CN111321128A

CN111321128A - Entada phaseolome cytochrome P450 gene and obtaining method and application thereof

Info

Publication number: CN111321128A
Application number: CN202010139618.1A
Authority: CN
Inventors: 廖卫芳; 缪礼鸿; 王科; 王珍珍
Original assignee: Wuhan Polytechnic University
Current assignee: Wuhan Polytechnic University
Priority date: 2020-03-03
Filing date: 2020-03-03
Publication date: 2020-06-23

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

Entada phaseolome cytochrome P450 gene and obtaining method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an entada phaseolol P450 gene and an obtaining method and application thereof.

Background

Entada phaseoloides (Entada phaseoloides) is a plant of Entada of Leguminosae, also called caulis Entadae, Acer truncatum, Spatholobus lanceolata, etc. Entada phaseoloides as a traditional Chinese herbal medicine has the effects of resisting inflammation, treating gouty arthritis, resisting oxidation, resisting diabetes and the like. The main pharmacological active component is ketenin, namely triterpenoid saponin. The ketengwood saponin obtained naturally can not meet the clinical requirement because of the scarce ketengwood plant resource and the extremely low ketengwood saponin content. Establishing a synthetic biology technology of entada saponin is an important way for solving the contradiction between supply and demand.

In recent years, it has become a hot spot of scientific research to analyze the molecular mechanism of the triterpene saponin synthesis pathway and synthesize triterpene saponin by metabolic engineering means. The biosynthetic pathway of entagenin saponins is divided into three phases: (1) synthesizing terpenoid precursor compounds IPP and DMAPP; (2) constructing an entasaponin skeleton; (3) post-modification of the backbone. Most enzyme genes in the first two stages are identified, but cytochrome P450 genes participating in post-modification of the triterpene skeleton structure of entada phaseolol and the functions thereof are not completely analyzed, which is a main difficulty in comprehensively establishing an entada saponin synthetic biology technology at present.

Disclosure of Invention

The invention aims to provide an entada phaseoloides biosynthetic cytochrome P450 gene and an obtaining method and application thereof.

According to the invention, the full length of Entada phaseoloides EpCYP93E1 gene is obtained by gene screening, amplification by a PCR method, sequencing and splicing of the product; subsequently, bioinformatics analysis is carried out on the beta-phaseoloside-alpha-phaseoloside-beta-phaseoloside, the change of the expression quantity of the gene in roots, stems and leaves of different organs of the beta-phaseoloside-beta-phaseoloside is determined, and the response condition of the gene to methyl jasmonate (MeJA) is detected, so that the function of the gene in the biosynthesis of the beta-phaseoloside-beta-phaseolo.

Specifically, the first aspect of the present invention provides an entada cytochrome P450 gene EpCYP93E1, the nucleotide sequence of which is as set forth in SEQ ID NO: 1 is shown. The total length of CDS is 1542bp, and 513 amino acids are coded.

A second aspect of the present invention provides an entada phaseolol biosynthesis-associated protein encoded by the entada cytochrome P450 gene EpCYP93E1, and having an amino acid sequence shown in SEQ ID NO: 2, respectively. The amino acid sequence of the entada saponin biosynthesis-related protein comprises a typical conserved domain of cytochrome P450 oxidase.

The third aspect of the present invention provides a method for obtaining the entada phaseolol P450 gene EpCYP93E1, comprising the steps of:

1) extracting total RNA of the sample of the young Entada phaseoloides leaves;

2) carrying out PCR amplification by taking cDNA obtained by reverse transcription of the RNA extracted in the step 1) as a template to obtain a cDNA full-length fragment of Entada phaseoloides EpCYP93E1 gene, wherein the nucleotide sequence of a specific primer used in the PCR amplification is as follows:

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

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

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

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

The technical scheme of the invention achieves the following beneficial effects:

1) the Entada phaseoloides cytochrome P450 gene EpCYP93E1 is an Entada phaseoloides saponin biosynthesis related cytochrome P450 oxidase gene in Entada phaseoloides, which is an active ingredient, and the invention determines the expression quantity change of the gene in roots, stems and leaves of different organs of the Entada phaseoloides and the response condition of the gene to methyl jasmonate.

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

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows the result of agarose gel electrophoresis detection of RNA integrity of young leaves of wild Entada phaseoloides.

Fig. 2 shows the result of agarose gel electrophoresis detection of the CDS full-length sequence of the entada EpCYP93E1 gene.

Figure 3 shows the conserved domain prediction of EpCYP93E 1.

Figure 4 shows the transmembrane domain prediction of EpCYP93E 1.

Fig. 5 shows the expression level difference of EpCYP93E1 in the roots, stems, and leaves of different organs of entada phaseoloides.

Fig. 6 shows the difference in the expression level of EpCYP93E1 induced by methyl jasmonate.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, 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, a cytochrome P450 oxidase gene was cloned by using a PCR method using a wild-type entada phaseoloides cDNA as a template, and the specific steps were as follows:

1) taking young Entada phaseoloides leaves as a material, and extracting the total RNA of the Entada phaseoloides. Total RNA extraction was performed according to the procedures described in the Promega RNA extraction kit, and then detected by electrophoresis on 1.5% agarose gel containing staining solution EB (shown in FIG. 1), which indicated successful extraction of total RNA. cDNA was synthesized according to the procedures described in the Thermo reventaid First Strand cDNAsSynthesis Kit, and was stored in a-20 ℃ freezer after being dispensed.

2) Primer design and PCR amplification:

in the aspect of primer design, partial cytochrome P450 enzyme gene sequences are screened by sequencing of transcription groups of roots, stems and leaves of different organs of the Entada phaseoloides at the early stage of a project group, and the P450 oxidase family genes are found to play an important role in the biosynthesis Pathway of Entada phaseoloides by combining the analysis of expression quantity difference, GO annotation, Pathway metabolism and the like. Therefore, based on an entada phaseolol cytochrome P450 oxidase gene sequence, the Primer 6.0 software is used to design a specific Primer, and the nucleotide sequence is as follows:

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

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

the specific method of PCR is as follows:

A) PCR reaction (25. mu.L):

10 × PCR Buffer 2.5. mu.L, dNTPs (10mmol/L, 2.5mmol each) 2. mu.L, forward and backward primers (10mmol/L) 1. mu.L each, DNA template (50ng/L) 1. mu.L, Takara-LA Hi-Fi enzyme (5U/L) 0.5. mu.L, using ddH₂And O is supplemented to 25 mu L.

B) And (3) PCR reaction conditions:

pre-denaturation at 94 ℃ for 4 min; denaturation at 95 deg.C for 1min, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 30s, and circulating for 35 times; extending for 10min at 72 ℃;

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

detecting a PCR product by using 1% agarose gel containing staining solution EB, generating a specific target band at 1542bp (figure 2), recovering a target fragment by using a Tiangen centrifugal column type agarose gel DNA recovery kit, connecting the target fragment with a PMD19-T carrier, connecting the target fragment at 16 ℃ for 2-3 hours, transforming the target fragment into escherichia coli DH5 α competent cells, coating the escherichia coli DH5 α competent cells on an LB agar plate culture medium containing AMP, inverting the cells overnight for culture at 37 ℃ to form a single colony, randomly selecting the single colony, inoculating the single colony in a liquid LB culture medium containing AMP, shaking the culture overnight, amplifying the single colony by using a carrier general M13 primer provided by Shanghai's engineering, identifying positive clones, sequencing and splicing to obtain a CDS full-length sequence of Entada EpCYP93E1 gene.

Example 2

This example carried out bioinformatics analysis of the Entada phaseoloides gene cloned in example 1, and confirmed by cluster analysis and amino acid sequence alignment that the gene belongs to the cytochrome P450 oxidase gene family and has high homology with cytochrome P450 oxidase gene in pea. The method comprises the following specific steps:

a cDNA sequence is obtained by cloning through a PCR technology, the length is 1542bp, 513 amino acids are coded and named as EpCYP93E1, and the nucleotide sequence of the cDNA sequence is shown as SEQ ID NO: 1 is shown. The predicted protein has a molecular weight of about 57.97kDa and an isoelectric point of 8.97, and has the amino acid sequence shown in SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. The conserved region of the amino acid sequence of EpCYP93E1 was predicted by Blastp (FIG. 3), and the sequence was shown to contain the typical conserved domain of cytochrome P450. The transmembrane domain was predicted by TMHMM2.0Server (http:// www.cbs.dtu.dk/services/TMHMM /), and the result showed that the protein contained 1 transmembrane region (7-25 aa) and had a predicted number of transmembrane helices of 24.07617, so that the protein was presumed to belong to a transmembrane protein (FIG. 4).

Example 3

In this example, real-time fluorescent quantitative PCR analysis indicates that the expression level of EpCYP93E1 is related to the content of ketenaside in ketena plants, and the function thereof is further confirmed. The method comprises the following specific steps:

1) respectively cutting root, stem and leaf of different organs of Entada phaseoloides, quickly freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C. Three replicates were used.

2) Preparing 0.1mmol/L methyl jasmonate solution, uniformly spraying onto plant leaves, picking the plant leaves after 6h, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 deg.C for later use. The control group was prepared from leaves of plants sprayed with ethanol. Three replicates were used.

3) Before reverse transcription, the concentration of the extracted total RNA is adjusted, the RNA to be subjected to reverse transcription of each sample is diluted to the same concentration, the reverse transcription is carried out to obtain cDNA, β -actin gene is taken as an internal reference gene to carry out real-time fluorescence quantitative PCR, each experiment is repeated for three times, and the expression difference of EpCYP93E1 in roots, stems and leaves of different organs of a ketchup and the response condition of induction of methyl jasmonate are detected, wherein the nucleotide sequences of the newly designed quantitative primers of the ketchup β -actin and EpCYP93E1 are as follows:

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)

fluorescent quantitative PCR reaction system (20 μ L)2 × SYBR Green qPCR Master Mix 10 μ L, upstream and downstream primers (10mmol/L) each 1 μ L, template 1 μ L, and ddH₂And O is supplemented to 20 mu L.

Fluorescent quantitative PCR reaction conditions: pre-denaturation at 95 ℃ for 4min, denaturation at 95 ℃ for 1min, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, and circulation for 40 times; three replicates per sample.

The results shown in fig. 5 indicate that EpCYP93E1 is expressed in high amounts in the stem. The preliminary research results of the subject groups show that the content of entada saponin in stems is high, which indicates that the expression quantity of EpCYP93E1 is related to the content of entada saponin. Methyl jasmonate can enter the plant body from the stomata of the plant due to the volatility and molecular characteristics of the methyl jasmonate, so that the synthesis of secondary metabolites of the plant is promoted. The results shown in fig. 6 indicate that the expression level of EpCYP93E1 gene was significantly increased after methyl jasmonate induction, and thus, it was confirmed that the gene was involved in the biosynthesis process of entada phaseoloides.

The EpCYP93E1 gene cloned by the invention not only provides a valuable candidate functional gene for revealing the molecular mechanism of the biosynthesis pathway of entagenin saponin, but also provides an important theoretical basis for producing the entagenin saponin in a large scale and at low cost by utilizing the synthetic biology technology.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many 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

1. An entada cytochrome P450 gene EpCYP93E1, characterized in that the nucleotide sequence is as shown in SEQ ID NO: 1 is shown.

2. An entada phaseoloside biosynthesis-related protein encoded by the entada cytochrome P450 gene EpCYP93E1 of claim 1, having an amino acid sequence shown in SEQ ID NO: 2, respectively.

3. The method for obtaining an entada cytochrome P450 gene EpCYP93E1 according to claim 1, comprising the steps of:

1) extracting total RNA of the sample of the young Entada phaseoloides leaves;

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

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

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

4. Use of the entada cytochrome P450 gene EpCYP93E1 of claim 1 or the entada saponin biosynthesis-associated protein of claim 2 in the biosynthesis of entada saponin.