Isopentenyl transferase gene PcPT11, its coding product and applications

Abstract

The present invention discloses an isopentenyltransferase gene PcPT11 of the UbiA family, belonging to the technical field of genetic engineering. Its gene sequence is shown in SEQ ID NO.1, and the protein sequence encoded by it is shown in SEQ ID NO.2. This gene can be heterologously expressed in yeast, catalyze 22 kinds of flavonoids and 1 kind of coumarin, and the molecular biological mechanism of isopentenyl modification of psoralen isopentenyl flavonoids and coumarin is analyzed. The substrate promiscuity of PcPT11 also explains the diversity of isopentenyl compounds in Psoralea corylifolia, and also lays a foundation for the in vitro biosynthesis of such compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and relates to key enzyme genes involved in the synthesis pathway of psoralen prenyl compounds. More specifically, it relates to the prenyltransferase gene PcPT11, its encoded product and applications. Background Art

[0002] Prenyltransferases (PTs) of the UbiA family are widely present in the biological world and are responsible for catalyzing or modifying substrates with phenolic structures to form more than 63,000 prenylated compounds, including primary metabolites and secondary metabolites. Therefore, they play important roles in primary and secondary metabolism processes.

[0003] Prenylphenolic compounds are a large class of secondary metabolites present in plants, including flavonols, flavones, isoflavones, anthocyanins, etc. They play important roles in the growth and development of plants and can also help plants resist various biotic and abiotic stresses in the natural environment. Prenylation is a special phenolic modification method, and prenylated phenolic compounds are mainly present in plants such as Leguminosae and Moraceae.

[0004] Psoralea corylifolia L. is an annual plant of the Leguminosae family, and its fruits are used as medicine. There are more than 90 compounds in Psoralea corylifolia, and prenyl compounds are the main active ingredients in Psoralea corylifolia, including flavonoids, coumarins, etc., which have anti-inflammatory, antibacterial, anti-aging, neuroprotective and anti-tumor effects.

[0005] The prenyl group can significantly increase the lipophilicity of phenolic compounds, increase the affinity with biological membranes, and is more likely to penetrate biological membranes and then produce stronger pharmacological functions, including antibacterial, antioxidant, anti-tumor, etc. However, the mechanism of prenylation modification of phenolic compounds in Psoralea corylifolia is not fully understood at present. In view of the important functions of prenyltransferases, cloning and identifying Psoralea corylifolia UbiA family prenyltransferase genes with special catalytic functions and biological activities are technical problems that need to be urgently solved by those skilled in the art. Summary of the Invention

[0006] The present invention provides the prenyltransferase gene PcPT11 and its encoded protein. This gene has regioselectivity and substrate promiscuity and can catalyze 23 compounds. In addition, the mechanism of prenylation modification of prenylphenolic compounds in Psoralea corylifolia is also analyzed, which is of great significance for the biosynthesis of prenyl compounds in Psoralea corylifolia.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] The isopentenyltransferase gene PcPT11, the gene sequence of which is shown in SEQ ID NO.1.

[0009] As an invention concept identical to the above technical solution, the present invention also claims the protein encoded by the isopentenyltransferase gene PcPT11, the amino acid sequence of the protein is shown in SEQ ID NO.2, and its N-terminus is a transit peptide sequence containing 44 amino acids.

[0010] As an invention concept identical to the above technical solution, the present invention also claims the truncated sequence of the isopentenyltransferase protein PcPT11, which is the PcPT11△44 sequence formed by removing the 44 amino acid transit peptide sequence at the N-terminus on the basis of the PcPT11 sequence.

[0011] As an invention concept identical to the above technical solution, the present invention also claims the application of the isopentenyltransferase gene PcPT11 in the synthesis of isopentenyl compounds. This gene catalyzes the isopentenylation of 22 kinds of flavonoids and 1 kind of coumarin.

[0012] As an invention concept identical to the above technical solution, the present invention also claims the application of the isopentenyltransferase gene PcPT11 in constructing engineering bacteria and plants containing the PcPT11 gene.

[0013] As an invention concept identical to the above technical solution, the present invention also claims an expression vector pDR196GW-PcPT11 / PcPT11△44, which is characterized by containing the isopentenyltransferase gene PcPT11 / PcPT11△44 and the pDR196GW vector.

[0014] From the above technical solution, the technical effects achieved by the present invention are as follows: The present invention has discovered the isopentenyltransferase gene PcPT11, and the isopentenyltransferase gene PcPT11 is involved in the biosynthesis of psoralen isopentenyl phenolic compounds. It can not only be applied to heterologous synthesis and production of isopentenyl compounds by synthetic biology, but also provide theoretical guidance for molecular breeding of psoralen. Brief Description of the Drawings

[0015] Figure 1 It is the phylogenetic tree analysis map of the psoralen isopentenyltransferase sequence of the present invention and the isopentenyltransferase sequences related to other plants;

[0016] Figure 2 It is the comparative analysis map of the amino acid sequences of the psoralen isopentenyltransferase of the present invention and the isopentenyltransferases of other plants. Among them, the two conserved motifs NQxxDxxxD and KDxxDx(D / E)GD of the plant isopentenyltransferase are represented by wireframes;

[0017] Figure 3 Chemical structure diagrams of all substrates that can be catalyzed by the PcPT11 recombinant protein;

[0018] Figure 4 In this invention, the UPLC and UPLC-MS / MS techniques were used to identify the main enzyme activity product map of the recombinant protein PcPT11, as well as the MS and MS / MS spectra of biochanin A and its product glycyrrhizin A;

[0019] Figure 5 In this invention, the UPLC and UPLC-MS / MS were used to identify the map of all enzyme activity products of the recombinant protein PcPT11;

[0020] Figure 6 Schematic diagram of the synthetic pathway of psoralen isoprenoid compounds designed in this invention. Detailed implementation manners

[0021] The present invention will be described clearly and completely below in conjunction with the accompanying drawings. Unless otherwise specified, the technical means used in the present invention are all well-known methods to those skilled in the art.

[0022] The materials, reagents and instruments used in the following examples of the present invention are all conventional materials, reagents and instruments in the art and can be obtained through commercial channels unless otherwise specified.

[0023] Plant materials and growth environment in the present invention: Psoralea corylifolia, planted in the Beijing Medicinal Plant Garden. The leaves of 4-week-old Psoralea corylifolia were picked, washed with double-distilled water, wrapped with tin foil, and immediately frozen in liquid nitrogen. They were stored in a -80°C refrigerator for subsequent RNA extraction. Different tissues (roots, stems, leaves, flowers, young seeds and mature seeds) of Psoralea corylifolia were collected from the Guangxi Medicinal Plant Garden, washed with double-distilled water, wrapped with tin foil, immediately frozen in liquid nitrogen, and then sent to Beijing Zhongxing Bomei Technology Co., Ltd. for transcriptome sequencing.

[0024] Standard products Biochanin A, genistein, 7-methoxy-4′-hydroxyisoflavone, daidzein, formononetin, irisolidone, 3′,4′,7-trihydroxyisoflavone, 2′-hydroxygenistein, 6,7,4′-trihydroxyisoflavone, glycitein, 5-methyl-7-hydroxyisoflavone, 7-hydroxyisoflavone, soyisoflavone, isorhamnetin, kaempferol, quercetin, mearnsetin, fisetin, morin hydrate, kaempferide, tamarixetin, myricetin, galangin, butein, naringenin chalcone, 2′,4′,6′,3,4-pentahydroxychalcone, isoliquiritigenin, echinatin, apigenin, luteolin, tricetin, tricin, naringenin, eriodictyol, liquiritigenin, hesperitin, dihydroflavones, dihydroquercetin, hydromyricetin, coumarin, umbelliferone, and coumestrol were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; bavachin, corylifolinin, 6-prenylnaringenin, wighteone, and 6-prenylkaempferol were purchased from Wuhan Zhongbiao Technology Co., Ltd.; 6-prenylapigenin was purchased from Yunnan Westlich Biotechnology Co., Ltd. DMAPP was purchased from Sigma-Aldrich. The yeast expression vector pDR196GW was from the research group of Wang Guodong at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0025] Example 1 Cloning of Psoralen Prenyltransferase PcPT11

[0026] 1. Screening of candidate genes

[0027] Based on the assembled Psoralea corylifolia gene data, combined with the NCBI Batch CD-search online database, bioinformatics methods were used to obtain the candidate gene sequence of Psoralea corylifolia isopentenyltransferase. The TMHMM 2.0 (http: / / www.cbs.dtu.dk / services / TMHMM / ) online website was used to predict the protein transmembrane domain. The ChloroP 1.1 (http: / / www.cbs.dtu.dk / services / ChloroP / ) and PSORT (http: / / psort1.hgc.jp / form.html) online websites were used to predict and analyze the protein transit peptides.

[0028] 2. Sequence characteristics and phylogenetic relationship analysis of Psoralea corylifolia isopentenyltransferase gene PcPT11

[0029] In the genomic data of Psoralea corylifolia, a candidate isopentenyltransferase gene sequence (referred to as PcPT11 in this invention) was screened, with a length of 1332 bp, containing a complete ORF, and predicted to encode a polypeptide with a length of 443 amino acids. Sequence comparison with other plant isopentenyltransferases with known functions found that the protein encoded by PcPT11 had a high similarity with these proteins. In this invention, the MAFFT software was used to align the protein sequences of PcPT11 and other plant isopentenyltransferases with known functions, and then the maximum likelihood method was used in MEGA 7.0 to construct a phylogenetic tree, with a bootstrap value of 1000 ( Figure 1 ). It can be seen from Figure 1 that PcPT11 clustered with the known legume flavonoid isopentenyltransferases.

[0030] The PcPT11 protein contains the conserved motifs NQxxD xxxD and KDxxDx(D / E)GD of plant flavonoid isopentenyltransferase ( Figure 2 ). The prediction by the TMHMM 2.0 program showed that the protein contained a transmembrane structure with 9 α-helices. The plant isopentenyltransferases reported so far generally have 7-9 transmembrane structures, and the number of transmembrane structures of the PcPT11 protein is within this range. Plant isopentenyltransferase is a membrane-bound protein located in plastids, and there is a transit peptide sequence targeting to plastids at the N-terminus. The PSORT prediction showed that there was a 44-amino acid transit peptide sequence at the N-terminus of this protein. The above data analysis indicated that PcPT11 encoded a typical legume isopentenyltransferase.

[0031] 3. Total RNA extraction and reverse transcription of plants

[0032] Take fresh Psoralea corylifolia leaves and use The total RNA of plants was extracted using the Super Total RNA Extraction Kit, and the integrity, concentration, and purity of the obtained RNA solution were detected. The reverse transcription experiment was carried out using the FastKing gDNA Dispelling RT SuperMix, and the reverse transcription product (cDNA reaction solution) was stored in a -20°C refrigerator for later use.

[0033] 4. Design nested primers for the candidate prenyltransferase gene PcPT11

[0034] Nested primers were designed according to the PcPT11 gene sequence, and the primer sequences are as follows:

[0035] PcPT11F1: 5′-CTCTTAATGGTCTATATAAAATATCT-3′, SEQ ID NO.3;

[0036] PcPT11R1: 5′-AGCCCATGATAGAGGCAACATT-3′, SEQ ID NO.4;

[0037] PcPT11R2: 5′-ACCTTCTTATCTAATTAAAGGCATG-3′, SEQ ID NO.5;

[0038] The primers were synthesized by Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.

[0039] 5. Amplification of the full-length sequence of the psoralen prenyltransferase gene PcPT11

[0040] Using highfidelity DNA Polymerases (New England BioLabs, MA, USA) for nested PCR amplification. Using cDNA as a template, PCR amplification was carried out with the first-round primers. After completion, using the PCR product as a template, the second-round primers were used for the second-round PCR amplification. The PCR product was purified using the AxyPrep DNA Gel Extraction Kit (Corning, NY, USA), and then cloned into the pTOPO-Blunt simple vector (LANY, Beijing, China). The competent cells of Escherichia coli Trans1-T1 (TransGen Biotech, Beijing, China) were transformed, and positive clones were selected for sequencing (Beijing Sanbo Yuanzhi Biotechnology Co., Ltd.). The PcPT11 gene clones with correct sequences were selected and preserved for the construction of subsequent expression vectors.

[0041] The open reading frame (ORF) of the psoralen prenyltransferase gene PcPT11 obtained by sequencing is 1332 bp in length, and the nucleotide sequence is as shown in SEQ ID NO.1; it encodes 443 amino acids, and the amino acid sequence is as shown in SEQ ID NO.2.

[0042] Example 2 Yeast genetic transformation of psoralen prenyltransferase PcPT11

[0043] 1. Amplification of the complete protein coding sequence of the PcPT11 gene and the truncated sequence without transit peptide

[0044] According to the open reading frame of PcPT11 obtained by cloning, primers were redesigned to amplify the complete protein coding sequence of the gene: the 5′ end of the forward primer needs to carry "CACC". According to the predicted size of the transit peptide of PcPT11, primers were designed to amplify the truncated sequence PcPT11△44 without transit peptide, and the 5′ end of the forward primer needs to carry "CACCATG". The complete protein coding sequence PcPT11 of the candidate gene and the truncated gene sequence PcPT11△44 without transit peptide were amplified from the Escherichia coli plasmid transfected into the pTOPO-Bluntsimple-PcPT11 vector with correct sequencing, and the method was the same as 1.5.

[0045] The primer sequences are as follows:

[0046] PcPT11F: 5′-CACCATGGTCTATATAAAATATCTCCTC-3′, SEQ ID NO.6;

[0047] PcPT11R: 5′-TTGCTCATGCCTTTAATTAGATAA-3′, SEQ ID NO.7;

[0048] PcPT11Δ44F: 5′-CACCATGTCTACTAATGCATGTTCAGTGAC-3′, SEQ ID NO.8;

[0049] 2. Construction of yeast expression vectors

[0050] The entry vectors pENTR / D-TOPO-PcPT11 and pENTR / D-TOPO-PcPT11△44 were constructed by TOPO cloning reaction, and then the yeast expression vectors pDR196GW-PcPT11 and pDR196GW-PcPT11△44 were constructed by LR reaction. After the positive clones were determined to have correct sequences by sequencing, plasmids were extracted for subsequent experimental operations such as yeast genetic transformation.

[0051] 3. Preparation of yeast competent cells

[0052] (1) Take a small amount of the stored DD104 yeast solution from the -80 °C refrigerator, streak it on a YP AD plate, and incubate it upside down in an incubator at 28 °C for 3 - 4 days;

[0053] (2) Pick yeast colonies with a diameter of 2 - 3 mm from the plate and transfer them into 5 mL of YPAD liquid medium. Incubate them overnight (12 - 16 hours) on a shaker at 28 °C with a rotation speed of 210 rpm;

[0054] (3) Pipette an appropriate amount of the yeast solution incubated overnight into 100 mL of YPAD medium, measure the OD 600 value between 0.2 - 0.3, and incubate it on a shaker at 28 °C with a rotation speed of 210 rpm until the OD 600 is between 0.4 - 0.6 (detect the OD 600 value of the bacterial solution at intervals);

[0055] (4) Place the conical flask containing the yeast solution on ice for 30 minutes, dispense it into 2 50 mL centrifuge tubes, and centrifuge at 4 °C and 4,500 rpm for 5 minutes;

[0056] (5) Discard the supernatant, add 20 - 25 mL of sterile distilled water to each centrifuge tube, mix well to suspend the bacterial cell precipitate, then combine the suspended bacterial solutions and centrifuge at 4 °C and 4,500 rpm for 5 minutes;

[0057] (6) Discard the supernatant and suspend the precipitate with 1 mL of freshly prepared sterile 100 mM LiAc;

[0058] (7) Centrifuge at 12,000 rpm for 30 seconds, discard the supernatant, suspend the bacterial solution with 1 mL of 100 mM LiAc, and incubate it on a shaker at 28 °C with a rotation speed of 210 rpm for 30 minutes. Then place it on ice for 10 minutes to prepare yeast competent cells.

[0059] 4. Transformation of yeast competent cells with plasmids

[0060] Use the lithium acetate transformation method to transfer the pDR196GW empty vector (blank control), pDR196GW - PcPT11, and pDR196GW - PcPT11△44 plasmids into the DD104 yeast strain. Extract yeast plasmids using the TIANprep Yeast Plasmid DNA Kit, and perform yeast plasmid PCR with gene - specific primers to verify whether the transfer into yeast is successful.

[0061] Example 3 Enzyme Activity Test

[0062] Perform the yeast - substrate incubation reaction according to the following method:

[0063] (1) Weigh a certain amount of the substrate, dissolve it with a certain volume of DMSO or chromatographic methanol to prepare a mother liquor of the substrate standard with a concentration of 10 mM;

[0064] (2) Inoculate the correctly identified DD104-type yeast cells transformed into the pDR196GW empty vector (blank control) and the pDR196GW-PcPT11 / PcPT11△44 vector respectively into 5 mL of uracil-deficient liquid medium (SD / -Ura), and culture them overnight in a shaker at 28 °C with a rotation speed of 210 rpm;

[0065] (3) Pipette 780 μL of the SD / -Ura liquid medium, add 20 μL of the substrate with a concentration of 10 mM, and 200 μL of the yeast cell solution cultured overnight, and mix well. Culture it in a shaker at 28 °C with a rotation speed of 210 rpm for 3 days for incubation to test the enzyme activity;

[0066] (4) After the incubation reaction is completed, add 1 mL of ethyl acetate to the reaction solution and extract it by ultrasonic wave for 20 minutes;

[0067] (5) After ultrasonic treatment, centrifuge at 12,000 rpm for 10 minutes, transfer the ethyl acetate part (upper layer) to a new centrifuge tube, and repeat 3 times;

[0068] (6) Place the centrifuge tube in the fume hood to evaporate the ethyl acetate solvent;

[0069] (7) Dissolve it with 100 μL of chromatographic methanol, centrifuge at 12,000 rpm for 10 minutes, pipette the supernatant and transfer it to a new centrifuge tube, repeat three times, and finally transfer it to a liquid phase vial for UPLC detection. The detection conditions are as follows: The mobile phase includes phase A as 0.1% formic acid water and phase B as acetonitrile, and the flow rate is 0.3 mL / min. Elution gradient: 0 - 6 min, 35% - 70% B; 6 - 8 min, 70% - 100% B; 8 - 10 min, 100% B; 11 - 12 min, 100% - 35% B. The detection wavelength is 270 nm;

[0070] Chromatographic conditions:

[0071] The compound was separated and detected by UPLC-MS. The separation column model was ACQUITY UPLC HSS C18 column (1.7 μm, 100×2.1 mm; Waters). Mobile phase: Phase A was 0.1% formic acid aqueous solution, and Phase B was acetonitrile. Elution gradient: 0 - 1 min, 5% B; 1 - 8 min, 5% - 30% B; 8 - 12 min, 30% - 40%; 12 - 16 min, 40% - 95% B; 16 - 17 min, 95% - 100% B; 17 - 21 min, 100% B. Flow rate was 0.3 mL / min, column temperature was 32 °C, injection volume was 5 μL, and the PDA detection wavelength was 270 nm.

[0072] Mass spectrometry analysis conditions:

[0073] The positive ion mode was adopted, with an electrospray ionization ion source (ESI) at a flow rate of 900 L / h. In the positive ion mode, the capillary voltage was 2.5 kV, the cone voltage was 30 V, and the molecular weight scanning range was 100 - 1000. The instrument used was Waters ACQUITY UPLC I-Class / Xevo G2-XS QTOF (Waters, Milford, MA, USA), and the obtained detection data was analyzed using software MassLynx (version 4.1).

[0074] Purification and identification of the product

[0075] Semi-preparative RP-HPLC conditions:

[0076] The enzyme activity product was separated and purified by semi-preparative liquid chromatography. The instrument used was Lumtech K-501, and the separation column model was YMC Pack ODS-A column (250 mm×10 mm, 5 μm, YMC Co., Ltd., Kyoto, Japan). Mobile phase: Phase A was pure water, and Phase B was chromatographic acetonitrile. Elution gradient: 0 - 20 min, 70% - 100% of Phase B. The UV detection wavelengths were 254 nm and 280 nm. Approximately 1 mg of the substrate and product were respectively evaporated to dryness under N2 gas, resuspended in deuterated acetone, and analyzed using 1 1H NMR and 13 13C NMR. The nuclear magnetic resonance spectra were detected on a Bruker DRX600 spectrometer. The detection results are shown in Figure 3 , and the chemical structures of all substrates that the PcPT11 recombinant protein can catalyze are shown in Figure 3 , and the enzyme activity results are shown in Figure 4 .

[0077] Example 4 Extraction of yeast microsomes and enzyme activity test

[0078] To further determine the enzyme activity results of the feeding substrate, the crude yeast membrane protein extract was prepared for the enzyme activity experiment.

[0079] Extraction of Yeast Microsomes

[0080] (1) Pick a pDR196GW-PcPT11 / PcPT11△44 positive clone into 10 mL of yeast auxotrophic medium (-Ura) and culture it overnight at 30 °C with shaking; transfer 10 mL of the yeast culture into 800 mL of the medium and culture it overnight at 30 °C with shaking.

[0081] (2) Use a urine glucose test strip to detect the consumption of glucose in the medium. After the glucose is exhausted, centrifuge at 4 °C and 4,000 g for 15 minutes to collect the yeast cells into two 50 mL centrifuge tubes.

[0082] (3) Discard the supernatant, add 50 mL of extraction buffer, shake to suspend the precipitate, let it stand at room temperature for 10 minutes, and then centrifuge at 4 °C and 4,000 g for 15 minutes.

[0083] (4) Discard the supernatant, invert the centrifuge tube on a piece of paper, and suck out the remaining liquid. Use a spatula pre-cooled with liquid nitrogen to break the yeast precipitate into small pieces and quickly transfer it to a 50 mL centrifuge tube pre-cooled with liquid nitrogen for freezing.

[0084] (5) Transfer the collected yeast into a grinding jar pre-cooled with liquid nitrogen, place it on a grinder, set the frequency to 30 and the time to 30 seconds, and break it.

[0085] (6) Re-precool the grinding jar with liquid nitrogen and break it again.

[0086] (7) Transfer the yeast powder into a 50 mL centrifuge tube, add 30 mL of extraction buffer, vortex to mix evenly, and centrifuge at 4 °C and 10,000 g for 10 minutes.

[0087] (8) Transfer the supernatant into a clean ultracentrifuge tube (Nalgene3118-0030), fill it up and balance it; centrifuge at 4 °C and 100,000 g for 90 minutes.

[0088] (9) After ultracentrifugation, discard the supernatant. Transfer the oily microsome precipitate into a homogenizer pre-cooled with ice water, add 1 - 1.5 mL of buffer (a mixture of pH 7.5, 0.1 M Tris-Hcl, and 20% glycerol), and homogenize.

[0089] (10) Aliquot the microsomes into 100 μL per tube, freeze them quickly with liquid nitrogen, and store them in an -80 °C refrigerator.

[0090] (11) Take 5 μL of microsomal protein and measure the protein concentration by the Bradford method (Bradford, 1976).

[0091] Composition and final concentration of extraction buffer: 20 mM Tris-Hcl, pH 7.5; 0.6 M sorbitol; 10 mM Dithiothreitol (DTT); 1 mM phenylmethylsulfonyl fluoride (PMSF), which needs to be dissolved with absolute ethanol, with a half-life of 30 minutes and should be freshly prepared before use.

[0092] In vitro enzyme activity assay method

[0093] The enzyme activity test system includes 40 μg microsomal protein, with a final concentration of 100 mM Tris-HCl, 1 mM DTT, 10 mM Mg 2+ , 300 μM DMAPP and 100 μM substrate. The reaction volume is 50 μL, and the reaction is carried out at 30 °C for 1 hour, then 50 μL of chromatographic methanol is added to terminate the reaction. After terminating the reaction, centrifuge (12,000 g, 10 minutes), transfer the supernatant to a new centrifuge tube, repeat 3 times, and detect by UPLC (UPLC I-class; Waters, Milford, MA, USA). The detection method is the same as that in Example 3, and the results are as Figure 5 shown.

[0094] In summary, the Psoralea PcPT11 gene encodes a prenyltransferase, and the PcPT11 protein catalyzes various flavonoids and a coumarin. PcPT11 has great differences in sequence and function from the flavonoid prenyltransferases of other known plants. PcPT11 is a brand-new plant prenyltransferase; Psoralea accumulates abundant prenyl phenolic compounds and is an ideal plant for studying prenyltransferases. The PcPT11 gene can be expressed in yeast, and the expressed recombinant protein has high activity. Given that PcPT11 has a high catalytic efficiency for various flavonoids, it can be used in plant or microbial metabolic engineering to produce active prenyl compounds. Based on the above experimental processes and results, we speculated the molecular biological mechanism of prenylation in Psoralea, such as Figure 6 .

[0095] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0096] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The prenyltransferase gene PcPT11, characterized in that, The nucleotide sequence of the isopentenyl transferase gene PcPT11 is shown in SEQ ID NO.

1.

2. The protein encoded by the isopentenyl transferase gene PcPT11, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.2, and the N-terminal of the protein is a transit peptide sequence of 44 amino acids.

3. The prenyltransferase protein PcPT11△44, characterized in that, The amino acid sequence of the isopentenyl transferase protein PcPT11△44 is obtained by removing the 44 amino acids at the end of the N-terminal of the amino acid sequence shown in SEQ ID NO.

2.

4. Use of the prenyltransferase gene PcPT11 as described in claim 1 in the synthesis of prenyl compounds, characterized in that, The isopentenyl transferase gene PcPT11 catalyzes 23 substrates to isopentenylate the substrates; The chemical structural formula of the substrate is as follows: 。 5. The application of the isopentenyl transferase gene PcPT11 as described in claim 1 in constructing an engineered bacterium and a plant containing the isopentenyl transferase gene PcPT11, wherein the plant is Psoralea corylifolia L.

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