CN103571860B - Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes and coded product thereof and application - Google Patents
Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes and coded product thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (PLIspF) gene and proteins encoded thereof and purposes, this gene is for utilizing construction cDNA library to clone from Chinese herbaceous peony first and obtaining, fill up the blank cloneing and isolateing 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes from China traditional Chinese medicine material Chinese herbaceous peony.Does 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (PLIspF) gene provided by the present invention have SEQ? nucleotide sequence shown in IDNO.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.2-methyl D-tetrahydroxybutane-2 provided by the invention, 4-ring diphosphate synthase (PLIspF) gene can improve the content of 4-cytidine diphosphate (CDP)-2-C-methyl D in Chinese herbaceous peony-tetrahydroxybutane-2-phosphate metabolism product 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 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes 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, 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).
2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (2-C-methyl-D-erythritol2,4-cyclodiphosphatesynthase, IspF) be the key gene of deoxy-D-xylulose sugar-5-phosphate pathway (DXP) of the terpenoid being arranged in plastid, its expression amount may be closely related with the content of the ter penoidses such as peoniflorin.IspF energy catalysis 4-cytidine diphosphate (CDP)-2-C-methyl D-tetrahydroxybutane-2-phosphoric acid (2-Phospho-4-(cytidine5 '-diphospho)-2-C-methyl-D-erythritol, CDP-ME2P) 2-C-methyl D-tetrahydroxybutane 2 is generated, 4 ring bisphosphate (2-C-Methyl-D-erythritol2, 4-cyclodiphosphate, ME-2, 4CPP) and CMP, again through 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the effect of isopentenyl monophosphate kinase (IPK) generates isopentenyl pyrophosphate (IPP) or dimethylallylpyrophosphate (DMAPP), synthesize various different terpenoid (Zhang Changbo further again, Sun Hongxia, Gong Zhongjun, Zhu Zengrong.The natural route of synthesis of plant terpene compound and relevant synthase [J] thereof.Plant Physiology Communications, 2007,43 (4): 779-786).Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2, the clone of 4-ring diphosphate synthase (PLIspF) gene, improve Chinese herbaceous peony active component content for utilizing genetically engineered and important foundation 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 reported Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes mentioned in present patent application and aminoacid sequence thereof.
Summary of the invention
The object of the present invention is to provide a kind of Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (PLIspF) 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 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (PLIspF) 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 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase (PLIspF) 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.
2-methyl D-tetrahydroxybutane-2 provided by the present invention, 4-ring diphosphate synthase (PLIspF) gene is clone's preparation from Chinese herbaceous peony first, experiment in vitro shows, IspF energy catalysis 4-cytidine diphosphate (CDP)-2-C-methyl D-tetrahydroxybutane-2-phosphoric acid (2-Phospho-4-(cytidine5 '-diphospho)-2-C-methyl-D-erythritol, CDP-ME2P) 2-C-methyl D-tetrahydroxybutane 2 is generated, 4 ring bisphosphate (2-C-Methyl-D-erythritol2,4-cyclodiphosphate, ME-2,4CPP) and the activity of CMP.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: PLIspF functional domain forecast analysis (deriving from ncbi database);
Fig. 2: PLIspF systematic evolution tree (adjacent method);
Fig. 3: expression vector pTrc-PLIspF 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) 100mmolL 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 (Ca.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, PLIspF gene
The length of Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2, the 4-ring diphosphate synthase genes full-length cDNA that the present invention relates to is 696bp, and detailed sequence is shown in the sequence 1 in sequence table, and wherein opening code-reading frame is positioned at 1-696bp.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 IspF in other species on amino acid levels, has typical MECDP_synthase (2-C-methyl-D-erythritol-2,4-cyclodiphosphatesynthase, IspF) structural domain simultaneously.As Fig. 1.
The research of embodiment 4, PLIspF gene function
1. the structure of expression vector
Be template with PLIspFcDNA, utilize primer P1:5 '-GGATCCATGGCCACGGCGACTCCGCTATA-3 ', P2:5 '-TCTAGACTACTTCTTCATAAGAAGAACCACA-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 708bp.With BamhI and XbaI double digestion amplified production 2 hours at 37 DEG C, utilize and reclaim test kit (Takara company, China) purifying digestion products.Utilize BamHI and XbaI 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 PLIspF clone is identified and DNA sequence analysis through PCR and digestion with restriction enzyme, preserves the recombinant plasmid pMD-PLIspF with correct target sequence.
BamHI and XbaI double digestion carries pMD-19TVector plasmid and the pTrc-AtIPI plasmid of PLIspF, reclaim target fragment 708bp and 3700bp 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 PLIspF containing correct sequence, this expression vector called after pTrc-PLIspF (Fig. 3).
2. engineering bacteria builds
PTrc-PLIspF is transformed and carries the intestinal bacteria XL1-Blue of pAC-BETA plasmid, and on the LB substratum containing Amp (150 μ g/ml) and Cm (50 μ g/ml) screening positive clone.
3.PLIspF gene function analysis
Owing to there is MEP approach in intestinal bacteria, can manufacture containing plasmid pAC-BETA and accumulate β-carotene in strain X L1-Blue, and forming yellow color colonies.When pTrc-PLIspF is transformed into this can accumulation after in the intestinal bacteria XL1-Blue of β-carotene, the color yellowing or orange-yellow of bacterium, shows that PLIspF can accelerate the accumulation of β-carotene.
The Escherichia coli clones of picking of the present invention respectively containing XL1-Blue+pTrc-PLIspF, XL1-Blue+pTrc-PLIspF+pAC-BETA, on the LB substratum containing Amp (150 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-BETA, XL1-BIue+pTrc+pAC-BETA Escherichia coli clones for contrast.
Experimental result shows, after within 2-3 days, cultivating, compared with control strain, the escherichia coli cloning color containing PLIspF becomes yellow, thus demonstrates the accumulation can accelerating β-carotene at PLIspF.
4. suddenly change IspF checking
Be template with PLIspFcDNA, utilize mutant primer P3:5 '-GGATCCATGGCCACGGC
aaCTCCGCTATA-3 ', P45 '-TCTAGACTACTTCTTCATAAGAAGAACCACA-3 ' carries out PCR reaction, structure and the function verification method of expression vector are the same, and result shows that sudden change PLIspF transgenic engineering bacterial strain and normal PLIspF do not have significant difference.
Claims (3)
1. Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes, it is characterized in that, this gene nucleotide series is as shown in SEQIDNo.1.
2. Chinese herbaceous peony 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate 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 2-methyl D-tetrahydroxybutane-2,4-ring diphosphate synthase genes complete sequence according to claim 1.
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Non-Patent Citations (3)
| Title |
|---|
| Arabidopsis thaliana 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ISPF) mRNA, complete cds;Theologis,A.等;《NCBI Reference Sequence：NM_180640.2》;20110528;序列说明 * |
| Functional Diversity of Genes for the Biosynthesis of Paeoniflorin and Its Derivatives in Paeonia;Yuan Yuan等;《Int. J. Mol. Sci.》;20130909;18502-18519 * |
| 芍药科植物的研究概况;翁小刚等;《中国实验方剂学杂志》;20030220;55-58 * |
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