Auricularia auricula terpene synthetic route key enzyme isopentene diphosphate delta isomerase

Abstract

The invention discloses a key enzyme isopentene diphosphate delta isomerase in a black fungus terpene 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 IDI protein of black fungus. The amino acid sequence of the IDI 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 isopentenyl diphosphate delta isomerase, a key enzyme in the terpene synthesis pathway of black fungus. Background Technology

[0002] Triterpenoids in black fungus have also gradually attracted attention. Patent studies have shown that extracts of black fungus triterpenoids have a significant regulatory effect on a high-fat diet-induced hyperlipidemia model in mice. Differences in the intracellular bioactive substance content after fermentation among different black fungus strains revealed that strain H29 had a significantly higher intracellular triterpenoid content than other strains. Screening of relevant genes in the black fungus genome identified 52 genes involved in terpene synthesis, among which the gene encoding δ24 sterol reductase, GLEAN0002067, belongs to a family unique to black fungus. Researching key enzyme genes in the mevalonate pathway (MVA) synthesis is of great significance. 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 IDI protein from black fungus, the amino acid sequence of which is shown in SEQ ID NO2.

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

[0006] The present invention provides a recombinant vector containing the above-mentioned IDI gene, wherein the IDI 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-described IDI gene, which 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 IDI protein, the above-mentioned IDI gene, the above-mentioned recombinant vector, and the above-mentioned recombinant host cell in the expression of isopentenyl diphosphate δ isomerase.

[0014] Beneficial effects: Bioinformatics analysis of IDI protein in black fungus lays a solid foundation for further in-depth research on the function of key genes in triterpenoid synthesis, and also provides 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 IDI 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. Methods for obtaining the gene: The gene and protein sequence of IDI protein were obtained by whole-genome sequencing and annotation analysis, and then the differential expression of IDI protein was analyzed by transcriptome sequencing.

[0017] Research Methods: Table 1 Bioinformatics Analysis Websites

[0018] The gene sequence of the IDI protein was screened from the genome of black fungus, as shown below: (SEQ ID NO.1) ATGGCGACAGCTGCAGCAGACATCCCGCGGTCGCTGGCGACCATCGACCTCAGCGAGTACGACCCCGAGCAGTCGCGTCTCATGGACGAGCGCTGCATCGTCGTCGACGAGCAGGACAGGCCCATCGGCGCGCTTGACAAGAAGACCTGCCATCTTATGGAGAACATCAACAAGGGCCTGCTGCACCGCGCGTTCTCCGCCTTTGTCTTCCGTCCGAGCGACGGCAAGCTGCTCCTGCAGCAGCGCGCGTCCGAGAAAATCACATTCCCGGACATGTGGACCAACACGTGCTGCTCGCACCCTTTGGACGATTTTGAGGAGGAGAAAATCGAAAAGGATCAGTACGGCGTCAAGGTCGCCGCGTCGCGCAAGCTCGAGCACGAGCTCGGCATCCCGAAGATGGACTACATTCTCTTCATTACCGCGGATGTGGACGTCACGCCCAATGTGAACGAGATCCGAGACCACAAATACGTCGACAAGGCCGAGCTCCAGGCCATGTTTGACGCGCCGGGCAACAGCTTCACACCATGGTTCAAGCTCATTGCGCGCGATTTCTTGTTCGGCTGGTGGGACGAGCTTCTCAAACGCAAGACCGCCGCAGGCACGGTCGACGCGAAAACTCTTACGGACCTCGTAGAAGACAAGGTTCACAAGATGGTCT; The sequence of the IDI protein screened from the Auricularia auricula genome: (SEQ ID NO.2) MATAAADIPRSLATIDLSEYDPEQSRLMDERCIVVDEQDRPIGALDKKTCHLMENINKGLLHRAFSAFVFRPSDGKLLLQQRASEKITFPDMWTNTCCSHPLDDFEEEKIEKDQYGVKVAASRKLEHELGIPKMDYILFITADVDVTPNVNEIRDHKYVDKAELQAMFDAPGNSFTPWFKLIARDFLFGWWDELLKRKTAAGTVDAKTLTDLVEDKVHKMVX。

[0019] 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 mycelia. Differential expression of IDI protein was screened from the transcriptome results, such as... Figure 1 As shown.

[0020] 3. Bioinformatics Analysis (1) Gene structure analysis, such as Figure 2 As shown: Gene cDNA and DNA sequences were analyzed using Gene Structure Display Server 2.0 and NCBI blastn software. The IDI sequence contained 3 exons (nucleotide sequence range: 0-147bp, 296-448bp, 646-909bp) and 2 introns (nucleotide sequence range: 147-194bp, 448-585bp).

[0021] (2) Prediction of transcription start site 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.

[0022] Table 2

[0023] (3) Prediction of promoter active elements Using plantCARE software to predict promoter functional elements, as shown in Tables 1 and 2, the IDI promoter sequence contains a significant number of ABRE, CAAT-box, CGTCA-motif, 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 of the MEJA response. In addition, the promoter sequence also contains dozens of other cis-regulatory elements such as A-box, ACE, and LTR. The presence of these elements suggests that the expression of these two genes may be regulated by multiple environmental signals and hormonal pathways, including abscisic acid response, light response, and low-temperature response.

[0024] Table 3

[0025] (4) Gene domain analysis The conserved domains of the gene amino acid sequence were analyzed using NCBI's Conserved Domains tool. The results are shown in the figure. IDI contains a NUDIX hydrolase domain, which consists of amino acids from position 21 to 209 and belongs to the NUDIX hydrolase superfamily.

[0026] (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 input 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 IDI protein has a molecular weight of approximately 25.28 kDa, a theoretical isoelectric point of 5.11, consists of 3542 atoms, and has the molecular formula C1. 1127 H 1766 N 300 O 338 S 11 The total number of negatively charged residues (Asp + Glu) is 39, and the total number of positively charged residues (Arg + Lys) is 29. Among the 20 amino acids that make up FPPS, aspartic acid (Asp) has the highest proportion, reaching 10.4%, while tryptophan (Trp), tyrosine (Tyr) and cysteine ​​(Cys) have the lowest proportions, all at 1.8%. The instability index is 34.32, predicting that this protein is a stable protein.

[0027] (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 IDI proteins is 0.11 indicates a hydrophilic protein.

[0028] (7) Prediction of protein transmembrane structure Analyzing protein transmembrane structures using TMHMM 2.0 software, such as... Figure 5 As shown, no transmembrane helices were found in either FPPS or IDI proteins. The 222 amino acids of the IDI protein are also primarily located extracellularly, with an internal probability of 13.341%. The probability curves further confirm that the proteins are 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. Neither is a transmembrane protein and they may function in the cytoplasm.

[0029] (8) Prediction results of protein phosphorylation sites are as follows Figure 6 As shown: Phosphorylation mostly occurs on amino acid residues such as serine, threonine, and tyrosine. Using NetPhos 3.1 software, the phosphorylation sites of the protein were analyzed. The results are shown in the figure. The IDI protein has 9 serine kinase phosphorylation sites, 13 threonine kinase phosphorylation sites, and 4 tyrosine kinase phosphorylation sites. It can be phosphorylated by all three amino acid kinases, with a relatively large number of threonine kinase phosphorylation sites, indicating that protein expression is more easily regulated by threonine.

[0030] (9) Prediction of protein secondary structure The GOR4 tool in PRABI-GERLAND was used to predict protein secondary structures. For example... Figure 7 and Figure 8 As shown, the IDI protein is mainly composed of three secondary structures: α-helix, extended chain, and random coil. The α-helix is ​​the most predominant structure, accounting for 45.95% with 102 residues; the extended chain (Ee) accounts for 16.22% with 36 residues; and the random coil (Cc) accounts for 37.84% with 84 residues. Other secondary structures such as 3_10 helices (Gg), π helices (Ii), β-fold bridges (Bb), β-turns (Tt), and curved 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 or other states, indicating a high degree of certainty in the prediction results.

[0031] (10) Prediction of protein tertiary structure 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.

[0032] like Figure 9As shown, the IDI protein tertiary structure model used A0A2H3IVB0.1.A as a template, which is an isopentenyl pyrophosphate Delta-isomerase. The oligomeric state of the model is monomeric, and the GMQE score is 0.95, indicating that the overall quality of the model is very high. The template is derived from the A0A2H3IVB0_WOLCO gene in the AlphaFold database, which belongs to Wolfiporia cocos (strain MD-104, i.e., brown rot fungus). The sequence identity between the model and the template is 79.64%, and the coverage is 100%. This model has a high similarity to known structures, and its overall quality is very reliable.

Claims

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

2.

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

1.

3. A recombinant vector containing the IDI gene as described in claim 2, characterized in that, The IDI 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 IDI gene of claim 2, characterized in that, The IDI 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 IDI protein of claim 1, the IDI 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 isopentenyl diphosphate delta isomerase.

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