Mulberry-derived morusin isopentenyl transferase as well as coding gene and application thereof
By providing mulion isopentyl transferase from mulion and its encoding genes, catalyzing the isopentyl modification reaction of mulion substrates, the problem of incomplete understanding of the biosynthesis pathway of isopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsopentylsop
Patent Information
- Application Number
- CN202510226716.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-05-23
AI Technical Summary
The prior art has not yet studied the biosynthesis of isopentylmacin in mulberry trees, resulting in an incomplete understanding of the isopentyl modification pathway of mulberry trees.
It provides mulberry isoprene transferase and its encoding gene named MaPT21/22/23/24/25/27/28/29/30/31 protein, which can catalyze the isoprene modification reaction at the C-5/7/2’/4’ site of the mulberry substrate.
Through the catalysis of this enzyme, the isoprenyl modified santine and distyrene products produced have good anti-inflammatory and anti-tumor activities and have good application prospects.
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Figure CN120025999A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to mulberry-derived mulberry isopentenyl transferase and a coding gene and application thereof. Background Art
[0002] Stilbene is a non-flavonoid phenylpropanoid compound widely distributed in the plant kingdom. It is a phytoalexin secreted by plants when they are damaged by external pathogens. The most common stilbene in plants is resveratrol, which has magical effects in antifungal, antioxidant, antitumor, anticancer, anti-obesity, nerve protection, and cardiovascular protection. Stilbene is a type of characteristic secondary metabolite mainly found in Moraceae plants. It has the basic structure of 2-arylbenzofuran and is a downstream product of stilbene in the biogenic pathway. Studies have shown that stilbene compounds have good antibacterial, antioxidant, anti-inflammatory, anti-diabetic and phosphodiesterase inhibition activities.
[0003] Prenylation is an important modification mode in natural products and is widely found in various organisms including plants, animals, fungi and bacteria. Prenylation enhances the lipophilicity and bioavailability of substrates by coupling substrates with prenyl chains of different lengths. In recent decades, the biological activities and health benefits of prenylated products have attracted widespread attention. For example, the psychoactive effects of cannabinoids, the antidepressant activity of polycyclic polyisopentenyl phloroglucinol, and the antitumor and anti-inflammatory activities of prenylated flavonoids and stilbenes.
[0004] Mulberry trees are rich in mulberry-derived sulcin and stilbene metabolites, which are commonly modified by isopentenyl in structure. The isopentenyl modification of mulberry-derived sulcin compounds can occur on all unsubstituted carbon atoms on the benzene ring, including C-4, C-5, C-7, C-2', C-4' and C-6'. The isopentenyl modification of stilbene compounds occurs on C-2' and C-4', and the modified ligands are mainly isopentenyl and geranyl. Studies have shown that isopentenyl modification can improve the lipid solubility of compounds and enhance their anti-inflammatory and anti-tumor biological activities. The isopentenylation reaction of plant small molecules is catalyzed by isopentenyl transferase (PT), which often occurs on the electron-rich carbon atoms on the substrate, and also occurs on -OH substitution. So far, only MaIDT, which catalyzes the transfer of isopentenyl groups from the C-6 position of flavonoid substrates, and MaOGT, which catalyzes the transfer of geranyl groups from the C-4' position of stilbene, have been reported in mulberry, and no research has been conducted on the biosynthesis of isopentenyl sulphurin. Therefore, in-depth research on mulberry isopentenyl transferase is of great significance for the analysis of the synthetic pathway of isopentenyl modified products and heterologous biosynthesis. Summary of the invention
[0005] The purpose of the present invention is to solve the deficiencies of the prior art, and specifically provide a mulberry-derived mulberry isopentenyl transferase and its encoding gene and application. The mulberry isopentenyl transferase is named MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 protein, which can catalyze the isopentenyl modification reaction of the C-5 / 7 / 2' / 4' site of the mulberry substrate.
[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a mulberry-derived isopentenyltransferase, which is used to catalyze mulberry-type substrates. The amino acid sequence of the mulberry-derived isopentenyltransferase satisfies one of the following characteristics:
[0008] (a) having an amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.10;
[0009] (b) an amino acid sequence having the same catalytic function formed by substituting and / or deleting and / or adding one or more amino acids to the amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.10;
[0010] (c) an amino acid sequence derived from a plant of the genus Morus that has an identity of greater than 95% with the amino acid sequence of any one of SEQ ID NO. 1 to SEQ ID NO. 10 and has the same catalytic function.
[0011] In a second aspect, the present invention provides a mulberry-derived ...
[0012] (a) having a nucleotide sequence shown in any one of SEQ ID NO.11 to SEQ ID NO.20;
[0013] (b) a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.10;
[0014] (c) a nucleotide sequence encoding the isopentenyl transferase according to claim 1 obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence shown in any one of SEQ ID NO.11 to SEQ ID NO.20;
[0015] (d) A nucleotide sequence derived from a plant of the genus Morus, having a nucleotide sequence identity of greater than 90% with the nucleotide sequence shown in any one of SEQ ID NO. 11 to SEQ ID NO. 20 and encoding the isopentenyl transferase according to claim 1.
[0016] In a third aspect, the present invention provides a recombinant expression vector obtained by inserting the sangsin isopentenyl transferase gene described in the second aspect into an expression vector; the expression vector is pEAQ-HT or pESC-HIS.
[0017] In a fourth aspect, the present invention provides a heterologous expression host comprising the recombinant expression vector described in the third aspect.
[0018] Preferably, Nicotiana benthamiana or Saccharomyces cerevisiae YPH499 is used as the host.
[0019] In a fifth aspect, the present invention provides a method for preparing mulsin isopentenyl transferase, wherein the mulsin isopentenyl transferase gene described in the second aspect is cloned into a recombinant expression vector, introduced into a host cell, and the recombinantly expressed mulsin isopentenyl transferase is obtained.
[0020] Preferably, the recombinant expression vector is an Escherichia coli expression vector, a yeast expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector; the host cell is an Escherichia coli host cell, a yeast host cell, a plant cell, an insect cell or a mammalian cell.
[0021] In the sixth aspect, the present invention provides an application of mulberry isopentenyl transferase, wherein the mulberry isopentenyl transferase described in the first aspect or the mulberry isopentenyl transferase obtained by the preparation method described in the fifth aspect is co-incubated with an isopentenyl donor, an isopentenyl acceptor, and magnesium ions, wherein the isopentenyl acceptor is mulberry and diphenylethylene substances, to obtain isopentenyl-modified mulberry and diphenylethylene products.
[0022] Preferably, the sangsin isopentenyl transferase is the MaPT21 protein with an amino acid sequence as shown in SEQ ID NO.1, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol, oxidized resveratrol and pinus erythrin, and the product is sangsin and diphenylethylene modified with isopentenyl at C-4' position;
[0023] The sangsin isopentenyl transferase is the MaPT22 protein with an amino acid sequence as shown in SEQ ID NO.2, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and oxidized resveratrol, and the product is sangsin and diphenylethylene modified with isopentenyl at the C-5 position;
[0024] The mulsin isopentenyl transferase is the MaPT23 protein with an amino acid sequence as shown in SEQ ID NO.3, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is mulsin M, and the product is mulsin modified with isopentenyl groups at the C-4' and C-2' positions;
[0025] The mulsin isopentenyl transferase is the MaPT24 protein with an amino acid sequence as shown in SEQ ID NO.4, the isopentenyl donor is dimethylallyl pyrophosphate, the isopentenyl acceptor is mulsin M, resveratrol and pinus erythrin, and the product is mulsin and diphenylethylene modified with isopentenyl groups at the C-4' and C-2' positions;
[0026] The sangsin isopentenyl transferase is the MaPT25 protein whose amino acid sequence is shown in SEQ ID NO.5, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and pinospermum officinale, and the product is sangsin and diphenylethylene modified with isopentenyl groups at the C-4' and C-2' positions.
[0027] Preferably, the sangsin isopentenyl transferase is the MaPT27 protein with an amino acid sequence as shown in SEQ ID NO.6, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and oxidized resveratrol, and the product is sangsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions;
[0028] The mulsin isopentenyl transferase is the MaPT28 protein with an amino acid sequence as shown in SEQ ID NO.7, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is mulsin M and oxidized resveratrol, and the product is mulsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions;
[0029] The mulsin isopentenyl transferase is the MaPT29 protein with an amino acid sequence as shown in SEQ ID NO.8, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is mulsin M, and the product is mulsin modified with an isopentenyl group at the C-5 position;
[0030] The sansin isopentenyl transferase is a MaPT30 protein with an amino acid sequence as shown in SEQ ID NO.9, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is sansin M, resveratrol and oxidized resveratrol, and the product is sansin and diphenylethylene modified with isopentenyl at position C-7;
[0031] The mulberry isopentenyl transferase is the MaPT31 protein with an amino acid sequence as shown in SEQ ID NO.10, the isopentenyl donor is geranyl pyrophosphate, the isopentenyl acceptor is mulberry M, and the product is mulberry modified with an isopentenyl group at the C-4' position.
[0032] Compared with the prior art, the present invention has the following advantages:
[0033] 1) The isopentenyl transferase in the present invention is the first isopentenyl transferase found in mulberry that can catalyze mulberry-like substrates.
[0034] 2) The isopentenyl transferase in the present invention can specifically catalyze the isopentenyl modification reaction at the C-5 / 7 / 2' / 4' sites of sansin-type substrates.
[0035] 3) The isopentenyl-modified sangsin and stilbene products produced by the isopentenyl transferase in the present invention have good anti-inflammatory and anti-tumor activities and have considerable application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is the identification of the mulberry isopentenyl transferase gene in the whole genome of white mulberry in Example 1.
[0037] Figure 2 The chromatograms of the reactions of mulberry M with different isopentenyl donors catalyzed by different mulberry isopentenyl transferases in Example 4 are shown in FIG. Figure 2 a is a dimethylallyl pyrophosphate (DMAPP) donor; Figure 2 b is the geranyl pyrophosphate (GPP) donor.
[0038] Figure 3 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyltransferase MaPT21 protein in Example 4 are as follows: Figure 3 a is to catalyze the reaction of sansin M and DMAPP, Figure 3 b is to catalyze the reaction of sansin M and GPP, Figure 3 c is the catalytic oxidation reaction of resveratrol and DMAPP, Figure 3 d is the catalytic oxidation reaction of resveratrol and GPP, Figure 3 e is catalyzing the reaction between resveratrol and DMAPP, Figure 3 It catalyzes the reaction between pinospermum officinale and DMAPP.
[0039] Figure 4 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyltransferase MaPT22 protein in Example 4 are as follows: Figure 4 a is to catalyze the reaction of sansin M and DMAPP, Figure 4 b is to catalyze the reaction of sansin M and GPP, Figure 4 c catalyzes the reaction of resveratrol and GPP.
[0040] Figure 5 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT23 protein in Example 4 are as follows: Figure 5 a is to catalyze the reaction of sansin M and DMAPP, Figure 5 b is to catalyze the reaction of sansin M and GPP.
[0041] Figure 6 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT24 protein in Example 4 are as follows: Figure 6 a is to catalyze the reaction of sansin M and DMAPP, Figure 6 b is to catalyze the reaction between resveratrol and DMAPP, Figure 6 c catalyzes the reaction between pinospermum officinale and DMAPP.
[0042] Figure 7 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyltransferase MaPT25 protein in Example 4 are as follows: Figure 7 a is to catalyze the reaction of sansin M and DMAPP, Figure 7 b is to catalyze the reaction of sansin M and GPP, Figure 7 c is to catalyze the reaction of sansin M and FPP, Figure 7 d is to catalyze the reaction of resveratrol and GPP.
[0043] Figure 8 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT27 protein in Example 4 are as follows: Figure 8 a is to catalyze the reaction of sansin M and DMAPP, Figure 8 b is to catalyze the reaction of sansin M and GPP, Figure 8 c is the catalytic oxidation reaction of resveratrol and DMAPP, Figure 8 d is catalyzing the reaction of resveratrol and DMAPP.
[0044] Fig. 9The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT28 protein in Example 4 are as follows: Fig. 9 a is to catalyze the reaction of sansin M and DMAPP, Fig. 9 b is to catalyze the reaction of sansin M and GPP, Fig. 9 c is the catalytic oxidation reaction of resveratrol and DMAPP.
[0045] Fig.10 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT29 protein in Example 4 are as follows: Fig.10 a is to catalyze the reaction of sansin M and DMAPP, Fig.10 b is to catalyze the reaction of sansin M and GPP, Fig.10 c catalyzes the reaction of sansin M and FPP.
[0046] Fig.11 The results of chromatography and mass spectrometry of the reactions of different substrates catalyzed by the mulberry isopentenyl transferase MaPT30 protein in Example 4 are as follows: Fig.11 a is to catalyze the reaction of sansin M and DMAPP, Fig.11 b is to catalyze the reaction of sansin M and GPP, Fig.11 c is to catalyze the reaction of sansin M and FPP, Fig.11 d is the catalytic oxidation reaction of resveratrol and GPP, Fig.11 e catalyzes the reaction of resveratrol with GPP.
[0047] Fig.12 These are the results of chromatography and mass spectrometry detection of the reaction between mulberryine M and GPP catalyzed by mulberryine isopentenyltransferase MaPT31 protein in Example 4. DETAILED DESCRIPTION
[0048] In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.
[0049] Example 1: Identification and analysis of the mulberry isopentenyl transferase gene in the whole genome of white mulberry
[0050] The MaPT gene has a complex structure and 9 introns. Most of the sequences annotated by the algorithm in the genome and transcriptome data of white mulberry are incomplete. In this example, the amino acid sequences encoded by the MaIDT (GeenBank No.: KM262659.1) and MaOGT (GeenBank No.: MG869704.1) genes of white mulberry were used as templates, and the tblastn sequence alignment algorithm was used to manually annotate 33 complete MaPT encoding genes from the genome data of white mulberry, such as Figure 1 shown.
[0051] The amino acid sequences encoded by the 33 MaPT genes were clustered into two main branches (Cluster I and Cluster II) on the phylogenetic tree. MaIDT and MaOGT were in one of the branches, catalyzing flavonoid (Cluster I) and stilbene (Cluster II) substrates, respectively. In addition, Cluster II was further divided into two evolutionary subclades (II-1 and II-2), which had high sequence similarity to each other. Given the structural similarity and correlation of stilbene and sulcin substrates in their synthesis pathways, it was speculated that MaPT in Cluster II-2 had the function of catalyzing sulcin substrates.
[0052] Cluster II-2 contains 10 isopentenyltransferases MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 that catalyze mulberry-like substrates, whose amino acid sequences are shown in SEQ ID NO.1 to SEQ ID NO.10, and the nucleotide sequences of the corresponding mulberry-like isopentenyltransferase encoding genes are shown in SEQ ID NO.11 to SEQ ID NO.20.
[0053] Example 2: Heterologous expression of serranoside isopentenyltransferase in Nicotiana benthamiana
[0054] In this example, the MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 genes in Example 1 were heterologously expressed in Nicotiana benthamiana leaves.
[0055] Using the gene sequence annotated in the white mulberry genome as a template, the company was commissioned to chemically synthesize the MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 genes and construct recombinant pEAQ-HT-MaPT vectors respectively.
[0056] The recombinant pEAQ-HT-MaPT vector was electroporated into Agrobacterium tumefaciens GV3101 competent cells to screen positive single clones. The positive single clones were picked and inoculated into liquid LB medium containing rifampicin and kanamycin, cultured overnight at 28°C and 220rpm, and then transferred to new medium and continued to culture until OD 600 = 0.8-1.0. Collect the cells by low-speed centrifugation and resuspend in infection buffer (10 mM MES, 10 mM MgCl 2 , 100 μM acetosyringone, pH = 5.6), and adjust the OD 600 =0.6, and stand at room temperature in the dark for 2-3 hours. Inject the bacterial solution from the back into 5-6 week-old Nicotiana benthamiana leaves, and continue to culture in an artificial climate box for 4 days before sampling.
[0057] Cut and weigh 4.0g of transiently transformed tobacco leaves, add 0.4g of polyvinyl pyrrolidone and 20mL of pre-cooled extraction buffer (100mM Tris-HCl, 10mM DTT, pH 7.5) in sequence, grind and homogenize on ice. Centrifuge at 4℃ and 12000g for 20min, collect the supernatant and repeat the centrifugation once, centrifuge the supernatant at 4℃ and 100000g for 60min, and resuspend the precipitate in 1ml of extraction buffer, which is the recombinant MaPT microsomal protein.
[0058] Example 3: Heterologous expression of serranoside isopentenyltransferase in Saccharomyces cerevisiae
[0059] In this example, the MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 genes in Example 1 were heterologously expressed in Saccharomyces cerevisiae YPH499.
[0060] Using the gene sequence annotated in the white mulberry genome as a template, the company was commissioned to chemically synthesize the MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 genes and construct recombinant pESC-HIS-MaPT vectors respectively.
[0061] The recombinant pESC-HIS-MaPT vector was electroporated into the competent yeast YPH499, and the positive single clones were screened on the His-deficient medium. The positive clones were inoculated into the liquid medium for expansion and culture, and galactose was added to induce the expression of the MaPT gene. The cells were collected by centrifugation, and an equal volume of acid-washed glass beads and an appropriate amount of extraction buffer were added. The cells were repeatedly shaken and centrifuged at 12000g for 20min. The supernatant was taken and centrifuged at 100000g for 60min. The precipitate was the microsomal protein, which was resuspended in the extraction buffer to obtain the recombinant sansin isopentenyl transferase MaPT microsomal protein.
[0062] Example 4: In vitro enzymatic reaction of serranoside isopentenyltransferase
[0063] In this example, the recombinant mulberine isopentenyltransferase MaPT microsomal protein expressed in Nicotiana benthamiana obtained in Example 2 was used to perform in vitro enzymatic reaction detection.
[0064] The in vitro enzymatic reaction system (100 μL) of recombinant sangrin isopentenyltransferase MaPT microsomal protein includes: 100 mM Tris-HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 100 μM prenyl donor substrate (dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), or farnesyl pyrophosphate (FPP)), 100 μM prenyl acceptor substrate (sansin M, resveratrol, oxidized resveratrol, or styrax), 10 μg microsomal protein (MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 protein).
[0065] After the system was reacted at 30°C for 1 hour, an equal volume of methanol was added to inactivate the reaction, and the supernatant was centrifuged at 17000g for 20 minutes and the product was detected by UPLC-UV and LC-MS / MS. The chromatographic column used was Thermo accucore C18 (150×2.1mm, 2.6μm), the mobile phase A was 0.1% (v / v) formic acid / acetonitrile, B was 0.1% (v / v) formic acid / water, the column temperature was 40°C, the flow rate was 0.5mL / min, and the chromatographic conditions were: 0-1min, 10% A; 1-13min, 10%-80% A; 13-14.5min, 80% A; 14.5-17min, 10% A. The UV detection wavelength was 320nm, and the mass spectrometry detection range was 100-600m / z.
[0066] like Figure 2 As shown in a, compared with the negative control group expressing EGFP protein, MaPT21 protein, MaPT25 protein, MaPT27 protein and MaPT30 protein catalyzed the reaction of sangsin M with DMAPP to produce four different products PM1-4, among which PM3 and PM4 co-eluted with standard sangsin C and sangsin N, and the structures of PM1 and PM2 were identified as 7 / 2'-isopentenyl sangsin M by NMR.
[0067] like Figure 2 As shown in b, MaPT21 protein, MaPT25 protein, MaPT27 protein and MaPT30 protein respectively catalyze the reaction of geranine M with GPP to produce four different products GM1~4, whose structures were identified by NMR as 7 / 2' / 4' / 5-geranylgeranine M.
[0068] The chromatographic and mass spectrometric detection results of the reactions of different isopentenyl donors and isopentenyl acceptors catalyzed by MaPT21 / 22 / 23 / 24 / 25 / 27 / 28 / 29 / 30 / 31 proteins are shown in Figure 2 Figures 3 to 12 shown.
[0069] like Figure 3 As shown, the isopentenyl donors are dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol, oxidized resveratrol and pinus erythrin, and the products obtained by MaPT21 protein catalysis are identified as sangsin and diphenylethylene modified with isopentenyl at the C-4' position.
[0070] like Figure 4 As shown, the isopentenyl donors are dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol and oxidized resveratrol, and the products obtained after catalysis by MaPT22 protein are sangsin and diphenylethylene modified with isopentenyl at the C-5 position.
[0071] like Figure 5 As shown, the isopentenyl donors are dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sansin M, and the product obtained after catalysis by MaPT23 protein is sansin modified with isopentenyl groups at the C-4' and C-2' positions.
[0072] like Figure 6 As shown, the isopentenyl donor is dimethylallyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol and pinocytol, and the products obtained after catalysis by MaPT24 protein are sangsin and diphenylethylene modified with isopentenyl groups at the C-4' and C-2' positions.
[0073] like Figure 7 As shown, the isopentenyl donors are dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol and pinospermone, and the products obtained after catalysis by MaPT25 protein are sangsin and stilbene modified with isopentenyl groups at the C-4' and C-2' positions.
[0074] like Figure 8 As shown, the isopentenyl donors are dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol and oxidized resveratrol, and the products obtained after catalysis by MaPT27 protein are sangsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions.
[0075] like Fig. 9As shown, the isopentenyl donors are dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptors are sangsin M and oxidized resveratrol, and the products obtained after catalysis by MaPT28 protein are sangsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions.
[0076] Fig.10 As shown, the isopentenyl donors are dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is sansin M, and the product obtained after catalysis by MaPT29 protein is sansin modified with isopentenyl at the C-5 position.
[0077] like Fig.11 As shown, the isopentenyl donors are dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptors are sangsin M, resveratrol and oxidized resveratrol, and the products obtained after catalysis by MaPT30 protein are sangsin and diphenylethylene modified with isopentenyl at the C-7 position.
[0078] like Fig.12 As shown, the isopentenyl donor is geranyl pyrophosphate, the isopentenyl acceptor is sansin M, and the product obtained by catalysis by MaPT31 protein is sansin modified with isopentenyl at the C-4' position.
[0079] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.
Claims
1. Morin isopentenyl transferase from white mulberry, characterized in that The mulberry isopentenyl transferase is used to catalyze mulberry substrates, and the amino acid sequence of the mulberry isopentenyl transferase satisfies one of the following characteristics: (a) having an amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.10; (b) an amino acid sequence having the same catalytic function formed by substituting and / or deleting and / or adding one or more amino acids to the amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.10; (c) an amino acid sequence derived from a plant of the genus Morus that has an identity of greater than 95% with the amino acid sequence of any one of SEQ ID NO. 1 to SEQ ID NO. 10 and has the same catalytic function.
2. A mulberry-derived mulberry isopentenyl transferase gene, characterized in that: The nucleotide sequence of the mulberry isopentenyl transferase gene satisfies one of the following characteristics: (a) having a nucleotide sequence shown in any one of SEQ ID NO.11 to SEQ ID NO.20; (b) a nucleotide sequence encoding an amino acid sequence as shown in any one of SEQ ID NO.1 to SEQ ID NO.10; (c) a nucleotide sequence encoding the isopentenyl transferase according to claim 1 obtained by substituting and / or deleting and / or adding one or more nucleotides to the nucleotide sequence shown in any one of SEQ ID NO.11 to SEQ ID NO.20; (d) A nucleotide sequence derived from a plant of the genus Morus, having a nucleotide sequence identity of greater than 90% with the nucleotide sequence shown in any one of SEQ ID NO. 11 to SEQ ID NO. 20 and encoding the isopentenyl transferase according to claim 1.
3. A recombinant expression vector, characterized in that: The method is obtained by inserting the sansin isopentenyl transferase gene according to claim 2 into an expression vector; the expression vector is pEAQ-HT or pESC-HIS.
4. A heterologous expression host containing the recombinant expression vector according to claim 3.
5. The heterologous expression host according to claim 4, characterized in that Nicotiana benthamiana or Saccharomyces cerevisiae YPH499 were used as hosts.
6. A method for preparing mulberry isopentenyl transferase, characterized in that: The mulberry isopentenyl transferase gene described in claim 2 is cloned into a recombinant expression vector and introduced into a host cell to obtain recombinantly expressed mulberry isopentenyl transferase.
7. The method for preparing the mulberry isopentenyl transferase according to claim 6, characterized in that: The recombinant expression vector is an Escherichia coli expression vector, a yeast expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector; the host cell is an Escherichia coli host cell, a yeast host cell, a plant cell, an insect cell or a mammalian cell.
8. An application of mulberry isopentenyl transferase, characterized in that: The mulsin isopentenyl transferase described in claim 1 or the mulsin isopentenyl transferase obtained by the preparation method described in claim 7 is co-incubated with an isopentenyl donor, an isopentenyl acceptor, and magnesium ions, wherein the isopentenyl acceptor is mulsin and diphenylethylene substances, to obtain isopentenyl-modified mulsin and diphenylethylene products.
9. The use of sansin isopentenyl transferase according to claim 8, characterized in that: The mulberry isopentenyl transferase is a MaPT21 protein with an amino acid sequence as shown in SEQ ID NO.1, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is mulberry M, resveratrol, oxidized resveratrol and pinus erythrin, and the product is mulberry and diphenylethylene modified with isopentenyl at C-4' position; The sangsin isopentenyl transferase is the MaPT22 protein with an amino acid sequence as shown in SEQ ID NO.2, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and oxidized resveratrol, and the product is sangsin and diphenylethylene modified with isopentenyl at the C-5 position; The mulsin isopentenyl transferase is the MaPT23 protein with an amino acid sequence as shown in SEQ ID NO.3, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is mulsin M, and the product is mulsin modified with isopentenyl groups at the C-4' and C-2' positions; The mulsin isopentenyl transferase is the MaPT24 protein with an amino acid sequence as shown in SEQ ID NO.4, the isopentenyl donor is dimethylallyl pyrophosphate, the isopentenyl acceptor is mulsin M, resveratrol and pinus erythrin, and the product is mulsin and diphenylethylene modified with isopentenyl groups at the C-4' and C-2' positions; The sangsin isopentenyl transferase is the MaPT25 protein whose amino acid sequence is shown in SEQ ID NO.5, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and pinospermum officinale, and the product is sangsin and diphenylethylene modified with isopentenyl groups at the C-4' and C-2' positions.
10. The use of sansin isopentenyl transferase according to claim 8, characterized in that: The sangsin isopentenyl transferase is the MaPT27 protein with an amino acid sequence as shown in SEQ ID NO.6, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is sangsin M, resveratrol and oxidized resveratrol, and the product is sangsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions; The mulsin isopentenyl transferase is the MaPT28 protein with an amino acid sequence as shown in SEQ ID NO.7, the isopentenyl donor is dimethylallyl pyrophosphate and geranyl pyrophosphate, the isopentenyl acceptor is mulsin M and oxidized resveratrol, and the product is mulsin and diphenylethylene modified with isopentenyl groups at the C-5 and C-7 positions; The mulsin isopentenyl transferase is the MaPT29 protein with an amino acid sequence as shown in SEQ ID NO.8, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is mulsin M, and the product is mulsin modified with an isopentenyl group at the C-5 position; The sansin isopentenyl transferase is a MaPT30 protein with an amino acid sequence as shown in SEQ ID NO.9, the isopentenyl donor is dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesyl pyrophosphate, the isopentenyl acceptor is sansin M, resveratrol and oxidized resveratrol, and the product is sansin and diphenylethylene modified with isopentenyl at position C-7; The mulberry isopentenyl transferase is the MaPT31 protein with an amino acid sequence as shown in SEQ ID NO.10, the isopentenyl donor is geranyl pyrophosphate, the isopentenyl acceptor is mulberry M, and the product is mulberry modified with an isopentenyl group at the C-4' position.