhibition rate was calculated.

[0053] The results are as follows Figure 4 As shown in Figure 2, after three days of cultivation, the wild-type Trichoderma strain had an inhibition rate of 44.87% against Fusarium graminearum compared to the control, while the inhibition rates of transformants Δvpt1-37 and Δvpt1-43 were significantly reduced, being 22.76% and 29.81%, respectively. Figure 4After five days of cultivation, the wild-type Trichoderma strain had an inhibition rate of 28.60% against Fusarium graminearum compared to the control, while the inhibition rates of transformants Δvpt1-37 and Δvpt1-43 were significantly lower, at 11.52% and 18.31%, respectively ( Figure 4 C and D).

[0054] These results indicate that knocking out the TrVPT1 gene significantly reduced the inhibitory effect of Trichoderma metabolites on Fusarium graminearum, suggesting that the TrVPT1 gene plays an important role in Trichoderma's antagonism against Fusarium graminearum. The TrVPT1 gene may indirectly affect Trichoderma's biocontrol function by regulating phosphorus metabolism and affecting the synthesis or secretion of secondary metabolites.

[0055] This example reveals the key role of the TrVPT1 gene in Trichoderma antagonism against Fusarium graminearum through antibacterial activity assay, providing an important basis for in-depth analysis of the function of the TrVPT1 gene and its application in biological control.

[0056] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1, characterized in that The nucleotide sequence of the Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1 is shown in SEQ ID NO:

1.

2. The Trichoderma vacuolar phosphate transporter gene TrVPT1 according to claim 1, characterized in that The sequence of the expressed protein is shown in SEQ ID NO:

2.

3. Use of the Trichoderma harzianum vacuolar phosphate transporter gene TrVPT1 according to claim 1 in regulating Trichoderma phosphate accumulation and / or resistance to ergot.

4. The use according to claim 3, characterized in that The regulation of Trichoderma phosphate accumulation is to control the total phosphorus content of Trichoderma mycelium.

5. The use according to claim 3, characterized in that The regulation of Trichoderma phosphatase accumulation is to transport phosphorus from the vacuole to the cytoplasm.

6. The use according to claim 3, characterized in that The fusarium wilt is a plant disease caused by Fusarium graminearum.

7. The use according to claim 6, characterized in that The plant is wheat.

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