CN106636142A - Clone identification and application of 2-oxoglutarate-dependent dioxygenase (2OGD-5) gene participating in tanshinone synthesis - Google Patents
Clone identification and application of 2-oxoglutarate-dependent dioxygenase (2OGD-5) gene participating in tanshinone synthesis Download PDFInfo
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Abstract
The invention discloses an encoding gene sequence of 2-oxoglutarate-dependent dioxygenase (2OGD-5) gene participating in tanshinone synthesis. The provided 2OGD-5 gene has a nucleotide sequence shown as SEQ ID No.1, and protein encoded by the gene has an amino acid sequence shown as SEQ ID No.2. Hairy roots of salvia miltiorrhiza bunge are subjected to genetic transformation by constructing a 2OGD-5-RNAi vector, and compared with the contrast, the content of miltirone, cryptotanshinone and tanshinone is remarkably reduced. The provided 2OGD-5 has the capability of producing tanshinone compounds under catalysis. The compounds have the function of treating cardiovascular disease. The research thought of tanshinone synthesis is innovated, and the encoding gene sequence lays a foundation for artificial synthesis of tanshinone compounds and has huge research and application prospect.
Description
Technical field
The invention belongs to molecular biology of plants and genetic engineering field, more particularly to a kind of 2- for participating in tanshinone synthesis
Ketoglutaric acid dependence dioxygenase gene clone identification and application.
Background technology
Salviamiltiorrhizabung be the Salvia labiate red sage root (Salvia miltiorrhiza Bunge) dry root and
Rhizome, is one of the most frequently used bulk medicinal materials, inducing meastruation to relieve menalgia with promoting blood circulation, relieving restlessness that clears away heart-fire, the effects such as cool blood to disappear carbuncle,
Clinical treatment cardiovascular and cerebrovascular disease has higher use value.Red sage root annidation is stronger, East China, North China, northwest, southwest,
Central-South to wait regional widely distributed, nowadays red sage root wild resource is greatly decreased, and is mainly derived from artificial cultivation product, in Hebei, river
Plant in a large number on the ground such as Soviet Union, Anhui, Liaoning, Shaanxi, Shandong, Henan, Shanxi, Hubei, Sichuan.Red sage root main active is liposoluble
Property tanshinone compound and water-soluble phenolic compounds.Red sage root base plant has that vitality is strong, the generation cycle is short, gene
The features such as organizing few little, chromosome number, tissue cultures and transgenic technology maturation, it is considered to be the idealized model life of traditional Chinese medicine research
Thing.Recently, the biological relations of red sage root main active tanshinone are just progressively parsed.Due to the high oxidation activity of tanshinone,
Its biosynthesis pathway is participated in by a series of oxidizing ferment, has reported that multiple CYP450s participate in the synthesis of tanshinone.2OGD superfamilies
It is the second largest gene family in Plant Genome, its function relates generally to oxidation and hydroxylation, such as gibberellin, flavonoids
Deng synthesis, but at present 2OGDs whether participate in tanshinone biosynthesis it is still unknown.
The content of the invention
Present invention aim at exploring function of the 2OGD family members in tanshinone synthesis, there is provided one kind participates in tanshinone
Biosynthetic 2OGD encoding genes and application.
The object of the invention can be achieved through the following technical solutions：One kind is based on red sage root full-length genome and different red sage root groups
Knit the coding base of the tanshinone route of synthesis correlation 2OGD superfamily gene members 2OGD-5 of organ transcriptome differences expression analysis
Cause, its nucleotide sequence is as shown in SEQ ID No.1.
The amino acid residue sequence of the gene 2OGD coded proteins such as SEQ ID No.2.
A kind of forward and reverse fragment sequence plant RNA i binary expression vectors containing 2OGD-5 encoding genes.
The present invention infects red sage root blade by agrobacterium rhizogenes, obtains positive 2OGD-5-RNAi hairy roots, hairy root Jing
0.1mg/L IAA induce a large amount of lateral roots to generate.
The present invention adopts the Cryptotanshinone and pellet of metabolism group (LC-MS) technical appraisement 2OGD-5-RNAi transgenic hairy roots
The content of ginseng ketone IIA is significantly reduced compared with the control.The life of 2OGD-5 catalysis Cryptotanshinones and tanshinone IIA that the present invention is provided
Thing synthesizes, and is that the parsing of tanshinone biosynthesis pathway lays the foundation.
Description of the drawings
Fig. 1 show red sage root genome 2OGD family member phylogenetic evolution trees.
Fig. 2 show differential expression of the red sage root 2OGD family members encoding gene in red sage root different tissues organ.
Fig. 3 show differential expressions of the 2OGD-5 in red sage root different tissues organ.
Fig. 4 show red sage root 2OGD-5 encoding genes structure and and protein three-dimensional structure prediction.
Fig. 5 show the red sage root hairy that the genetic transformation of agrobacterium rhizogenes ACCC10060 mediations obtains 2OGD-5-RNAi
Root.
Fig. 6 show the inhibitory action figure of pK7GWIWG2D (II) -2OGD-5 transgenic hairy roots.
Fig. 7 show tanshinone content difference between the different transgenic hairy roots of UPLC analyses.
Fig. 8 show tanshinone content difference between the different transgenic hairy roots of LC-MS analyses.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The full-length genome of embodiment 1 screening red sage root 2OGD families
1) based on red sage root genome-wide screening 2OG-FeII_Oxy domains (PF03171), the super families of red sage root 2OGD are screened
Race member 132, length amino acid sequence is distributed from 120aa to 504aa.
2) compared and phylogenetic tree construction using MEGA, red sage root 2OGD superfamilies are divided into the sub- families of DOXB and DOXC two
Race, wherein DOXB include 14 2OGD members, and DOXC includes 116 2OGD members, as shown in Figure 1.
3) the transcript profile sequencing data of red sage root different organ and tissue and process is collected, it is soft using Tophat and Cufflinks
Part analyzes differential expression spectrum of the red sage root 2OGD member in the case where red sage root different organ and tissue and MeJA are processed, as shown in Figure 2.
4) according to tanshinone red sage root different parts synthesis and accumulation, the consistent red sage root 2OGD of screening-gene differential expression
Family member, wherein 2OGD-5 specifically expressing and are significantly induced in red sage root by MeJA, as shown in Figure 3.
The clone of the red sage root 2OGD-5 encoding genes of embodiment 2 and structural analysis
1) the 2OGD-5 sequence alignments filtered out in embodiment 1 are designed into total length primer, is carried out from the cDNA of red sage root
PCR is expanded, and obtains nucleotide sequence of the length for 1089bp, such as SEQ ID No.1.According to derivation after full length cDNA sequence translation
Go out the amino acid sequence of red sage root 2OGD-5, totally 362 amino acid residues, such as SEQ ID No.2.
2) using PyMOL software prediction 2OGD-5 protein structures, with reference to the protein three-dimensional structure model of arabidopsis AtLDOX
(PDBID：1GP5), as shown in Figure 4.
The red sage root 2OGD-5 Function Identifications of embodiment 3
1) Gateway design of primers and fragment amplification.RNAi fragment primer (the genes of 186bp are designed to 2OGD-5 genes
Position：304-489bp), 5 ' end addition attB1 sequences：GGGGACAAGTTTGTACAAAAAAGCAGGCT, 3 ' end addition attB2
Sequence GGGGACCACTTTGTACAAGAAAGCTGGGT.In order to avoid the RNAi regiospecificities of design during designated rna i primers
Difference causes transgene silencing target spot not single, and the RNAi regions of each gene are compared with genome BLAST, chooses specificity most
Good RNAi regions, primer sequence is as follows：
F-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCCTCGGAGACGATGGACG
R-GGGGACCACTTTGTACAAGAAAGCTGGGTGTCGTCGCTGAAGACGCAC
2) Gateway builds RNAi carrier.BP reacts：Take the attB PCR recovery products and 75ng pDONR221 of 25ng
Entry vector, the mixing that adds water is subsequently adding the BPclonase II enzyme of 1 μ L to 4 μ L, mixes, 25 DEG C of incubation 1h, adds
The Proteinase K of 0.5 μ L, 37 DEG C of incubation 10m, proceed to DH5 α competent cells, in 50mg/L Kan resistance LB solid cultures
Screening positive clone on base, and PCR detections；LR reacts：Take pDONR-RNAi and 75ng pK7GWIWG2D (II) acceptor of 75ng
Carrier, the mixing that adds water is subsequently adding the LR clonase II enzyme of 1 μ L to 4 μ L, mixes, 25 DEG C of incubation 1h, adds 0.5 μ
The Proteinase K of L, 37 DEG C of incubation 10m, proceed to DH5 α competent cells, in 50mg/L Spec resistance LB solid mediums
Upper screening positive clone simultaneously send survey, successful positive colony is sequenced and extracts recombinant plasmid pK7GWIWG2D (II) -2OGD-5 importings
Agrobacterium rhizogenes ACCC10060.
3) Agrobacterium ACCC10060 infects red sage root blade (leaf disk method).Design control experiment, selects to contain recombinant plasmid respectively
The agrobacterium rhizogenes ACCC10060 of PK7GWIWG2D (II) and pK7GWIWG2D (II) -2OGD-5 infects red sage root blade.Containing weight
The agrobacterium rhizogenes of group plasmid is inoculated in the YEB culture mediums of 50mg/L Spec+50mg/L Rif, 28 DEG C of shaking tables to OD600 about
For 0.5；After centrifugation, with the resuspended precipitation (MS-plasmid) of isopyknic MS fluid nutrient mediums；By red sage root aseptic seedling young leaflet tablet
It is cut into 0.5cm2Leaf dish, MS solid medium precultures 2-3 days；Pre-incubated leaf dish soaks 10min in MS-plasmid,
After aseptic paper is blotted, blank MS solid cultures are based on 25 DEG C of dark co-cultivation 48-72h；Leaf dish after co-cultivation is in containing 500mg/L
10min is cleaned in the sterilized water of Car (carbenicillin), the MS solid mediums of the Kan of Car+50mg/L containing 500mg/L are proceeded to
In, screen in 25 DEG C of dark, per 10 days subcultures once；Select growing way it is fast, resistance hairy root, cut 2-3cm numbering and with contain
Cultivate on the MS solid mediums of 30mg/L Kan；The MS solids of the IAA of Kan+0.1mg/L containing 30mg/L are selected in incubation
Stimulate 2-3d on culture medium, then continue on the MS solid mediums of the 30mg/L Kan without IAA, it is to be generated to grow a large amount of lateral roots
After be transferred to liquid 6,7-V cultures are based on shaking table culture in 25 DEG C of dark of 120rpm；The expression of GFP is detected under fluorescence microscope,
And Amplification Culture, as shown in Figure 5.
The tanshinone content detection of embodiment 4
The present invention is detected to the tanshinone content of transgenic hairy root using UPLC and LC-MS technologies, mainly taken
Following steps：1) Hairy Root Cultures of Salvia miltiorrhiza is dried and with mortar grinder into powder, and 100mg powder is extracted with 0.5mL methyl alcohol, super to extract
Twice, 0.2 μm of polytetrafluoroethylene syringe filter filtration of 1,500g centrifugation 10min, supernatant Jing is to be detected for sonication；2)UPLC
Condition, using Waters ACQUITY UPLC BCH C18 chromatographic columns, mobile phase：Methyl alcohol (A)-water (B), 75%A: 25%B washes
De- 10min, post 5min is washed after the completion of each chromatographic process with 100% methyl alcohol, then the balance columns of initial proportion 75%A: 25%B
Sub- 5min；Column temperature, 30 DEG C；Flow velocity, 0.3mL/min；Detection wavelength, 270nm；Sample size, 5 μ L；3) LC-MS/MS conditions：LC：
Shim-pack UFLC SHIMADZU CBM20A UPLC systems, chromatographic column：WatersACQUITY UPLC HSS T3C18
(1.8 μm, 2.1mm × 100mm), sample size：5 μ L, mobile phase：A=acetonitriles (0.04% acetic acid), B=0.04% acetic acid is water-soluble
Liquid, linear gradient：5%-95%A (0-12min), 95%A (12-15min), flow velocity：0.4mL/min, column temperature：40℃；MS/
MS：AB SCIEX QTRAP 4500, electro-spray ionization temperature：550 DEG C, mass spectrum voltage：5500V, blowback gas curtain gas：
25psi, collision induced dissociation：It is high.The results are as follows：The miltionone of 2OGD-5-RNAi transgenic hairy roots, Cryptotanshinone
Significantly reduce with the content of tanshinone IIA, be 0.16,0.56 and 0.56 times of control；The content of dihydrotanshinone and Tanshinone I
Slightly reduce, as shown in Figure 6,7.
The present invention screens first and clones 2OGD-5 genes based on red sage root full-length genome, and checking finds that 2OGD-5 participates in the red sage root
The biosynthesis of ketone, to parse tanshinone biosynthesis pathway new approaches are provided, and are studied for the external synthetic biology of tanshinone
Lay the foundation.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, on the premise of without departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (4)
1. it is a kind of participate in tanshinone biosynthesis pathway 2-oxoglutaric acid dependence dioxygenase (2OGD-5) encoding gene, its
Nucleotide sequence is as shown in SEQ ID No.1.
2. tanshinone synthesis related gene 2OGD-5 according to claim 1, it is characterised in that the gene 2OGD-5 is compiled
The amino acid residue sequence of code protein is as shown in SEQ ID No.2.
3. a kind of plant RNA i binary expression vectors, it is characterised in that the RNAi carrier contains the forward and reverse of 2OGD-5
Fragment sequence.
4. a kind of method that induction Hairy Root Cultures of Salvia miltiorrhiza generates a large amount of lateral roots, it is characterised in that the Hairy Root Cultures of Salvia miltiorrhiza incubation
Stimulate 2-3d on the MS solid mediums of middle selection Kan containing 30mg/L (kanamycins)+0.1mg/L IAA (heteroauxin), with
Proceed to afterwards on the MS solid mediums of the 30mg/L Kan without IAA, to be generated growing be transferred to after a large amount of lateral roots liquid 6,7-V trainings
Support based on shaking table culture in 120rpm25 DEG C of dark.
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Cited By (2)
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| CN108866016A (en) * | 2017-05-16 | 2018-11-23 | 中国中医科学院中药研究所 | Protein and its preparing the application in Cryptotanshinone |
| CN108893482A (en) * | 2018-06-22 | 2018-11-27 | 中国医学科学院药用植物研究所 | Radix Salviae Miltiorrhizae Terpene synthase gene SmTPS8, its cloning primer, expression vector, catalysate and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866016A (en) * | 2017-05-16 | 2018-11-23 | 中国中医科学院中药研究所 | Protein and its preparing the application in Cryptotanshinone |
| CN108866016B (en) * | 2017-05-16 | 2021-11-02 | 中国中医科学院中药研究所 | Protein and application thereof in preparation of cryptotanshinone |
| CN108893482A (en) * | 2018-06-22 | 2018-11-27 | 中国医学科学院药用植物研究所 | Radix Salviae Miltiorrhizae Terpene synthase gene SmTPS8, its cloning primer, expression vector, catalysate and application |
| CN108893482B (en) * | 2018-06-22 | 2021-11-05 | 中国医学科学院药用植物研究所 | Salvia miltiorrhiza terpene synthase gene SmTPS8, cloning primer, expression vector, catalytic product and application thereof |
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Application publication date: 20170510 |