Key enzyme farnesyl pyrophosphate synthase for black fungus terpene synthetic route

Abstract

The invention discloses a key enzyme farnesyl pyrophosphate synthase of a black fungus terpenoid synthetic route, and belongs to the technical field of protein. The invention aims to provide a key enzyme gene of a mevalonic acid pathway synthesis pathway in black fungus. The invention provides an FPPS protein of black fungus. The amino acid sequence of the FPPS protein is as shown in SEQ ID NO. 2. A solid foundation is laid for deeply exploring functions of key genes for triterpene synthesis, and meanwhile important data support is provided for systematically explaining specific effects of the genes in terpene synthesis routes.

Description

Technical Field

[0001] This invention belongs to the field of protein technology, specifically relating to a key enzyme in the terpene synthesis pathway of black fungus, Fanesyl pyrophosphate synthase. Background Technology

[0002] Farmland weeds refer to wild plants that grow in farmland without intentional human cultivation. It is estimated that approximately 250 species of weeds worldwide harm major food crops, with the most damaging including Bermuda grass, purslane, goosegrass, and lambsquarters. Farmland weeds can gradually evolve with changes in natural conditions and agricultural development, exhibiting strong adaptability, wide reproductive methods, and high seed production. They not only affect the environment but also have a serious impact on the economy and production. Farmland weed control generally includes agricultural control, chemical control, and biological control. Compared with manual mechanical weeding, chemical herbicides are more efficient and timely, but they cause environmental pollution and harm to humans and livestock. Therefore, the search for greener and more environmentally friendly microbial herbicides has become an inevitable trend. Developing new microbial products that can gradually replace chemical pesticides is of great significance for achieving sustainable development that coordinates agricultural yield and quality safety with agricultural ecological environmental protection. Summary of the Invention

[0003] The purpose of this invention is to provide the key enzyme gene for the mevalonate pathway (MVA) synthesis pathway in black fungus.

[0004] This invention provides an FPPS protein from black fungus, the amino acid sequence of which is shown in SEQ ID NO.2.

[0005] This invention provides an FPPS gene for black fungus, as shown in SEQ ID NO.1.

[0006] The present invention provides a recombinant vector containing the above-mentioned FPPS gene, wherein the FPPS gene is shown in SEQ ID NO.1.

[0007] Further specifying, the launch vehicle can be any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.

[0008] The present invention provides a recombinant host cell containing the above-mentioned FPPS gene, wherein the FPPS gene is shown in SEQ ID NO.1.

[0009] Further specifying, the recombinant host cell is a plant cell.

[0010] To further specify, the plant cells are black fungus cells.

[0011] Further specifying, the host cell is a microbial cell.

[0012] Further specifying, the microbial cells are Escherichia coli or Agrobacterium.

[0013] The present invention provides the application of the above-mentioned FPPS protein, the above-mentioned FPPS gene, the above-mentioned recombinant vector, and the above-mentioned recombinant host cell in the expression of farnesyl pyrophosphate synthase.

[0014] Beneficial effects: This study conducted bioinformatics analysis on the FPPS protein of black fungus, laying a solid foundation for further in-depth research on the function of key genes in triterpenoid synthesis, and also providing important data support for systematically elucidating the specific role of these genes in the terpene synthesis pathway. Attached Figure Description

[0015] Figure 1 This is a graph showing the differential expression of FPPS proteins in the transcriptome. Figure 2 This is a gene structure diagram; Figure 3 A conservative structural domain analysis diagram; Figure 4 This is a graph showing the results of protein hydrophilicity analysis; Figure 5 This is a diagram showing the results of protein transmembrane structure analysis; Figure 6 Figure showing the results of protein phosphorylation site analysis; Figure 7 This is an amino acid sequence diagram; Figure 8 This is a diagram showing the predicted results of protein secondary structure. Figure 9 This is a diagram showing the predicted tertiary structure of the protein. Detailed Implementation

[0016] Example 1. 1. Research Methods: By using whole-genome sequencing and annotation analysis, the gene and protein sequences of FPPS proteins were screened, and then the differential expression of FPPS proteins was analyzed by transcriptome sequencing.

[0017] The gene sequence of FPPS protein was screened from the genome of black fungus, as shown below: (SEQ ID NO.1) Table 1 Bioinformatics Analysis Websites

[0018] FPPS protein was screened from the genome of black fungus: (SEQ ID NO.2) MTDLDARAALAKTRFLGMFPRVRDELVEHMRSHKMPEDACSRFSRNLDYNVPTGKLNRGITVVDSAEILLGRPLADEEYSRAAILGWCVELLQATFLVTDDMMDSCHMRRGEPSWYRLSGIGTDAINDSFLLEASIYYLLKTYFRHEPYYVDLLELFQETTLKTGLGQLIDLLTAPQHQVDLSRFSLDKHQLIVVYKTAYYSFFLPVALA MHFARITDSAAYEHAAKILIPLGEYFQVQDDYLDCYGAPEQTGKIGTDIVDSKCSWLINVALAHAGESDRAILDANYGRKDAESVQRVKRVFERLGVKALYHEYE KRCFKELSALIDQIPETEDPQQAPTLRREVFRSLLRKIYRHNKLSTISQDIKLKIFLNMSQPVAGGCVQMLNNHWQKIHKNANPIVYGDPTPVPGQRNGWTVVLT RNTGKVFTGTGGSKQDAKESAARKALVDLKLISAX; 2. Using untreated black fungus "Hei 29" as the control group, and black fungus "Hei 29" treated with 100 μmol / L MeJA for 6 days as the experimental group, transcriptome sequencing was performed on the mycelium. Differential expression of FPPS protein was screened from the transcriptome results, such as... Figure 1 As shown.

[0019] 3. Bioinformatics analysis: (1) Gene structure analysis, such as Figure 2 As shown: The gene cDNA and DNA sequences were analyzed using Gene Structure Display Server 2.0 and NCBI blastn software. The FPPS sequence contains 6 exons (nucleotide sequence range: 0-134 bp, 138-276 bp, 226-1018 bp, 1304-1357 bp, 1370-1510 bp, 1565-1666 bp) and 5 introns (nucleotide sequence range: 134-194 bp, 276-329 bp, 1018-2453 bp, 2453-2515 bp, 2515-2569 bp).

[0020] (2) Prediction of transcription start sites The transcription start site of the promoter sequence was predicted using the Berkeley Drosophila Genome Project tool. Both the FPPS and IDI promoter sequences contain transcription start sites, and the location and sequence of the transcription start site (SEQ ID NO. 3) are shown in Table 2.

[0021] Table 2 Transcription Initiation Sites

[0022] Table 3 Prediction results of promoter elements

[0023] (3) Prediction of promoter active elements Using plantCARE software to predict promoter functional elements, Table 3 shows that the FPPS promoter sequence contains a large number of ABRE, CAAT-box, G-Box, and TGACG-motif elements, with CAAT-box being one of the core elements of the promoter region. CGTCA-motif and TGACG-motif are the core cis-regulatory elements for the MEJA response. In addition, the FPPS promoter sequence also contains dozens of cis-regulatory elements such as A-box, ACE, and LTR, indicating that their promotion and expression may be related to the abscisic acid response, light response, and low-temperature response. Furthermore, two other promoter sequences also contain dozens of cis-regulatory elements such as A-box, ACE, and LTR, suggesting that the expression of these two genes may be regulated by multiple environmental signals and hormonal pathways, including the abscisic acid response, light response, and low-temperature response.

[0024] (4) Gene domain analysis The conserved domains of the gene amino acid sequence were analyzed using NCBI's Conserved Domains tool, and the results are as follows: Figure 3 As shown, the amino acid sequence of FPPS contains two prominent conserved domains. The first domain is associated with polyprenyl synthetase, specifically located at positions 43 to 303. This domain likely plays a crucial catalytic role in FPPS, participating in the synthesis of isoprene units. The second domain is associated with the double-stranded RNA binding motif (DSRM), located at positions 394 to 446. The DSRM domain is typically associated with RNA binding and may play a role in the regulatory mechanism of FPPS.

[0025] (5) Analysis of the physicochemical properties of proteins The physicochemical properties of the protein were analyzed using the ProParam tool. The amino acid sequence of the gene was entered into a form in FASTA format. After submission, the protein's molecular weight, theoretical isoelectric point, molecular formula, and the proportion of each amino acid were obtained. The results showed that the FPPS protein has a molecular weight of approximately 51.59 kDa, a theoretical isoelectric point of 7.20, consists of 7251 atoms, and has the molecular formula C2. 2303 H 3626 N 634 O 671 S 17 The total number of negatively charged residues (Asp + Glu) is 56, and the total number of positively charged residues (Arg + Lys) is 56. Among the 20 amino acids that make up FPPS, leucine (Leu) accounts for the highest proportion at 11.5%, and tryptophan (Trp) accounts for the lowest proportion at 1.1%. The instability index is 36.98, predicting that this protein is a stable protein.

[0026] (6) Protein hydrophilicity / hydrophobicity analysis Protein hydrophobicity was analyzed using ProtScale software. A vertical axis greater than 0 indicates a hydrophobic protein, while a vertical axis less than 0 indicates a hydrophilic protein. For example... Figure 4 As shown, the overall average hydrophilicity of PFFS protein is 0.09, indicating a hydrophilic protein. (7) Prediction of protein transmembrane structure, such as Figure 5 As shown: Using TMHMM 2.0 software, transmembrane structure analysis revealed no transmembrane helices in either FPPS or IDI proteins. The 455 amino acids of the FPPS protein are primarily located extracellularly, with only 22.134% occurring internally. Probability curves further confirm that the protein is mainly located outside the cell, with the probability of the "outside" state approaching 1, while the probabilities of the "inside" and "transmembrane" states are close to 0. This suggests that the protein is a non-transmembrane protein and may function within the cytoplasm.

[0027] (8) Prediction of protein phosphorylation sites, such as Figure 6 As shown: Phosphorylation mostly occurs on amino acid residues such as serine, threonine, and tyrosine. NetPhos 3.1 software was used to analyze the phosphorylation sites of the protein. The results are shown in the figure. The PFFS protein has 25 serine kinase phosphorylation sites, 26 threonine kinase phosphorylation sites, and 19 tyrosine kinase phosphorylation sites, all of which can be phosphorylated by the three amino acid kinases. The greater number of threonine kinase phosphorylation sites indicates that the expression of these three proteins is more easily regulated by threonine.

[0028] (9) Prediction of protein secondary structure The GOR4 tool in PRABI-GERLAND was used to predict protein secondary structures. For example... Figure 7 and 8 As shown, the secondary structure of the PFFS protein is mainly composed of α-helices and random coils, accounting for 50.55% and 39.56% of the total residues, respectively. In addition, 9.89% of the residues were predicted as extended strands. Other secondary structures such as 3_10 helices (Gg), π helices (Ii), β-fold bridges (Bb), β-turns (Tt), and bent regions (Ss) were not predicted in this sequence, indicating that these structures may be absent or very rare in this protein. Furthermore, no residues were predicted as ambiguous states or other states, indicating a high degree of certainty in the prediction results.

[0029] (10) Protein tertiary structure prediction, such as Figure 9 As shown: Three-dimensional homology modeling of PFFS and IDI proteins was performed using the SWISS-MODEL homology comparison mode. GMQE is a quality assessment value for the 3D model, with scores ranging from 0 to 1; the closer the score is to 1, the higher the reliability of the model. QMEAN is an assessment value for the matching degree between the test protein and the template protein; QMEAN scores range from -4 to 0, with scores closer to 0 indicating a better match.

[0030] As shown in the figure, the tertiary structure model of the PFFS protein used 5cg5.1.A as a template, which is the neutron crystal structure of the human farnesyl pyrophosphate synthase complex with risedronate. The sequence identity between the model and the template was 48.54%, achieving 100% coverage. In terms of model quality assessment, the GMQE score was 0.61, and the QMEANDisCo global score was 0.74, indicating that the overall model quality is relatively reliable. Further local quality assessment showed that although the similarity between the model and the target structure fluctuated at different residue positions, the similarity in most regions remained high.

Claims

1. A type of FPPS protein from black fungus, characterized in that, The amino acid sequence of the FPPS protein is shown in SEQ ID NO.

2.

2. An FPPS gene for black fungus, characterized in that, The FPPS gene is shown in SEQ ID NO.

1.

3. A recombinant vector containing the FPPS gene as described in claim 2, characterized in that, The FPPS gene is shown in SEQ ID NO.

1.

4. The recombinant vector according to claim 3, characterized in that, The launch carrier can be any one of the pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K or pPIC9K series.

5. A recombinant host cell containing the FPPS gene of claim 2, characterized in that, The FPPS gene is shown in SEQ ID NO.

1.

6. The recombinant host cell according to claim 5, characterized in that, The recombinant host cell is a plant cell.

7. The recombinant host cell according to claim 6, characterized in that, The plant cells mentioned are black fungus cells.

8. The recombinant host cell according to claim 5, characterized in that, The host cell is a microbial cell.

9. The recombinant host cell according to claim 8, characterized in that, The microbial cells are Escherichia coli or Agrobacterium.

10. The use of the FPPS protein of claim 1, the FPPS gene of claim 2, the recombinant vector of claim 3 or 4, and the recombinant host cell of claims 5-9 in the expression of farnesyl pyrophosphate synthase.

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