CN113846106B - 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 transcription factors interacted with the promoter fragments from the pseudo-ginseng cDNA library by utilizing a yeast single hybridization method, provides a new way for regulating and controlling the synthesis of pseudo-ginseng saponin, and lays a foundation for exploring the transcriptional regulation of the biosynthesis way of pseudo-ginseng saponin.
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
Notoginseng radix [ Panax notoginseng (Burk.) F.H.Chen ] is a perennial upright herb of Panax (Panax) of Araliaceae, and is one of the traditional and rare medicinal materials in China. Notoginseng radix saponin (Panax notoginseng saponins, PNS) is a main medicinal active ingredient of Notoginseng radix, and consists of multiple tetracyclic triterpene saponins. The existing researches show that the panax notoginseng saponins have better pharmacological activity in the aspects of central nervous system, cardiovascular and cerebrovascular systems, blood systems, immune systems, anti-fibrosis, anti-aging, anti-tumor and the like. The pseudo-ginseng has strict requirements on planting environment, long growth cycle and serious rotation obstacle, and the yield of the pseudo-ginseng is difficult to meet the market demand, so that the sustainable development of the pseudo-ginseng industry is seriously restricted. The notoginsenoside is mainly synthesized by a Mevalonate (MVA) pathway, and squalene epoxidase (Squalene epoxidase, SE) is a key enzyme, and has important regulation and control effects on triterpenes and sterols in plants.
In recent years, gene regulation of medicinal plant secondary metabolism synthesis has become a research hotspot, and completion of notoginseng genome sequencing provides a powerful research and application foundation for elucidation of notoginseng saponin biosynthesis regulation mechanism and development of traditional Chinese medicine. Transcription factors can act on promoter sequences upstream of multiple genes to achieve "multi-point regulation" affecting expression of multiple genes associated with secondary metabolite synthesis. Transcription activation of the transcription factor (Transcription factor) on the genes is an important regulation link of the secondary metabolic process of plants, and has the advantage of 'multi-point regulation'.
Studies have shown that transcription factors can influence the expression of key enzyme genes directly involved in the synthesis of notoginsenoside, thereby realizing the regulation of anabolism of the saponin. The gene PnSE1 is a key enzyme gene of the biological synthesis path of the triterpenoid saponin of the pseudo-ginseng. PnSE1 is another key enzyme in the triterpene saponin biosynthesis pathway. Two types of PnSE genes exist in pseudo-ginseng, pnSE1 codes 537 amino acids, pnSE2 codes 545 amino acids which are expressed in various plant tissues, but only expressed in flowers is obvious, and the rest tissues are weaker. PnSE1 and PnSE2 are presumed to have different expression patterns, 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 PnDCD and application thereof in regulating and controlling saponin synthesis, wherein the gene PnDCD is a development and cell death related protein coding gene, and has interaction with a promoter directly participating in a key enzyme gene PnSS for synthesizing pseudo-ginseng saponin, thereby influencing the synthesis of pseudo-ginseng saponin.
The specific technical scheme is as follows:
the invention provides a gene PnDCD, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
The present invention provides a recombinant expression vector comprising the gene PnDCD as described above.
The 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 as described above or of the protein as described above for interacting with the promoter of the gene PnSE 1.
The invention also provides application of the gene PnDCD or the genetically engineered bacterium in regulating and controlling the synthesis of saponin.
Further, the regulation and control path is as follows: the synthesis of saponins is regulated by the combination of the protein encoded by the gene PnDCD and the promoter of the gene PnSE 1.
The invention also provides the use of a protein as described above for modulating 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 transcription factors interacted with the promoter fragments from the pseudo-ginseng cDNA library by utilizing a yeast single hybridization method, provides a new way for regulating and controlling the synthesis of pseudo-ginseng saponin, and lays a foundation for exploring the transcriptional regulation of the biosynthesis way of pseudo-ginseng saponin.
Drawings
FIG. 1 shows the predicted results of the secondary structure of PnDCD protein in example 3;
wherein, α -helix: a longest vertical line; extension chain: a second long vertical line; beta-turning angle: a third long vertical line; random coil: a shortest vertical line.
FIG. 2 shows the predicted result of the tertiary structure of PnDCD protein in example 3.
FIG. 3 is a phylogenetic tree of the PnDCD proteins of example 3.
FIG. 4 is a graph showing the result of verifying interaction between PnSE1 (CK) and PnDCD by the X-alpha-gal chromogenic reaction in example 4.
FIG. 5 shows the results of inducible expression of the PnDCD protein in example 5.
FIG. 6 is a map of the subcellular localization of the PnDCD protein of example 6 (CK: 35S-sGFP).
Detailed Description
The invention will be further described with reference to the following examples, which are given by way of illustration only, but the scope of the invention is not limited thereto.
EXAMPLE 1 construction of Yeast bait Strain
Randomly selecting sequence fragments on the PnSE1 promoter, wherein each fragment contains at least one main cis-acting element, and constructing the sequence fragments into the pAbAi vector by a PCR cloning mode. Specific primers were designed using primer premier 5.0 and selected SmaI and XhoI restriction enzyme sites using SnapGene 3.2.1 to predict optional cleavage sites within the sequence. The target fragment was amplified using KOD high fidelity enzyme and the PCR product was separated electrophoretically on a 1% agarose gel. And then cutting and recycling the target fragment. The target fragment and pAbAi vector were double digested with SmaI and XhoI, digested at 37℃for 6h, and then the digestion reaction was stopped by adding 5. Mu.L of 10X DNA loading buffer. And (3) carrying out electrophoresis separation on the enzyme digestion product on 1% agarose gel, and then carrying out gel digestion recovery treatment.
The fragment of interest was ligated overnight with the vector using T4 ligase and the ligation products were all transformed into E.coli DH 5. Alpha. Competent (100. Mu.L). Monoclone is selected and put into LB liquid culture medium (50 mg/L Amp), and is cultivated in an enlarged way, and then 1 mu L of bacterial liquid is taken as a template for bacterial liquid PCR verification. The positive clones obtained were verified to be extracted from the plasmid using TIANGEN TIANpure Mini Plasmid Kit II (Code No. DP107).
The bait vector constructed above was subjected to enzyme-tangential restriction with BstBI restriction enzyme. The linearized plasmid after purification is transferred into a yeast Y1H strain. Yeast bait strain identification was performed using the Matchmaker Insert Check PCR Mix I kit. Identification of successful Yeast colonies for expansion culture, selection of the appropriate Yeast decoy strain YE697 capable of being inhibited by AbA.
Example 2 selection of genes that interact with the PnSE1 Gene promoter
Total Notoginseng RNA was extracted with reference to TIANGEN RNAprep Pure plant total RNA extraction kit instructions. Using total notoginseng RNA as template, through SMART reverse transcription to synthesize cDNA first chain, using long distance PCR (LD-PCR) amplification technique to synthesize double-chain cDNA, using 1.2% agarose gel electrophoresis to detect, finally using CHROMA spin+TE-400 chromatographic column to purify, referring to Matchmaker Gold Yeast One-Hybrid Library Screening System reagent box instruction to construct notoginseng cDNA library.
The purified pseudo-ginseng cDNA library and pGADT7 vector are transformed into positive decoy yeast strain competent cells, and cultured for 3d at 30 ℃ on SD/-Leu/AbA (500 ng/mL) medium, and positive clones are selected and expanded to YPDA liquid medium. The small amount of extraction kit of the Biyundian yeast plasmid is used for extracting the yeast plasmid. PCR amplification was performed using the extracted yeast plasmid as a template, and the primers were universal primers T7 (5'-AATA CGACTCACTATAGGGCG-3') and 3-AD (5'-AGATGGTGCACGATGCACAG-3'). The PCR product obtained was subjected to sample-feeding sequencing. And (3) comparing the data obtained by the sequencing result in NCBI, pseudo-ginseng genome, transcriptome data and an Arabidopsis thaliana database to obtain a development and cell death related protein coding gene (development and cell death domain protein, DCD) which interacts 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 bioinformatics analysis
The nucleotide 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 for 321 amino acids. The molecular weight was predicted to be 36583.21Daltons and the isoelectric point (pI) to be 8.95 using ExpASY on-line software (https:// web. ExPASy. Org/computer_pi /), indicating that the protein was an alkaline protein. Of these, 44 strongly basic amino acids (K, R), 38 strongly acidic amino acids (D, E), 94 hydrophobic amino acids (Hydrophobic Amino Acids) (A, I, L, F, W, V), and 99 polar amino acids (Polar Amino Acids) (N, C, Q, S, T, Y). The instability index (instability index, II) of the PnDCD protein was 37.79, the total average hydrophilic value (Grand average of hydropathicity, GRAVY) was-0.737, and the protein was stable hydrophilic. SMART on-line software (http:// SMART. Embl-heidelberg. De /) predictions show that the protein has no transmembrane structure (transmembrane domains), but has two low-copy regions (low complexness) located at 106-115 aa and 122-143 aa, respectively, of the predicted amino acid sequence.
The secondary structure of the PnDCD protein was predicted using on-line software SOPMA (https:// npsa-prabi.ibcp. Fr/cgi-bin/npsa_Automat. Pl. The three-dimensional structure of the PnDCD protein is predicted by utilizing on-line software SWISS-MODEL (http:// swissmodel. Expasy. Org /), and the using method is X-ray, and the respective rate is thatThe results are shown in FIG. 2. The template used was 2p5d.1.A, the Identity of the sequence (Seq Identity) was 15.53%, the status of the oligonucleotide (Oligo-state) was Monomer, the similarity of the sequence to the template sequence (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 was aligned multiple times with the amino acid sequence of the gene in other plants in the NCBI database by software Clustal X and MEGA6.0 and a evolutionary tree was constructed, and specific species and protein sequence numbers are shown in Table 1. From the results of the evolutionary tree (FIG. 3), pnDCD is evolutionarily similar to the Rosaceae PdDCD and Salicaceae PdDCD.
TABLE 1 nucleotide sequences for constructing evolutionary trees
Example 4 Yeast in vivo validation
Based on the results of the yeast single hybridization, the gene sequence that interacts with the PnSE1 promoter was selected as the in vivo verification target for yeast. Specific primers (upstream primer: 5' -CG) were designed based on Notoginseng radix transcriptome dataGAATTCATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGGATCCTCAACTTCCAAGCTTGCGCT-3'), ecoRI and BamHI cleavage sites were introduced into the sequence, and the gene was constructed into pGADT7 vector by PCR amplification using pseudo-ginseng cDNA as a template. The constructed recombinant vectors were transferred into yeast strain YE697, respectively, and interaction between PnDCD and PnSE1 promoter was verified by X-alpha-gal chromogenic reaction (FIG. 4).
Example 5 in vitro validation
Specific primers (upstream primer: 5' -CG) were designed based on Notoginseng radix transcriptome dataGGATCCATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGAATTCTCAACTTCCAAGCTTGCGCT-3'), bamHI and EcoRI cleavage sites were introduced into the sequence, and PnDCD was constructed into the prokaryotic expression vector pET-32a by PCR amplification using Notoginseng radix cDNA as a template. And (3) extracting positive recombinant plasmids, and transforming competent cells of escherichia coli BL21 (DE 3). Positive clones were selected with LB medium (50 mg/L Amp) containing antibiotics. 200 mu L of positive clone bacterial liquid is inoculated into 5mL of LB liquid culture medium for expansion culture, when the bacterial liquid reaches a logarithmic growth phase (OD 600 = 0.5), IPTG is added to induce and express recombinant protein, the concentration of the IPTG is 1mmol/L, the proper induction time is 6h, and the induction temperature is 25 ℃. Finally, the obtained protein is subjected to gel electrophoresis by adopting SDS-PAGE gel electrophoresis technology, and the result is shown in figure 5. The size of the PnDCD fusion protein is 50KD, and after His tag proteins are removed, the band size is matched with the predicted molecular weight size of the PnDCD protein, namely about 36KD.
EXAMPLE 6 subcellular localization
According to the data of the pseudo-ginseng transcriptomeMeter-specific primer (upstream primer: 5' -TCC)CCCGGGATGGAGAACATGAATAGCTTTTGG-3', downstream primer: 5' -CGGGATCCACTTCCAAGCTTGCGCTTTAC-3'), smaI and BamHI cleavage sites were introduced into the sequence, and the gene was constructed into a 35S-sGFP vector by PCR amplification using Notoginseng cDNA as a template. And (3) transforming the constructed recombinant vector into DH5 alpha competence of the escherichia coli. Picking up monoclone to LB liquid culture medium (50 mg/L Kan), enlarging culture, then sucking 1 mu L bacterial liquid as template for bacterial liquid PCR verification. The positive clone obtained was verified to be plasmid-extracted with TIANGEN TIANpure Mini Plasmid Kit II (Code No. DP107) and then transformed with Agrobacterium tumefaciens strain GV3101. Agrobacterium H 2 B-RFP Strain and GV3101 Strain containing the target Gene according to 1:1 are injected into the same tobacco leaf after being evenly mixed according to the proportion, H 2 B-RFP strains can cause nuclei to appear red. The tobacco after injection was cultured for 3 days, and then sampled and placed under a laser confocal microscope for observation. Subcellular localization results showed that the green fluorescence expressed by the PnDCD protein was weak, which was distributed in both the nucleus and cytoplasm, indicating its localization in the cytoplasm and cell membrane (fig. 6).
Sequence listing
<110> university of Zhejiang
Zhejiang university of technology limited company Zhejiang of Hemsleya shapewear
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Claims (6)
1. GenePnDCDThe nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. A gene comprising the gene of claim 1PnDCDIs a recombinant expression vector of (a).
3. A gene comprising the gene of claim 1PnDCDIs a genetically engineered bacterium of the strain.
4. A gene as claimed in claim 1PnDCDThe coded protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
5. The gene according to claim 1PnDCDOr the use of the genetically engineered bacterium of claim 3 in regulating the synthesis of saponins, wherein the saponins are notoginsenoside.
6. The use of the protein of claim 4 for regulating the synthesis of saponin, wherein said saponin is notoginsenoside.
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