CN114058632A - Gene PnCOX11 and application thereof in regulating and controlling synthesis of notoginsenoside - Google Patents

Gene PnCOX11 and application thereof in regulating and controlling synthesis of notoginsenoside Download PDF

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CN114058632A
CN114058632A CN202111181146.7A CN202111181146A CN114058632A CN 114058632 A CN114058632 A CN 114058632A CN 202111181146 A CN202111181146 A CN 202111181146A CN 114058632 A CN114058632 A CN 114058632A
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pncox11
notoginsenoside
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夏鹏国
胡婉莹
郑宇婕
张宇
梁宗锁
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Zhejiang Sci Tech University ZSTU
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    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01021—Squalene synthase (2.5.1.21), i.e. farnesyl-disphosphate farnesyltransferase

Abstract

The invention discloses a gene PnCOX11 and application thereof in regulating and controlling synthesis of notoginsenoside, wherein the nucleotide sequence of the gene PnCOX11 is shown as SEQ ID NO. 1. The invention takes the PnSS upstream promoter sequence of the gene as a research object, screens the transcription factor interacting with the promoter fragment from the panax notoginseng cDNA library by using a yeast single hybrid 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

Gene PnCOX11 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 PnCOX11 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, 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 Synthase (SS) is a key enzyme, and has important regulation and control effects on triterpenes and sterols in plant body.
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 is a key enzyme gene of a biosynthesis pathway of the triterpenoid saponin of 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 inhibited the phenotype of the ginseng SS geneAfter that, it was found that the decrease of the amount of triterpene saponin synthesized and the increase of the expression level of SS in the adventitious roots of ginseng simultaneously increased the contents of phytosterol and ginsenoside. When SS and DS in the pseudo-ginseng cell line are co-overexpressed, the saponin content is obviously improved, wherein the ginsenoside Re content is 5-6 times of that of a control。
Disclosure of Invention
The invention provides a gene PnCOX11 and application thereof in regulating and controlling synthesis of notoginsenoside, wherein the gene PnCOX11 is a cytochrome C oxidase 11 gene and has interaction with a promoter directly participating in a key enzyme gene PnSS for synthesizing notoginsenoside, so that synthesis of notoginsenoside is influenced.
The specific technical scheme is as follows:
the invention provides a gene PnCOX11, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The present invention provides a recombinant expression vector comprising the gene PnCOX11 described above.
The invention provides a genetically engineered bacterium comprising the gene PnCOX11 described above.
The invention provides cytochrome C oxidase 11, wherein the amino acid sequence of the cytochrome C oxidase 11 is shown as SEQ ID No. 2.
The present invention provides a cytochrome C oxidase 11, wherein the cytochrome C oxidase 11 is obtained by encoding a gene PnCOX11 having a nucleotide sequence shown in SEQ ID NO. 1.
The invention also provides the use of the gene PnCOX11 as described above or of the cytochrome C oxidase 11 as described above in the interaction with the promoter of the gene PnSS.
The invention also provides the application of the gene PnCOX11 or the genetic engineering bacteria in regulating and controlling the synthesis of notoginsenoside.
Further, the regulatory pathways are: the protein coded by the gene PnCOX11 is combined with the promoter of the gene PnSS to regulate and control the synthesis of notoginsenoside.
The invention also provides application of the cytochrome C oxidase 11 in regulation and control of notoginsenoside synthesis.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the PnSS upstream promoter sequence of the gene as a research object, screens the transcription factor interacting with the promoter fragment from the panax notoginseng cDNA library by using a yeast single hybrid 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 result of predicting the secondary structure of PnCOX11 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 PnCOX11 protein in example 3.
FIG. 3 is a phylogenetic tree of the PnCOX11 protein of example 3.
FIG. 4 shows the results of the X- α -gal chromogenic reaction in example 4, which verifies the interaction between PnSS (CK) and PnCOX 11.
FIG. 5 shows the results of the induced expression of PnCOX11 protein in example 5.
FIG. 6 is a subcellular localization map of PnCOX11 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 PnSS promoter, 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.
The constructed bait carrier is digested and linearized by BstBI restriction enzyme, and the purified linearized plasmid is transferred into a yeast Y1H strain. Yeast decoy strain identification was performed using the Matchmaker Insert Check PCR Mix I kit. Successfully identified yeast colonies were expanded and selected to give the appropriate yeast decoy strain YS1262 that was inhibited by AbA.
Example 2 screening of genes interacting with PnSS 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'-AATACGACTCACTA TAGGGCG-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 cytochrome C oxidase 11(cytochrome C oxidase 11, COX11) gene interacted with the PnSS 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 above gene PnCOX11 were submitted to NCBI for analysis. The Open Reading Frame (ORF) sequence of PnCOX11 has 642bp, and encodes 213 amino acids. ExPASY online software (https:// web. ExPASy. org/computer _ pI /) was used to predict a molecular weight of 24335.88Daltons with an isoelectric point (pI) of 8.49, indicating that the protein is a basic protein. Wherein, 26 strong basic Amino Acids (K, R), 23 strong acidic Amino Acids (D, E), 67 Hydrophobic Amino Acids (hydrophosphonic Amino Acids) (A, I, L, F, W, V) and 63 Polar Amino Acids (Polar Amino Acids) (N, C, Q, S, T, Y). PnCOX11 protein had an Instability Index (II) of 53.33, a Total average hydrophilicity (GRAVY) of-0.305, and was a labile hydrophilic protein. The prediction result of SMART online software (http:// SMART. embl-heidelberg. de /) shows that the protein has a transmembrane structure (trans domains) and is positioned at 37-59 aa of the predicted amino acid sequence.
The secondary structure of PnCOX11 protein was predicted using the online software SOPMA (https:// npsa-prabi.ibcp.fr/cgi-bin/npsa _ Automat.pl. The three-dimensional structure of PnCOX11 protein was predicted using the on-line software SWISS-MODEL (http:// swissmodel. expasy. org /), and the results are shown in FIG. 2, using the method NMR. The template number used was 1sp0.1.A, the sequence Identity (Seq Identity) was 49.66%, the oligonucleotide state (Oligo-state) was Monomer, the sequence-to-template sequence similarity (Seq similarity) was 0.45, the Coverage (Coverage) was 0.70, and the predicted sequence was described as a solution structure of apoCOX 11.
The gene PnCOX11 was cloned and analyzed in many species. The amino acid sequence of PnCOX11 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 (FIG. 3), PnCOX11 is grouped with NaCOX11 of the model plant Nicotiana, both dicotyledonous plants.
TABLE 1 nucleotide sequences for construction of evolutionary trees
Figure BDA0003297221280000041
Figure BDA0003297221280000051
Example 4 in vivo yeast validation
Based on the result of the single hybridization of yeast, the gene sequence interacting with the PnSS promoter was selected as the target of in vivo yeast assay. Specific primers (upstream primer: 5'-CGGAATTCATGCCACGATGTGATTTGCCA-3' and downstream primer: 5'-CGGGATCCTTATTCTTCAGAAACCTTGAAAAA-3') are designed according to panax notoginseng transcriptome data, EcoRI and BamHI enzyme cutting sites are introduced into sequences, panax notoginseng cDNA is used as a template, and genes are constructed into pGADT7 vectors through PCR amplification. The constructed recombinant vectors were transferred into the yeast strain YS1262, respectively, and the interaction between PnCOX11 and PnSS promoter was verified by X- α -gal color reaction (FIG. 4).
Example 5 in vitro validation
Designing a specific primer (an upstream primer: 5'-CGGGATCCATGCCACGATGTGATTTGCCA-3' and a downstream primer: 5'-CGGAATTCTTATTCTTCAGAAACCTTGAAAAA-3') according to the panax notoginseng transcriptome data, introducing BamHI and EcoRI enzyme cutting sites into a sequence, and constructing PnCOX11 into a prokaryotic expression vector pET-32a by PCR amplification by taking panax notoginseng cDNA as a template. 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 medium for amplification culture, and adding IPTG (isopropyl-beta-D-thiogalactoside) to induce and express the recombinant protein when the bacterial liquid reaches logarithmic growth period (OD600 is 0.5), wherein the concentration of the IPTG is 1mmol/L, the appropriate induction time is 6h, and the induction temperature is 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 PnCOX11 fusion protein was 37kD, and after removal of the His tag protein, the band size matched the predicted size of the PnCOX11 protein, which was approximately 24 kD.
Example 6 subcellular localization
Specific primers (upstream primer: 5'-TCCCCCGGGATGCCACGATGTGATTTGCCA-3' and downstream primer: 5'-CGGGATCCTTCTTCAGAAACCTTGAAAAAGGTA-3') are designed according to panax notoginseng transcriptome data, SmaI and BamHI enzyme cutting sites are introduced into sequences, and genes are constructed into a 35S-sGFP vector by PCR amplification by taking panax notoginseng 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. The agrobacterium H2B-RFP strain and GV3101 strain containing the target gene were mixed according to the ratio of 1: 1, and injecting the mixture into the same tobacco leaf, wherein the H2B-RFP strain can make the cell nucleus 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 the fluorescence of PnCOX11 protein was as highly similar to chloroplast fluorescence, indicating that it is predominantly localized in chloroplasts (fig. 6).
Sequence listing
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Claims (9)

1.A gene PnCOX11 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 PnCOX11 of claim 1.
3. A genetically engineered bacterium comprising the gene PnCOX11 of claim 1.
4. A cytochrome C oxidase 11, wherein the amino acid sequence of the cytochrome C oxidase 11 is represented by SEQ ID NO. 2.
5. Cytochrome C oxidase 11 as claimed in claim 4, which is obtained by encoding the gene PnCOX11 having the nucleotide sequence shown in SEQ ID No. 1.
6. Use of the gene PnCOX11 according to claim 1 or of the cytochrome C oxidase 11 according to claim 4 for interacting with the promoter of the gene PnSS.
7. The use of the gene PnCOX11 as defined in claim 1 or the genetically engineered bacterium as defined in claim 3 for regulating and controlling the synthesis of notoginsenoside.
8. The use of claim 7, wherein the regulatory pathway is: the protein coded by the gene PnCOX11 is combined with the promoter of the gene PnSS to regulate and control the synthesis of notoginsenoside.
9. Use of cytochrome C oxidase 11 as claimed in any of claims 4 or 5 for regulating the synthesis of notoginsenoside.
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Publication number Priority date Publication date Assignee Title
CN113652426A (en) * 2021-08-20 2021-11-16 昆明理工大学 Panax notoginseng inducible promoter R1 and application thereof

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CN102517297A (en) * 2011-12-22 2012-06-27 四川农业大学 Rice rhizoctonia solani effector gene AG1IA06910 and application thereof
WO2016109758A2 (en) * 2014-12-30 2016-07-07 Symbiota, LLC Seed endophytes across cultivars and species, associated compositions, and methods of use thereof
CN106497939A (en) * 2016-10-14 2017-03-15 昆明理工大学 A kind of Radix Notoginseng transcription factor gene PnMYB1 and its application

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Publication number Priority date Publication date Assignee Title
CN102517297A (en) * 2011-12-22 2012-06-27 四川农业大学 Rice rhizoctonia solani effector gene AG1IA06910 and application thereof
WO2016109758A2 (en) * 2014-12-30 2016-07-07 Symbiota, LLC Seed endophytes across cultivars and species, associated compositions, and methods of use thereof
CN106497939A (en) * 2016-10-14 2017-03-15 昆明理工大学 A kind of Radix Notoginseng transcription factor gene PnMYB1 and its application

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CN113652426A (en) * 2021-08-20 2021-11-16 昆明理工大学 Panax notoginseng inducible promoter R1 and application thereof
CN113652426B (en) * 2021-08-20 2023-06-20 昆明理工大学 Pseudo-ginseng inducible promoter R1 and application thereof

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