CN106434704A - Cytochrome P450 gene CYP76AH12 involved in tanshinone compound biosynthesis and coding product and application thereof - Google Patents
Cytochrome P450 gene CYP76AH12 involved in tanshinone compound biosynthesis and coding product and application thereof Download PDFInfo
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- AIGAZQPHXLWMOJ-UHFFFAOYSA-N tanshinone IIA Natural products C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)=CO1 AIGAZQPHXLWMOJ-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 16
- 229930183118 Tanshinone Natural products 0.000 title claims abstract description 13
- -1 tanshinone compound Chemical class 0.000 title claims abstract description 7
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 title description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 240000007164 Salvia officinalis Species 0.000 claims description 12
- 235000005412 red sage Nutrition 0.000 claims description 11
- 241000196324 Embryophyta Species 0.000 claims description 10
- 150000001413 amino acids Chemical group 0.000 claims description 5
- 239000002299 complementary DNA Substances 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 238000009395 breeding Methods 0.000 claims description 4
- 230000001488 breeding effect Effects 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 241000894006 Bacteria Species 0.000 claims description 2
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 claims description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 claims description 2
- 238000009396 hybridization Methods 0.000 claims description 2
- 108010087432 terpene synthase Proteins 0.000 claims 1
- 230000009261 transgenic effect Effects 0.000 claims 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 abstract description 4
- 229930004069 diterpene Natural products 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 238000012269 metabolic engineering Methods 0.000 abstract description 2
- 230000002194 synthesizing effect Effects 0.000 abstract description 2
- 238000009402 cross-breeding Methods 0.000 abstract 1
- QXNWVJOHUAQHLM-AZUAARDMSA-N ferruginol Chemical compound CC([C@@H]1CC2)(C)CCC[C@]1(C)C1=C2C=C(C(C)C)C(O)=C1 QXNWVJOHUAQHLM-AZUAARDMSA-N 0.000 abstract 1
- HOJWCCXHGGCJQV-YLJYHZDGSA-N ferruginol Natural products CC(C)c1ccc2c(CC[C@@H]3C(C)(C)CCC[C@]23C)c1O HOJWCCXHGGCJQV-YLJYHZDGSA-N 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 108700026220 vif Genes Proteins 0.000 abstract 1
- 239000000047 product Substances 0.000 description 8
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- CZEPBGSMIRTHKN-UHFFFAOYSA-N (+-)-Kryptojaponol Natural products O=C1CC2C(C)(C)CCCC2(C)C2=C1C=C(C(C)C)C(OC)=C2O CZEPBGSMIRTHKN-UHFFFAOYSA-N 0.000 description 3
- CZEPBGSMIRTHKN-KKSFZXQISA-N (4as,10as)-5-hydroxy-6-methoxy-1,1,4a-trimethyl-7-propan-2-yl-3,4,10,10a-tetrahydro-2h-phenanthren-9-one Chemical compound CC([C@@H]1CC2=O)(C)CCC[C@]1(C)C1=C2C=C(C(C)C)C(OC)=C1O CZEPBGSMIRTHKN-KKSFZXQISA-N 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- GVKKJJOMQCNPGB-UHFFFAOYSA-N Cryptotanshinone Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)CO2 GVKKJJOMQCNPGB-UHFFFAOYSA-N 0.000 description 2
- GVKKJJOMQCNPGB-JTQLQIEISA-N Cryptotanshinone Chemical compound O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1[C@@H](C)CO2 GVKKJJOMQCNPGB-JTQLQIEISA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 150000004141 diterpene derivatives Chemical group 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 230000037353 metabolic pathway Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- HARGZZNYNSYSGJ-UHFFFAOYSA-N 1,2 dihydrotanshinquinone Natural products C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2C(C)CO1 HARGZZNYNSYSGJ-UHFFFAOYSA-N 0.000 description 1
- XDUXBBDRILEIEZ-UHFFFAOYSA-N 6-(hydroxymethyl)-1,6-dimethyl-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(C)(CO)C3=CC=C2C2=C1C(C)=CO2 XDUXBBDRILEIEZ-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 235000001405 Artemisia annua Nutrition 0.000 description 1
- 240000000011 Artemisia annua Species 0.000 description 1
- 240000001829 Catharanthus roseus Species 0.000 description 1
- HARGZZNYNSYSGJ-JTQLQIEISA-N Dihydrotanshinone I Chemical compound C1=CC2=C(C)C=CC=C2C(C(=O)C2=O)=C1C1=C2[C@@H](C)CO1 HARGZZNYNSYSGJ-JTQLQIEISA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100039556 Galectin-4 Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 101150053185 P450 gene Proteins 0.000 description 1
- 244000131316 Panax pseudoginseng Species 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 241000208966 Polygala Species 0.000 description 1
- 208000001431 Psychomotor Agitation Diseases 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 206010038743 Restlessness Diseases 0.000 description 1
- 241000304195 Salvia miltiorrhiza Species 0.000 description 1
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 1
- HYXITZLLTYIPOF-UHFFFAOYSA-N Tanshinone II Natural products O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000005899 aromatization reaction Methods 0.000 description 1
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000020335 dealkylation Effects 0.000 description 1
- 238000006900 dealkylation reaction Methods 0.000 description 1
- 238000005695 dehalogenation reaction Methods 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 238000006477 desulfuration reaction Methods 0.000 description 1
- 230000023556 desulfurization Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- KXNYCALHDXGJSF-UHFFFAOYSA-N dihydroisotanshinone I Natural products CC1=CC=CC2=C(C(C=3OCC(C=3C3=O)C)=O)C3=CC=C21 KXNYCALHDXGJSF-UHFFFAOYSA-N 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 238000006735 epoxidation reaction Methods 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 230000000640 hydroxylating effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000024053 secondary metabolic process Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- AZEZEAABTDXEHR-UHFFFAOYSA-M sodium;1,6,6-trimethyl-10,11-dioxo-8,9-dihydro-7h-naphtho[1,2-g][1]benzofuran-2-sulfonate Chemical compound [Na+].C12=CC=C(C(CCC3)(C)C)C3=C2C(=O)C(=O)C2=C1OC(S([O-])(=O)=O)=C2C AZEZEAABTDXEHR-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a CYP450 gene CYP76AH12 involved in tanshinone biosynthesis and a coding product and application thereof, belonging to the field of medicinal plant genetic engineering. The gene is cloned from radix salviae miltiorrhizae; in the encoded protein of the gene, a carbonyl can be added to the position 7 of ferruginol or derivative thereof; and the encoded protein is a key enzyme in the path of tanshinone biosynthesis. The CYP76AH12 gene has a nucleotide sequence shown by SEQ ID No.1, and the encoded protein of the gene has an amino acid sequence shown by SEQ ID No.2. The CYP76AH12 gene provided by the invention can be used for heterologously synthesizing and producing diterpene compounds through synthetic biology or metabolic engineering technology and also can be used for adjusting the biosynthesis of plant diterpene compounds and increasing the content of tanshinone compounds through biotechnology. Meanwhile, the gene also can be applied to the crossbreeding and good variety selection of radix salviae miltiorrhizae.
Description
Technical field
The invention belongs to Gene Engineering of Medicinal Plants field, relate generally to tanshinone metabolic pathway relevant cell cytochrome p 450
The screening of gene C YP76AH12, identifies and application.
Background technology
Secondary metabolite is the main source of medicinal plant bioactive ingredients.In recent years, medicinal plant genomics
The fast development of research is that the excavation of secondary metabolite synthesis related gene has established early-stage Study basis with qualification.Secondary generation
Thank to the clone of Product formation related gene and metabolic engineering and the synthetic biology for natural products is studied and provided by functional verification
Also the seed selection breeding and quality-improving for medicinal plant is provided and instructs simultaneously by required biological elements.
The red sage root (Salvia miltiorrhiza Bunge) is that Lamiaceae Salvia belongs to herbaceos perennial, has work
Blood stimulates the menstrual flow, and tired pain relieving of dispelling, clear away heart-fire the effects such as relieving restlessness.Tanshinone is diterpene quinone, is the main liposoluble active of the red sage root
Composition, mainly includes tanshinone IIA, Tanshinone II B, Tanshinone I, Cryptotanshinone, dihydrotanshinone, different Cryptotanshinone etc., tool
Have expansion blood vessel, antitumor, anti-inflammation and antithrombotic, the multiple pharmacological effect such as anti-oxidant.Tanshinone is thought at present research
Route of synthesis can be divided into three steps：The precursor substance of first step synthesis terpene, second step forms tanshinone skeleton structure, the 3rd step
Tanshinone skeleton structure is modified, including aoxidize, methylate, aromatization etc. is modified.Speculate according to tanshinone route of synthesis,
Cytochrome P450 is to participate in the main enzyme that tanshinone skeleton structure is modified.
Cytochrome P450 is widely present in living nature, which is the memebrane protein containing ferroheme, has monooxygenase activity.
Cytochrome P450 can be catalyzed polytype reaction, mainly include hydroxylating, epoxidation, isomerization, dealkylation, desulfurization,
Dehalogenation, dehydrogenation etc..Although Cytochrome P450 can be catalyzed polytype reaction, but has identical catalyst mechanism,
I.e. by NADPH or NADH be Cytochrome P450 transmission electronics, excited oxygen molecule, one of them oxygen is inserted into substrate
On, generate a molecular water simultaneously.Utilize the technology such as heterogenous expression and RNA interference in succession at artemisia annua, catharanthus roseus, ginseng in recent years
Etc. multiple medicinal plants identify the Cytochrome P450 participating in secondary metabolism approach.
The present invention passes through genescreen, and heterogenous expression, the P450 to a red sage root for the technology such as Enzyme assay has carried out work(
Can identify, this enzyme can add a carbonyl at 7 of ferruginal and derivative thereof, according to cytochrome P450 gene nomenclature
Named CYP76AH12, this gene can be applicable to biosynthesis and regulation and control and the red sage root breeding of diterpene-kind compound.
Content of the invention
It is an object of the invention to provide a cytochrome P450 gene participating in tanshinone route of synthesis
CYP76AH12, the albumen of its coding can add a carbonyl at 7 of ferruginal and derivative thereof.
The invention provides a gene relevant with tanshinone compound anabolism：CYP76AH12, it is following
One of nucleotide sequence：
1) cDNA sequence shown in SEQ ID No.1 in sequence table；Or
2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks or increases one or more nucleotides, and expresses
The nucleotide sequence of identical function protein：Or
3) nucleotide sequence with sequence hybridization shown in SEQ ID No.1 under strict conditions；Described stringent condition is：?
In 0.1 × SSPE containing 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and washed by this solution
Film.
A kind of protein being encoded by said gene, is characterized by：
I) there is the amino acid residue sequence of SEQ ID No.2 in sequence table；Or
Ii) amino acid sequence shown in SEQ ID No.2 is substituted, lacks or adds the tool that one or several amino acid produces
There is the albumen of identical function.
The DNA sequence dna of SEQ ID No.1 of the present invention is by 1494 base compositions, in polynucleotide SEQ ID No.2
Protein is made up of 497 amino acid residues.
Expression vector containing gene of the present invention, clone, transfer-gen plant and Host Strains and this gene of use are in regulation
With the application producing in plant diterpene-kind compound and red sage root breeding also within protection scope of the present invention.By SEQ ID
Gene shown in No.1 is cloned between two restriction enzymes of BamHI and EcoRI of expression vector pYES2-URA (pYES2),
Build the recombinant expression carrier pYES2-CYP76AH12 with CYP76AH12 gene；Transformed saccharomyces cerevisiae bacterial strain WAT11, gala
Sugar inducible gene expression, and in nutrient solution, add substrate ferruginal or derivatives thereof.With n-hexane to it after inducing 24 hours
Expression product carries out extracting and carries out GC-MS analysis.Analysis result shows that CYP76AH12 can be at ferruginal or derivatives thereof
7 add a carbonyl.
The present invention also provides the specific primer pair for the described CYP76AH12 encoding gene cDNA sequence of PCR amplification, bag
Include：
Forward primer：ATGGATTCCTTCTTCTTAT
Reverse primer：TTAAATTTTAAATGGAATA
Brief description
Fig. 1 CYP76AH12 gene coded protein catalysis iron rust alcoholase promotees the GC-MS analysis result (chromatogram) of product
Fig. 2 CYP76AH12 gene coded protein catalysis iron rust alcoholase promotees the mass spectrogram of product and standard items cryptojaponol
Fig. 3 CYP76AH12 gene coded protein catalysis ferruginal is to the diagram of cryptojaponol
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
All according to conventional laboratory conditions, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning：A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.
The clone of cytochrome P450 gene in embodiment 1 red sage root
1st, according to red sage root BAC (the bacterial artificial chromosome) data having checked order, by splicing,
Annotation, screening etc. operate, it is thus achieved that the candidate cell cytochrome p 450 gene cDNA sequence in tanshinone route of synthesis.
2nd, primer-design software Lasergene PrimerSelect is used to design this candidate cell cytochrome p 450 gene
Primer, primer sequence is：
Forward primer：ATGGATTCCTTCTTCTTAT
Reverse primer：TTAAATTTTAAATGGAATA
Primer is won the synthesis of polygala root biotechnology Co., Ltd by Beijing three.
3rd, take eugonic red sage root plant leaf, use QIAGENMini kit extracts total serum IgE, utilizes
PROMEGA Reverse Transcriptase kit carries out reverse transcription and obtains eDNA, the gene order being template amplification CYP76AH12 with cDNA.
4th, agarose gel electrophoresis occurs specific band at 1500bp, carries out cutting glue to target stripe and reclaims, and glue returns
Receiving product and being connected to pMD18T carrier (TaKaRa), and convert escherichia coli DH5a, picking positive colony carries out (Beijing agriculture of checking order
Industry academy of sciences order-checking center), select the CYP76AH12 gene clone correct with saving sequence for the structure of follow-up expression vector
Build.
The bioinformatics of embodiment the 2nd, CYP76AH12 gene order
The red sage root diterpene metabolic pathway of synthesizing cytochrome P450 gene CYP76AH12 that the present invention relates to, this full length gene
A length of 1494 nucleotides of opening code-reading frame (ORF), encode 497 amino acid, and detailed sequence is shown in the SEQ ID in sequence table
No.1 and SEQ ID No.2.CYP76AH12 total length opening code-reading frame blast program is carried out homology in ncbi database
Property retrieval, this gene on amino acid levels comparison analyze display, the gal4 amino acid sequence of red sage root CYP76AH12 gene code
Arrange relatively low with the homology of other species.
Embodiment the 3rd, CYP76AH12 gene eucaryon expression and functional analysis
1st, the structure of Yeast expression carrier
Coded sequence according to gene C YP76AH12 and restriction enzyme site analysis result, to CYP76AH12 design with
The primer of BamHI and EcoRI restriction enzyme site, is expanded by the ORF to CYP76AH12 for the primer with restriction enzyme site, and amplification is produced
Thing carries out sequence verification after being connected to pMD18T, finally by digestion method, genes of interest CYP76AH12 is connected to yeast
On expression vector pYES2, determined the correctness of carrier pYES2-CYP76AH12 by order-checking.
2nd, yeast conversion
Lithium acetate transformation method is utilized to proceed to pYES2-CYP76AH12 carrier, in Wine brewing yeast strain WAT11, pass through bacterium colony
PCR method selects positive colony.
3rd, abduction delivering
Picking positive monoclonal is inoculated in 5ml SD fluid nutrient medium, 30 DEG C, 200rmin-1Cultivate 24h；According to 1: 50
Inoculum concentration access in 50ml SD fluid nutrient medium, 30 DEG C, 200rmin-1, cultivate 2-4h and centrifuge goes to mid-log phase, 2000g
Supernatant, is washed with deionized thalline and transfers in the SD inducing culture containing galactolipin twice afterwards, 30 DEG C, 200rmin-1, lure
Add ferruginal as substrate after leading two hours, continue to cultivate 24h.
4th, the extraction of catalysate and qualification
Equal-volume n-hexane is added to carry out extraction of ocean eddies to catalysate.The product extracting carries out GC-MS analysis, and result is sent out
Now contain the catalysis group of CYP76AH12 gene recombinant vectors pYES2-CYP76AH12 compared with containing unloaded pYES2 control group
There is novel substance to produce, show that this new product is cryptojaponol through analyzing.
Claims (8)
1. CYP450 gene C YP76AH12 related to tanshinone compound biosynthesis, it is characterised in that：
1) reading frame as shown in SEQ ID No.1 is 1-1494 position nucleotide sequence；Or
2) nucleotide sequence shown in SEQ ID No.1 is substituted, lacks or increases one or more nucleotides and expresses identical
The nucleotide sequence of functional protein；Or
3) nucleotide sequence with sequence hybridization shown in SEQ ID No.1 under strict conditions；Described stringent condition is：Containing
In the 0.1 × SSPE of 0.1%SDS or the 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and wash film with this solution.
2. a Terpene synthase CYP76AH12 related to tanshinone compound biosynthesis, it is characterised in that：
I) albumen of the 1-497 amino acids Sequence composition as shown in SEQ ID No.2；Or
Ii) amino acid sequence shown in SEQ ID No.2 be substituted, lack or add one or several amino acid produce there is phase
The albumen of congenerous.
3. contain the expression vector of gene described in claim 1.
4. contain transgenic cell line and the genetically modified plants of gene described in claim 1.
5. contain the engineering bacteria of gene described in claim 1.
6. albumen described in gene described in claim 1 and claim 2 regulation and produce in plant diterpene-kind compound should
With.
7. application in red sage root breeding for the albumen described in gene described in claim 1 and claim 2.
8. the specific primer pair for encoding gene cDNA sequence described in PCR amplification claim 1, including：
Forward primer：ATGGATTCCTTCTTCTTAT
Reverse primer：TTAAATTTTAAATGGAATA .
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| CN102676549A (en) * | 2012-01-09 | 2012-09-19 | 中国中医科学院中药研究所 | CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and coded product as well as application thereof |
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| WO2018015512A1 (en) * | 2016-07-20 | 2018-01-25 | Evolva Sa | Biosynthesis of 13r-manoyl oxide derivatives |
| CN108795960A (en) * | 2018-06-22 | 2018-11-13 | 中国医学科学院药用植物研究所 | A method of using a variety of sesquiterpenoids of Radix Salviae Miltiorrhizae SmTPS3 gene chemical synthesis |
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