Use of glycine / glutamine rich sequences to improve xylanase activity

Abstract

This invention discloses the application of glycine / glutamine enriched sequences in enhancing xylanase activity. The application of the glycine / glutamine enriched sequence shown in SEQ ID NO.1 in enhancing the xylanase activity of BCG1 and its homologous proteins is described. The xylanase activity of the originating BCG1 protein is enhanced by incorporating the glycine / glutamine enriched sequence shown in SEQ ID NO.1 into the C-terminus of a BCG1 protein lacking the glycine / glutamine enriched sequence; or the xylanase activity of the BCG1 protein is reduced by knocking out the glycine / glutamine enriched sequence of a BCG1 protein with xylanase activity. This finding indicates that glycine / glutamine enriched sequences have significant application potential in agricultural production for enhancing xylanase activity.

Description

Technical Field

[0001] This invention belongs to the field of plant protection and relates to the application of glycine / glutamine enriched sequences (G / Q-rich motif) in enhancing xylanase activity. Background Technology

[0002] Xylanase is an enzyme that specifically hydrolyzes the xylan backbone, breaking down large xylan molecules into xylooligosaccharides, xylobiose, and small amounts of xylose. Xylanases produced by many beneficial microorganisms are widely used in bread baking, starch processing, animal feed, and pulp biobleaching. Improving xylanase activity is of great significance for solving energy problems. Xylanase activity is related to the temperature and pH of the environment, and some activators and inhibitors can also affect enzyme activity. Adjusting the ambient temperature and pH to their optimal values, while simultaneously adding some enhancers, can enhance xylanase activity. However, effective enhancers are relatively few. Therefore, currently, there is a lack of effective methods to enhance xylanase activity.

[0003] Fusarium graminearum, the scab disease of wheat, poses a serious threat to wheat production in my country. Toxins produced in diseased wheat grains, such as zearalenone (ZEN) and deoxynivalenol (DON), severely threaten human and animal health. The virulence factor BCG1 of Fusarium graminearum exhibits extremely high xylanase activity. Its C-terminus contains a 54-amino acid-rich glycine / glutamine sequence (G / Q-rich motif) with an unknown function; the specific function of this sequence has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing the application of glycine / glutamine-rich sequences (G / Q-rich motif) in enhancing xylanase activity.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] Application of the glycine / glutamine enriched sequence shown in SEQ ID NO.1 in enhancing the xylanase activity of BCG1 homologous protein.

[0007] As a preferred embodiment of the present invention, the BCG1 homologous protein is a BCG1 protein that does not contain a glycine / glutamine enriched sequence at its C-terminus.

[0008] As a preferred embodiment of the present invention, the glycine / glutamine enrichment sequence shown in SEQ ID NO.1 is incorporated into the C-terminus of the BCG1 protein, which does not have a glycine / glutamine enrichment sequence, to enhance the xylanase activity of the starting BCG1 protein.

[0009] Application of glycine / glutamine enriched sequences in regulating the activity of BCG1 homologous xylanase.

[0010] As a preferred embodiment of the present invention, the xylanase activity of the BCG1 protein is enhanced by incorporating the glycine / glutamine enrichment sequence shown in SEQ ID NO.1 into the C-terminus of the BCG1 protein, which does not have a glycine / glutamine enrichment sequence.

[0011] As a preferred embodiment of the present invention, the xylanase activity of the BCG1 protein is reduced by knocking out the glycine / glutamine enrichment sequence of the BCG1 protein with xylanase activity.

[0012] Beneficial effects:

[0013] The virulence factor FgBCG1 from *Fusarium graminearum* and its homologs, FoBCG1 from *Fusarium oxysporum* and FvBCG1 from *Fusarium verticillioides*, all exhibited high xylanase activity, indicating that BCG1 in *Fusarium* species possesses conserved xylanase activity. We also tested the xylanase activity of BCG1 homologs lacking G / Q-rich motifs, including MoBCG1 from *Magnaporthe grisea* and AfBCG1 from *Aspergillus flavus*. Compared to FgBCG1, FoBCG1, and FvBCG1, these G / Q-rich motif-deficient MoBCG1 and AfBCG1 proteins showed weaker xylanase activity. We further tested the G / Q-rich motif-deficient truncated protein FgBCG1. ΔGQ FoBCG1 ΔGQ FvBCG1 ΔGQ The xylanase activity was measured. The results showed that xylanase activity induced by the truncated protein lacking the G / Q-rich motif was significantly reduced.

[0014] We incorporated a G / Q-motif from the Fg gene into the C-terminus of two proteins, MoBCG1 and AfBCG1, which do not contain G / Q-rich motifs. The results showed a significant increase in xylanase activity, leading to better degradation of plant cell walls and infection of plants. This indicates that G / Q-rich motifs can act as enhancers, significantly increasing the xylanase activity of proteins. This finding suggests that G / Q-rich motifs have promising applications in agricultural production for significantly enhancing xylanase activity. Attached Figure Description

[0015] Figure 1 The G / Q-rich motif of BCG1 is highly conserved only in Fusarium genus. (A) Sequence features of BCG1 protein; (B) Phylogenetic tree constructed based on RPB2; (C) Weblogo 3 showing the G / Q-rich motif sequences of each protein.

[0016] Figure 2 The absence of G / Q-rich motifs leads to reduced xylanase activity in the protein.

[0017] Figure 3 Increased G / Q-rich motif significantly enhances xylanase activity of the protein. Detailed implementation method:

[0018] Example 1: The G / Q-rich motif of BCG1 is highly conserved only in Fusarium species.

[0019] Experimental methods:

[0020] The protein sequence was aligned using ClustalW, and the alignment results were visualized using the ENDscript / ESPript 3.0 website.

[0021] Experimental Results: BCG1 from *F. graminearum* contains a novel G / Q-rich motif. We constructed a phylogenetic tree based on the RPB2 protein sequence to investigate the phylogenetic distribution of BCG1 in different organisms. The results showed that the G / Q-rich motif sequence is highly conserved and widely present in *F.* genera, such as FoBCG1 from *F. oxysporum* and FvBCG1 from *F. verticillioides*, both of which contain G / Q-rich motif sequences. However, no homologous proteins containing G / Q-rich motifs were found in other organisms.

[0022] Example 2: The absence of G / Q-rich motifs led to a decrease in xylanase activity of the protein.

[0023] Experimental methods:

[0024] Protein expression and purification: The NCBI accession number of the protein in Table 1 (https: / / www.ncbi.nlm.nih.gov / ) was sent to Nanjing Zhongding Biotechnology Co., Ltd., and they were commissioned to perform protein expression and purification.

[0025]

[0026]

[0027] DNS method for determining xylanase activity of proteins

[0028] (1) Reagent preparation

[0029] Acetic acid-sodium acetate (CH3COOH-CH3COONa, 3M, pH 5.2):

[0030] Weigh 40.8g of CH3COONa 3H2O, dissolve it in pure water, add 40mL of glacial acetic acid, adjust the pH to 5.2, and bring the volume to 100mL.

[0031] DNS: Purchased from Solarbio, part number D7800.

[0032] (2) Xylanase activity assay

[0033] Add 150 μL of 1% beech wood xylan acetate-sodium acetate solution (pH 5.2) to a sterile 1.5 mL EP tube as the reaction substrate, and incubate at 40 °C in a metal bath for 10 min. Add 30 μL of the above-mentioned purified protein dilution to the reaction substrate, and react precisely at 40 °C in a water bath for 10 min. Immediately afterward, add 350 μL of DNS reagent to terminate the reaction, boil at 100 °C in a water bath for 5 min, cool, and then add 500 μL of ddH2O and mix well. Pipette 250 μL of the reaction solution into a 96-well plate and measure the OD using a DTX800 microplate reader (Beckman Coulter Inc., http: / / www.beckman.com). 540 .

[0034] (3) Draw the standard curve of xylose

[0035] Weigh 1.0 g of xylose, dissolve it in water, and bring the volume to 100 mL to prepare a 10 mg / mL xylose solution. Prepare xylose standard solutions with concentrations of 0, 0.2, 0.4, 0.5, 0.6, 0.8, and 1 mg / mL using acetate-sodium acetate buffer. OD 540 Plot a standard curve with the Y-axis value and xylose concentration as the X-axis.

[0036] Based on the xylose standard curve, the xylose content in the protein was calculated, and the xylanase activity of each protein was then determined. At 40℃ and pH 5.2, 1 μmol of xylose produced per minute was defined as one unit of enzyme activity, denoted as 1 U. Each sample was measured three times, and the average value was taken.

[0037] Experimental Results: The company successfully purified the protein to the expected size. Xylanase activity assays showed that proteins containing G / Q-rich motifs, including FgBCG1 from *Fusarium graminearum*, FoBCG1 from *Fusarium oxysporum*, and FvBCG1 from *Fusarium verticillatum*, exhibited high xylanase activity. Proteins lacking G / Q-rich motifs, such as MoBCG1 from *Magnaporthe oryzae* and AfBCG1 from *Aspergillus flavus*, showed weaker xylanase activity. The truncated protein FgBCG1 lacking the G / Q-rich motif was also observed. ΔGQ FoBCG1 ΔGQ FvBCG1 ΔGQ The xylanase activity was significantly reduced ( Figure 2 In summary, our findings indicate that G / Q-rich motifs are specifically present only in Fusarium species and make a significant contribution to the xylanase activity of the BCG1 protein.

[0038] Example 3: Using G / Q-rich motif as an enhancer to enhance the xylanase activity of the protein.

[0039] Experimental methods:

[0040] Protein expression and purification: Nanjing Zhongding Biotechnology Co., Ltd. was commissioned to express and purify the two proteins by fusing the G / Q-motif sequence of FgBCG1 (i.e. the last 54 amino acids of the FgBCG1 sequence) to the C-terminus of MoBCG1 and AfBCG1 proteins, which do not have G / Q-rich motifs.

[0041] The DNS method for determining xylanase activity was the same as in Example 2.

[0042] Experimental Results: The company successfully purified the protein to the expected size. Xylanase activity assays showed that the C-terminus of the protein without G / Q-rich motifs incorporated G / Q-rich motifs from *Fusarium graminearum*, significantly enhancing the protein's xylanase activity. Figure 3This indicates that the G / Q-rich motif can act as an enhancer, increasing the xylanase activity of the protein.

Claims

1. The application of the glycine / glutamine enriched sequence shown in SEQ ID NO.1 in enhancing the xylanase activity of BCG1 homologous protein, characterized in that, The application involves incorporating the glycine / glutamine enrichment sequence shown in SEQ ID NO.1 into the C-terminus of a BCG1 homolog that lacks a glycine / glutamine enrichment sequence to enhance the xylanase activity of the BCG1 homolog; the BCG1 homolog is selected from MoBCG1 shown in SEQ ID NO.5 and AfBCG1 shown in SEQ ID NO.

6.

2. The application according to claim 1, characterized in that, The BCG1 homologous protein is a BCG1 protein that does not contain a glycine / glutamine enriched sequence at its C-terminus.

3. A method for enhancing the xylanase activity of BCG1 homologous protein, characterized in that, The xylanase activity of the starting BCG1 homolog was enhanced by incorporating the glycine / glutamine enrichment sequence shown in SEQ ID NO.1 into the C-terminus of a BCG1 homolog that does not have a glycine / glutamine enrichment sequence. The BCG1 homolog is selected from MoBCG1 shown in SEQ ID NO.5 and AfBCG1 shown in SEQ ID NO.6.

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