CN106349352A - Artemisia apiacea translocator AaPDR3 and application thereof - Google Patents
Artemisia apiacea translocator AaPDR3 and application thereof Download PDFInfo
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Abstract
The invention relates to an artemisia apiacea translocator AaPDR3 and an application thereof. The amino acid sequence is shown as SEQ ID No.2; the artemisia apiacea translocator AaPDR3 can affect the synthesizing of sesquiterpenes in artemisia apiacea non-secreting type glandular hair; the translocator is encoded by the nucleotide sequence shown as SEQ ID No.1. The invention further provides a method for realizing transgenosis artemisia apiacea seedlings with the artemisia apiacea translocator AaPDR3.
Description
Technical field
The present invention relates to a kind of technology of bioengineering field, specifically a kind of Herba Artemisiae Annuae transport protein aapdr3 and its
Application.
Background technology
Herba Artemisiae Annuae (artemisia annua l.) is the annual herb plant of Compositae artemisia.Its aerial parts is extracted
Sesquiterpene lactoness oxide arteannuin containing peroxide bridge, is most widely used at present, the best anti-malaria medicaments of curative effect,
Particularly more efficient to encephalic malaria and anti-chlorine quinoline malaria.At present, arteannuin conjoint therapy (acts) is World Health Organization (WHO)
The method of the maximally effective treatment malaria recommended.But content in plant ginghao for its arteannuin is low, method is there is no to fully meet entirely
The market demand of ball.Herba Artemisiae Annuae has secreting type glandular hair (glandular trichomes) and nonsecreting type glandular hair
(nonglandular trichomes).In the front and back of Herba Artemisiae Annuae blade, stalk, take and all there is secreting type gland in a large number
Hair, is the accumulation place of a large amount of secondary metabolitess, arteannuin is recognized as being stored in herein here.And study and be reported in non-secretion
In glandular hair, there are a large amount of terpene substances synthesis.Experiment proves stone column alkene, and the terpene substances such as β farnesene are only in Herba Artemisiae Annuae nonsecreting type gland
Synthesize in hair.
Abc (atp binding cassette) transport protein is the greatly very special super family of a big class
Race.Most of abc transport protein participates directly in the transdermal delivery of various molecules.This transport protein is using hydrolysis atp release energy
Amount carries out transdermal delivery to various biomolecules in Cytoplasm, and transhipment substrate includes: lipid, aminoacid, alkaloid, terpene substances
Deng.Picked-up transport protein (importer) and outer row's transport protein can be roughly classified into according to for cytoplasmic transhipment direction
(exporter).The feature of abc transport protein is with atp calmodulin binding domain CaM (atp binding cassette), also referred to as
Nucleic acid binding region nbd (nucleotide binding domain), has some very conservative motif, including walker
A and walker b sequence, abc signature motif, h loop and q loop.There is substantial amounts of abc transport protein in plant,
Recent studies indicate that plant abc transport protein not exclusively to phytohormone, esters, metal ion, secondary metabolitess,
The transhipment of exogenous chemical substances is relevant, and has built up important work for plant and pathogen interaction and ion channel
With.Pdr (pleiotropic dr μ g resistance) transport protein belongs to abc transport protein family g subfamily, comprises anti-
To nbd tmd type transport protein.If by Herba Artemisiae Annuae secreting type glandular hair transcript profile database analysises, filtering out participation
The correlation transport protein of artemisinin synthesis approach, and verify its function, then just can improve transhipment using genetic engineering means
The transport efficacy of albumen, thus improve the content of Artemisinin in Artemisia annuna.Aapdr3 is to clone the abc obtaining from Herba Artemisiae Annuae to turn
The transport protein of pdr (pleiotropic dr μ g resistance) subfamily in fortune albumen.Using genetic engineering means, will
This aapdr3 transport protein rnai interference carrier and Overexpression vector conversion Herba Artemisiae Annuae, sesquiterpene in rnai interference of transgene plant
The synthesis of stone column alkene is significantly suppressed, and overexpression transfer-gen plant has part to improve in sesquiterpene stone column alkene.Additionally, aapdr3
Transport protein rnai interference carrier converts Herba Artemisiae Annuae, and in rnai interference of transgene plant, artemislnin content is significantly increased.Therefore,
The research of aapdr3 gene is significant for improving sesquiterpene stone column alkene genetic engineering breeding in Herba Artemisiae Annuae.
Content of the invention
The present invention is directed to deficiencies of the prior art, proposes a kind of Herba Artemisiae Annuae transport protein aapdr3 and its application,
This transport protein aapdr3 expression in Herba Artemisiae Annuae old leaf, root and alabastrum is higher, and with leaf senile, expression constantly carries
High.Its promoter merges gus and turns the transhipment that Herba Artemisiae Annuae proves that aapdr3 is expression in Herba Artemisiae Annuae nonsecreting type glandular hair specifically expressing and root
Albumen；Nicotiana tabacum L. and yeast Subcellular Localization prove that aapdr3 is positioned on cell membrane (Fig. 1).Using transgenic technology by Herba Artemisiae Annuae
Aapdr3 transport protein interference carrier and Overexpression vector conversion Herba Artemisiae Annuae, sesquiterpene stone column alkene in rnai interference of transgene plant
Synthesis significantly suppressed, and overexpression transfer-gen plant sesquiterpene stone column alkene have part improve.Additionally, aapdr3 transhipment egg
White rnai interference carrier conversion Herba Artemisiae Annuae, in rnai interference of transgene plant, artemislnin content is significantly increased.Aapdr3 gene
Research is significant for improving sesquiterpene stone column alkene genetic engineering breeding in Herba Artemisiae Annuae.
The present invention is achieved by the following technical solutions:
The present invention relates to a kind of Herba Artemisiae Annuae transport protein aapdr3, its aminoacid sequence is as shown in seq id no.2.
Described Herba Artemisiae Annuae transport protein aapdr3 affect in Herba Artemisiae Annuae nonsecreting type glandular hair sesquiterpene stone column alkene synthesis and
Transhipment.
Described transport protein is by nucleotide sequence coded as shown in seq id no.1.
The invention further relates to a kind of polypeptide, the aminoacid sequence of described polypeptide is as shown in seq id no.4.
The present invention relates to a kind of transgene abrotanum Seedling implementation method with Herba Artemisiae Annuae transport protein aapdr3, walk including following
Rapid:
Step one, the albumen coded sequence of Herba Artemisiae Annuae transport protein aapdr3 is respectively connected to plant rnai interference and crosses scale
Reach on carrier, build the plant rnai interference obtaining the transport protein coding sequence of aapdr3 containing Herba Artemisiae Annuae and Overexpression vector；
Step 2, plant rnai interference expression vector and Overexpression vector is proceeded to Agrobacterium, then Agrobacterium is proceeded to
Herba Artemisiae Annuae；
Described proceed to particularly as follows: being proceeded to using freeze-thaw method.
Described Agrobacterium is Agrobacterium tumefaciems (agrobacterium tumefaciens) bacterial strain eh105, and this bacterial strain can
With open purchase from the market (from Australian cambia company, strain number is gambar 1).
Step 3, obtains Herba Artemisiae Annuae resistance Seedling by antibiotic-screening, then the plant through pcr test positive is transgenic
Herba Artemisiae Annuae Seedling.
Described pcr detection refers to, separately designs the detection primer of synthesis aapdr3 gene, carries out dna amplification, ultraviolet
Under observe purpose band positive strain be transgene abrotanum plant.
The present invention relates to a kind of application based on above-mentioned transgene abrotanum Seedling, use it for raising Herba Artemisiae Annuae terpene substances and contain
Amount.
Described Herba Artemisiae Annuae terpene substances, measure analysis by described transgene abrotanum Seedling is carried out with gc ms, and then obtain again
Hemiterpene stone column alkene, β farnesene and gima ethylenic d；Analysis artemislnin content is measured by hplc.
In the present invention, can be selected for various carrier known in the art, such as commercially available carrier, including plasmid, cosmid etc..?
During the Herba Artemisiae Annuae aapdr3 protein polypeptide of the production present invention, Herba Artemisiae Annuae aapdr3 albumen coded sequence can be operably coupled to express
Regulating and controlling sequence, thus form Herba Artemisiae Annuae aapdr3 protein expression vector.
" being operably coupled to " refers to such a situation, that is, some parts of linear dna sequence can affect same linear
The activity of dna sequence other parts.For example, if signal peptide dna as precursor expression and participates in the secretion of polypeptide, then signal
Peptide (secretion targeting sequencing) dna is exactly to be operably coupled to polypeptide dna；If the transcription of promoter control sequence, then it is
It is operably coupled to coded sequence；If ribosome binding site is placed in when can make position that it is translated, then it is to grasp
Make ground and be connected in coded sequence.Typically, " it is operably coupled to " to mean adjacent, and secretion targeting sequencing is then meaned readding
Adjacent in frame.
Technique effect
Compared with prior art, the present invention is by Herba Artemisiae Annuae nonsecreting type glandular hair transcript profile data analysiss, from Herba Artemisiae Annuae gram
Grand aapdr3 gene, builds the plant overexpression containing aapdr3 gene and interference expression vector, is situated between with Agrobacterium tumefaciems eh105
Lead, aapdr3 gene overexpression and interference expression vector are converted by Herba Artemisiae Annuae using leaf disk method；Pcr detects external source genes of interest
The integration of aapdr3, measures sesquiterpene content in Herba Artemisiae Annuae by gas chromatogram and mass spectrometry (gc ms), shows to obtain
In rnai interference of transgene plant, the synthesis of sesquiterpene stone column alkene is significantly suppressed, and overexpression transfer-gen plant sesquiterpene stone
Post alkene improves.Additionally, aapdr3 transport protein rnai interference carrier conversion Herba Artemisiae Annuae, in rnai interference of transgene plant, arteannuin contains
Amount is significantly increased.
Brief description
Fig. 1 is the pcr positive test symbol figure of transgene abrotanum plant；
In figure: m: molecular weight marker；Swimming lane 1 is negative control；Swimming lane 2 is positive control；Swimming lane 3 13 is 11 plants respectively
Transgene abrotanum plant genome is masterplate, carries out the product that pcr obtains；
Fig. 2 is aapdr3 gene overexpression and interference carrier converts Herba Artemisiae Annuae, measures Herba Artemisiae Annuae by liquid chromatograph (hplc)
Middle artemislnin content；
In figure: a is aapdr3 gene expression amount in aapdr3 interference of transgene Herba Artemisiae Annuae, and b converts for aapdr3 interference carrier
Herba Artemisiae Annuae, measures sesquiterpene content in Herba Artemisiae Annuae by gas chromatogram and mass spectrometry (gc ms), c turns base for aapdr3 overexpression
Because of aapdr3 gene expression amount in Herba Artemisiae Annuae, d converts Herba Artemisiae Annuae for aapdr3 Overexpression vector, by gas chromatogram and mass spectrometry
(gc ms) measures sesquiterpene content in Herba Artemisiae Annuae, and e is aapdr3 gene overexpression and interference carrier conversion Artemisinin in Artemisia annuna contains
Amount.
Specific embodiment
Embodiment 1
The clone of Herba Artemisiae Annuae aapdr3 gene
1. the extraction of the total rna of Herba Artemisiae Annuae genome
Take Herba Artemisiae Annuae leaf tissue, be placed in liquid nitrogen and grind, add 1.5ml eppendorf (ep) centrifugation filling lysate
Guan Zhong, after fully vibrating, the description according to tiangen test kit extracts total rna.Identify total rna matter with agarose gel electrophoresis
Amount, then measures rna content on spectrophotometer.
2. the clone of Herba Artemisiae Annuae aapdr3 gene
With total rna of being extracted as template, synthesize cdna in the presence of powerscript reverse transcription；According to
The sequential design gene-specific primer of aapdr3 gene, expands aapdr3 gene by pcr from total cdna, and is sequenced.
By above-mentioned steps, obtain total length 4278bp of this transcription factor in Herba Artemisiae Annuae, coded sequence, i.e. nucleotide sequence
As shown in seq id no.1, and derive its albumen coded sequence as shown in seq id no.2, wherein, start codon is
Atg, termination codon is taa.
Table 1 is the positive anti-primer of said gene specificity, i.e. aapdr3 fp1 (seq id no.3) and aapdr3 rp1 (seq
id no.4)
| Primer | Primer sequence (5 ' → 3 ') |
| aapdr3‐fp1 | ttctcgtagggctttttgagcatta |
| aapdr3‐rp1 | gccatacaaagacgctaatagaactca |
Table 2 is the reaction system of above-mentioned pcr
| Herba Artemisiae Annuae cdna | 1μl |
| 10×kod plus buffer | 5μl |
| dntp | 5μl |
| mgso4 | 2μl |
| aapdr3‐fp1 | 1μl |
| aapdr3‐rp1 | 1μl |
| kod plus | 1μl |
| ddh2o | 34μl |
| Cumulative volume | 50μl |
Embodiment 2
The structure of the plant interference expression vector containing aapdr3 gene
1. the structure of intermediate carrier ptopo aapdr3
In non-conservative region design forward primer and the downstream primer of aapdr3 gene, in order to build interference carrier.Upper
Add tetra- bases of cacc to build gateway entry vector before trip primer.According to invitrogen company pentrtm/The operating procedure of cloning kit, first obtains the fragment of aapdr3, passes through after recovery purifying with flush end enzymatic amplification
Gateway clone technology is connected to pentr/d topo carrier.
2. plant expresses the structure of interference carrier phellsgate1.2 aapdr3
Lr according to invitrogen companyThe operation of ii enzyme test kit, ptopo aapdr3 is carried
Recombinate two of rna interference carrier phellsgate1.2 of the interference fragment of the aapdr3 in body can form hairpin structure
In recombination site, obtain the rna interference carrier phellsgate1.2 aapdr3 of aapdr3.
Herba Artemisiae Annuae aapdr3 gene is operatively connectable to expression regulation sequence by the present embodiment, forms hair fastener containing aapdr3
The plant expression interference carrier of structure, this carrier can be used for regulating and controlling the content of Artemisinin in Artemisia annuna by metabolic engineering strategies.
Required primer is as shown in table 3:
Table 3 builds adopted pcr primer aapdr3 by above-mentioned interference carrier phellsgate1.2 aapdr3 body
Rnai fp (seq id no.5) and aapdr3 rnai rp (seq id no.6)
| Primer | Primer sequence (5 ' → 3 ') |
| aapdr3‐rnai‐fp | caccatggatggaagtgatatttataa |
| aapdr3‐rnai‐rp | catcaacttcttctgaaggtccag |
Embodiment 3
The structure of the plant Overexpression vector containing aapdr3 gene
By gene constructed for aapdr3 on Overexpression vector, the structure of expression vector, draws in forward primer for convenience
Enter the restriction enzyme site of bamhi, in reverse primer, introduced the restriction enzyme site of xbai, primer is as shown in table 4；
Table 4 builds adopted pcr primer bamhi aapdr3 fp (seq id no.7) by above-mentioned Overexpression vector
With aapdr3 xbai rp (seq id no.8)
| Primer | Primer sequence (5 ' → 3 ') |
| bamhi‐aapdr3‐fp | cgggatccatggatggaagtgatattta |
| aapdr3‐xbai‐rp | gctctagactatctcttttggaaattaaatgc |
Embodiment 4
Agrobacterium tumefaciens mediated aapdr3 overexpression and interference carrier genetic transformation Herba Artemisiae Annuae obtain transgene abrotanum and plant
Strain
1. the acquisition of the Agrobacterium tumefaciems engineering bacteria of overexpression containing aapdr3 and interference expression vector
The plant binary interference expression vector containing aapdr3 in embodiment 2 is proceeded to commercially available crown gall agriculture bar using freeze-thaw method
Bacterium (such as eha105, can buy from Australian cambia company, and strain number is gambar 1), and carry out pcr checking.Knot
Fruit shows, the plant binary interference expression vector containing aapdr3 has successfully been building up in Agrobacterium tumefaciens strain.
2. Agrobacterium tumefaciens mediated aapdr3 gene transformation Herba Artemisiae Annuae
2.1. the preculture of explant
Seeds of southernwood soaks 1min with 75% ethanol, then soaks 20min, aseptic water washing 34 times with 20%naclo, uses
Surface moisture is blotted in aseptic absorbent paper, is inoculated in ms (murashige and skoog, the 1962) solid medium of no hormone
In, 25 DEG C, 16h/8h (light/dark) illumination cultivation, you can obtain Herba Artemisiae Annuae aseptic seedling.After Seedling length to 5cm about after, clip
Tests for sterility explant is used for converting.
2.2. the co-cultivation of Agrobacterium and explant
By described leaf explant, go in co-cultivation culture medium (100 μm of ol/l of 1/2ms+as), Deca contains activation
The 1/2ms suspension of the Agrobacterium tumefaciems engineering bacteria of the good described overexpression of plant containing aapdr3 and interference expression vector, makes outer
Implant is fully contacted with bacterium solution, 28 DEG C of light culture 3d.With Deca the Agrobacterium tumefaciems without genes of interest 1/2ms liquid
The leaf explant of culture medium suspension is comparison.
2.3. the screening of resistance regeneration plant
The Herba Artemisiae Annuae explant of described co-cultivation 3d is transferred to germination screening culture medium (ms+6 ba 0.5mg/l+
Naa0.05mg/l+hyg 100mg/l+cb 500mg/l) in 25 DEG C, 16h/8h illumination cultivation, successive transfer culture one every two weeks
Secondary, hyg resistance Multiple Buds can be obtained after 23 subcultures.Well-grown resistance Multiple Buds are cut and proceeds to training of taking root
Foster base (1/2ms+cb 125mg/l) goes up culture to taking root, thus obtaining hyg resistance regeneration Herba Artemisiae Annuae plant.
3. the pcr detection of transgene abrotanum plant
35s promoter region according to genes of interest place expression cassette upstream and aapdr3 separately design forward primer design
Phellsgate 35s fp (seq id no.9) and reverse primer aapdr3 rnai rp (seq id no.6) turns base to interference
Because plant is detected；Forward primer design primer 35sf (seq id no.10) and reverse primer aapdr3 rp (seq id
No.11) overexpression transfer-gen plant is detected.Result shows, using designed pcr special primer, can amplify
Special dna fragment.And during with non-transformed Herba Artemisiae Annuae genome dna for template, do not amplify any fragment.
Described plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, obtain for convert Herba Artemisiae Annuae containing aapdr3
Plant interference expression vector and the Agrobacterium tumefaciens strain of Overexpression vector, using constructed Agrobacterium tumefaciens strain conversion
Herba Artemisiae Annuae, obtains the transgene abrotanum plant through pcr detection.
Embodiment 5
Measure sesquiterpene stone column alkene, β farnesene and gima ethylenic d content in transgene abrotanum using gc ms
1.gc ms condition and the preparation of system suitability and standard solution
Gc ms analytical tool is: agilent (7890a gc/5975c ms, usa), and analysis pillar is agilent db
5ms column(30m×0.25mm×0.25μm).First furnace temperature is preheated to 270 DEG C, takes 1 μ l sample feeding, sample introduction pattern is
No shunt mode；Heating schedule is: 60 DEG C of 3min, and process of heating from 60 DEG C to 300 DEG C is 10 DEG C/min, after EP (end of program) of heating
Keep 9min；The total time of whole measurement process is 50min；Electron impact ionization mass spectrographic ionization energy record from 70ev to
1765v, scanning of the mass spectrum scope is m/z 33to 500.Concentration according to standard substance and calculated by peak area go out the sesquiterpene in sample
Stone column alkene, β farnesene and gima ethylenic d content, then divided by Herba Artemisiae Annuae lyophilized powder dry weight, thus calculating stone column alkene, β farnesene and
Gima ethylenic d accounts for the content of sample fresh weight.Accurately use dchloromethane stone column alkene, β farnesene and gima ethylenic d standard substance
(sigma company), obtains stone column alkene, β farnesene and gima ethylenic d standard solution, be stored in 20 DEG C standby.
2.gc ms sample data is analyzed
Data analysis software is: msd chemstation software (version is e.02.02.1431), by sample
The common identification of comparing of the retention time of middle separation product and the same data base of mass spectrum (nist 11) n-compound separates and produces
The composition of thing.
3. the preparation of sample
Upper in Herba Artemisiae Annuae plant, neutralization bottom takes fresh Herba Artemisiae Annuae blade altogether.Powder in Herba Artemisiae Annuae blade and young tender sprig liquid nitrogen
After broken, 50 DEG C of lyophilization 8h.Accurately weigh 50mg sample powder to put in 10mll teat glass, plus 4ml normal hexane and 100 μ l
Trans farnesol titer (77.6 μ g/ml) supersound extraction 40min, is cooled to centrifuging and taking supernatant after room temperature, supernatant nitrogen
Dissolved with 200 μ l dichloromethane after drying up, loading is analyzed.
For sesquiterpene stone column alkene, β farnesene and gima ethylenic d assay, result as shown in Fig. 2 result shows,
In rnai interference of transgene plant, the synthesis of sesquiterpene stone column alkene is significantly suppressed, and overexpression transfer-gen plant sesquiterpene stone column
Alkene has part to improve.
Embodiment 6
Measure artemislnin content in transgene abrotanum using hplc
Hplc condition and the preparation of system suitability and standard solution
Hplc: using water alliance 2695 system, using waters c18 post, arteannuin measures mobile phase and makes
With methanol: water volume ratio 60%:40%, dihydroartemisinic acid measures mobile phase and is suitable for acetonitrile: 0.1% glacial acetic acid (ph 3.2) volume
Compare 60%:40%；Flow velocity is 1.0ml/min；Elsd detecting system is water alliance 2420, evaporative light scattering detection
Device drift tube temperature is 40 DEG C, nebulizer gas pressure 5bar；Arteannuin appearance time is 7min.Arteannuin sampling volume is
20ul.Concentration according to standard substance and calculated by peak area go out the artemislnin content in sample, then divided by Sweet Wormwood Herb dry weight, thus
Calculate the content that arteannuin accounts for sample dry weight.It is complete that precision weighs arteannuin standard substance (sigma company) 2.0mg 1ml methanol
CL, obtains 2mg/ml arteannuin standard solution, be stored in 20 DEG C standby.
The preparation of sample
Upper in Herba Artemisiae Annuae plant, neutralization bottom takes fresh Herba Artemisiae Annuae blade altogether, dries to constant weight in 45 DEG C of baking ovens.Then from
Strike lower blade on the branch dried, clay into power.Weigh about 0.1g dry powder in 2ml eppendorf pipe, add 2ml ethanol,
With 40w ultrasonic Treatment 30min, 5000rpm is centrifuged 10min, takes 0.22 μm of membrane filtration of supernatant, you can survey for hplc
Fixed.
For artemislnin content measure, result as shown in Fig. 2 result shows, arteannuin in rnai interference of transgene plant
Content is significantly increased.
Above-mentioned be embodied as can by those skilled in the art on the premise of without departing substantially from the principle of the invention and objective with difference
Mode local directed complete set is carried out to it, protection scope of the present invention is defined by claims and is not embodied as institute by above-mentioned
Limit, each implementation in the range of it is all by the constraint of the present invention.
Sequence table
<110>Shanghai Communications University
<120>Herba Artemisiae Annuae transport protein aapdr3 and its application
<130>11
<170> patentin version 3.5
<210> 1
<211> 4278
<212> dna
<213>Herba Artemisiae Annuae (artemisia annua l.)
<400> 1
atggatggaa gtgatattta taaagctagt agtagcttaa ggttagggag taatagtgga 60
agaatgggaa gtattagagc tggaagctct acccgatgga ggaacactgg catggatgtt 120
ttctcgagat caactcgtga agaagatgac gaggaagctt tgaaatgggc tgctttagaa 180
aagcttccga cttacgaccg tttaaagaaa ggtttgatct ttgggtcaac tggaccttca 240
gaagaagttg atgtagctag tcttggtttt gaagaacgaa aacgattact tgagaggctt 300
gttcgtagtg ctgatgaaga taatgagaag ttcttgctaa agttcaggaa cagaattgat 360
agggttgggc ttgatttgcc aaaaattgaa gtcaagtttg agcatttgac tgttgaggcc 420
gatattaata caggaagcag agctttacct agttttataa acttccatat tgatatattt 480
gagatgttct tgagcttatt ccgtctactt ccaaatacga aaaaacatat aacaatcctt 540
gacgatgtta gcgggcttgt taagcctagc agaatgacgt tacttttggg acctccaagt 600
tctgggaaga cgacattgtt gttagccttg gctggaaagc tcgataagga gcttaagagc 660
tcagggaagg tgacatacaa tgggcatgag ttacatgaat ttgtacctca aagaacctct 720
gcttatatca gtcaagatga tgtgcatatc ggagaaatga ctgtcagaga aaccttggct 780
ttctctgcac gatgccaagg ggtcggatcg cgttatgaga tgttggccga gctgtcaaga 840
agagaaaaag atgcaaacat taaacctgat cctgatgttg atgtcttcat gaagtctgcg 900
gcatcagaag gtcaagaagc gaatgtggtg acagattaca ctcttaagat gttggggttg 960
gacgtttgtg cagataccat ggtaggggat caaatgatta gggggatatc cggtgggcaa 1020
aagaagcgtg ttacaacagg tgaaatgata gttggaccat cgaatgttct tctcatggat 1080
gagatttcaa ctggtttgga tagttctaca acttttcaaa ttgtgaaatc gtttagacaa 1140
tatcttcata tccttgaagc aactgctgtt atttctcttc tccaaccagc acccgagaca 1200
tataatttat ttgatgacat tatactttta actgagggga aaatagtgta ccaaggacca 1260
cgtgacaatg tgctagagtt ttttgaattt atggggttca aatgccccga gaggaaaggc 1320
gttgcggatt ttttgcaaga agtgacttcg agaaaagatc aacagcaata ctggatgaga 1380
agaaatgagg attacagatt cgtatcagcc aaggaattcg ctgattcttt ccaatcattc 1440
catattggaa agagactgaa ggaggatcta gccaccccgt atgacaaatc cagaagccac 1500
ccggctgctc tcactacaga gaagtacggt ttaaataaga aagagctctt gaaagcttgc 1560
atcgagagag agatcttgct tatgaagaga aactcatttg tttactactt caaattgtcc 1620
caactacttt tgatgtcgct agttgctatg actgtatttt tccgaaccga gatgagcaaa 1680
gataacgtgg aagatggagg gatatatatg ggtgctctat tctttggtgt tattatgatc 1740
atgtttaatg ggatggctga gatttcaatg acaattgcca agcttcctgt gttctacaaa 1800
caacgagact tcctgtttta cccctcgtgg gcatacgctc ttccatcatg gatagtcaag 1860
atccccgttt cgtttattga agttgccctg tggacgattc tcacttacta tgtgattgga 1920
tttgatccca atatcacaag attcttccgg cagtactttt tactcttaat tgtaaaccag 1980
atgtctgctg cattgtttag attcattgga gcaatgggac ggaacatgat tgttgcaaat 2040
acattcggtt catttgccct tctcataatg tttgcattgg gtggctttgt cctagcacga 2100
gatgatgtaa agaagtggtg gttatggggt tactggacgt cgccaatgat gtatgcgatg 2160
aatgggattg tagttaatga atttctctca aacagctgga ataagcctat aaatgatact 2220
acactaggga aaagtatcat cacctctcga ggcttcttca cggatgctta ctggtattgg 2280
cttggtgttg tggcctcagt tggatttatt tttttcttca acttgtgttt cggtttgtct 2340
cttgcgtttc tcaacccatt tgggaagtcc cgatctactg tatcacagaa tgatagtgac 2400
aaagattcag ttgagttatc atctagtgac gagagaaata aaaacaagaa gaaaggaatg 2460
gttcttcctt ttgagccaca ttcaattacc ttcaacgatg tcaaatactc ggtcgatatg 2520
ccacaggaaa tgagagagca aggaacgaac gaagctagat tgacgctact taagagtgtg 2580
agcggagctt ttcgacccgg tgttctaact gcgctaatgg gggtgagtgg cgcgggtaaa 2640
actactttga tggatgtgct agcgggtaga aaaactggtg ggtatataga gggggacatc 2700
cgaatttccg ggtatcctaa gaaacaagaa acattcgctc gaatttctgg atattgtgag 2760
caaaatgata ttcactcacc tcatgtgacg gtttacgagt ctttgctcta ctcagcttgg 2820
ctaaggttgt caaccgatga tgaaggaacc cgcaagatgt ttgtcgatga agtgatggaa 2880
cttgtggaac tgaacccatt gagggacgcg ctagttgggt tgcctggtgt caacggactc 2940
tcaacagaac agcgaaaaag gttaaccata gcagttgagc tcgttgctaa tccgtctata 3000
atatttatgg acgaaccaac atctgggctt gatgctagag cagctgctat tgtgatgaga 3060
accgttagaa acacagttga cacaggaaga acggttgtgt gcaccattca tcaacctagc 3120
atcgatatat ttgaagcttt tgatgagcta ttcttgctga aaagaggggg agaagagata 3180
tatgtaggac ctgtaggtcg caattcttgc gagttgataa agtactttga ggatatcaac 3240
ggggtaagta agattcaaga tggatataac cctgcaactt ggatgctgga agttagtact 3300
tcggcgcaag aactaatgct aggcattgat tttgcagaca tctaccggaa ttcagaacta 3360
tttaagagaa acaaggccct tattgcagaa ttaagtgtac cacgtcctgg tacacaagac 3420
ctctattttc ctacccgata ctcgcagtct ttttctgtcc agtgtattgc ttgcctttgg 3480
aaacaacgac tgtcttactg gagaaaccct ccttatactg ctgtccgttt tgcattcacc 3540
acctttatcg gcatcatgtt cggtacaatg ttctgggata ttggccgtaa acggaaaaac 3600
caacaagaac tgtttaatgc aatgggttcc atgtatacag caagtctctt cctaggagtt 3660
caaaacgcat cagcagtgca gcctgttgta gacatagaac gcactgtatt ttacagagaa 3720
cgagctgcag gaatgtattc cgctttacca tacgcctgtg ctcagattct ggtggagata 3780
ccttacatct tctcacaaac aatagtttat tgtctaatag tttatgccat gattggattc 3840
gaatggacag cttccaaatt cttttggtac gtcttcttcc agttctgctc tttactctac 3900
atgacatact atggcatgat gactgtcgcc atcaccccaa atgccaacat tgccgctatc 3960
gtcgcagctc tattttacgg tttctttaat ctattctctg gtttcatcat cccacgaacc 4020
aaaattccgg tttggtggcg atggtactat tggtgtaacc cgctagcttg gaccatctac 4080
ggcatggtgg tgtcacagtt cggggactat aaggatttgc ttgtaaacgg tgagaccgtg 4140
aaggagtacc tagatcgata ttttggctac aaacatgatt ttcttggtat tatcgcggga 4200
gtgcatgtgg gattagtact tgctttcggg tttatatttg cttattgcat tagagcattt 4260
aatttccaaa agagatag 4278
<210> 2
<211> 1429
<212> prt
<213>Herba Artemisiae Annuae (artemisia annua l.)
<400> 2
met asp gly ser asp ile tyr lys ala ser ser ser leu arg leu gly
5 10 15
ser asn ser gly arg met gly ser ile arg ala gly ser ser thr arg
20 25 30
trp arg asn thr gly met asp val phe ser arg ser thr arg glu glu
35 40 45
asp asp glu glu ala leu lys trp ala ala leu glu lys leu pro thr
50 55 60
tyr asp arg leu lys lys gly leu ile phe gly ser thr gly pro ser
65 70 75 80
glu glu val asp val ala ser leu gly phe glu glu arg lys arg leu
85 90 95
leu glu arg leu val arg ser ala asp glu asp asn glu lys phe leu
100 105 110
leu lys phe arg asn arg ile asp arg val gly leu asp leu pro lys
115 120 125
ile glu val lys phe glu his leu thr val glu ala asp ile asn thr
130 135 140
gly ser arg ala leu pro ser phe ile asn phe his ile asp ile phe
145 150 155 160
glu met phe leu ser leu phe arg leu leu pro asn thr lys lys his
165 170 175
ile thr ile leu asp asp val ser gly leu val lys pro ser arg met
180 185 190
thr leu leu leu gly pro pro ser ser gly lys thr thr leu leu leu
195 200 205
ala leu ala gly lys leu asp lys glu leu lys ser ser gly lys val
210 215 220
thr tyr asn gly his glu leu his glu phe val pro gln arg thr ser
225 230 235 240
ala tyr ile ser gln asp asp val his ile gly glu met thr val arg
245 250 255
glu thr leu ala phe ser ala arg cys gln gly val gly ser arg tyr
260 265 270
glu met leu ala glu leu ser arg arg glu lys asp ala asn ile lys
275 280 285
pro asp pro asp val asp val phe met lys ser ala ala ser glu gly
290 295 300
gln glu ala asn val val thr asp tyr thr leu lys met leu gly leu
305 310 315 320
asp val cys ala asp thr met val gly asp gln met ile arg gly ile
325 330 335
ser gly gly gln lys lys arg val thr thr gly glu met ile val gly
340 345 350
pro ser asn val leu leu met asp glu ile ser thr gly leu asp ser
355 360 365
ser thr thr phe gln ile val lys ser phe arg gln tyr leu his ile
370 375 380
leu glu ala thr ala val ile ser leu leu gln pro ala pro glu thr
385 390 395 400
tyr asn leu phe asp asp ile ile leu leu thr glu gly lys ile val
405 410 415
tyr gln gly pro arg asp asn val leu glu phe phe glu phe met gly
420 425 430
phe lys cys pro glu arg lys gly val ala asp phe leu gln glu val
435 440
thr ser arg lys asp gln gln gln tyr trp met arg arg asn glu asp
450 455 460
tyr arg phe val ser ala lys glu phe ala asp ser phe gln ser phe
465 470 475 480
his ile gly lys arg leu lys glu asp leu ala thr pro tyr asp lys
485 490 495
ser arg ser his pro ala ala leu thr thr glu lys tyr gly leu asn
500 505 510
lys lys glu leu leu lys ala cys ile glu arg glu ile leu leu met
515 520
lys arg asn ser phe val tyr tyr phe lys leu ser gln leu leu leu
530 535 540
met ser leu val ala met thr val phe phe arg thr glu met ser lys
545 550 555 560
asp asn val glu asp gly gly ile tyr met gly ala leu phe phe gly
565 570 575
val ile met ile met phe asn gly met ala glu ile ser met thr ile
580 585 590
ala lys leu pro val phe tyr lys gln arg asp phe leu phe tyr pro
595 600
ser trp ala tyr ala leu pro ser trp ile val lys ile pro val ser
610 615 620
phe ile glu val ala leu trp thr ile leu thr tyr tyr val ile gly
625 630 635 640
phe asp pro asn ile thr arg phe phe arg gln tyr phe leu leu leu
645 650 655
ile val asn gln met ser ala ala leu phe arg phe ile gly ala met
660 665 670
gly arg asn met ile val ala asn thr phe gly ser phe ala leu leu
675 680
ile met phe ala leu gly gly phe val leu ala arg asp asp val lys
690 695 700
lys trp trp leu trp gly tyr trp thr ser pro met met tyr ala met
705 710 715 720
asn gly ile val val asn glu phe leu ser asn ser trp asn lys pro
725 730 735
ile asn asp thr thr leu gly lys ser ile ile thr ser arg gly phe
740 745 750
phe thr asp ala tyr trp tyr trp leu gly val val ala ser val gly
755 760
phe ile phe phe phe asn leu cys phe gly leu ser leu ala phe leu
770 775 780
asn pro phe gly lys ser arg ser thr val ser gln asn asp ser asp
785 790 795 800
lys asp ser val glu leu ser ser ser asp glu arg asn lys asn lys
805 810 815
lys lys gly met val leu pro phe glu pro his ser ile thr phe asn
820 825 830
asp val lys tyr ser val asp met pro gln glu met arg glu gln gly
835 840
thr asn glu ala arg leu thr leu leu lys ser val ser gly ala phe
850 855 860
arg pro gly val leu thr ala leu met gly val ser gly ala gly lys
865 870 875 880
thr thr leu met asp val leu ala gly arg lys thr gly gly tyr ile
885 890 895
glu gly asp ile arg ile ser gly tyr pro lys lys gln glu thr phe
900 905 910
ala arg ile ser gly tyr cys glu gln asn asp ile his ser pro his
915 920
val thr val tyr glu ser leu leu tyr ser ala trp leu arg leu ser
930 935 940
thr asp asp glu gly thr arg lys met phe val asp glu val met glu
945 950 955 960
leu val glu leu asn pro leu arg asp ala leu val gly leu pro gly
965 970 975
val asn gly leu ser thr glu gln arg lys arg leu thr ile ala val
980 985 990
glu leu val ala asn pro ser ile ile phe met asp glu pro thr ser
995 1000
gly leu asp ala arg ala ala ala ile val met arg thr val arg asn
1010 1015 1020
thr val asp thr gly arg thr val val cys thr ile his gln pro ser
1025 1030 1035 1040
ile asp ile phe glu ala phe asp glu leu phe leu leu lys arg gly
1045 1050 1055
gly glu glu ile tyr val gly pro val gly arg asn ser cys glu leu
1060 1065 1070
ile lys tyr phe glu asp ile asn gly val ser lys ile gln asp gly
1075 1080
tyr asn pro ala thr trp met leu glu val ser thr ser ala gln glu
1090 1095 1100
leu met leu gly ile asp phe ala asp ile tyr arg asn ser glu leu
1105 1110 1115 1120
phe lys arg asn lys ala leu ile ala glu leu ser val pro arg pro
1125 1130 1135
gly thr gln asp leu tyr phe pro thr arg tyr ser gln ser phe ser
1140 1145 1150
val gln cys ile ala cys leu trp lys gln arg leu ser tyr trp arg
1155 1160
asn pro pro tyr thr ala val arg phe ala phe thr thr phe ile gly
1170 1175 1180
ile met phe gly thr met phe trp asp ile gly arg lys arg lys asn
1185 1190 1195 1200
gln gln glu leu phe asn ala met gly ser met tyr thr ala ser leu
1205 1210 1215
phe leu gly val gln asn ala ser ala val gln pro val val asp ile
1220 1225 1230
glu arg thr val phe tyr arg glu arg ala ala gly met tyr ser ala
1235 1240
leu pro tyr ala cys ala gln ile leu val glu ile pro tyr ile phe
1250 1255 1260
ser gln thr ile val tyr cys leu ile val tyr ala met ile gly phe
1265 1270 1275 1280
glu trp thr ala ser lys phe phe trp tyr val phe phe gln phe cys
1285 1290 1295
ser leu leu tyr met thr tyr tyr gly met met thr val ala ile thr
1300 1305 1310
pro asn ala asn ile ala ala ile val ala ala leu phe tyr gly phe
1315 1320
phe asn leu phe ser gly phe ile ile pro arg thr lys ile pro val
1330 1335 1340
trp trp arg trp tyr tyr trp cys asn pro leu ala trp thr ile tyr
1345 1350 1355 1360
gly met val val ser gln phe gly asp tyr lys asp leu leu val asn
1365 1370 1375
gly glu thr val lys glu tyr leu asp arg tyr phe gly tyr lys his
1380 1385 1390
asp phe leu gly ile ile ala gly val his val gly leu val leu ala
1395 1400
phe gly phe ile phe ala tyr cys ile arg ala phe asn phe gln lys
1410 1415 1420
arg
1425
<210> 3
<211> 25
<212> dna
<213> aapdr3-fp1
<400> 3
ttctcgtagg gctttttgag catta 25
<210> 4
<211> 27
<212> dna
<213> aapdr3-rp1
<400> 4
gccatacaaa gacgctaata gaactca 27
<210> 5
<211> 27
<212> dna
<213> aapdr3-rnai-fp
<400> 5
caccatggat ggaagtgata tttataa 27
<210> 6
<211> 24
<212> dna
<213> aapdr3-rnai-rp
<400> 6
catcaacttc ttctgaaggt ccag 24
<210> 7
<211> 28
<212> dna
<213> bamhi-aapdr3-fp
<400> 7
cgggatccat ggatggaagt gatattta 28
<210> 8
<211> 28
<212> dna
<213> aapdr3-xbai-rp
<400> 8
gctctagact atctcttttg gaaattaaat gc 32
<210> 9
<211> 28
<212> dna
<213> phellsgate-35s-fp
<400> 9
cgaaaggaca gtagaaaagg aaggtggc 28
<210> 10
<211> 22
<212> dna
<213> 35sf
<400> 10
gaagatgcct ctgccgacag tg 22
<210> 11
<211> 22
<212> dna
<213> aapdr3-rp
<400> 11
aagcccgcta acatcgtcaa gg 22
Claims (10)
1. a kind of Herba Artemisiae Annuae transport protein aapdr3 is it is characterised in that its aminoacid sequence is as shown in seq id no.2；This Herba Artemisiae Annuae
Transport protein aapdr3 affects the synthesis of sesquiterpene stone column alkene in Herba Artemisiae Annuae nonsecreting type glandular hair.
2. Herba Artemisiae Annuae transport protein aapdr3 according to claim 1, is characterized in that, described transport protein is by such as seq id
Nucleotide sequence coded shown in no.1.
3. Herba Artemisiae Annuae transport protein aapdr3 according to claim 1 and 2, is characterized in that, the transport protein of aapdr3 containing Herba Artemisiae Annuae
The plant rnai interference expression vector of coded sequence can suppress the synthesis of sesquiterpene stone column alkene and improve artemisinin synthesis, plant
Rnai Overexpression vector can improve the synthesis of sesquiterpene stone column alkene.
4. a kind of polypeptide is it is characterised in that aminoacid sequence is as shown in seq id no.4.
5. a kind of transgene abrotanum Seedling implementation method with Herba Artemisiae Annuae transport protein aapdr3 is it is characterised in that include following walking
Rapid:
Step one, the albumen coded sequence of Herba Artemisiae Annuae transport protein aapdr3 is respectively connected to plant rnai interference and overexpression carries
On body, build the plant rnai interference obtaining the transport protein coding sequence of aapdr3 containing Herba Artemisiae Annuae and Overexpression vector；
Step 2, plant rnai interference expression vector and Overexpression vector is proceeded to Agrobacterium, then Agrobacterium is proceeded to Herba Artemisiae Annuae；
Step 3, obtains Herba Artemisiae Annuae resistance Seedling by antibiotic-screening, then the plant through pcr test positive is transgene abrotanum
Seedling.
6. method according to claim 5, is characterized in that, described plant rnai interference expression vector is:
Phellsgate1.2 aapdr3, obtains in the following manner:
The structure of step 1. intermediate carrier ptopo aapdr3: the non-conservative region of aapdr3 gene design forward primer and under
Trip primer, in order to build interference carrier, adds tetra- bases of cacc to build gateway entry vector, first before forward primer
Obtain the fragment of aapdr3 with flush end enzymatic amplification, after recovery purifying, pentr/d topo is connected to by gateway clone technology
Carrier；
Step 2. plant expresses the structure of interference carrier phellsgate1.2 aapdr3: by ptopo aapdr3 carrier
The interference fragment of aapdr3 is recombinated two restructuring positions that can form hairpin structure of rna interference carrier phellsgate1.2
In point, obtain the rna interference carrier that can suppress the synthesis of sesquiterpene stone column alkene and the aapdr3 of raising artemisinin synthesis
phellsgate1.2‐aapdr3.
7. method according to claim 5, is characterized in that, described Overexpression vector is: phb aapdr3, by inciting somebody to action
Aapdr3 gene constructed on Overexpression vector, the structure of expression vector for convenience, forward primer bamhi aapdr3 fp
In introduce the restriction enzyme site of bamhi, introduce the restriction enzyme site of xbai in reverse primer aapdr3 xbai rp, through double enzymes
Connect after cutting, obtain improving the recombiant plasmid phb aapdr3 of sesquiterpene stone column alkene synthesis.
8. method according to claim 5, is characterized in that, described proceeds to particularly as follows: being proceeded to using freeze-thaw method.
9. method according to claim 5, is characterized in that, described pcr detection refers to, separately designs synthesis aapdr3 base
The detection primer of cause, carries out dna amplification, and the positive strain of viewed under ultraviolet radiation to purpose band is transgene abrotanum plant.
10. a kind of application of the transgene abrotanum Seedling realized based on arbitrary methods described in claim 59 it is characterised in that
Use it for improving Herba Artemisiae Annuae terpene substances content；
Described Herba Artemisiae Annuae terpene substances, measure analysis by described transgene abrotanum Seedling is carried out with gc ms, and then obtain sesquiterpene
Stone column alkene, β farnesene and gima ethylenic d.
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| CN113755504A (en) * | 2021-09-29 | 2021-12-07 | 西南大学 | Artemisia apiacea mediator AaMED25 gene and application thereof |
| CN114106120A (en) * | 2021-09-29 | 2022-03-01 | 上海交通大学 | Heavy metal transport protein PhHMA5II-1 and related product and application thereof |
| WO2023041649A1 (en) * | 2021-09-20 | 2023-03-23 | Basf Plant Science Company Gmbh | Coumarin transporters and uses thereof |
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| CN102604987A (en) * | 2012-01-17 | 2012-07-25 | 上海交通大学 | Method for improving artemisinin content in Artemisia annua L. by DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase) gene transfer |
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