CN111321128A - A kind of Fructus vine cytochrome P450 gene and its obtaining method and application - Google Patents

Abstract

The invention relates to an entada phaseolol P450 gene and an obtaining method and application thereof. The nucleotide sequence of the entada cytochrome P450 gene is shown as SEQ ID NO: 1 is shown. The invention determines the expression quantity change of the gene in different organ roots, stems and leaves of the entada phaseoloides and the response condition of the gene to methyl jasmonate, experiments show that the expression quantity of the gene is positively correlated with the content of entada phaseoloides in entada plants, show that the gene participates in the biosynthesis process of entada phaseoloides, and the cloning and research of the gene have reference value for analyzing the molecular mechanism of the biosynthesis route of the entada phaseoloides.

Description

A kind of Fructus vine cytochrome P450 gene and its obtaining method and application

technical field

The present invention belongs to the field of biotechnology, and more particularly, relates to a Fructus vine cytochrome P450 gene and its obtaining method and application.

Background technique

Entada phaseoloides (Entada phaseoloides) is a plant of the genus Entada in the leguminous family, also known as big blood vine, Guoshan maple, flat dragon and so on. As a traditional Chinese herbal medicine, fenugreek has anti-inflammatory, gouty arthritis, anti-oxidant, anti-diabetic and other effects. Its main pharmacologically active ingredient is serratia saponins, which are triterpenoid saponins. Due to the scarcity of the resources of the genus Fructus Fructus and the extremely low content of Fructus Fructus saponins, the naturally obtained Fructus Fructus Saponins are far from meeting the clinical needs. The establishment of the synthetic biology technology of mentax saponins will be an important way to solve the contradiction between supply and demand.

In recent years, analyzing the molecular mechanism of triterpenoid saponins synthesis pathway and using metabolic engineering to synthesize triterpenoid saponins has become a hotspot of scientific research. The biosynthetic pathway of Fructus Fructus saponins is divided into three stages: (1) synthesis of terpenoid precursor compounds IPP and DMAPP; (2) construction of Fructus Fructus Fructus skeleton; (3) post-modification of the skeleton. Most of the enzyme genes in the first two stages have been identified, but the cytochrome P450 genes involved in the post-modification of the triterpenoid skeleton structure of Fructus chinensis and their functions have not been fully analyzed. The main difficulty of the technology.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of biosynthetic cytochrome P450 gene of mentanoside saponin and its obtaining method and application.

The present invention obtains the full length of the EpCYP93E1 gene of Fructus chinensis through gene screening, amplification by PCR method, sequencing and splicing of the product; then, bioinformatics analysis is performed on it, and the gene is detected in the roots, stems and leaves of different organs of Fructus chinensis. The changes of expression in the gene and the response of the gene to methyl jasmonate (MeJA) were also detected, which confirmed the role of the gene in the biosynthesis of Fructus Fructus.

Specifically, the first aspect of the present invention provides EpCYP93E1, a cytochrome P450 gene of Fructus chinensis, the nucleotide sequence of which is shown in SEQ ID NO: 1. The full length of its CDS is 1542bp, encoding 513 amino acids.

The second aspect of the present invention provides a protein related to the biosynthesis of quince saponins, the protein related to the biosynthesis of juniper saponins is encoded by the above-mentioned lotus cytochrome P450 gene EpCYP93E1, and its amino acid sequence is shown in SEQ ID NO: 2. The amino acid sequence of the protein related to the biosynthesis of mentanoside contains a typical conserved domain of cytochrome P450 oxidase.

A third aspect of the present invention provides a method for obtaining the above-mentioned Fructus chinensis cytochrome P450 gene EpCYP93E1, comprising the following steps:

1) Extract the total RNA of the young leaf samples of Fructus chinensis;

2) The cDNA obtained by reverse transcription of the RNA extracted in step 1) is used as a template, and PCR amplification is carried out to obtain the full-length cDNA fragment of the EpCYP93E1 gene of Fructus chinensis, and the nucleosides of the specific primers used in the PCR amplification The acid sequence is as follows:

93E1-F: 5'-ATGCTAGATATCCAAGGCTACTTCG-3' (SEQ ID NO: 3)

93E1-R: 5'-TCAGGCAGCAGAAAATGGAAC-3' (SEQ ID NO: 4)

3) Recover the PCR product obtained in step 2).

The fourth aspect of the present invention provides the application of the Fructus Fructus cytochrome P450 gene EpCYP93E1 or the Fructus Fructus saponin biosynthesis-related protein in the Fructus Fructus saponin biosynthesis.

The technical scheme of the present invention has achieved the following beneficial effects:

1) The cytochrome P450 gene of the present invention, EpCYP93E1, is a cytochrome P450 oxidase gene related to the biosynthesis of the active ingredient in Fructus Fructus, and the present invention has determined the expression of this gene in the roots, stems and leaves of different organs of Fructus Fructus and the response of the gene to methyl jasmonate. Experiments showed that the expression of the gene was positively correlated with the content of mentanoside in Fructus Fructus, indicating that the gene was involved in the biosynthesis of Fructus Fructus. The cloning of the gene And the research has reference value for analyzing the molecular mechanism of the biosynthetic pathway of Fructus chinensis.

2) The present invention discloses the role of the gene in the biosynthesis of Fructus Fructus, and the acquisition of the gene lays a foundation for the large-scale and low-cost production of Fructus Fructus by synthetic biology technology.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of the exemplary embodiments of the present invention in conjunction with the accompanying drawings.

Figure 1 shows the results of agarose gel electrophoresis detection of RNA integrity in young leaves of C. chinensis.

Fig. 2 shows the results of agarose gel electrophoresis detection of the CDS full-length sequence of the EpCYP93E1 gene of Fructus chinensis.

Figure 3 shows the predicted conserved domains of EpCYP93E1.

Figure 4 shows the predicted transmembrane domain of EpCYP93E1.

Figure 5 shows the differences in the expression levels of EpCYP93E1 in roots, stems and leaves of different organs of Fructus chinensis.

Figure 6 shows the difference in the expression levels of EpCYP93E1 induced by methyl jasmonate.

Detailed ways

Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

Example 1

In this example, the wild-type Fructus chinensis cDNA was used as the template, and the cytochrome P450 oxidase gene was cloned by the PCR method, and the specific steps were as follows:

1) Using the young leaves of Fructus Fructus as the material, the total RNA of Fructus Fructus was extracted. The total RNA was extracted according to the operation steps in the instructions of the Promega RNA extraction kit, and then electrophoresed on a 1.5% agarose gel containing staining solution EB (as shown in Figure 1). The results showed that the total RNA was successfully extracted. For cDNA synthesis, refer to the operation steps in the manual of the Thermo RevertAid First Strand cDNASynthesis Kit, and store in a -20°C refrigerator for later use.

2) Primer design and PCR amplification:

In terms of primer design, the research group screened some cytochrome P450 enzyme gene sequences by sequencing the transcriptomes of different organs of the root, stem, and leaf of Fructus Fructus in the early stage. The family genes play an important role in the biosynthetic pathway of Fructus chinensis. Therefore, on the basis of a cytochrome P450 oxidase gene sequence of Fructus chinensis, Primer 6.0 software was used to design specific primers, and the nucleotide sequences were as follows:

93E1-F: 5'-ATGCTAGATATCCAAGGCTACTTCG-3' (SEQ ID NO: 3)

93E1-R: 5'-TCAGGCAGCAGAAAATGGAAC-3' (SEQ ID NO: 4)

Among them, the specific method of PCR is:

A) PCR reaction system (25 μL):

10×PCR Buffer 2.5μL, dNTPs (10mmol/L, 2.5mmol each) 2μL, upstream and downstream primers (10mmol/L) 1μL each, DNA template (50ng/L) 1μL, Takara-LA high-fidelity enzyme (5U/L) ) 0.5 μL, supplemented to 25 μL with ddH ₂ O.

B) PCR reaction conditions:

Pre-denaturation at 94 °C for 4 min; denaturation at 95 °C for 1 min, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, 35 cycles; extension at 72 °C for 10 min;

C) Recovery, sequencing and sequence analysis of PCR products:

The PCR product was detected by 1% agarose gel containing staining solution EB, and a specific target band appeared at 1542bp (Figure 2). The target fragment was recovered by Tiangen spin column agarose gel DNA recovery kit. It was ligated with PMD19-T vector, ligated at 16°C for 2-3 hours, transformed into E. coli DH5α competent cells, spread on LB agar plate medium containing AMP, and cultured overnight at 37°C upside down to form a single colony. A single colony was randomly selected, inoculated into liquid LB medium containing AMP, cultured overnight with shaking, and amplified using the universal M13 primer for the carrier provided by Shanghai Sangon. full-length sequence.

Example 2

In this example, bioinformatics analysis was carried out on the Fructus chinensis gene cloned in Example 1. Through cluster analysis and amino acid sequence comparison, it was determined that the gene belongs to the cytochrome P450 oxidase gene family, which is similar to the cytochrome P450 oxidase gene in pea. Gene homology is high. details as follows:

A cDNA sequence with a length of 1542 bp and encoding 513 amino acids was obtained by cloning by PCR technology, which was named EpCYP93E1. The nucleotide sequence of the cDNA sequence is shown in SEQ ID NO: 1. The predicted molecular weight of the protein is about 57.97kDa, the isoelectric point is 8.97, and the protein has the amino acid sequence shown in SEQ ID NO:2. The conserved region of the amino acid sequence of EpCYP93E1 was predicted by Blastp (Fig. 3), and the results showed that the sequence contained a typical conserved domain of cytochrome P450. Using TMHMM2.0Server (http://www.cbs.dtu.dk/services/TMHMM/) to predict its transmembrane domain, the results show that it contains one transmembrane domain (7-25aa), and its expected number of transmembrane helices is 24.07617, so it is speculated that this protein is a transmembrane protein (Fig. 4).

Example 3

In this example, real-time fluorescence quantitative PCR analysis shows that the expression level of EpCYP93E1 is correlated with the content of Fructus Fructus saponins in Fructus Fructus plants, and its function is further confirmed. details as follows:

1) Cut out the roots, stems and leaves of different organs of the vine plant respectively, and store them in a -80°C refrigerator for later use after being quick-frozen in liquid nitrogen. Three repetitions.

2) A 0.1 mmol/L methyl jasmonate solution was prepared and sprayed evenly on the leaves of the plants. After 6 hours, the leaves were picked and quick-frozen in liquid nitrogen and stored in a -80°C refrigerator for later use. The leaves of the plants sprayed with ethanol evenly were used as the control group. Three repetitions.

3) Extract the total RNA of each sample respectively, and measure the concentration and purity by Nanodrop 2000 nucleic acid analyzer. Before reverse transcription, the concentration of the extracted total RNA was adjusted so that the RNA to be reverse transcribed in each sample was diluted to the same concentration, and cDNA was obtained by reverse transcription. Using β-actin gene as the internal reference gene, real-time quantitative PCR was performed, and each experiment was repeated three times to detect the expression differences of EpCYP93E1 in roots, stems and leaves of different organs of C. chinensis and the response to methyl jasmonate induction. Among them, the nucleotide sequences of the newly designed quantitative primers for β-actin and EpCYP93E1 are:

β-actin-F: 5'-TTGGACTGTGCCTCATCACC-3' (SEQ ID NO: 5)

β-actin-R: 5'-CTTCCATCACCCTCGGCATT-3' (SEQ ID NO: 6)

dl-93E1-F: 5'-CTTTGTCAGCATTCGCGAGG-3' (SEQ ID NO:7)

dl-93E1-R: 5'-ATCAGTGGTCCCGCTACTCT-3' (SEQ ID NO: 8)

Fluorescence quantitative PCR reaction system (20 μL): 10 μL of 2×SYBR Green qPCR Master Mix, 1 μL of upstream and downstream primers (10 mmol/L), 1 μL of template, supplemented to 20 μL with ddH ₂ O.

Fluorescence quantitative PCR reaction conditions: pre-denaturation at 95 °C for 4 min, denaturation at 95 °C for 1 min, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, 40 cycles; each sample was repeated three times.

The results shown in Figure 5 indicate that EpCYP93E1 is highly expressed in stems. The previous research results of the research group showed that the content of Fructus Fructus in stems was higher, indicating that the expression of EpCYP93E1 was related to the content of Fructus Fructus. Because of its volatility and molecular properties, methyl jasmonate can enter plants from the stomata of plants and promote the synthesis of secondary metabolites in plants. The results shown in FIG. 6 show that the expression of the EpCYP93E1 gene is significantly increased after being induced by methyl jasmonate, therefore, it is proved that this gene is involved in the biosynthesis of mentanoside.

The EpCYP93E1 gene cloned in the present invention not only provides a valuable candidate functional gene for revealing the molecular mechanism of the biosynthetic pathway of Fructus Fructus, but also provides an important theory for the large-scale and low-cost production of Fructus Fructus by synthetic biology technology Base.

Various embodiments of the present invention have been described above, and the foregoing descriptions are exemplary, not exhaustive, and not limiting of the disclosed embodiments. Numerous modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

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Claims (4)

1. A Lentinus syringae cytochrome P450 gene EpCYP93E1, characterized in that, its nucleotide sequence is as shown in SEQ ID NO: 1. 2. A related protein of the biosynthesis of Fructus Fructus, it is characterized in that, this related protein of Fructus Fructus biosynthesis is encoded by the EpCYP93E1 gene of Fructus Fructus Cytochrome P450 according to claim 1, and its amino acid sequence is as shown in SEQ ID NO:2 Show. 3. the obtaining method of the Fructus chinensis cytochrome P450 gene EpCYP93E1 according to claim 1, comprises the steps: 1) Extract the total RNA of the young leaf samples of Fructus chinensis; 2) The cDNA obtained by reverse transcription of the RNA extracted in step 1) is used as a template, and PCR amplification is performed to obtain the full-length cDNA fragment of the EpCYP93E1 gene of Fructus chinensis, and the nucleosides of the specific primers used in the PCR amplification The acid sequence is as follows: 93E1-F: 5'-ATGCTAGATATCCAAGGCTACTTCG-3' (SEQ ID NO: 3) 93E1-R: 5'-TCAGGCAGCAGAAAATGGAAC-3' (SEQ ID NO: 4) 3) Recover the PCR product obtained in step 2). 4. The application of the Fructus chinensis cytochrome P450 gene EpCYP93E1 according to claim 1 or the related protein of Fructus Fructus saponin biosynthesis in the biosynthesis of Fructus chinensis saponins.

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