CN114058628A

CN114058628A - Gene PnWRKY1 and application thereof in regulating and controlling synthesis of notoginsenoside

Info

Publication number: CN114058628A
Application number: CN202111181722.8A
Authority: CN
Inventors: 夏鹏国; 郑宇婕; 胡婉莹; 张晓丹; 梁宗锁
Original assignee: Zhejiang Sci Tech University ZSTU
Current assignee: Zhejiang Sci Tech University ZSTU
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-02-18

Abstract

The invention discloses a gene PnWRKY1 and application thereof in regulating and controlling notoginsenoside synthesis, wherein the nucleotide sequence of the gene PnWRKY1 is shown as SEQ ID No. 1. The invention takes the promoter sequences of the gene PnSS and the gene PnSE1 as research objects, screens the transcription factors interacting with the promoter fragments from the panax notoginseng cDNA library by using a yeast single hybridization method, and lays a foundation for researching the transcription regulation of the biosynthesis pathway of the notoginsenoside.

Description

Gene PnWRKY1 and application thereof in regulating and controlling synthesis of notoginsenoside

Technical Field

The invention relates to the technical field of genetic engineering, and mainly relates to a gene PnWRKY1 and application thereof in regulating and controlling synthesis of notoginsenoside.

Background

Pseudo-ginseng (Panax notoginseng (Burk.) F.H.Chen) is a perennial upright herbaceous plant of Panax of Araliaceae (Araliaceae), and is one of the traditional and rare medicinal materials in China. Notoginsenoside (PNS) is the main medicinal active component of notoginseng, and is composed of various tetracyclic triterpenoid saponins. The research shows that the panax notoginseng saponins have better pharmacological activity in the aspects of central nervous system, cardiovascular and cerebrovascular systems, blood system, immune system, fibrosis resistance, aging resistance, tumor resistance and the like. The requirements of the pseudo-ginseng on the planting environment are strict, the growth cycle is long, the crop rotation obstacle is serious, the yield of the pseudo-ginseng is difficult to meet the market demand, and the sustainable development of the pseudo-ginseng industry is severely restricted. Notoginsenoside is mainly synthesized by mevalonic acid (MVA), and Squalene Synthase (SS) and Squalene Epoxidase (SE) are key enzymes, and have important regulation and control effects on triterpenes and sterols in plants.

In recent years, the gene regulation of the secondary metabolic synthesis of medicinal plants becomes a research hotspot, and the completion of panax notoginseng genome sequencing provides a powerful research and application basis for the elucidation of the biosynthesis regulation mechanism of notoginsenoside and the development of traditional Chinese medicine. The transcription factor can act on promoter sequences at the upstream of a plurality of genes to realize 'multi-point regulation', and influence the expression of the plurality of genes related to the synthesis of secondary metabolites. The transcriptional activation of a Transcription factor (Transcription factor) on a gene is an important regulation link in the secondary metabolic process of plants, and has the advantage of 'multi-point regulation'.

The research shows that the transcription factor can influence the expression of key enzyme gene directly participating in the synthesis of notoginsenoside so as to realize the regulation and control of saponin anabolism。The gene PnSS and the gene PnSE1 are key enzyme genes of the biosynthesis pathway of the triterpenoid saponins in panax notoginseng. PnSS is a key enzyme which is exclusively used in the branch metabolic flow of triterpenoid and sterol substance synthesis, and plays an important regulation role in the biosynthesis of triterpenoid and sterol compounds in plants. Patel et al, which inhibited the expression of the ginseng SS gene, found that the amount of triterpene saponin synthesized was decreased and that increasing the expression level of SS in ginseng adventitious roots increased the contents of phytosterol and ginsenoside at the same time. When pseudo-ginseng cell lineWhen SS and DS in the product are co-overexpressed, the content of the saponin is obviously improved, wherein the content of the ginsenoside Re is 5-6 times of that of a control_。PnSE1 is another key enzyme in the triterpene saponin biosynthetic pathway. Two types of PnSE genes are found in panax notoginseng, PnSE1 encodes 537 amino acids and is expressed in each plant tissue, PnSE2 encodes 545 amino acids but is significantly expressed only in flowers, with the rest of the tissues being weaker. PnSE1 is presumed to have a different expression pattern than PnSE2, with PnSE1 being involved in the triterpene saponin synthesis pathway and PnSE2 being involved in the sterol synthesis pathway.

Disclosure of Invention

The invention provides a gene PnWRKY1 and application thereof in regulation and control of notoginsenoside synthesis, wherein the gene PnWRKY1 is a transcription factor, and has interaction with a gene PnSS promoter and a gene PnSE1 promoter which directly participate in notoginsenoside synthesis key enzyme gene, so that notoginsenoside synthesis is influenced.

The specific technical scheme is as follows:

the invention provides a gene PnWRKY1, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The present invention provides a recombinant expression vector comprising the gene PnWRKY1 described above.

The invention provides a genetically engineered bacterium containing the gene PnWRKY 1.

The invention provides a protein coded by the gene PnWRKY1, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.

The invention also provides the use of the gene PnWRKY1 or the protein as described above in interaction with the promoter of the gene PnSS and the promoter of the gene PnSE 1.

The invention also provides the application of the gene PnWRKY1 or the gene engineering bacteria in regulating and controlling the synthesis of notoginsenoside.

Further, the pathways of modulation are: the synthesis of notoginsenoside is regulated by the combination of the protein coded by gene PnWRKY1 and the promoter of gene PnSS or the promoter of gene PnSE 1.

Further, the application of the protein in regulating and controlling the synthesis of notoginsenoside.

Compared with the prior art, the invention has the following beneficial effects:

the invention takes the promoter sequences of the gene PnSS and the gene PnSE1 as research objects, screens the transcription factors interacting with the promoter fragments from the panax notoginseng cDNA library by using a yeast single hybridization method, and lays a foundation for researching the transcription regulation of the biosynthesis pathway of the notoginsenoside.

Drawings

FIG. 1 is the prediction result of the secondary structure of PnWRKY1 protein in example 3;

wherein, α -helix: the longest vertical line; extension chain: a second long vertical line; beta-turn: a third long vertical line; random curl: the shortest vertical line.

FIG. 2 shows the predicted results of the tertiary structure of PnWRKY1 protein in example 3.

FIG. 3 is a phylogenetic tree of PnWRKY1 protein in example 3.

FIG. 4 shows the results of X- α -gal chromogenic reaction verifying the interaction between PnSS (CK) and PnSE1(CK) and PnWRKY1 in example 4.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

EXAMPLE 1 construction of Yeast decoy strains

The sequence fragments of the Pnss and PnSE1 promoters, each containing at least one of the major cis-acting elements, were randomly selected and constructed into the pAbAi vector by means of PCR cloning. Specific primers were designed using primer premier 5.0 and alternative restriction sites within the sequence were predicted using SnapGene 3.2.1, selecting SmaI and XhoI restriction enzyme sites. Amplifying the target fragment by using KOD high-fidelity enzyme, and carrying out electrophoretic separation on the PCR product on 1% agarose gel. Then, the target fragment is cut and recovered. The target fragment and the pAbAi vector were digested simultaneously with SmaI and XhoI for 6 hours at 37 ℃ and then 5. mu.L of 10 XDNA loading buffer was added to terminate the digestion reaction. And (3) carrying out electrophoretic separation on the enzyme digestion product on 1% agarose gel, and then carrying out gel cutting recovery treatment.

The fragment of interest was ligated to the vector overnight using T4 ligase and the ligation products were all transformed into E.coli DH5 alpha competent (100. mu.L). And (3) picking the single clone into an LB liquid culture medium (50mg/L Amp), carrying out amplification culture, and then sucking 1 mu L of bacterial liquid as a template to carry out bacterial liquid PCR verification. Verification of the resulting positive clones Plasmid extraction was performed using TIANPure Mini Plasmid Kit II (Code No. DP107) from TIANGEN.

And carrying out enzyme digestion linearization on the constructed bait vector by BstBI restriction enzyme. The purified linearized plasmid was transferred into the yeast strain Y1H. Yeast decoy strain identification was performed using the Matchmaker Insert Check PCR Mix I kit. Successful yeast colonies were identified and expanded and appropriate yeast decoy strains YS1262 and YE697, which were inhibited by AbA, were selected.

Example 2 screening of transcription factors interacting with the PnSS/SE1 Gene promoter

Panax notoginseng total RNA is extracted according to the instruction of a TIANGEN RNAprep Pure plant total RNA extraction kit. Using total RNA of panax notoginseng as a template, synthesizing a first cDNA chain by SMART reverse transcription, synthesizing double-stranded cDNA by using a long-distance PCR (LD-PCR) amplification technology, detecting by using 1.2% agarose gel electrophoresis, and finally purifying by using a CHROMA SPIN + TE-400 chromatographic column, wherein the panax notoginseng cDNA Library is constructed by referring to the use instruction of a Matchmaker Gold Yeast One-Hybrid Screening System kit. Co-transforming the purified notoginseng cDNA library and pGADT7 vector into positive bait yeast strain competent cells, culturing at 30 ℃ for 3d on SD/-Leu/AbA (500 ng/mL) culture medium, and selecting positive clones to YPDA liquid culture medium for amplification culture. Extracting the yeast plasmid by using a small extraction kit of the Biyuntian yeast plasmid. PCR amplification was performed using the extracted yeast plasmid as a template, using the universal primers T7 (5'-AATACGACTCACTATAGGGCG-3') and 3-AD (5'-AGATGGTGCACGATGCACAG-3'). The obtained PCR product is sent for sequencing. And comparing the data obtained by the sequencing result in NCBI and panax notoginseng genomes, transcriptome data and an arabidopsis database to obtain a transcription factor PnWRKY1 interacted with a PnSS/SE1 gene promoter, wherein the base sequence is shown as SEQ ID No.1, and the amino acid sequence of the encoded protein is shown as SEQ ID No. 2.

Example 3 bioinformatic analysis

The nucleotide and amino acid sequences of the transcription factor PnWRKY1 were submitted to NCBI for analysis. The Open Reading Frame (ORF) sequence of PnWRKY1 has 804bp, and codes 267 amino acids. ExPASY online software (https:// web. ExPASy. org/computer _ pI /) was used to predict a molecular weight of 32575.96Daltons and an isoelectric point (pI) of 10.08, indicating that the protein is a basic protein. Wherein, 47 strong-alkaline Amino Acids (K, R), 23 strong-acid Amino Acids (D, E), 64 Hydrophobic Amino Acids (hydrophosphonic Amino Acids) (A, I, L, F, W, V) and 82 Polar Amino Acids (Polar Amino Acids) (N, C, Q, S, T, Y) are included. The PnWRKY1 protein has an Instability Index (II) of 64.84, a total average hydrophilicity (GRAVY) of-0.803, and is an unstable hydrophilic protein. The prediction result of SMART online software (http:// SMART. embl-heidelberg. de /) shows that the protein has no transmembrane structures (transmembrane domains), but has a low copy area (low complexity) which is 94-107 aa of the predicted amino acid sequence.

The secondary structure of transcription factor PnWRKY1 protein was predicted using the online software SOPMA (https:// npsa-prabi.ibcp.fr/cgi-bin/npsa _ Automat.pl. Predicting the three-dimensional structure of PnWRKY1 protein by using online software SWISS-MODEL (http:// swi ssmodel. expasy. org /), wherein the using method is X-ray, and the respective rate is

The results are shown in FIG. 2. The template number used was 2ayd.1.A, the sequence identity (Seq Id entry) was 44.44%, the oligonucleotide state (Oligo-state) was Monomer, the sequence-to-template sequence similarity (Seq similarity) was 0.43, the Coverage (Coverage) was 0.27, andthe predicted sequence is described as WRKY transcription factor 1.

The transcription factor PnWRKY1 was cloned and analyzed in many species. The amino acid sequence of PnWRKY1 and the amino acid sequence of the gene in other plants in the NCBI database are subjected to multi-sequence alignment through software Clustal X and MEGA6.0 to construct an evolutionary tree, and specific species and protein sequence numbers are shown in Table 1. The evolutionary tree result shows that (shown in figure 3) the panax notoginseng PnWRKY1 is evolutionarily closer to the panax japonicus PjWRKY in the same genus, and is further from the panax ginseng PgWRKY1 and the American ginseng PqWRKY1 in the same genus.

TABLE 1 nucleotide sequences for construction of evolutionary trees

Example 4 in vivo yeast validation

Based on the results of the single hybridization of yeast, transcription factor sequences interacting with the promoters of PnSS and PnSE1 were selected as targets for in vivo yeast validation. Designing specific primer (upstream primer: 5' -CG) according to panax notoginseng transcriptome dataGAATTCATGGAAAATCATGTTGGGAT-3', downstream primer: 5' -CGGGATCCTCATTTCGACTCTACTAGTATACC-3'), introducing EcoRI and BamHI enzyme cutting sites into the sequence, and constructing the gene into pGADT7 vector by PCR amplification with pseudo-ginseng cDNA as a template. The constructed recombinant vectors were transferred into yeast strains YS1262 and YE697, respectively, and the interaction between PnWRKY1 and PnSS and PnSE1 promoters was verified by X- α -gal color reaction (FIG. 4).

Example 5 in vitro validation

Designing specific primer (upstream primer: 5' -CG) according to panax notoginseng transcriptome dataGGATCCATGGAAAATCATGTTGGGAT-3', downstream primer: 5' -CGGAATTCTCATTTCGACTCTACTAGTATACC-3'), introducing BamHI and EcoRI enzyme cutting sites into sequences, taking pseudo-ginseng cDNA as a template, and constructing PnMYB2 into a prokaryotic expression vector pET by PCR amplification-32 a. Extracting positive recombinant plasmid, and transforming competent cells of Escherichia coli BL21(DE 3). Positive clones were selected with LB medium (50mg/L Amp) containing antibiotics. Inoculating 200 mu L of the positive clone bacterial liquid into 5mL of LB liquid culture medium for amplification culture, adding IPTG (isopropyl-beta-D-thiogalactoside) to induce and express the recombinant protein when the bacterial liquid reaches a logarithmic growth period (OD600 is 0.5), wherein the concentration of the IPTG is 1mmol/L, the appropriate induction time is 6h, the induction temperature is 25 ℃, and finally performing gel electrophoresis on the obtained protein by adopting an SDS-PAGE gel electrophoresis technology. The size of the PnWRKY1 fusion protein is 39KD, and after the His tag protein is removed, the size of the band is identical with the predicted size of the PnWRKY1 protein and is about 29 KD.

Sequence listing

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tcttctcagt ctgaaccatt tacaccgatt cagctcaaac caatccctaa accgtgcgac 240

tcaaaaatat ctgaagaatg taaaaccaaa aatactccga tatcgtccgg gagctcgtcg 300

atcaccggag aggaagggac cgtttccaat ggtaagcgag gattattaaa caccgcagca 360

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agatgccgtg accttgagcc caccgacgga atttctggca aacgttcaat ttcacgcggc 480

tgccactgtt gcaagagaag gaaaacagtg gagattagaa gagtaacaac aacaaaagga 540

ggttcgtcat ccattcctgc ggatgagtat tcatggagga agtactatca aaagttaatc 600

ccgggcactc tcttcccaag aggatattac aaatgcagta gcgtaaaggg atgcccggcg 660

aggaagcacg cggtgagatc ccaagatgat ccaacggtgc tagtcgtgac atacgaagga 720

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Gln Ile Gln Leu Asp Phe Ser Lys Lys Ala Val Asn Arg Thr Gly His

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Ser Lys Ile Ser Glu Glu Cys Lys Thr Lys Asn Thr Pro Ile Ser Ser

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Gly Ser Ser Ser Ile Thr Gly Glu Glu Gly Thr Val Ser Asn Gly Lys

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Arg Gly Leu Leu Asn Thr Ala Ala Ala Pro Ala Pro Arg Val Tyr Ser

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Ser Arg Lys Pro Pro Leu Pro Ser Ser His Arg Lys Arg Cys Arg Asp

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Leu Glu Pro Thr Asp Gly Ile Ser Gly Lys Arg Ser Ile Ser Arg Gly

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Claims

1. The gene PnWRKY1 is characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

2. A recombinant expression vector comprising the gene PnWRKY1 of claim 1.

3. A genetically engineered bacterium comprising the gene PnWRKY1 of claim 1.

4. A protein encoded by the gene PnWRKY1 as claimed in claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.

5. Use of the gene PnWRKY1 as claimed in claim 1 or the protein as claimed in claim 4 for interacting with the promoter of the gene PnSS and the promoter of the gene PnSE 1.

6. Use of the gene PnWRKY1 as claimed in claim 1 or the genetically engineered bacterium as claimed in claim 3 in regulating and controlling notoginsenoside synthesis.

7. The use of claim 6, wherein the regulated pathway is: the synthesis of notoginsenoside is regulated by the combination of the protein coded by gene PnWRKY1 and the promoter of gene PnSS or the promoter of gene PnSE 1.

8. Use of the protein of claim 4 for modulating notoginsenoside synthesis.