Parthenin C2 hydroxylase, encoding gene thereof, preparation method and application

Abstract

The present invention relates to the field of biotechnology, and discloses a parthenin C2 hydroxylase, its encoding gene, preparation method and application. The protein with parthenin C2 hydroxylase activity is any one of the following a), b), c) or d): a) a protein with an amino acid sequence as shown in SEQ ID NO.2; b) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2; c) a protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.2; d) a protein with 75% or more homology with the amino acid sequence shown in SEQ ID NO.2 and having the same function. The parthenin C2 hydroxylase provided by the present invention can catalyze the hydroxylation reaction at the C2 position of parthenin to form 2-hydroxy parthenin, and the generated 2-hydroxy parthenin is acidified to form xanthinin, providing a key gene resource for the production of xanthinin by enzymatic method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a parthenin C2 hydroxylase, its encoding gene, preparation method and application. Background Art

[0002] Parthenin is a sesquiterpene lactone compound, mainly synthesized in the Xanthium sibiricum of the Compositae family. Parthenin has a strong inhibitory effect on tumor cells such as lung cancer and liver cancer. The research on its mechanism of action shows that parthenin mainly induces apoptosis of tumor cells by activating signaling pathways such as PERK-eIF2α-ATF4. In addition, parthenin is expected to be developed into a new type of plant-derived fungicide in agriculture. For example, the 30% parthenin emulsifiable concentrate developed by Gansu Agricultural University has strong antibacterial activity against Botrytis cinerea of tomato and Cladosporium cucumerinum of cucumber, and its control effect reached more than 60% 14 days after field application.

[0003] At present, the source of parthenin is mainly extracted and purified from Xanthium plants. However, due to the limited resources of wild Xanthium plants, its large-scale development and utilization are restricted. On the other hand, although certain progress has been made in the research on tissue culture and plant regeneration system of Xanthium plants, since the content of parthenin in Xanthium plants is only a few ten-thousandths, its extraction cost is very expensive.

[0004] Therefore, there is an urgent need for a new way to synthesize parthenin. Summary of the Invention

[0005] In order to solve the above problems existing in the source of parthenin, the present invention provides a parthenin C2 hydroxylase, its encoding gene, preparation method and application, providing a new way for the synthesis of parthenin.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect of the present invention, there is provided a parthenin C2 hydroxylase, and the protein having the activity of the parthenin C2 hydroxylase is a protein of any one of the following a), b), c) or d):

[0008] a) a protein with an amino acid sequence as shown in SEQ ID NO.2;

[0009] b) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.2;

[0010] c) a protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in SEQ ID NO.2 and having the same function;

[0011] d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO. 2 and having the same function.

[0012] Preferably, the tag is a 6×histidine tag, Flag tag, MBP tag, HA tag or c-Myc tag.

[0013] In the second aspect of the present invention, there is provided a gene encoding the above-mentioned protein with Parthenin C2 hydroxylase activity, and its nucleic acid molecule is a gene of the following (a) or (b) or (c):

[0014] (a) Its coding sequence is the cDNA molecule or genomic DNA molecule shown in SEQ ID NO: 1;

[0015] (b) A cDNA molecule or genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in (a) and encoding the protein shown in SEQ ID NO: 2;

[0016] (c) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide defined in (a) or (b) under stringent conditions and encodes the protein shown in SEQ ID NO: 2.

[0017] Preferably, the stringent conditions are hybridization in a solution of 6×SSC (containing 0.5% SDS) at 68 °C, and then washing the membrane once with 2×SSC (containing 0.1% SDS) and once with 1×SSC (containing 0.1% SDS).

[0018] Preferably, the gene is derived from Xanthium sibiricum.

[0019] In the third aspect of the present invention, there is provided an expression cassette containing the nucleic acid molecule of the above-mentioned gene encoding the protein with Parthenin C2 hydroxylase activity.

[0020] In the fourth aspect of the present invention, there is also provided an expression vector containing the nucleic acid molecule of the above-mentioned gene encoding the protein with Parthenin C2 hydroxylase activity.

[0021] Preferably, the expression vector is a yeast expression vector or an Escherichia coli expression vector.

[0022] In the fifth aspect of the present invention, there is also provided a transformed cell containing the nucleic acid molecule of the above-mentioned gene encoding the protein with Parthenin C2 hydroxylase activity.

[0023] Preferably, the transformed cell is a bacterial cell, mammalian cell, fungal cell, yeast cell, insect cell or plant cell.

[0024] The sixth aspect of the present invention further provides a method for preparing the protein with parthenin C2 hydroxylase activity, which at least includes the following steps:

[0025] (1) Amplify the gene fragment encoding the protein with parthenin C2 hydroxylase activity;

[0026] (2) Clone the gene fragment into a yeast expression vector to obtain a yeast expression plasmid;

[0027] (3) Transfer the yeast expression plasmid into Pichia pastoris or Saccharomyces cerevisiae to obtain a strain expressing parthenin C2 hydroxylase, and induce the expression of parthenin C2 hydroxylase.

[0028] The seventh aspect of the present invention further provides the application of the protein with parthenin C2 hydroxylase activity in any of the following:

[0029] (a1) Preparing a product containing 2-hydroxy parthenin;

[0030] (a2) Preparing a product containing xanthinin;

[0031] (a3) Preparing a product containing parthenin C2 hydroxylase.

[0032] The present invention also provides a method for synthesizing xanthinin, including the following steps:

[0033] (b1) Using the gene encoding the above-mentioned protein with parthenin C2 hydroxylase activity as the target gene to construct a yeast expression system;

[0034] (b2) Using the yeast expression system to prepare yeast microsomes containing the protein with parthenin C2 hydroxylase activity;

[0035] (b3) Using parthenin as a substrate and yeast microsomes to form 2-hydroxy parthenin;

[0036] (b4) Treating 2-hydroxy parthenin with acidification to form xanthinin.

[0037] The present invention has the following advantages:

[0038] The present invention for the first time clones the full-length sequence of a parthenin C2 hydroxylase gene from Xanthium plants, and uses biochemical techniques to identify that the parthenin C2 hydroxylase can use parthenin as a substrate to hydroxylate at its C2 position to form 2-hydroxy parthenin; 2-hydroxy parthenin can be dehydrated by acidification treatment to form xanthinin; therefore, the present invention provides a key gene resource for the enzymatic synthesis of xanthinin.

[0039] In view of the high production cost and serious environmental pollution of directly extracting and purifying xanthocerin from plants, the discovery of the gene of the present invention provides the necessary gene resources for producing the compound xanthocerin or 2-hydroxyguayulin by metabolic engineering technology, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The LC-MS detection spectrum of TomH2 protein expressed by yeast cells of the embodiment of the present invention catalyzing the formation of 2-hydroxy-guayulin using guayulin as a substrate;

[0041] Figure 2 This is the HPLC detection spectrum of xanthocerin generated by acidification of 2-hydroxyguayulin formed by TomH2 in an example of the present invention. DETAILED DESCRIPTION

[0042] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Example 1

[0044] Obtaining the full-length cDNA sequence of the gene encoding TomH2 from Xanthium sibiricum

[0045] 1. Extraction of total RNA from leaves of Xanthium sibiricum. The specific method is as follows:

[0046] About 100 mg of young leaves of Xanthium sibiricum were weighed and quickly ground in liquid nitrogen, and total RNA was extracted from the ground sample powder using EASYspin plus Plant RNA Rapid Extraction Kit (Adlai).

[0047] 2. Reverse transcribe RNA into cDNA. The specific method is as follows:

[0048] Add the following components to a RNase-free PCR tube: RNA: 8 μL (about 1.5 μg), DNase I: 1 μL, 10×DNase I buffer: 1 μL, RNase inhibitor: 0.5 μL. Mix the above substances well and incubate at 37 °C for 30 min. Then add 1 μL of 100 mM EDTA and incubate at 65 °C for 10 min to terminate the reaction. Add 1 μL of Oligo(dT)18 and 1 μL of dNTPMIX (10 mM) to the PCR tube of the above mixture, pre-denature at 65 °C for 5 min, and then immediately place on ice. Then sequentially add 4 μL of 5× reverse transcriptase buffer, 0.5 μL of RNase inhibitor and 1 μL of reverse transcriptase, mix well and incubate at 42 °C for 1 hour to synthesize the first-strand cDNA. Then terminate the reaction at 70 °C for 10 min. Store the cDNA at -80 °C in a refrigerator.

[0049] 3. Obtain the cDNA sequence of TomH2 by conventional RT-PCR reaction.

[0050] Amplify the Parthenium hysterophorus C2 hydroxylase gene TomH2 by PCR technology, and the primer pair used is:

[0051] TomH2-F: 5'-ATGTTTTTAGCCTTGCAAGTTTTTCTC-3';

[0052] TomH2-R: 5'-TTATTTGGCTTTTGAATTGTGC-3'.

[0053] The 20 μL PCR reaction system is: 2 μL of cDNA template, 1 μL each of primers TomH2-F and TomH2-R, 10 μL of 2× PrimerSTAR Max Premix (Takara), 6 μL of ddH2O.

[0054] PCR reaction conditions: Pre-denature at 98 °C for 2 min; the cycling program is denaturation at 95 °C for 20 s, annealing at 58 °C for 20 s, extension at 72 °C for 1.5 min, 30 cycles, and finally extension at 72 °C for 5 min. After the reaction product is recovered by a common agarose gel DNA recovery kit (purchased from Beijing Aidlab) and ligated to the pEASY-Blunt Simple vector (Beijing TransGen Biotech), the ligation product is transformed into Escherichia coli DH5α competent cells, and then colony PCR is carried out. After the reaction, take 5 μL of the reaction product for agarose gel electrophoresis detection to determine the positive clone, and extract the plasmid of the positive clone and send it to a sequencing company (Beijing Tsingke) for sequencing. The results show that the length of this fragment is 1479 bp, its deoxyribonucleotide sequence is as shown in SEQ ID NO: 1, and the amino acid sequence it encodes is as shown in SEQ ID NO: 2.

[0055] Example 2

[0056] Application of Parthenin C2-hydroxylase (TomH2) gene

[0057] The Parthenin C2-hydroxylase (TomH2) gene with correct sequencing results in Example 1 was cloned into the yeast expression vector pESC-Ura through BamHI and SalI sites to construct the yeast expression plasmid pESC-Ura-TomH2 containing the Parthenin C2-hydroxylase gene. The recombinant plasmid pESC-Ura-TomH2 was transferred into the Saccharomyces cerevisiae WAT11 strain by the conventional lithium acetate transformation method. The yeast cells with empty vector (pESC-Ura) were used as the control group for the experiment. The transgenic yeast was plated on the SD-Ura auxotrophic solid medium for culture at a temperature of 30 °C. Multiple positive yeast colonies identified by colony PCR were picked into 5 ml of SD-Ura auxotrophic liquid medium and cultured at 30 °C and 250 rpm for 48 hours. The bacterial liquid was collected by centrifugation at 5000 rpm, washed once with double-distilled water, and then resuspended in 500 ml of SD-Ura medium containing 2% galactose (the OD600 after resuspension was controlled between 0.4 and 0.6), and induced to culture at 30 °C for 48 hours. The cells obtained from the above culture were collected by centrifugation at 5000 rpm, resuspended in 50 ml of potassium phosphate buffer (0.1 M; pH = 8.0) containing 1 M sorbitol and 14 mM β-mercaptoethanol, and 20 ml of glass beads with a diameter of 0.45 mm were added for oscillatory disruption (the frequency was 30 Hz). The disrupted yeast cells were centrifuged at 12,000 xg and 4 °C for 10 minutes, the precipitate was discarded, and the supernatant was collected. The supernatant was centrifuged at 14,000 g and 4 °C for 90 minutes, the precipitate was collected and dissolved in 1 ml of potassium phosphate buffer (0.1 M; pH = 8.0) as yeast microsomes.

[0058] Take 100 μg of the above microsomes containing TomH2 or control protein, 1 mM parthenin, and 2 mM NADPH and react in 100 μL of potassium phosphate buffer (0.1 M; pH = 8.0) at 30 °C for 3 hours. The reaction system was extracted with an equal volume of ethyl acetate. After the ethyl acetate extract was evaporated to dryness, it was dissolved in 200 μL of methanol solution for LC-MS detection. The LC-MS detection results are as Figure 1 shown. The control microsomes containing the empty vector pESC-Ura could not convert parthenin, while the microsomes containing TomH2 converted parthenin into 2-hydroxy parthenin.

[0059] Adjust the pH value of the above reaction system to 2.0 with 1 M HCl, and place it at room temperature for 30 min. After extracting the reaction system with ethyl acetate of the same volume and evaporating to dryness, dissolve it in 200 μL of methanol solution for LC-MS detection. The LC-MS detection results are as Figure 2 shown, and the generated 2-hydroxy parthenin has been completely converted into xanthinin.

[0060] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A guayule C2 hydroxylase, characterized in that The guayule C2 hydroxylase is a protein of a) or b) as follows: a) a protein with an amino acid sequence as shown in SEQ ID NO.2; b) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO.

2.

2. The gene encoding the guayule C2 hydroxylase according to claim 1, characterized in that: The nucleic acid molecule is the gene of (a) or (b) or (c): (a) the coding sequence is a cDNA molecule or a genomic DNA molecule as shown in SEQ ID NO: 1; (b) a cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in (a) and encodes the protein shown in SEQ ID NO: 2; (c) a cDNA molecule or a genomic DNA molecule which hybridizes with the nucleotide sequence defined in (a) or (b) under stringent conditions and encodes the protein shown in SEQ ID NO:

2.

3. The gene according to claim 2, characterized in that The gene is derived from Xanthium sibiricum.

4. An expression cassette, characterized in that It contains the nucleic acid molecule of the gene as claimed in claim 2.

5. An expression vector, characterized in that: It contains the nucleic acid molecule of the gene as claimed in claim 2.

6. A transformed cell, characterized in that: It contains the nucleic acid molecule of the gene as claimed in claim 2.

7. A transformed cell according to claim 6, characterized in that: The transformed cells are bacterial cells, mammalian cells, fungal cells, insect cells or plant cells.

8. The method for preparing guayule C2 hydroxylase according to claim 1, characterized in that: At least the following steps are included: (1) amplifying a gene fragment encoding the guayule C2 hydroxylase; (2) cloning the gene fragment into a yeast expression vector to obtain a yeast expression plasmid; (3) The yeast expression plasmid is transferred into Pichia pastoris or Saccharomyces cerevisiae to obtain a strain expressing guayule C2 hydroxylase, and the guayule C2 hydroxylase is induced to be expressed.

9. The use of guayule C2 hydroxylase according to claim 1, characterized in that: Used for (a1) preparing a product containing 2-hydroxyguayulin; and / or (a2) preparing products containing xanthocerin; and / or (a3) preparing a product containing guayule C2 hydroxylase.

10. A method for synthesizing xanthocerin, characterized in that: The steps include: (b1) constructing a yeast expression system using the gene encoding the guayule C2 hydroxylase as claimed in claim 1 as the target gene; (b2) using a yeast expression system to prepare yeast microsomes containing guayule C2 hydroxylase active protein; (b3) using guayule as a substrate and using yeast microsomes to form 2-hydroxyguayule; (b4) 2-Hydroxyguayulin is converted into xanthocerin by acidification.

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