CN105925577A - Promoter for regulating and controlling predominant expression of gene in glandular secretory trichome based cells and application of promoter - Google Patents
Promoter for regulating and controlling predominant expression of gene in glandular secretory trichome based cells and application of promoter Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 230000000762 glandular Effects 0.000 title claims abstract description 37
- 230000003248 secreting effect Effects 0.000 title claims abstract description 24
- 230000001276 controlling effect Effects 0.000 title abstract 3
- 230000001105 regulatory effect Effects 0.000 title abstract 3
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 238000010353 genetic engineering Methods 0.000 claims abstract description 7
- 238000009395 breeding Methods 0.000 claims abstract description 6
- 230000001488 breeding effect Effects 0.000 claims abstract description 6
- 210000004209 hair Anatomy 0.000 claims description 30
- 241000589158 Agrobacterium Species 0.000 claims description 13
- 210000000270 basal cell Anatomy 0.000 claims description 11
- 239000007943 implant Substances 0.000 claims description 9
- 108020004414 DNA Proteins 0.000 claims description 8
- 108091062157 Cis-regulatory element Proteins 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
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- 108010072454 CTGCAG-specific type II deoxyribonucleases Proteins 0.000 claims description 3
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- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 description 7
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- 239000012634 fragment Substances 0.000 description 6
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- OTYVBQZXUNBRTK-UHFFFAOYSA-N 3,3,6-trimethylhepta-1,5-dien-4-one Chemical compound CC(C)=CC(=O)C(C)(C)C=C OTYVBQZXUNBRTK-UHFFFAOYSA-N 0.000 description 2
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- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 description 1
- 235000003826 Artemisia Nutrition 0.000 description 1
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- 240000000011 Artemisia annua Species 0.000 description 1
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- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 241000223260 Trichoderma harzianum Species 0.000 description 1
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- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
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- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
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- 238000012772 sequence design Methods 0.000 description 1
- 229930009674 sesquiterpene lactone Natural products 0.000 description 1
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- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8225—Leaf-specific, e.g. including petioles, stomata
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Abstract
The invention discloses a promoter for regulating and controlling predominant expression of a gene in glandular secretory trichome based cells and an application of the promoter. The gene promoter disclosed by the invention belongs to the field of plant biotechnology has a nucleotide sequence as shown in SEQ ID NO: 1, and the gene promoter is capable of regulating and controlling the predominant expression of the target gene in the glandular secretory trichome based cells. Meanwhile, the invention also relates to an application of the gene promoter in genetic engineering breeding of expressing and producing metabolic products by virtue of plant glandular trichome tissues. The promoter disclosed by the invention, which is free from damage to the growth and development of a plant when used for conducting genetic operation on the glandular trichome system of the plant on the basis of the specific expression of plant glandular trichome, has an important significance to the genetic engineering breeding of expressing and producing the metabolic products by virtue of the plant glandular trichome tissues.
Description
Technical field
The present invention relates to plant biotechnology field, particularly relate to a kind of controlling gene excellent in secreting type glandular hair basal cell
The promoter of gesture expression and application thereof.
Background technology
Herba Artemisiae Annuae (Artemisia annua L.) is Compositae artemisia annual herb plant, and plant has strong volatility fragrant
Gas, containing many secondary metabolites in its plant: arteannuin, volatile oil, australene, Camphora, artemisia ketone etc.,
Additionally contain flavone compound.Arteannuin (Artemisinin) is to contain from the isolated one of its aerial parts
The sesquiterpene lactones compound of peroxide bridge structure, the anti-malaria medicaments associating recommended as World Health Organization (WHO) (WHO)
The principle active component for the treatment of (Artemisinin-based combination therapies, ACTs).Arteannuin at present
Main source be to extract from the aerial parts of Herba Artemisiae Annuae, but the content of Artemisinin in Artemisia annuna the lowest (0.01%-1%),
This makes the large-scale commercial of this medicine produce and receives greatly restriction.
The blade surface of Herba Artemisiae Annuae has two kinds of glandular hairs: secreting type glandular hair (glandular secretory trichome, GST) and
Nonsecreting type glandular hair (T shaped trichome, TST), two kinds of glandular hairs for plant growth, grow, defend, pollen
Propagate to wait and all play vital effect.Wherein, arteannuin only synthesizes in the secreting type glandular hair of Herba Artemisiae Annuae, secreting type
Glandular hair is that Herba Artemisiae Annuae blade surface one has ten cyto-architectural specialization organs, be by two basal cells, two stalk cells,
The sack cavity structure of six secreting type cells and outside is constituted.Promoter is in the specific of structural gene 5' end upstream
Nucleotide sequence, it is possible to the expression of regulation and control downstream gene, promoter just as one " switch ", by cis acting element with
The interaction of trans acting factor plays its specific function.Promoter one is divided into three kinds: constitutive promoter,
Specific promoter, inducible promoter.In the genetic engineering research of current Herba Artemisiae Annuae, use constitutive promoter more,
Such as cauliflower mosaic virus 35 S promoter (CaMV35S), this promoter can drive exogenous gene all
Tissue and organ in express, intracellular matter and energy can be consumed excessively, and can not adjust over time and space
The expression of control genes of interest, can cause certain burden and harm to the normal growth of plant most probably.Therefore it is badly in need of looking for
Constitutive promoter is replaced to promoter specific expressed in the histoorgan of plant, thus preferably to plant
The expression of gene regulates and controls.Owing to the promoter of glandular hair specifically expressing carries out genetic manipulation for the glandular hair system of plant,
The growth promoter of plant will not be worked the mischief, all drawbacks of constitutive promoter can be overcome.Therefore, the present invention
Endeavour to provide a kind of promoter being cloned into plant glandular hair organizing specific expression.
Summary of the invention
Because the drawbacks described above of prior art, the invention provides a kind of controlling gene in secreting type glandular hair basal cell
The promoter of predominant expression, can guiding gene specifically expressing in transgenic plant secreting type glandular hair.Above-mentioned promoter
For MYB family transcription factor gene promoter, its nucleotide sequence is as shown in SEQ ID NO.1.
Further, above-mentioned promoter is inducible promoter.
Present invention also offers the application in the genetic engineering breeding of plant production metabolite of the above-mentioned promoter.
Further, above-mentioned promoter is specific promoter, it is possible to replace constitutive promoter to guide external source base
Because of specifically expressing in plant secretion type glandular hair.
Present invention also offers a kind of recombinant expression carrier, it comprises the nucleotide sequence as shown in SEQ ID NO.1.
Present invention also offers a kind of recombinant expressed transformant, it comprises the nucleotides sequence as shown in SEQ ID NO.1
Row.
Present invention also offers a kind of controlling gene preparation side of the promoter of predominant expression in secreting type glandular hair basal cell
Method, comprises the following steps:
Step one, cultivation Herba Artemisiae Annuae aseptic seedling；
Step 2, clone's controlling gene promoter gene group sequence of predominant expression in secreting type glandular hair basal cell；
Step 3, analyze controlling gene cis acting above the promoter of predominant expression in secreting type glandular hair basal cell
Element, determines above-mentioned promoter and enhancer；
Step 4, the above-mentioned promoter being cloned into is connected in pCAMBIA1391z carrier by enzyme action；
Step 5, the vector Agrobacterium tumefaciems that will build in step 4；
Step 6, by Agrobacterium tumefaciems stable conversion Herba Artemisiae Annuae with carrier in step 5；
Step 7, PCR detect transfer-gen plant；
Step 8, determine gus reporter gene expressive site in plant that above-mentioned promoter is guided.
Further, above-mentioned steps two, with genomic DNA as template, uses the amplification of two-wheeled nested PCR method to start
Subsequence.
Further, the cis acting element in above-mentioned steps three includes: TATA box, CAAT box, G-box,
ARE、GATA-motif、GAG-motif、ABRE、HES。
In promoter sequence, controlling element see table 1:
Table 1: controlling element analysis in promoter sequence
Further, in above-mentioned steps four, for building pCAMBIA1391z carrier, forward primer introduces PstI
Restriction enzyme site, introduces BamHI restriction enzyme site in reverse primer, primer sequence is as follows:
1391z-proAaMIXTA1-F:AACTGCAGAAAGGCCCCCTTAAAAGATACG
1391z-proAaMIXTA1-R:CGGGATCCAATGAAGACGAAAACCGTCGC
Further, above-mentioned steps six specifically includes:
1) preculture of outer implant；
2) the co-culturing of Agrobacterium and outer implant；
3) screening of resistance regeneration plant.
The beneficial effects of the present invention is, utilize effective Herba Artemisiae Annuae epidermal hair tissue-specific promoter to replace composing type to open
Mover may be used for building in molecular biology at the fusion gene of Herba Artemisiae Annuae secreting type glandular hair specifically expressing genes of interest,
Utilize genetic transfoumation to be proceeded in the genome of other plant, thus the directional operation realizing genes of interest obtains
Obtain transgenic plant, and the growth promoter of plant will not be worked the mischief, can be widely used in and utilize plant glandular hair tissue
Express and in the genetic engineering breeding of production metabolite.
Below with reference to accompanying drawing, the invention will be further described, with absolutely prove the purpose of the present invention, technical characteristic and
Technique effect.
Accompanying drawing explanation
By the detailed description non-limiting example made with reference to the following drawings of reading, the further feature of the present invention,
Purpose and advantage will become more apparent upon:
Fig. 1 shows the AaMIXTA1 gene promoter sequence in a preferred embodiment of the present invention and cis work thereof
Use element；
Fig. 2 shows the PCR positive test symbol figure of transgene abrotanum plant；
Fig. 3 shows the carrier utilizing AaMIXTA1 gene promoter to merge with gus gene
After pCAMBIA1391z-pro AaMIXTA1 is by agriculture bacillus mediated stable conversion Herba Artemisiae Annuae, the transgenic obtained
GUS tissue staining figure in Herba Artemisiae Annuae；
Fig. 4 shows that AaMIXTA1 secreting type glandular hair base portion in Herba Artemisiae Annuae expresses schematic diagram.
Detailed description of the invention
Elaborating the present invention below in conjunction with instantiation, following instance will assist in the technology of this area
Personnel are further appreciated by the present invention and limit the present invention the most in any form.It should be pointed out that, to this area
For ordinary skill, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, this
Broadly fall into protection scope of the present invention.
The experimental technique of unreceipted actual conditions in following embodiment, generally according to normal condition, such as Sambrook
Equimolecular is cloned: laboratory manual is shown in the 1989 of New York:Cold Spring Harbor Laboratory Press
Condition described in year version, or according to the condition proposed by manufacturer.
The Agrobacterium tumefaciems EHA105 that the present embodiment relates to " Huang Yali, Jiang Xiliang, Yunlong, field, Guo Ping,
Zhu Changxiong；The research of Agrobacterium tumefaciens mediated trichoderma harzianum genetic transformation, Chinese biological engineering magazine,
2008,28 (3): 38-43 " disclosed in document.Agrobacterium tumefaciems EHA105, plasmid pCAMBIA1391z
Can obtain by disclosing commercially available commercial channel, as buied from CAMBIA company of Australia, bacterial strain is compiled
Number it is Gambar1.
Embodiment
The present embodiment relates to the acquisition of AaMIXTA1 gene promoter, specifically includes following steps:
Step one, cultivation Herba Artemisiae Annuae aseptic seedling
Seeds of southernwood volume fraction is soak with ethanol 1min of 75%, then soaks with the NaClO of 20% (w/v)
After bubble 20min, aseptic water washing 3 times～4 times, blots surface moisture with aseptic absorbent paper, is inoculated in without any
On the MS solid medium of hormone, 25 DEG C, 16h/8h (light/dark) illumination cultivation, can obtain after 14 days
Herba Artemisiae Annuae aseptic seedling；
The clone of promoter sequence in step 2, genomic DNA
1. the extraction of genomic DNA
In 1.5mL centrifuge tube, add 2 steel balls, put a piece of Herba Artemisiae Annuae blade (1cm wherein2Left and right size,
Contain with ice chest and take).Add 300 μ L TPS buffer (operation in fume hood, containing 2% mercaptoethanol in TPS),
55-60Hz, shakes 90 seconds.Add 300 μ L TPS buffer (fume hood).65 DEG C of water-bath 1h are (every 20min
Shake), can the proper extension time, the longest 1.5h.Being cooled to room temperature, 4 DEG C of 12000rpm are centrifuged 15min.
Take 300 μ L-400 μ L of supernatant liquid.Equal-volume adds 300 μ L-400 μ L isopropanol (isopropanol is-20 DEG C of pre-coolings).
In-20 DEG C of refrigerators, 10-15min (1h can be extended to) is placed after mixing.Take out 4 DEG C of 12000rpm to be centrifuged
10min, sucks supernatant, is inverted 10-15min in fume hood.Add 75% ethanol 500-600 μ L, will precipitation
Blow and beat or upspring with finger, being placed on shaking table and shake 15-20min, be repeated once.Blot liquid, be placed on 37 DEG C
Middle drying, until precipitation becomes transparent.Add 50 μ L ddH2O back dissolving, 4 DEG C of preservations.
2.PCR expands
With genomic DNA as template, PCR method is utilized to expand secreting type glandular hair specific promoter sequence.For
Improve the specificity of product, use two-wheeled nest-type PRC (Nested PCR) amplification, according to this laboratory base
As shown in table 2 because organizing the promoter sequence design nest-type PRC primer of the AaMIXTA1 gene that order-checking obtains, first
Wheel PCR reaction system is as shown in table 3.PCR condition is: 95 DEG C of denaturations 5min；94 DEG C of degeneration 30s；55
DEG C annealing 30s；72 DEG C extend 3min, 35 circulations；72 DEG C extend 10min.In the agarose gel of 1%
Electrophoresis detection PCR primer.
Table 2 nest-type PRC design of primers
Table 3 first round PCR reaction system
By the product dilution 100 times of first run PCR, carrying out second as template and take turns PCR, second takes turns PCR reaction
System is shown in Table 4.PCR condition is: 95 DEG C of denaturations 5min；94 DEG C of degeneration 30s；55 DEG C of annealing 30s；72℃
Extend 3min, 35 circulations；72 DEG C extend 10min.In the agarose gel of 1%, electrophoresis detection PCR is produced
Thing, cuts glue and reclaims purpose fragment, purification DNA.It is then attached to pMD18-T (purchased from TaKaRa company)
Carrier is used for checking order, and the sequence of this fragment is spliced with gene order, it is thus achieved that AaMIXTA1 upstream region of gene
The fragment of 2569bp.
PCR reaction system taken turns by table 4 second
The cis acting element of the AaMIXTA1 gene promoter that step 3, analysis obtain, determines AaMIXTA1
The type of gene promoter
The a length of 2569bp of AaMIXTA1 gene promoter sequence obtained in the present invention.In order to find promoter
Cis acting element above, with Plantcare (http://bioinformatics.psb.ugent.be/webtools/
Plantcare/html/) promoter of AaMIXTA1 gene is analyzed.Analyze the startup finding that polyclone arrives
Son has except TATA box and CAAT box above, also have a lot of cis acting element: G-box, ARE,
ABRE-motif, GATA-motif, HES etc.；G-box is found in a lot of plant promoters, and it is
Element necessary to a lot of response promoter functionatings, and plants light, oxygen-free environment at plant promoter
The response process of thing hormone plays critically important effect；Additionally, the regulation and control of GA-motif light, ABRE-motif
Abscisic acid can be responded in plant；Result above analysis shows, AaMIXTA1 gene promoter is an induction
The promoter of type, can be induced by multiple hormone and natural cause.AaMIXTA1 gene promoter sequence and cis
Functional element is shown in Fig. 1.
Step 4, the promoter obtained is connected into pCAMBIA-1391z carrier, merges gus reporter gene.
In order to study gene promoter expression in plant different tissues position, by opening of AaMIXTA1 gene
Mover proAaMIXTA1 connect pCAMBIA-1391z carrier and merge gus reporter gene, in order to realize table
Reach the structure of carrier, forward primer introduce PstI restriction enzyme site, reverse primer introduces BamHI restriction enzyme site,
Primer sequence is as shown in the table:
Table 5pCAMBIA1391z-proAaMIXTA1 vector construction PCR primer
Step 5, by the pCAMBIA1391z-proAaMIXTA1 vector Agrobacterium tumefaciems that builds also
Detection.
Agrobacterium tumefaciems (EHA105), performing PCR of going forward side by side will be proceeded to containing the plant binary expression vector built
Checking.Result shows: the plant binary expression vector containing promoter gene fragment is successfully building up to crown gall agriculture
In bacillus strain, thus obtain the plant expression vector pCAMBIA merged containing gene promoter and gus gene
In the Agrobacterium tumefaciens strain of 1391z-proAaMIXTA1.
Step 6, by the Agrobacterium tumefaciens transformation Herba Artemisiae Annuae with pCAMBIA1391z-proAaMIXTA1 carrier
1) preculture of outer implant
Seeds of southernwood volume fraction is soak with ethanol 1min of 75%；Soak with the NaClO of 20% (w/v) again
Bubble 20min；Aseptic water washing 3～4 times；Surface moisture is blotted with aseptic absorbent paper；It is inoculated in without hormone
MS, this MS culture medium uses Murashige and Skoog at the solid medium of invention in 1962, this solid
Culture medium can be obtained by commercial channel；The night of 25 DEG C, the daylight of 16 hours and 8 hours cultivates,
Obtaining Herba Artemisiae Annuae aseptic seedling, after Seedling length to about 5cm, the outer implant of clip tests for sterility is used for converting；
2) the co-culturing of Agrobacterium and outer implant
By described leaf explant, forward 1/2MS and 100 μm ol/L AS composition to co-cultures in culture medium,
Dropping is containing the described Agrobacterium tumefaciems engineering bacteria containing DEL0 to DEL8 gene plant binary expression vector activated
1/2MS suspension, make outer implant be fully contacted with bacterium solution, 28 DEG C of light culture 3d, with dropping without purpose
The leaf explant of the 1/2MS fluid medium suspension of the Agrobacterium tumefaciems of gene is comparison；
3) screening of resistance regeneration plant
Outer for the described Herba Artemisiae Annuae co-culturing 3d implant is transferred to MS, 0.5mg/L 6-BA, 0.05mg/L NAA,
In the germination screening culture medium of 50mg/L Kan and 500mg/L Cb composition, in 25 DEG C, the daylight of 16 hours
(light) cultivating in the dark (dark) of and 8 hours, successive transfer culture is once every two weeks, through 2-3 subculture
After can obtain Kan resistance Multiple Buds, well-grown resistance Multiple Buds is cut and proceeds to 1/2MS0With
Cultivate to taking root on the root media of 125mg/L Cb composition, thus obtain Kan resistance regeneration Herba Artemisiae Annuae plant；
Step 7, PCR detect transfer-gen plant
Forward is separately designed according to genes of interest place expression cassette promoter-GUS sequence promoter and GUS
Primer (proAaMIXTA1F:CTCCGTATTATCTTATACTAGTA) and reverse primer (GUSR:
GACATCGGCTTCAAATGGCGTA) gus gene is detected；Result shows, utilizes designed
PCR special primer, specific DNA fragment can be amplified, and with non-transformed Herba Artemisiae Annuae genomic DNA as template
Time, do not amplify any fragment, as shown in Figure 2；
The determination of the gus reporter gene that step 8, promoter are guided expressive site in plant
Herba Artemisiae Annuae plant to PCR test positive, takes its blade and carries out GUS tissue staining, result such as Fig. 3
Shown in；Fig. 4 then shows that AaMIXTA1 secreting type glandular hair base portion in Herba Artemisiae Annuae expresses schematic diagram；Result shows,
Dyeing part is predominant expression in the secreting type glandular hair base portion of Herba Artemisiae Annuae, illustrates that proAaMIXTA1 gene promoter exists
Transgene abrotanum can guide exogenous gene specifically expressing in glandular hair, it can be seen that, the present invention is cloned
ProAaMIXTA1 gene promoter can be used for utilizing plant glandular hair tissue expression and producing the gene work of metabolite
In journey breeding and industrialization.
The preferred embodiment of the present invention described in detail above.Should be appreciated that the ordinary skill of this area is without wound
The property made work just can make many modifications and variations according to the design of the present invention.Therefore, all technology in the art
Personnel can be obtained by logical analysis, reasoning, or a limited experiment the most on the basis of existing technology
The technical scheme arrived, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. controlling gene promoter of predominant expression in secreting type glandular hair basal cell, it is characterised in that described promoter is MYB family transcription factor gene promoter, and the nucleotide sequence of described promoter is as shown in SEQ ID NO.1.
Promoter the most according to claim 1, it is characterised in that described promoter is inducible promoter.
The promoter the most according to claim 1 and 2 application in the genetic engineering breeding of plant production metabolite.
4. a recombinant expression carrier, it is characterised in that described recombinant expression carrier comprises the nucleotide sequence as shown in SEQ ID NO.1.
5. a recombinant expressed transformant, it is characterised in that described recombinant expressed transformant comprises the nucleotide sequence as shown in SEQ ID NO.1.
6. controlling gene preparation method of the promoter of predominant expression in secreting type glandular hair basal cell, it is characterised in that described preparation method comprises the following steps:
Step one, cultivation Herba Artemisiae Annuae aseptic seedling；
Step 2, clone's controlling gene promoter gene group sequence of predominant expression in secreting type glandular hair basal cell；
Step 3, analyze controlling gene cis acting element above the promoter of predominant expression in secreting type glandular hair basal cell, determine described promoter and enhancer；
Step 4, the described promoter being cloned into is connected in pCAMBIA1391z carrier by enzyme action；
Step 5, the vector Agrobacterium tumefaciems that will build in step 4；
Step 6, by Agrobacterium tumefaciems stable conversion Herba Artemisiae Annuae with carrier in step 5；
Step 7, PCR detect transfer-gen plant；
Step 8, determine gus reporter gene expressive site in plant that described promoter is guided.
Preparation method the most according to claim 6, it is characterised in that described step 2, with genomic DNA as template, uses two-wheeled nested PCR method amplification promoter sequence.
Preparation method the most according to claim 6, it is characterised in that the described cis acting element in described step 3 includes: TATA box, CAAT box, G-box, ARE, GATA-motif, GAG-motif, ABRE, HES.
Preparation method the most according to claim 6, it is characterised in that in described step 4, for building pCAMBIA1391z carrier, introduces PstI restriction enzyme site, introduces BamHI restriction enzyme site in reverse primer in forward primer, primer sequence is as follows:
1391z-proAaMIXTA1-F:AACTGCAGAAAGGCCCCCTTAAAAGATACG
1391z-proAaMIXTA1-R:CGGGATCCAATGAAGACGAAAACCGTCGC.
Preparation method the most according to claim 6, it is characterised in that described step 6 specifically includes:
1) preculture of outer implant；
2) the co-culturing of Agrobacterium and outer implant；
3) screening of resistance regeneration plant.
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