CN113846106A - Gene PnDCD and application thereof in regulating and controlling saponin synthesis - Google Patents
Gene PnDCD and application thereof in regulating and controlling saponin synthesis Download PDFInfo
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Abstract
The invention discloses a gene PnDCD and application thereof in regulating and controlling saponin synthesis, wherein the nucleotide sequence of the gene PnDCD is shown as SEQ ID No. 1. The invention takes the upstream promoter sequence of the gene PnSE1 as a research object, screens the transcription factor interacting with the promoter fragment from the panax notoginseng cDNA library by using a yeast single hybridization method, provides a new way for regulating and controlling the synthesis of notoginsenoside, and lays a foundation for researching the transcription regulation and control of the biosynthesis way of notoginsenoside.
Description
Technical Field
The invention relates to the technical field of genetic engineering, and mainly relates to a gene PnDCD and application thereof in regulating and controlling saponin synthesis.
Background
Pseudo-ginseng (Panax notoginseng (Burk.) F.H.Chen) is a perennial upright herbaceous plant of Panax of Araliaceae, and is one of the traditional famous and precious 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 Epoxidase (SE) is a key enzyme in the synthesis, and has important regulation and control effects on triterpenes and sterols in plant bodies.
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 PnSE1 is a key enzyme gene in the biosynthesis pathway of the triterpenoid saponins in panax notoginseng. 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 gene PnDCD is a protein coding gene related to development and cell death, and has interaction with a promoter directly participating in a key enzyme gene PnSS for synthesizing notoginsenoside so as to influence the synthesis of the notoginsenoside.
The specific technical scheme is as follows:
the invention provides a gene PnDCD, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
The present invention provides a recombinant expression vector comprising the gene PnDCD as described above.
The present invention provides a genetically engineered bacterium comprising the gene PnDCD described above.
The invention provides a protein coded by a gene PnDCD with a nucleotide sequence shown as SEQ ID NO.1, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
The invention also provides the use of the gene PnDCD or of the proteins described above for interacting with the promoter of the gene PnSE 1.
The invention also provides the application of the gene PnDCD or the gene engineering bacteria in regulating and controlling saponin synthesis.
Further, the regulatory pathways are: the protein encoded by gene PnDCD binds to the promoter of gene PnSE1 to regulate the synthesis of saponins.
The invention also provides the application of the protein as described above in regulating and controlling saponin synthesis.
Further, the saponin is notoginsenoside.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the upstream promoter sequence of the gene PnSE1 as a research object, screens the transcription factor interacting with the promoter fragment from the panax notoginseng cDNA library by using a yeast single hybridization method, provides a new way for regulating and controlling the synthesis of notoginsenoside, and lays a foundation for researching the transcription regulation and control of the biosynthesis way of notoginsenoside.
Drawings
FIG. 1 is the predicted result of the secondary structure of the PnDCD 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 PnDCD protein in example 3.
FIG. 3 is a phylogenetic tree of the PnDCD protein in example 3.
FIG. 4 shows the results of the X-. alpha. -gal coloration reaction in example 4, which verifies the interaction between PnSE1(CK) and PnDCD.
FIG. 5 shows the results of the induced expression of PnDCD protein in example 5.
FIG. 6 is a subcellular localization map of the PnDCD protein in example 6 (CK: 35S-sGFP).
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 PnSE1 promoter, each of which contains at least one major cis-acting element, 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 for expansion culture and the appropriate yeast decoy strain YE697 that was inhibited by AbA was selected.
Example 2 screening of genes interacting with the PnSE1 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 (500ng/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'-AATA CGACTCACTATAGGGCG-3') and 3-AD (5'-AGATGGTGCACGATGCACAG-3'). The obtained PCR product is sent for sequencing. Comparing the data obtained by the sequencing result in NCBI and panax notoginseng genome, transcriptome data and an arabidopsis database to obtain a development and cell death related protein coding gene (DCD) interacted with a PnSE1 gene promoter, wherein the base sequence is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2.
Example 3 bioinformatic analysis
The base and amino acid sequences of the above gene PnDCD were submitted to NCBI for analysis. The Open Reading Frame (ORF) sequence of PnDCD has 966bp, and codes 321 amino acids. ExPASY online software (https:// web. ExPASy. org/computer _ pI /) was used to predict a molecular weight of 36583.21Daltons and an isoelectric point (pI) of 8.95, indicating that the protein is a basic protein. Wherein, the number of strong-alkaline Amino Acids (K, R) is 44, the number of strong-acid Amino Acids (D, E) is 38, the number of Hydrophobic Amino Acids (hydrophosphonic Amino Acids) (A, I, L, F, W, V) is 94, and the number of Polar Amino Acids (Polar Amino Acids) (N, C, Q, S, T, Y) is 99. The PnDCD protein has an Instability Index (II) of 37.79 and a total average value of hydrophilicity (GRAVY) of-0.737, and is a stable 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 two low copy regions (low complexity) which are respectively positioned at 106-115 aa and 122-143 aa of the predicted amino acid sequence.
The secondary structure of the PnDCD protein was predicted using the online software SOPMA (https:// npsa-prabi.ibcp.fr/cgi-bin/npsa _ Automat.pl. Predicting the three-dimensional structure of PnDCD protein by using online software SWISS-MODEL (http:// swissmodel. expa sy. 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 2p5d.1.A, the sequence Identity (Seq Identity) was 15.53%, the oligonucleotide state (Oligo-state) was Monomer, the sequence-template sequence similarity (Seq similarity) was 0.31, and the Coverage (Coverage) was 0.32.
The gene PnDCD was cloned and analyzed in many species. The amino acid sequence of PnDCD 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. According to the evolutionary tree results (figure 3), PnDCD was evolutionarily similar to PdDCD of rosaceae and PdDCD of salicaceae.
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, the gene sequence interacting with the PnSE1 promoter was selected as the target for in vivo yeast assay. Designing specific primer (upstream primer: 5' -CG) according to panax notoginseng transcriptome dataGAATTCATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGGATCCTCAACTTCCAAGCTTGCGCT-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 individually transferred into yeast strain YE697 and the interaction between the PnDCD and PnSE1 promoters was verified by X- α -gal chromogenic reaction (FIG. 4).
Example 5 in vitro validation
Designing specific primer (upstream primer: 5' -CG) according to panax notoginseng transcriptome dataGGATCCATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGAATTCTCAACTTCCAAGCTTGCGCT-3'), introducing BamHI and EcoRI enzyme cutting sites into sequences, taking pseudo-ginseng cDNA as a template, and constructing PnDCD into a prokaryotic expression vector pET-32a through PCR amplification. 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 μ L of the bacterial liquid of the positive clone into 5mL of LB liquid medium for amplification culture until the bacterial liquid reachesAt the time of logarithmic growth (OD600 ═ 0.5), IPTG was added to the cells to induce expression of the recombinant protein, the concentration of IPTG was 1mmol/L, the induction time was suitably 6 hours, and the induction temperature was 25 ℃. Finally, the obtained protein was subjected to gel electrophoresis using SDS-PAGE gel electrophoresis technique, and the results are shown in FIG. 5. The size of the PnDCD fusion protein is 50KD, and after the His tag protein is removed, the size of the band is identical to the predicted size of the PnDCD protein, and is about 36 KD.
Example 6 subcellular localization
Designing specific primer (upstream primer: 5' -TCC) according to panax notoginseng transcriptome dataCCCGGGATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGGATCCACTTCCAAGCTTGCGCTTTAC-3'), introducing SmaI and BamHI enzyme cutting sites into sequences, and constructing the gene into a 35S-sGFP vector by PCR amplification by using pseudo-ginseng cDNA as a template. The constructed recombinant vector was transformed into E.coli DH5 alpha competence. And (3) picking the single clone into an LB liquid culture medium (50mg/L Kan), carrying out amplification culture, and then sucking 1 mu L of bacterial liquid as a template to carry out bacterial liquid PCR verification. The positive clones obtained were verified to be extracted with the Plasmid using TIANPure Mini Plasmid Kit II (Code No. DP107) from TIANGEN, and then transformed into Agrobacterium tumefaciens strain GV 3101. Agrobacterium H2The B-RFP strain and the GV3101 strain containing the target gene are mixed according to the ratio of 1: 1, then injecting the mixture into the same tobacco leaf, H2The B-RFP strain causes the nucleus to appear red. The tobacco after injection was cultured for 3 days, and then sampled and observed under a confocal laser microscope. Subcellular localization results showed that PnDCD protein expressed a weaker green fluorescence, which was distributed in both the nucleus and cytoplasm, indicating its localization in the cytoplasm and cell membrane (fig. 6).
Sequence listing
<110> Zhejiang university of science and engineering
Hangzhou radix tetrastigme agricultural science and technology limited company Zhejiang science and technology university
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Claims (9)
1.A gene PnDCD 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 PnDCD of claim 1.
3. A genetically engineered bacterium comprising the gene PnDCD according to claim 1.
4. A protein encoded by the gene PnDCD according to claim 1, characterized in that its amino acid sequence is represented by SEQ ID No. 2.
5. Use of the gene PnDCD according to claim 1 or of the protein according to claim 4 for interacting with the promoter of the gene PnSE 1.
6. Use of the gene PnDCD according to claim 1 or the genetically engineered bacterium according to claim 3 for regulating saponin synthesis.
7. The use of claim 6, wherein the regulatory pathway is: the protein encoded by gene PnDCD binds to the promoter of gene PnSE1 to regulate the synthesis of saponins.
8. Use of a protein according to claim 4 for modulating saponin synthesis.
9. The use according to claim 6 or claim 8, wherein the saponin is notoginsenoside.
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