CN103374582B - Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene and coded product thereof and application - Google Patents
Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene and coded product thereof and application Download PDFInfo
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- CN103374582B CN103374582B CN201210112952.3A CN201210112952A CN103374582B CN 103374582 B CN103374582 B CN 103374582B CN 201210112952 A CN201210112952 A CN 201210112952A CN 103374582 B CN103374582 B CN 103374582B
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Abstract
The invention discloses proteolytic enzyme and the purposes of a kind of Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene and coding thereof.Does Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) 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.
Description
Technical field
The invention belongs to biological technical field, relate generally to and utilize cDNA library to clone styracin-4-hydroxylation enzyme gene and coded product thereof and application, particularly relate to biosynthesizing and have the machine acids of pharmacological component, flavones fermentoid 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 formation of biosynthetic pathway and regulatory mechanism and explanation medical material quanlity for resolving medicinal plant activeconstituents is provided fundamental basis by the clone of these genes, 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, Japanese Honeysuckle LonicerajaponicaThunb is conventional Chinese medicine simply, be mainly used in various febrile disease, as the treatment of the symptoms such as body heat, dermexanthesis, spot, hot carbuncle carbuncle, swelling and pain in the throat.The main active ingredient of Japanese Honeysuckle comprises chlorogenic acid and wooden slippers grass glycosides etc., and its Content of Chlorogenic Acid belongs to organic acid, and galuteolin belongs to flavonoid compound.
Styracin-4-hydroxylase (cinnamate-4-hydroxylase, C4H), also known as trans-cinnamic acid-4-mono-oxygenase, one of member of cytopigment monooxygenase P450 superfamily, belong to CYP73 subfamily, 4-tonka-bean hydrochlorate can be produced by catalysis styracin hydroxylation, tonka bean camphor, chlorogenic acid etc. can be further converted to, also CoA ester can be formed, being further converted to the flavonoids such as wooden slippers grass glycosides again, is second key enzyme after phenylpropyl alcohol alkane approach relaying phenylalanine ammonia lyase.The clone of Japanese Honeysuckle styracin-4-hydroxylation enzyme gene, improving honey suckle active component content for utilizing genetically engineered provides important foundation.Before the present invention comes forth, not yet there is styracin-4-hydroxylation enzyme gene and aminoacid sequence thereof in any Japanese Honeysuckle openly or mentioned by reporting in present patent application.
Summary of the invention
The object of the present invention is to provide a kind of Japanese Honeysuckle styracin-4-hydroxylation enzyme gene (LJC4H).
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.
Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) 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 Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) 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.
Japanese Honeysuckle styracin-4-hydroxylase (LJC4H) gene provided by the present invention is cloned first from Japanese Honeysuckle, and experiment in vitro shows, LJC4H has the activity that catalysis styracin transforms.Utilize the present invention can be improved the content of organic acid substance chlorogenic acid in the plants such as Japanese Honeysuckle, Flavonoid substances galuteolin by genetic engineering technique.
Accompanying drawing illustrates:
Fig. 1: LJC4H functional domain forecast analysis (deriving from ncbi database);
Fig. 2: LJC4H systematic evolution tree (adjacent method);
Fig. 3: expression vector pYEUra3-LJC4H builds;
Fig. 4: enzymatic reaction product HPLC detected result.A, pYEUra3 catalysate; B, pYEUra3-LJC4H catalysate (without NADPH); C, pYEUra3-LJC4H catalysate; D, Isosorbide-5-Nitrae-coumaric acid; 2, trans-cinnamic acid standard substance.
Embodiment
The structure of embodiment 1, Japanese Honeysuckle cDNA library
1, the separation and detection of Japanese Honeysuckle total serum IgE
Extracting honeysuckle (LonicerajaponicaThunb) bud 2g, 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 2h 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 Japanese Honeysuckle 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.Each reagent all adds well, whizzer gets rid of, makes it to sink to the bottom, be placed in PCR instrument.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 the leakage of electricity swimming of 3ul bromine Finland, 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 Japanese Honeysuckle related gene sequence.
The bioinformatic analysis of embodiment 3, LJC4H gene
The length of Japanese Honeysuckle styracin-4-hydroxylase (C4H) the full length gene cDNA that the present invention relates to is 1518bp, and detailed sequence is shown in the sequence 1 in sequence table, and wherein opening code-reading frame is positioned at 1-1518bp.Japanese Honeysuckle 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 C4H in other species on amino acid levels, has typical cypXsuperfamily structural domain simultaneously.As Fig. 1.
The research of embodiment 4, LJC4H gene function
1. the structure of expression vector
Take LJC4HcDNA as template, utilize primer P1:5 '-GTCGACATGGATCTTCTCCTCTTAGA-3 ', P2:5 '-GTCGACTCAAAAAGATCTCTT-3 ' carries out PCR reaction, get 5ul amplified production and add the leakage of electricity swimming of 3ul bromine Finland, take a picture after half an hour, observe glue figure, amplified fragments is 1500bp.Cut amplified production 2 hours with SalI enzyme, utilize and reclaim test kit (Takara company, China) purifying digestion products.Utilize SalI enzyme at 37 DEG C to cut pYEUra3 carrier 2 hours simultaneously, add 5ul bromine Finland and carry out agarose gel electrophoresis, observe glue figure, and utilize recovery test kit to reclaim 6100bp fragment.
The two linked enzyme spends the night 16 DEG C of connections.Connect product and cut 2h with XhoI enzyme, reclaim about 7600bp fragment to reclaim test kit, 16 DEG C of connections are spent the night.Electroporated bacillus coli DH 5 alpha competent cell, is screening recon containing on 2% glucose USM flat board of penbritin.Containing the pYEUra3 plasmid of C4H clone through PCR and digestion with restriction enzyme electroresis appraisal and DNA sequence analysis, preserve there is correct target sequence recombinant plasmid pET-LJC4H for expressing conversion.This plant expression vector called after pYEUra3-LJC4H (Fig. 3).
2. abduction delivering and Enzyme activity assay
With pYEUra3-LJC4H plasmid electroporated Partial digestion yeast host bacterium AB1380, cultivate and within 5 days, screen positive yeast afterwards in 2ml containing on the USM substratum of 2% glucose, choose in mono-clonal and 2mLUSM liquid nutrient medium after 4-5 days, 30 DEG C of concuss overnight incubation.The centrifugal 5min of 5000rpm, abandons supernatant, dilutes 20 times with the USM liquid nutrient medium containing 2% glucose, and 30 DEG C of concuss cultivate 24h.Extract culturing cell total protein, detect heme peroxidase activity.Choose after high expression level transformant cultivates 20h with 50mL containing USM liquid nutrient medium 30 DEG C of concuss of 2% glucose, be inoculated into 1L and do not cultivate 40h containing USM liquid nutrient medium 30 DEG C of concuss of glucose, reclaim thalline, separating particles body.
Get 30-50mg microsomal protein 30 DEG C of incubation 10min in 600mLC4H enzymatic reaction system (NADPH final concentration is 0.5mM, Na3PO3 concentration 100mM, pH7.4, and styracin concentration is 0.1mm).With 40mL6MHCl termination reaction, with 600 μ L extraction into ethyl acetate twice, volatilize with making organic phase in vacuum freezing drying oven, and be dissolved in 800 μ L acetonitriles, with high-efficient liquid phase chromatogram technique analysis, condition is 3.9 × 300mm (10 μm) μ BondapakC18column (Waters), 996photodiodearraydetector (Waters, Milford, MA)), mobile phase A: 0.85% phosphoric acid [w/v]; Mobile phase B: acetonitrile. gradient elution 0-30minA80%-48%, B20%-52%, column temperature 30 DEG C, the results are shown in Figure 4.
The research of embodiment 5, sudden change LJC4H gene function
Take LJC4HcDNA as template, utilize primer P3:5 '-GTCGACATGGATCTTCTCCTCTTCGA-3 ', P4:5 '-GTCGACTCAAAAAGATCTCTT-3 ' carries out PCR reaction, get after 5ul amplified production adds 3ul bromine Finland leakage of electricity half an hour and take a picture, observe glue figure, obtain nonsynonymous mutation LJC4H.Expression vector establishment, abduction delivering and Enzyme activity assay are identical with embodiment 4, and result shows that sudden change LJC4H gene function and LJC4H do not have significant difference.
Claims (3)
1. a Japanese Honeysuckle styracin-4-hydroxylation enzyme gene LJC4H, is characterized in that, this gene nucleotide series is the DNA sequence dna of SEQIDNo.1.
2. a Japanese Honeysuckle styracin-4-hydroxylase LJC4H, is characterized in that, its aminoacid sequence is as shown in SEQIDNo.2.
3. recombinant vectors, is characterized in that: containing Japanese Honeysuckle styracin-4-hydroxylation enzyme gene LJC4H complete sequence according to claim 1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565709A (en) * | 2009-05-20 | 2009-10-28 | 华东理工大学 | 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof |
| CN101705239A (en) * | 2009-10-30 | 2010-05-12 | 上海交通大学 | CYP704B2 gene and protein coded by same |
| CN101830804A (en) * | 2010-03-16 | 2010-09-15 | 天津中新药业集团股份有限公司中新制药厂 | Method for extracting chlorogenic acid from honeysuckle by using compound enzyme method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565709A (en) * | 2009-05-20 | 2009-10-28 | 华东理工大学 | 3-sterone-9Alpha-hydroxylation enzyme gene, 3-sterone-9Alpha-hydroxylation enzyme reductase gene, relevant carriers, engineering bacteria and applications thereof |
| CN101705239A (en) * | 2009-10-30 | 2010-05-12 | 上海交通大学 | CYP704B2 gene and protein coded by same |
| CN101830804A (en) * | 2010-03-16 | 2010-09-15 | 天津中新药业集团股份有限公司中新制药厂 | Method for extracting chlorogenic acid from honeysuckle by using compound enzyme method |
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