CN103627717B - Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene and coded product thereof and application - Google Patents
Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene and coded product thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene and proteins encoded thereof and purposes, this gene, for utilizing construction cDNA library to clone from Chinese herbaceous peony first and obtaining, has filled up the blank cloneing and isolateing deoxy-D-xylulose sugar-5-phosphate synthase gene from China traditional Chinese medicine material Chinese herbaceous peony.Does Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene provided by the present invention have SEQ? ID? nucleotide sequence shown in NO.1 or add, replace, insert or delete the homologous sequence of one or more Nucleotide or its allelotrope and derivative nucleotide sequence thereof.Does the protein of described genes encoding have SEQ? ID? aminoacid sequence shown in NO.2 or add, replace, insert or delete one or more amino acid whose homologous sequence.Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene provided by the invention can improve the content of deoxy-D-xylulose sugar-5-phosphoric acid (DXP) approach meta-bolites in Chinese herbaceous peony by biotechnology, in the quality-improving, activeconstituents synthetic biology etc. of medicinal material Chinese herbaceous peony, there is good application prospect.
Description
Technical field
The invention belongs to biological technical field, relate generally to and utilize Chinese herbaceous peony cDNA library to clone deoxy-D-xylulose sugar-5-phosphate synthase gene and coded product thereof and application, particularly relate to biosynthesizing and have the organic acid of pharmacological component, terpene enzyme gene and coded product thereof and application, belong to Gene Engineering of Medicinal Plants field.
Background technology
The formation of medicinal plant activeconstituents is the product of peculiar gene group in Secondary Metabolism of Plant approach.Along with plant functional genomics research extensively with deeply, the research shown unique characteristics and have the medicinal plant secondary metabolism of broad prospect of application to synthesize correlation function gene becomes the focus of research gradually, the clone of these genes provides fundamental basis for the parsing biosynthetic pathway of active components in medicinal plant and the formation of regulatory mechanism and explanation medical material quanlity thereof, brings wide application space for utilizing biotechnology to improve target component content or direct production effective constituent or intermediate simultaneously.The complex chemical composition of medicinal plant, of a great variety, wherein mainly containing Multiple components such as organic acid, flavonoid, volatile oil, terpene, diterpenes, trace elements, Chinese herbaceous peony Paeonialactiflora is not only famous flower, still conventional Chinese medicine simply, and its root can hyoscine.Wherein the root of herbaceous peony there is antispastic, ease pain, the effect such as to stimulate the menstrual flow.Containing peoniflorin, M-nitro benzoic acid isoreactivity composition in Peony Root; wherein peoniflorin belongs to monoterpenes compound; have significant analgesia, calmness, anticonvulsion; (Yang Jun, Fang Hong compile in the effect such as spasmolysis, antipyretic, anti-inflammatory, antiulcer agent, antianaphylaxis, hypoglycemic, immunity moderation, protection liver.Chinese herbaceous peony [M].Beijing: China Traditional Chinese Medicine Publishing House, 2001,113).
Deoxy-D-xylulose sugar-5-phosphate synthase (1-deoxy-D-xylulose-5-phosphatesynthase, DXS) be the initial enzyme of terpenoid biosynthetic pathway deoxy-D-xylulose sugar-5-phosphoric acid (DXP) approach being arranged in plastid, pyruvic acid and glyceraldehyde-3-phosphate can be transformed and generate DXP, generally believe now that DXS is key enzyme and the rate-limiting enzyme of DXP approach, its expression amount may be closely related with the content of the ter penoidses such as peoniflorin.The clone of Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene, improve Chinese herbaceous peony active component content for utilizing genetically engineered and important substance basis is provided, in the quality-improving, activeconstituents synthetic biology etc. of medicinal material Chinese herbaceous peony, there is good application prospect.Before the present invention comes forth, not yet have any open or to report in this patent please in deoxy-D-xylulose sugar-5-phosphate synthase gene and aminoacid sequence thereof in mentioned Chinese herbaceous peony.
Summary of the invention
The object of the present invention is to provide a kind of Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene.
The present invention's second object is to provide the protein of this genes encoding.
Present invention also offers the recombinant vectors containing this gene and host cell.
Another object of the present invention is the application providing this gene.
Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene provided by the present invention is one of following nucleotide sequence:
(1) there is the nucleotide sequence shown in SEQIDNO.1;
(2) nucleotide sequence shown in SEQIDNO.1 add, replace, insert or delete the homologous sequence of one or more Nucleotide or its allelotrope and derivative nucleotide sequence thereof.
The protein of this coded by said gene is Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene, is one of following aminoacid sequence:
(1) there is the aminoacid sequence shown in SEQIDNO.2;
(2) SEQIDNO.2 add, replace, insert or delete one or more amino acid whose homologous sequence.
Deoxy-D-xylulose sugar-5-phosphate synthase (PLDXS) gene provided by the present invention is clone's preparation from Chinese herbaceous peony first, and experiment in vitro shows, PLDXS has the activity of catalysis pyruvic acid and glyceraldehyde-3-phosphate generation DXP.Utilize the present invention can be improved terpene substances content in the plants such as Chinese herbaceous peony by genetic engineering technique.
Accompanying drawing illustrates:
Fig. 1: PLDXS functional domain forecast analysis (deriving from ncbi database);
Fig. 2: PLDXS systematic evolution tree (adjacent method);
Fig. 3: expression vector pET32-PLDXS builds;
Embodiment
The structure of embodiment 1, Chinese herbaceous peony cDNA library
1, the separation and detection of Chinese herbaceous peony total serum IgE
Get the root 2g of Chinese herbaceous peony (Paeonialactiflora), with the quick grind into powder of liquid nitrogen in mortar, (CTAB (W/V) 2%, Tris-HCl (pH8.0) 100mmo1L in the 10mL Extraction buffer of fast transfer to 65 DEG C preheating
-1, EDTA25mmolL
-1, NaCl2.0molL
-1, PVP402%, spermidine 0.5g/L, mercaptoethanol 2%), mixing of fully vibrating; With equal-volume chloroform twice, 7500g centrifugal 15 minutes.Supernatant liquor adds the 10MLiCl of 1/4 volume, places 4 DEG C of precipitates overnight after mixing; Centrifugal 20 minutes of 7500g, precipitates with 500 μ LSSTE (SDS0.5%, NaCl1molL
-1, Tris-HCl (pH8.0) 10mmolL
-1, EDTA1mmolL
-1, dissolve 5 minutes at 65 DEG C.Use equal-volume chloroform, centrifugal 5 minutes of 13000g; Supernatant liquor adds 2 times of volume dehydrated alcohols, places 2 hours for-70 DEG C; Centrifugal 20 minutes of 4 DEG C of 13000g, precipitation drying at room temperature is dissolved in the water of 100 μ LDEPC process after 10 minutes, by the integrity of 1.0% agarose electrophoresis detection RNA, by GenQuant nucleic acid quantification instrument mensuration A260, A280 ratio and concentration.Be placed in-80 DEG C of refrigerators for subsequent use.
2, the structure of cDNA library
Adopt mRNA purification kit (QuichprepTMMicromRNAPurifiCationKit, Pharmacia company) after separating mRNA, adopt the CreatorSmartcDNALibraryConstructionKit (Cat.No.634903) of Clontech company to carry out building storehouse, principle is SMART (switchmechanismat5 ' endofmRNAtemplate).
Embodiment 2: the clone of Chinese herbaceous peony genes involved
Random picking 5000 mono-clonals carry out bacterium colony PCR qualification.Get appropriate PCR thin-walled tube, be placed on ice, often pipe first adds the aqua sterilisa of 17.3ul.Mono-clonal hickie is got in aqua sterilisa, vibration mixing with sterilized 10ul lancet choicest.Add successively: Taqbuffer2.5 μ L, MgCl
2(25mM) 1.8 μ L, dNTP (2.5mM) 1 μ L, M13+ primer (10pmol) 1 μ L, M13-primer (10pmol) 1 μ L, Taq enzyme 0.4 μ L.PCR reaction conditions is 94 DEG C of denaturations after 5 minutes, 94 DEG C 40 seconds, 54 DEG C 40 seconds, 72 DEG C 4 minutes, latter 72 DEG C of 35 circulations extend 10 minutes, 4 DEG C of preservations.After PCR reaction enters 4 DEG C, take off PCR thin-walled tube, get 7ulPCR product and add 3ul bromine Finland and carry out agarose gel electrophoresis, take a picture after half an hour, observe glue figure, identify roughly size and the small segment rate of Insert Fragment according to glue figure.Select the single amplified production of band to deliver to Hua Da genome company to check order, obtain Chinese herbaceous peony related gene sequence.
The bioinformatic analysis of embodiment 3, PLDXS gene
The length of the Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase gene full-length cDNA that the present invention relates to is 2160bp, and detailed sequence is shown in the sequence 1 in sequence table, and wherein opening code-reading frame is positioned at 1-2160bp.Chinese herbaceous peony full length cDNA sequence blast program is carried out nucleotide homology retrieval in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslation+PDB+Swissprot+Superda te+PIR database.This gene has higher homology with the DXS in other species on amino acid levels, has typical 1-deoxy-D-xylulose-5-phosphatesynthase structural domain simultaneously.As Fig. 1.
The research of embodiment 4, PLDXS gene function
1. the structure of recombinant plasmid
Be template with PLDXScDNA, utilize primer P1:5 '-GGATCCATGGGTACTGCTTCTGCTCATTAC-3 ', P2:5 '-GGTACCCTAGCACATCAAAAGGAGAGCTTCA-3 ' carries out PCR reaction, and reaction system is with experimental example 2.Get 5ul amplified production to add 3ul bromine Finland and carry out agarose gel electrophoresis, take a picture after half an hour, observe glue figure, amplified fragments is 2172bp.With BamHI and KpnI double digestion amplified production 2 hours at 37 DEG C, utilize and reclaim test kit (Takara company, China) purifying digestion products.Utilize BamHI and KpnI enzyme at 37 DEG C to cut pMD19-T carrier 2 hours simultaneously, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize recovery test kit to reclaim fragment.
Reclaim fragment to spend the night 8 DEG C of connections through T4 ligase enzyme.Electroporated bacillus coli DH 5 alpha competent cell, the LB flat board containing penbritin screens recon.PMD19 plasmid containing PLDXS clone is identified and DNA sequence analysis through PCR and digestion with restriction enzyme, preserves the recombinant plasmid pMD19-PLDXS with correct target sequence.
2. prokaryotic expression and vector construction
BamHI and KpnI double digestion pMD19-PLDXS plasmid and pET32a plasmid, reclaim the pET32a fragment of target fragment PLDXS and open loop respectively.Reclaim fragment PLDXS and pET32a to spend the night in 16 DEG C of connections, obtain recombinant plasmid, identify and DNA sequence analysis through PCR and digestion with restriction enzyme, prove the PLDXS containing correct sequence, this expression vector called after pET32-PLDXS (Fig. 3).
CaCl is utilized with target plasmid pET32-PLDXS
2method proceeds to E.coliBL21 competent cell, cultivates the positive strain of screening containing pET32-PLDXS plasmid.The engineering bacteria of picking list bacterium colony contains jolting overnight incubation in 100 μ g/ml penbritin LB substratum in 3ml, concentration by 1: 100 is drawn nutrient solution and is cultivated about 3 hours in new LB substratum (containing 100 μ g/ml penbritins), reach O.5 to OD600, get 1ml bacterium liquid as contrast before induction, then the isopropylthio-β-D-galactoside (IPTG) that final concentration is 1mmol/L is added, engineering bacterium expression is induced in 37 DEG C of shaking culture, 1ml is sampled, with the E.coliBL21 culture containing pET32a empty carrier for negative control after induction 3h.SDS-polyacrylamide gel electrophoresis result shows, is about 78.89kD place at molecular weight, occurs an obvious specific protein band of expression, consistent with theoretical value.
3.PLDXS gene function analysis
BamHI and KpnI double digestion pMD19-PLDXS plasmid and pTrc-AtIPI plasmid, reclaim target fragment PLDXS and large fragment pTrc skeleton respectively, connect in 16 DEG C and reclaim fragment, obtain recombinant plasmid, identify and DNA sequence analysis through PCR and digestion with restriction enzyme, prove the PLDXS containing correct sequence
PTrc-PLDXS is transformed and carries the intestinal bacteria XL1-Blue of pAC-LYC plasmid, and on the LB substratum containing Amp (100 μ g/ml) and Cm (50 μ g/ml) screening positive clone.
After carrying the prokaryotic expression carrier pAC-LYC importing coli strain XLl-Blue of Trans-Geranylgeranyl diphosphate synthase (crtE) on Lyeopene biosynthetic pathway, phytoene synthetase (crtB), phytoene desaturase (crtl) 3 key genes, Lyeopene can be expressed on background level.After pTrc-PLDXS is imported above-mentioned recombinant bacterium, owing to breaching the metabolism bottleneck of upstream pathway, promote metabolic fluxes to the direction flowing that Lyeopene synthesizes, make this bacterium can gather Lyeopene in a large number.
The Escherichia coli clones of picking of the present invention respectively containing XL1-Blue+pTrc-PLDXS, XL1-Blue+pTrc-PLDXS+pAC-LYC, on the LB substratum containing Amp (100 μ g/ml) and Cm (50 μ g/ml), be inverted light culture in 28 DEG C, within 2 ~ 3 days, observe colony colour changing conditions afterwards.With containing XL1-Blue, XL1-Blue-+pAC-LYC, XL1-BIue+pTrc+pAC-LYC Escherichia coli clones for contrast.
Experimental result shows, after 72h cultivates, the empty bacterium of XL1-Blue, XL1-Blue+pTrc-PLDXS, XL1-Blue+pAC-LYC can not grow; XL1-Blue+pTrc+pAC-LYC does not carry PLDXS, can not overexpression Lyeopene, so bacterium colony still presents the faint yellow of XLI-Blue itself; Only have XL1-Blue+pTrc-PLDXS+pAC-LYC due to energy great expression Lyeopene, after cultivating, bacterium colony presents the distinctive redness of Lyeopene.Thus demonstrate PLDXS and can promote metabolic fluxes to the flowing of direction that Lyeopene synthesizes, make this bacterium can gather Lyeopene in a large number.
4. suddenly change DXS determination of activity
Be template with PLDXScDNA, utilize mutant primer P3:5 '-GGATCCATGGGTAC
cgCTTCTGCTCATTAC-3 ', P4:5 '-GGTACCCTAGCACATCAAAAGGAGAGCTTCA-3 ' carries out PCR reaction, the structure of recombinant plasmid, prokaryotic expression and vector construction, PLDXS functional analysis approach are the same, and result shows that sudden change PLDXS transgenic engineering bacterial strain and normal PLDXS do not have significant difference.
Claims (3)
1. a Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase gene, its nucleotide sequence is as shown in SEQIDNo.1.
2. a Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase, is characterized in that its aminoacid sequence is as shown in SEQIDNo.2.
3. recombinant vectors, is characterized in that: containing Chinese herbaceous peony deoxy-D-xylulose sugar-5-phosphate synthase gene complete sequence according to claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102333866A (en) * | 2008-12-30 | 2012-01-25 | 丹尼斯科美国公司 | Produce the method for isoprene and common product |
| CN102899389A (en) * | 2012-10-12 | 2013-01-30 | 西北大学 | Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102333866A (en) * | 2008-12-30 | 2012-01-25 | 丹尼斯科美国公司 | Produce the method for isoprene and common product |
| CN102899389A (en) * | 2012-10-12 | 2013-01-30 | 西北大学 | Method for selecting 1-deoxy-D-xylulose-5-phosphate synthase inhibitor from plant extract |
Non-Patent Citations (3)
| Title |
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| 1-deoxyxylulose-5-phosphate synthase, putative [Ricinus communis];Chan,A et al;《GenBank 登录号 XP_002514364.1》;20090806;origin * |
| Ricinus communis 1-deoxyxylulose-5-phosphate synthase, putative, mRNA;Chan,A et al;《GenBank 登录号为 XP_002514318.1 》;20090806;origin * |
| 芍药科植物的研究概况;翁小刚;《中国实验方剂学杂志》;20031231;第9卷(第1期);55-59 * |
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