CN107699577A - A kind of screening, identification and the application of the SmAP2/ERF8 transcription factors of regulation and control danshinolic acid biosynthesis - Google Patents
A kind of screening, identification and the application of the SmAP2/ERF8 transcription factors of regulation and control danshinolic acid biosynthesis Download PDFInfo
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Abstract
The invention discloses the AP2/ERF transcription factors SmAP2/ERF8 of regulation and control danshinolic acid synthesis coding gene sequence；SmAP2/ERF8 genes provided by the present invention have the nucleotide sequence shown in SEQ ID No.1, and the DNA encoding the protein has the amino acid sequence shown in SEQ ID No.2.The present invention constructs SmAP2/ERF8 RNAi carriers and SmAP2/ERF8 over-express vectors, the genetic transformation red sage root, obtain transgenic hairy root, compared with compareing strain, the content of tanshin polyphenolic acid B significantly reduces in SmAP2/ERF8 RNAi strains, and the content that SmAP2/ERF8 is overexpressed tanshin polyphenolic acid B in strain significantly rises.SmAP2/ERF8 provided by the invention has the function of positive regulation and control pressure differential self biosynthesis, and such compound has the significant curative effect for the treatment of angiocardiopathy.The present invention has innovated the Research Thinking of danshinolic acid synthesis regulation molecular mechanism, is laid the foundation for the synthetic biology research of danshinolic acid, while provide target gene to carry out red sage root good variety selection.
Description
Technical field
The invention belongs to molecular biology of plants and genetic engineering field, and in particular to one kind regulation and control danshinolic acid biosynthesis
AP2/ERF transcription factors screening, identification and application.
Background technology
The red sage root (Salvia miltiorrhiza Bunge) is Salvia Labiatae medicinal plant, and its root is used as medicine, and is normal
With one of bulk medicinal materials, there is important medical value and economic value.Red sage root main active includes fat-soluble tanshinone
Compound and water-soluble pressure differential self, there is treatment cardiovascular and cerebrovascular disease, the function such as anti-oxidant, using the red sage root as mainly into
The compound danshen dripping pills annual sales amount divided is more than 3,900,000,000.The red sage root is because with genetic conversion system is ripe, genome is small, chromosome number
The features such as mesh is few, the generation cycle is short, tissue culture technique is ripe, it is chosen as the idealized model plant of traditional Chinese medicine research.At present, the red sage root
The parsing of the biosynthesis pathway of ketone and danshinolic acid is more deep, but on regulating and controlling the molecule of these reactive compound biosynthesis
Mechanism Study is but rarely reported.
AP2/ERF transcription factors are one of maximum transcription factor gene families of plant, and AP2/ERF protein structure domains can be with
This particular sequence of the cis-acting elements of target gene promoter region carries out selectivity combination.Have verified that AP2/ERF at present
The biosynthetic process of alkaloid and terpenoid can be regulated and controled in catharanthus roseus, tobacco and Chinese yew；And AP2/ERF is transcribed
Whether the factor can regulate and control the biosynthesis of pressure differential self in the red sage root, and there is not been reported.
The content of the invention
It is an object of the invention to provide a kind of the AP2/ERF transcription factor genes and its volume of regulation and control danshinolic acid biosynthesis
The protein of code.
It is another object of the present invention to the function of verifying AP2/ERF transcription factor family members.
SmAP2/ERF8 genes provided by the invention, its nucleotides sequence are classified as shown in SEQ ID No.1.
The protein of SmAP2/ERF8 gene codes provided by the invention, its amino acid sequence is as shown in SEQ ID No.2.
The purpose of the present invention can be achieved through the following technical solutions：Based on red sage root full-length genome and different red sage root devices
Official/tissue transcriptome differences expression analysis filters out the AP2/ERF gene family members SmAP2/ that may regulate and control danshinolic acid synthesis
ERF8 encoding genes.
Build a kind of plant RNA i double base tables of forward and reverse sequence containing SmAP2/ERF8 gene-specific fragments
Up to carrier.
Build a kind of plant containing SmAP2/ERF8 full length gene sequences and be overexpressed binary expression vector.
The present invention infects red sage root blade by agrobacterium rhizogenes, obtain SmAP2/ERF8-RNAi positive hairy roots and
SmAP2/ERF8- is overexpressed positive hairy root.
The present invention is shown using HPLC technical appraisement content of tanshin polyphenolic acid B into SmAP2/ERF8-RNAi transgenic hairy roots
Writing reduces, and the content that SmAP2/ERF8- is overexpressed tanshin polyphenolic acid B in strain significantly rises.SmAP2/ERF8 tools provided by the invention
There is the function of positive regulation and control danshinolic acid synthesis, the biosynthesis of danshinolic acid can be promoted, for point of parsing regulation and control danshinolic acid biosynthesis
Handset system lays the foundation.
Brief description of the drawings
Fig. 1 show the pellet of the SmAP2/ERF8-RNAi/ overexpression genetic transformations of agrobacterium rhizogenes ACCC10060 mediations
Join hairy root.
Fig. 2 show SmAP2/ERF8 in SmAP2/ERF8-RNAi transgenic hairy roots expression quantity reduce (A) and
SmAP2/ERF8- is overexpressed expression quantity in transgenic hairy root and rises (B).
Fig. 3 show HPLC analysis SmAP2/ERF8-RNAi transgenic hairy roots (A) and SmAP2/ERF8- is overexpressed and turned
The content of tanshin polyphenolic acid B in gene hairy root (B).
Fig. 4 show tanshin polyphenolic acid B content reduced in SmAP2/ERF8-RNAi transgenic hairy roots (A) and
SmAP2/ERF8-, which is overexpressed in transgenic hairy root, rises (B).
Embodiment
Below in conjunction with the example in detail present invention.Implementation is for a better understanding of the present invention, but to be not limited to the present invention.
Experimental method in following implementation is conventional method, and involved experiment reagent is routine biochemistry reagent.
Embodiment 1 is screened in red sage root full-length genome and identifies the member of AP2/ERF gene families
Utilize Pfam database HMMs HMM：PF00847 search red sage root genomes are annotated, so as to obtain
Obtain all AP2/ERF genes.Predict 170 AP2/ERF transcription factor genes altogether in red sage root genome, manual correction is simultaneously pre-
ORFs (ORF) is surveyed, is named as Sm001-Sm170, length protein scope is between 79aa to 595aa.Wherein, Sm008
SmAP2/ERF8 is named as in the present invention.
The clone of the red sage root SmAP2/ERF8 genes of embodiment 2
Total length primer is designed according to the ORFs of SmAP2/ERF8 sequences, using the cDNA of the red sage root as template, PCR amplifications
The nucleotide sequence of SmAP2/ERF8 genes is obtained, length is 660 bp, such as SEQ ID No.1.After nucleotide sequence is translated
SmAP2/ERF8 amino acid sequence is derived, comprising 220 amino acid residues, such as SEQ ID No.2.
The functional verification of the red sage root SmAP2/ERF8 genes of embodiment 3
1) RNAi design of primers and PCR amplifications.Select the specific piece that one section of length is 185 bp in SmAP2/ERF8 genes
Duan Zuowei RNAi target areas (the 95-279 bp for being located at gene), design both ends primer to target area, are made according to Gateway
With principle, addition attB sequences, wherein F primers addition attB1 sequences are held in primer 5 '：GGGGACAAGTTTGTACAAAAAAG
CAGGCT, R primer add attB2 sequences：GGGGACCACTTTGTACAAGAAAGCTGGGT.Primer sequence is as follows：
F-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCGCCGCCGGAGAGTACT
R-GGGGACCACTTTGTACAAGAAAGCTGGGTGCGCAGCATCATCGTATGC
2) design of primers and PCR amplifications are overexpressed.In the end of the total length primer 5 ' addition attB sequences of SmAP2/ERF8 genes.
Primer sequence is as follows：
F-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGAAGCATCGGCGCAGG
R-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCCGTTTGGGTTATTCG
3) SmAP2/ERF8-RNAi carriers are built.BP reacts：25 ng attB-PCR recovery is added in PCR reaction tubes
Product, 75ngpDONR221 entry vectors, 1 μ LBP clonase II enzyme, supplement ddH2O to 5 μ L；After gently mixing,
It is incubated more than 1 hour in 25 DEG C；0.5 μ L protein kinase K is added, 37 DEG C of 10 min of incubation after mixing；It is transferred to DH5 α impressions
State cell, PCR progress is utilized with the LB solid medium screening and culturings containing 50mg/LKan (kanamycins) resistance, then to clone
Detection.LR reacts：75ng pDONR221-RNAi recovery products, 75 ng pK7GWIWG2D (II) are added in PCR reaction tubes
Acceptor carrier, 1 μ L LR clonase II enzyme, supplement ddH2O to 5 μ L；Gently mix after 25 DEG C incubate 1 hour with
On；0.5 μ L protein kinase K is added, 37 DEG C of incubation 10min after mixing；DH5 α competent cells are transferred to, with containing 50mg/
The LB solid medium screening and culturings of LSpec (spectinomycin) resistance, positive colony is sent to survey after PCR is detected；Sequencing is correct
Clone extract recombinant plasmid pK7GWIWG2D (II)-SmAP2/ERF8, be transferred in agrobacterium rhizogenes ACCC10060.
4) SmAP2/ERF8- over-express vectors are built.BP reactions are the same as 3).LR reacts：75 ng are added in PCR reaction tubes
PDONR221- is overexpressed recovery product, 75ng pK7WG2D acceptor carriers, 1 μ L LR clonase II enzyme, supplement
ddH2O to 5 μ L；Gently mix and incubated more than 1 hour after 25 DEG C；0.5 μ L protein kinase K is added, is incubated for 37 DEG C after mixing
Educate 10min；DH5 α competent cells are transferred to, training is screened with the LB solid mediums containing 50mg/LSpec (spectinomycin) resistance
Support, positive colony is sent to survey after PCR is detected；Correctly clone extracts recombinant plasmid pK7WG2D-SmAP2/ERF8 for sequencing, turns
Enter in agrobacterium rhizogenes ACCC10060.
5) Agrobacterium ACCC10060 infects red sage root blade.With the root of hair for being transferred to pK7GWIWG2D (II)/pK7WG2D carriers
Agrobacterium is as control, Synchronous Infection red sage root blade；Eugonic red sage root tissue-cultured seedling is chosen, young leaflet tablet is taken, is cut into
0.5cm2Leaf dish, be placed on blank MS culture medium flat plates 25 DEG C of precultures 2-3 days；With 50mg/L Spec+50mg/LRif's
Liquid YEB culture mediums cultivate the agrobacterium rhizogenes containing recombinant plasmid and empty carrier respectively, and 28 DEG C are shaken training and reach 0.4- to OD600
0.6；Bacterium solution is centrifuged, is enriched with after thalline and thalline (MS-plasmid) is resuspended with isometric MS fluid nutrient mediums, by preculture
Leaf dish be placed in MS- plasmid, with aseptic filter paper suck unnecessary bacterium solution after soaking 10min, be placed on blank MS flat boards, 25
48-72h is co-cultured under DEG C dark condition；The leaf dish of co-cultivation is soaked in the sterilized water of 600mg/LCar (carbenicillin)
10min, it is placed on the Kan of Car+50mg/L containing 500mg/L MS flat boards after sucking excessive moisture, is sieved under 25 DEG C of dark conditions
Choosing culture, change a subculture within every 10 days；The fast positive hairy root of growing way is selected, cuts, puts after its length to 2.0-3.0cm
On 6 of the Kan containing 15mg/L, 7-V flat boards, after growing more lateral root, the expression that GFP is detected using fluorescence microscope is sentenced
Whether the hairy root of disconnected transgenosis is positive strain, as shown in Figure 3.Positive strain is moved in 6,7-V fluid nutrient mediums,
150rpm, expand culture under 25 DEG C of dark conditions.Detected using real time quantitative PCR method in transgenic positive strain, gene table
Up to/overexpression degree is suppressed, as shown in Figure 4.
The pressure differential self content detection of embodiment 4
The present invention carries out chemical composition detection using HPLC technologies to the hairy root of transgenosis, and step is as follows：1) by the red sage root
Weighed after the drying of hairy root, powder is beaten using ball milling instrument, extracted per 100mg hairies root with 0.5ml methanol, extract is ultrasonically treated
30min, 3,000g centrifugation 10min, supernatant is filtered into brown liquid phase bottle, treat sample introduction with 0.22 μm of nylon filter；2)
HPLC conditions：Using Waters XBridge C18 chromatographic columns, Detection wavelength, 286nm；Column temperature, 25 DEG C；Flow velocity, 1ml/min；
Sample size, 10 μ L, mobile phase：The phosphoric acid solution of acetonitrile -0.1% (22: 78).
The present invention is screened based on red sage root full-length genome and clones SmAP2/ERF8 genes first, and checking finds SmAP2/ERF8
Function with positive regulation and control danshinolic acid biosynthesis, the research cultivated to carry out danshinolic acid synthetic biology and elite germplasm are established
Basis.
Described above is only the preferred embodiment of the present invention, it should be pointed out that not for technical field ordinary person
On the premise of departing from the technology of the present invention principle, some improvements and modifications can also be made, these also should be regarded as the guarantor in the present invention
Protect scope.
Claims (5)
1. a kind of AP2/ERF transcription factors SmAP2/ERF8 of regulation and control danshinolic acid biosynthesis pathway encoding gene, its nucleosides
Acid sequence is as shown in SEQ ID No.1.
2. the related gene SmAP2/ERF8 of regulation and control danshinolic acid biosynthesis according to claim 1, it is characterised in that institute
The amino acid residue sequence of gene SmAP2/ERF8 encoding proteins matter is stated 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 positive and anti-of SmAP2/ERF8
To specific fragment sequence.
4. a kind of plant is overexpressed binary expression vector, it is characterised in that the over-express vector contains SmAP2/ERF8 total length
Sequence.
5. claim 1 encodes applications of the AP2/ERF transcription factors SmAP2/ERF8 in plant genetic engineering.It is characterized in that
The biology that SmAP2/ERF8 regulates and controls pressure differential self in bacterium, fungi and higher plant by genetic engineering means closes
Into.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548401A (en) * | 2020-06-04 | 2020-08-18 | 中国中医科学院中药研究所 | Salvia miltiorrhiza ERF-VII transcription factor SmERF73 participating in diterpene biosynthesis regulation and application thereof |
| CN112143734A (en) * | 2019-06-28 | 2020-12-29 | 中国医学科学院药用植物研究所 | SmbHLH92 gene cloning primer, expression vector, function of regulating and controlling salvianolic acid biosynthesis and application |
| CN112626075A (en) * | 2019-10-08 | 2021-04-09 | 中国医学科学院药用植物研究所 | Cloning primer, function and application of SmAP2/ERF152 gene for regulating and controlling tanshinone synthesis |
-
2017
- 2017-09-20 CN CN201710849712.4A patent/CN107699577A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112143734A (en) * | 2019-06-28 | 2020-12-29 | 中国医学科学院药用植物研究所 | SmbHLH92 gene cloning primer, expression vector, function of regulating and controlling salvianolic acid biosynthesis and application |
| CN112626075A (en) * | 2019-10-08 | 2021-04-09 | 中国医学科学院药用植物研究所 | Cloning primer, function and application of SmAP2/ERF152 gene for regulating and controlling tanshinone synthesis |
| CN112626075B (en) * | 2019-10-08 | 2022-08-09 | 中国医学科学院药用植物研究所 | Cloning primer, function and application of SmAP2/ERF152 gene for regulating and controlling tanshinone synthesis |
| CN111548401A (en) * | 2020-06-04 | 2020-08-18 | 中国中医科学院中药研究所 | Salvia miltiorrhiza ERF-VII transcription factor SmERF73 participating in diterpene biosynthesis regulation and application thereof |
| CN111548401B (en) * | 2020-06-04 | 2022-08-02 | 中国中医科学院中药研究所 | Salvia miltiorrhiza ERF-VII transcription factor SmERF73 and application thereof |
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