Application of trichoderma chitinase ech2 and mutant thereof in inhibiting plant pathogenic fungi

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of trichoderma chitinase ech2 and a mutant thereof in inhibiting plant pathogenic fungi. According to the present invention, the protein represented by the amino acid sequence such as SEQ ID NO: 2 is determined as the chitinase, and the chitinase ech2 shows excellent temperature adaptability and pH stability; experiments show that the activity of the enzyme is remarkably improved within the range of 20-35 DEG C, and the activity reaches the peak value at 35 DEG C; high enzyme activity is maintained in a wide range of pH 4-9, and the optimum pH is 5.5. The storage stability is good, the application range is wide, and the growth of plant pathogenic fungi botrytis cinerea and fusarium oxysporum is inhibited. The optimal temperature of the improved chitinase mutant ech2-1 is 45 DEG C, which is 10 DEG C higher than that of the original ech2; the ech2-1 has good stability at 4-40 DEG C, and is suitable for being developed into agricultural chitinase.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to the application of Trichoderma chitinase ech2 and its mutants in inhibiting plant pathogenic fungi. Background Technology

[0002] Fungal diseases are a significant factor affecting crop yield and quality. Chitin, as a structural polysaccharide in the fungal cell wall, is an ideal antifungal target. Chitinase (EC 3.2.1.14) specifically hydrolyzes the β-1,4 glycosidic bonds in chitin. Chitinases are a large family, mainly divided into two families based on their amino acid sequence and catalytic mechanism: the GH18 family and the GH19 family. Furthermore, chitinases exhibit diversity in type and function. Endochondrinases randomly cleave glycosidic bonds within the chitin chain, a crucial step in disrupting the structural integrity of the fungal cell wall. Only this type of enzyme can effectively and directly inhibit fungal growth. The effectiveness of a chitinase in inhibiting fungi depends on factors such as enzyme activity, substrate accessibility, and synergistic effects. Therefore, identifying highly active, antifungal chitinases is crucial for agricultural applications. Chitinase specifically catalyzes the hydrolysis of the N-acetyl-β-D-glucosamine glycosidic bond in chitin, generating N-acetyl-β-D-glucosamine. Cellulase specifically acts on polymers composed of glucose units linked by β-1,4-glycosidic bonds to produce glucose.

[0003] Chitinases from microbial sources (such as Trichoderma) Trichoderma Bacillus Bacillus Trichoderma harzianum (etc.) has been shown to have antagonistic effects against a variety of plant pathogenic fungi, and has broad application potential in agriculture, biological control, and medicine. As an important biocontrol fungus, Trichoderma harzianum can secrete various chitinases, which have a significant inhibitory effect on plant pathogenic fungi. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Trichoderma chitinase ech2 in inhibiting the plant pathogenic fungus Botrytis cinerea.

[0005] According to the technical solution of this application, the *Trichoderma harzianum* chitinase gene... ech2 It has the nucleotide sequence shown in SEQ ID NO:1.

[0006] SEQ ID NO:1:

[0007] The Trichoderma harzianum chitinase described herein has the amino acid sequence shown in SEQ ID NO: 2.

[0008] SEQ ID NO: 2: MSGDGYRSVAYFVNWAIYARKHRPQDLPVDKLTHILYAFANVRQDSGEVHMTDGWADTDIHWEGDSWNDTGNNMYGCLKQLNLLKKRNRNLKVLLSIGGWTYSGNFKGPASTQQGRETFAKSSLELLKNLGFDGLDIDWEYPQNADEARNFVELLATVRRELDAYSATLPTYSHFELTVACPAGATHFQKLDVPGMDQY LDFWNLMAYDYAGSWDQTSGHQANLHPSSDNPTSTPFSTDAAIDFYTRSGVAPSKIVLGMPIYGRAFENTDGPGRPYNGIGEGSWENGIFDYKVLPHPGSQEIWDRATGASYSYNPQTRKLVSYDTPHASRAKAGYIKEWGLGGGMWWESSGDKEGPDSLIGIVVNEFGGPGALQRKDNCIDYPQSKYDNLKNGFPNN.

[0009] According to the technical solution of this application, the *Trichoderma harzianum* chitinase gene... ech2 mutant ech2-1 Its nucleotide sequence is shown in SEQ ID NO:3.

[0010] SEQ ID NO:3:

[0011] The aforementioned Trichoderma harzianum chitinase mutant ech2-1 It has the amino acid sequence shown in SEQ ID NO: 4.

[0012] SEQ ID NO: 4: MSGDGYRSVAYFVNWAIYARKHRPQDLPVDKLTHILYAFANVRQDSGEVHLTDGWADTDIHWEGDSWNDTGNNMYGCLKQLNLLKKRNRNLKVLLSIGGWTYSGNFKGPASTQQGRETFAKSSLELLKNLGFDGLDIDWEYPQNADEARNFVELLATVRRELDAYSATLPTPSHFELTVACPAGATHFQKLDVPGMDQY LDFWNLMAYDYAGSWDQTSGHQANLHPSSDNPTSTPFSTDAAIDFYTRSGVAPSKIVLGMPIYGRAFENTDGPGRPYNGIGEGSWENGIWDYKVLPHPGSQEYWDRATGASYSYDPQTRKLVSYDTPHAARAKAGYIKEWGLGGGMWWESSGDKEGPDSLIGIVVNEFGGPGALQRKDNCIDYPQSKYDNLKAGFPNN.

[0013] This invention has positive and beneficial effects: This invention identifies the protein with the amino acid sequence shown in SEQ ID NO: 2 as a chitinase, which exhibits excellent temperature adaptability and pH stability. Experiments show that the enzyme's activity is significantly enhanced within the 20-35℃ range, reaching peak activity at 35℃; it maintains high enzyme activity over a wide pH range of 4-9, with an optimal pH of 5.5. It exhibits good storage stability, a wide range of applications, and inhibits the growth of plant pathogenic fungi Botrytis cinerea and Fusarium oxysporum. The optimal temperature for the improved chitinase mutant ech2-1 is 45℃, an increase of 10℃ compared to the original ech2; ech2-1 shows good stability between 4℃ and 40℃, making it suitable for development as an agricultural chitinase. Attached Figure Description

[0014] Figure 1 For the present invention ech2 Electrophoresis results of gene clones, where M1: DNA standard molecular weight DL5000; 2: PCR product; Figure 2SDS-PAGE results of expressed chitinase: M: color pre-stained protein marker, 1-2: chitinase ech2; Figure 3 Temperature curves showing chitinase activity; Figure 4 The temperature stability curve of chitinase; Figure 5 The pH curve for chitinase activity; Figure 6 The pH stability curve of chitinase; Figure 7 The diagram shows the inhibitory effect of chitinase on the plant pathogenic fungus Botrytis cinerea. Figure A shows the control effect of chitinase ECh2 on detached cucumber leaves, 1: Botrytis cinerea; 2: Botrytis cinerea + chitinase. Figure B shows the effect of chitinase ECh2 on the liquid fermentation of Botrytis cinerea, 3: Botrytis cinerea; 4: Botrytis cinerea + chitinase. Figure 8 The image shows the inhibitory effect of chitinase on the plant pathogenic fungus Fusarium oxysporum. Figure A shows the effect of chitinase ECh2 on the liquid fermentation of Fusarium oxysporum, and Figure B shows the microscopic image of chitinase ECh2 on Fusarium oxysporum. Figure 9 Comparison of enzyme activities among chitinase mutants; Figure 10 Showing the optimal temperature for the chitinase mutant ech2-1; Figure 11 The temperature stability of the chitinase mutant ech2-1 is shown.

[0015] Figure 12 This shows a comparison of the antibacterial effects of chitinase mutants ech2-1 and ech2. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Example 1: Cloning and Sequencing of the Chitinase ech2 Gene

[0017] Total RNA extracted from Trichoderma harzianum LTR-2 was amplified to encode chitinase. ech2 The primer pairs for the nucleotide sequence are: SEQ ID No:5:5'- agcaaatgggtcgcggatcc ATGTCGGGAGACGGCTATCGTTCA-3', SEQ IDNo:6: tggtggtggtggtgctcgagTTAGTTGTTTGGGAATCCATTCTT, respectively, introduce portions of homologous recombination with the vector (indicated by underline).

[0018] After PCR, the sample was subjected to 1% agarose gel electrophoresis. Figure 1 Chitinase was used to recover the target DNA fragment using a DNA gel extraction kit. ech2 Gene. Using restriction endonuclease B amHI and X ohI The vector pET28a(+) was double-digested, and the purified product was recovered and homologous recombination was performed to obtain the recombinant plasmid pET28a-ech2. Sequencing confirmed that the cloned gene sequence was correct. Example 2: Expression of chitinase gene

[0019] The recombinant plasmid obtained in Example 1 was transformed into... E. coli Fast-T1 was screened by colony PCR and sequenced to obtain the clone strain 28a-ech2-Fast-T1. This was heat-shock transformed into the expression strain BL21(DE3) and cultured overnight at 37°C to obtain the recombinant expression strain Ech2-Bl21(DE3). The recombinant expression strain Ech2-Bl21(DE3) was inoculated into LB liquid medium (with 50 μg / mL kanamycin) and cultured overnight at 35°C and 200 rpm. A 1% inoculum was then added to 100 mL of LB medium (with 50 μg / mL kanamycin) and cultured at 35°C until OD (Organic Dry Index) was reached. 600 =Approximately 0.7, add IPTG inducer (final concentration 1 mM), and induce culture at 16 ℃ for 16 h. The induced expression of *E. coli* bacterial culture was centrifuged at 12000 rpm for 2 min, and the bacterial cells were collected. The cells were resuspended in citrate-sodium dihydrogen phosphate buffer, sonicated for approximately 30 min, and the supernatant was collected by centrifugation to obtain crude chitinase solution. The crude chitinase solution was purified using a His-tag column. The purified recombinant protein solution was subjected to SDS-PAGE, yielding a single protein band of approximately 43.7 kDa (e.g., ...). Figure 2 (As shown), the concentration is around 1 mg / mL. Example 3: Determination of chitinase activity

[0020] Determination of chitinase activity: Using 10 g / L colloidal chitin as a substrate, the enzyme activity of chitinase ech2 prepared in Example 2 was determined. 200 μL of substrate (pH 6) was added to 100 μL of purified chitinase solution, and the mixture was reacted in a 35℃ water bath for 30 min. Then, 100 μL of DNS reagent was added, and the mixture was boiled in water for 5 min. The mixture was centrifuged at 12000 rpm for 10 min. An equal volume of citrate-disodium hydrogen phosphate (100 mM, pH 6) was used as a blank control. The absorbance of the supernatant was measured at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme that produces 1 μmol of N-acetyl-D-glucosamine per hour by decomposing chitin. The chitinase ech2 was found to have chitin endonuclease activity; the purified ech2 activity was 61.3 U / mg. Example 4: Enzymatic properties of chitinase

[0021] The enzymatic properties of the chitinase in Example 2 were determined, including optimal temperature, optimal pH, temperature stability, and pH stability. Enzyme activity was determined using 1% colloidal chitin as a substrate.

[0022] 4.1 Optimal Temperature: The reaction system included 200 μL of 1% colloidal chitin and 100 μL of pure chitinase solution; pH 6 (citric acid-sodium dihydrogen phosphate, 100 mM); the reaction was carried out for 0.5 h at temperature gradients of 4℃, 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, 70℃, and 80℃, respectively. An inactivated crude enzyme solution was used as a blank. After the reaction, the mixture was boiled in a water bath for 10 min to inactivate the enzyme. Then, 750 μL of DNS was added to the reaction system, and the mixture was boiled in water for 5 min. Finally, the enzyme was measured using an OD350 microplate reader. 540 The absorbance was measured to determine the chitinase activity at different temperatures; curves were plotted based on the relative enzyme activity at different temperatures to determine the optimal reaction temperature for chitinase; Figure 3 The optimal reaction temperature for chitinase ech2 is 35℃. 4.2 Optimal pH: The substrate was 1% colloidal chitin, and enzyme activity was determined in a buffer solution with a pH range of 2-11. The reaction system was the same as that used in the optimal reaction temperature test, and the reaction temperature was 35℃. Curves were plotted based on the relative activity of the enzyme at different pH values, as shown below. Figure 4 The optimal reaction pH for chitinase ech2 is 5.5.

[0023] 4.3 Temperature stability study: Chitinase pure enzyme solution was incubated at different temperatures (4℃, 20℃, 30℃, 35℃, 40℃) for 0, 10, 20, 30, 40, 50, and 60 min respectively. Residual enzyme activity was measured after reacting at 35℃ for 30 minutes and compared with the activity of untreated enzyme to calculate relative enzyme activity; the results are as follows: Figure 5 As shown, the results indicate that chitinase ech2 has a certain degree of temperature tolerance, exhibiting good thermal stability within the range of 4–30℃, and maintaining a residue of over 95% after 1 h of reaction.

[0024] 4.4 pH Stability: Chitin-purified enzyme solution was placed in 100 mM buffer solutions at different pH values ​​(2.0, 3.0, 4.0, 5.0, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, 11.0) and incubated at 30 °C for 1 h. Residual enzyme activity was then measured at the optimal reaction temperature (30 °C). The untreated enzyme activity was used as a blank control, and the relative enzyme activity was calculated. Figure 6 As shown, chitinase ech2 maintains an activity of over 50% after 1 hour of incubation in a pH range of 4.0–9.0, demonstrating high pH tolerance. Example 5: Study on the antifungal properties of chitinase ECh2

[0025] The antifungal activity of purified ech2 was measured using a hyphal extension inhibition test. Using a 5 mm diameter punch, mycelial cakes were cut from the edge of a cultured *Botrytis cinerea* colony and inoculated onto the center of the left and right halves of a cucumber leaf. 50 μL of chitinase solution from Example 2 was dropped onto the right half of the mycelial cake. 100 mM citrate-sodium dihydrogen phosphate buffer was used as a control (CK). After 3 days of culture, the hyphal growth inhibition rate (GI) was estimated using the following formula: GI = (Rr) / R × 100%, where R and r refer to the average diameter of the fungal hyphae on the control and experimental plates, respectively. Figure 7 As shown in Figure A, 0.05 mg / mL chitinase ech2 inhibited the growth of the plant pathogenic fungus *Botrytis cinerea*, with an inhibition rate of 100%. Furthermore, by adding 0.05 mg / mL chitinase solution to liquid potato medium, after 3 days of cultivation, *Botrytis cinerea* showed almost no growth, while the biomass of *Fusarium oxysporum* was significantly reduced. Figure 7 Figure B in the diagram, Figure 8 Figure A in the diagram. Under a microscope, the hyphae with added chitinase solution broke and bent, showing a significant inhibitory effect. Figure 8 (Figure B in the diagram). Example 6: Design of a chitinase ech2 mutant with enhanced enzyme activity

[0026] To further improve the enzyme activity and temperature stability of the extracted chitinase, the original chitinase sequence was used as a target for mutation to synthesize a mutant gene, namely the *Trichoderma harzianum* chitinase mutant. ech2-1 The amino acid sequence is shown in SEQ ID NO: 4. Based on the parent, six sites were mutated, specifically: M51L, Y172P, F289W, I302Y, N314D, and S329A. The synthesized gene was heterologously expressed in E. coli, and the mutant protein was purified by affinity chromatography.

[0027] The enzyme activity assay for the ech2 mutant was performed as follows: Using 10 g / L colloidal chitin as a substrate, 200 μL of substrate (pH 6) was added to 100 μL of purified chitinase solution. The mixture was incubated in a 45℃ water bath for 30 min. The remaining steps were the same as for the ech2 chitinase activity assay. The purified ech2-1 enzyme activity was 134.9 U / mg, 2.2 times that of the control. Figure 9 The method for determining the optimal temperature is the same as that for ech2-1. The optimal temperature for chitinase ech2-1 has been increased to 45℃, which is 10℃ higher than that of the original ech2. Figure 10 Temperature stability: Chitinase pure enzyme solution was incubated at different temperatures (4℃, 20℃, 30℃, 35℃, 40℃, 45℃) for 0, 10, 20, 30, 40, 50, and 60 min respectively. Residual enzyme activity was measured after reacting at 45℃ for 30 minutes and compared with the activity of untreated enzyme to calculate relative enzyme activity. ECH2-1 showed good stability at 4℃-40℃. Figure 11 It is suitable for development into an agricultural chitinase. Example 7: Study on the antifungal activity of chitinase ech2 mutant with enhanced enzyme activity

[0028] Using a sterile punch with a diameter of 5 mm, mycelial cakes of uniform thickness (approximately 2 mm) were collected from the edge of activated *Botrytis cinerea* agar plates. Purified chitinase ech2 and its mutant ech2-1 stock solutions (1 mg / mL) were serially diluted, and 100 μL of each enzyme solution was used to treat cucumber leaves, with final enzyme additions of 0, 2.5, 5, 10, 15, 25, 50, and 100 mg per leaf. The treatment method was as follows: ech2 was inoculated on the left side of the leaf, and ech2-1 on the right side, with a blank control (no enzyme added). Each concentration treatment was replicated in triplicate. All culture dishes were incubated at 20°C–22°C and 85%–90% relative humidity for 4 days. Under the same protein addition (≤25 mg), the inhibitory rate of mutant ech2-1 was significantly higher than that of wild-type ech2. For example, when the added amount was 15 mg, the antibacterial rates of ech2 and ech2-1 were 75.3% and 96.0%, respectively. Figure 12 ).

[0029] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. The application of Trichoderma chitinase for inhibiting plant pathogenic fungi, characterized in that, The amino acid sequence of the Trichoderma chitinase ech is shown in SEQ ID NO: 2 or SEQ ID NO:

4.

2. The application according to claim 1, characterized in that, The plant pathogenic fungus is Botrytis cinerea or Fusarium oxysporum.

3. A Trichoderma chitinase mutant, characterized in that, The amino acid sequence of the Trichoderma chitinase mutant is shown in SEQ ID NO: 4.

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