CN105177007A - Promoter for regulating and controlling expression of genes in non-secretory type glandular hairs and application of promoter - Google Patents
Promoter for regulating and controlling expression of genes in non-secretory type glandular hairs and application of promoter Download PDFInfo
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- CN105177007A CN105177007A CN201510729014.1A CN201510729014A CN105177007A CN 105177007 A CN105177007 A CN 105177007A CN 201510729014 A CN201510729014 A CN 201510729014A CN 105177007 A CN105177007 A CN 105177007A
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Abstract
The invention discloses a promoter for regulating and controlling expression of genes in non-secretory type glandular hairs. The nucleotide sequence of the promoter is shown in SEQ ID No:1. The invention further provides a preparation method of the promoter, a carrier containing the promoter and an expression case. The promoter can regulate and control the expression of target genes in time and space, therefore, morphogenesis and development of the non-secretory type glandular hairs and metabolism regulation of secondary metabolites in the non-secretory type glandular hairs can be intensively studied, and application in genetic engineering breeding for producing the metabolites can be achieved. Therefore, the promoter for regulating and controlling the expression of the genes in the non-secretory type glandular hairs has important significance on genetic engineering breeding for expressing and producing the metabolites by means of plant glandular hair tissue.
Description
Technical field
The present invention relates to plant biotechnology field, be specifically related to a kind of terpene synthase gene promoter (proTPS7) of specifically expressing and application in transgenic plant thereof in plant nonsecreting type glandular hairs.
Background technology
Sweet wormwood (ArtemisiaannuaL.) is composite family artemisia annual herb plant, plant has strong volatile aroma, and the artemisia annua essential oil extracted from sweet wormwood glandular hairs contains the terpenoid and flavonoid compound in a large number with pharmacology or biocidal activity.The blade surface of sweet wormwood has two kinds of glandular hairs: secretor type glandular hairs (glandularsecretorytrichome, and nonsecreting type glandular hairs (Tshapetrichome GST), TST), two kinds of glandular hairs all play vital effect for the growth of plant, growth, defence and pollen transmission etc.Artemisinin (Artemisinin) is from the isolated a kind of sesquiterpene lactones compound containing peroxide bridge structure of its over-ground part, is the medicine that in current antimalarial drug, effect is best, toxicity is minimum.Artemisinin conjoint therapy (Artemisinin-basedcombinationtherapies, ACTs) has become the treatment of world health organisation recommendations.The main source of current Artemisinin extracts from the over-ground part of sweet wormwood, but the content of Artemisinin in Artemisia annuna is very low (0.01%-1%), and this makes the large-scale commercial of this medicine produce and receives very big restriction.
Promotor is positioned at the specific nucleic acid sequence that structure gene 5' holds upstream, and can regulate and control the expression of downstream gene, promotor, just as one " switch ", plays its specific function by the interaction of cis-acting elements and trans-acting factor.Promotor one is divided into three kinds: constitutive promoter, specific promoter, inducible promoter.In the genetically engineered research of current sweet wormwood, adopt constitutive promoter more, such as cauliflower mosaic virus 35 S promoter (CaMV35S), this promotor can drive foreign gene to express in all tissues and organ, intracellular matter and energy can be consumed excessively, and the expression of goal gene can not be regulated and controled over time and space, certain burden and harm can be caused to the normal growth of plant most probably.
Therefore, those skilled in the art is devoted to a kind of promotor specific expressed in the histoorgan of plant of exploitation to replace constitutive promoter, thus better regulates and controls the expression of plant gene.
Summary of the invention
Because the above-mentioned defect of prior art, technical problem to be solved by this invention is to provide a kind of promotor specific expressed in the histoorgan of plant, thus regulate and control the expression of goal gene over time and space, thus in order to the metabolic regulation of the initiation and development of furtheing investigate nonsecreting type glandular hairs and secondary metabolite wherein.
An aspect of of the present present invention provides the promotor that a kind of regulatory gene is expressed in nonsecreting type glandular hairs, and the nucleotide sequence of this promotor is as shown in SEQIDNo:1.This promotor, for being inducible promoter, is again specific promoter.This promotor is terpene synthase gene promoter.
Further, the cis-acting elements in this promotor comprises: CGTCA-motif, HSE, MBS, MRE, TC-richrepeats, WUN-motif.
Another aspect provides a kind of method preparing the promotor that above-mentioned regulatory gene is expressed in nonsecreting type glandular hairs, comprise the following steps:
Step one, from sweet wormwood genome, clone the genome sequence of this promotor; Wherein, sweet wormwood genome obtains from the sweet wormwood aseptic seedling of cultivating;
Step 2, the cis-acting elements analyzed in this promotor, determine this promoter and enhancer;
Step 3, the sequence of this promotor obtained in step one to be connected with carrier;
Step 4, the vector agrobacterium tumefaciens will obtained in step 3;
Step 5, the agrobacterium tumefaciens stable conversion plant with above-mentioned carrier will obtained in step 4; This plant is sweet wormwood;
Step 6, detect proceeding to of above-mentioned promotor, and determine the expressive site of gene in plant that above-mentioned promotor guides; The method that detection promotor proceeds to is that PCR detects, and the gene that promotor guides is gus reporter gene.
Further, in above-mentioned preparation method, it is template that step one comprises with genomic dna, adopts two-wheeled nested PCR method to increase described promoter sequence; Wherein, the primer of nest-type PRC is:
Further, in above-mentioned preparation method, carrier in step 3 is pCAMBIA1391z, utilize primer pair 1391z-proTPS7-F and 1391z-proTPS7-R, in the promoter sequence obtained, restriction enzyme site PstI and NcoI is introduced by PCR, thus be connected on carrier pCAMBIA1391z, wherein primer sequence is as follows: 1391z-proTPS7-F:5 '-AACTGCAGAAGAGAAAGCTACATACAATGGGAA-3 '; 1391z-proTPS7-R:5 '-CATGCCATGGGGAGAAAGATCGGTTATGC-3 '.
Further, in above-mentioned preparation method, step 5 comprises: the 1) preculture of explant; 2) Dual culture of Agrobacterium and explant; 3) screening of resistance regeneration plant.
Present invention also offers a kind of carrier, this carrier is connected with the promotor that above-mentioned regulatory gene is expressed in nonsecreting type glandular hairs.
Present invention also offers a kind of expression cassette, this expression cassette comprises the promotor that above-mentioned regulatory gene is expressed in nonsecreting type glandular hairs.
Present invention also offers promotor that above-mentioned regulatory gene expresses in nonsecreting type glandular hairs for utilizing plant glandular hairs tissue expression and the application in the genetic engineering breeding of plant production meta-bolites.
Present invention also offers the application in the metabolic regulation research of the initiation and development of nonsecreting type glandular hairs and secondary metabolite wherein of promotor that above-mentioned regulatory gene expresses in nonsecreting type glandular hairs.
Compared with prior art, the invention has the beneficial effects as follows: because the promotor of glandular hairs specifically expressing carries out genetic manipulation for the glandular hairs system of plant, can not work the mischief to growing of plant, all drawbacks of constitutive promoter can be overcome.Therefore, the promotor being cloned into plant glandular hairs organizing specific expression is of great significance for Artemisinin metabolic engineering tool.The present invention utilizes effective sweet wormwood epidermis glandular hairs tissue-specific promoter to replace constitutive promoter, be structured in the fusion gene at sweet wormwood nonsecreting type glandular hairs specifically expressing goal gene in molecular biology, genetic transfoumation is utilized to be proceeded in the genome of other plant, thus the directional operation realizing goal gene is to obtain transgenic plant, and can not work the mischief to growing of plant, can be widely used in and utilize plant glandular hairs tissue expression and produce in the genetic engineering breeding of meta-bolites.
Be described further below with reference to the technique effect of accompanying drawing to design of the present invention, concrete structure and generation, to understand object of the present invention, characteristic sum effect fully.
Accompanying drawing explanation
Fig. 1 is the PCR positive detection of the transgene abrotanum plant of a preferred embodiment of the present invention.Wherein, M: molecular weight marker; 1-2 swimming lane: the PCR primer being template with the genomic dna of transgene negative sweet wormwood plant; 3-7: the PCR primer being template with transgene abrotanum plant genomic dna; +: positive control;-: negative control.
Fig. 2 be the carrier pCAMBIA1391z-proTPS7 utilizing AaTPS7 gene promoter and gus gene to merge of a preferred embodiment of the present invention by after agriculture bacillus mediated stable conversion sweet wormwood, GUS tissue staining figure in the transgene abrotanum obtained.
Embodiment
The experimental technique of unreceipted actual conditions in following embodiment, usual conveniently condition, " molecular cloning: laboratory manual " (NewYork:ColdSpringHarborLaboratoryPress of the people such as such as Sambrook, version in 1989) described in condition, or according to the condition that manufacturer advises.
The agrobacterium tumefaciens EHA105 that the present embodiment relates to is at " 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 " open in document.Agrobacterium tumefaciens EHA105 and plasmid pCAMBIA1391z obtains by openly commercially available commercial channel, and as buied from Australian CAMBIA company, strain number is Gambar1.
Embodiment 1
The genome sequence of cloning promoter from sweet wormwood genome
1, sweet wormwood aseptic seedling is cultivated:
Seeds of southernwood volume fraction is the alcohol immersion 1min of 75%, after using the NaClO of 20% (w/v) to soak 20min again, aseptic water washing 3 times ~ 4 times, surface-moisture is blotted with aseptic thieving paper, be inoculated on the MS solid medium without hormone, 25 DEG C, 16h/8h (daylight/night) illumination cultivation, can obtain sweet wormwood aseptic seedling after 14 days;
2, the extraction of genomic dna
A slice sweet wormwood blade (1cm is put in 1.5mL centrifuge tube
2left and right size, contains with ice chest and gets), add 2 steel balls.Add 300 μ LTPSbuffer (operation in stink cupboard, containing mercaptoethanol in TPS), 55-60Hz, shakes 100 seconds.Add 300 μ LTPSbuffer (stink cupboard) again.65 DEG C of water-bath 1h (shaking every 20min), can the proper extension time, the longest 1.5h.Be cooled to room temperature, 4 DEG C of centrifugal 15min of 10000rpm.Get 300 μ L-400 μ L supernatant liquors.Add 300 μ L-400 μ L Virahol (Virahol is-20 DEG C of precoolings).In-20 DEG C of refrigerators, 10-15min (1h can be extended to) is placed after mixing.Take out 4 DEG C of centrifugal 12000rpm, suck supernatant, in stink cupboard, be inverted 10-15min.Add 75% ethanol 500-600 μ L, precipitation blown and beaten or has upspring with finger, being placed on shaking table and shaking 15-20min, repeat once.Blot liquid, be placed in 37 DEG C and dry, until precipitation becomes transparent.Add 50 μ LddH
2o back dissolving, namely obtains genomic dna, in 4 DEG C of preservations.
3, pcr amplification
With above-mentioned genomic dna for template, utilize PCR method amplification nonsecreting type glandular hairs specific promoter sequence.In order to improve the specificity of product, adopt two-wheeled nested PCR amplification, the promoter sequence design nest-type PRC primer of the AaTPS7 gene obtained according to gene order-checking is as shown in table 1, and first round PCR reaction system is as shown in table 2.PCR condition is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s; 48 DEG C of annealing 30s; 72 DEG C extend 2min, 35 circulations; 72 DEG C extend 10min.Electrophoresis detection PCR primer in the sepharose of 1%.
Table 1 nest-type PRC primer
Table 2 first round PCR reaction system
By the product dilution after first round PCR 50 times, carry out second as template and take turns PCR, second takes turns PCR reaction system in table 3.PCR condition is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s; 54 DEG C of annealing 30s; 72 DEG C extend 1min20s, 35 circulations; 72 DEG C extend 10min.Electrophoresis detection PCR primer in the sepharose of 1%, cuts glue and reclaims object fragment, purify DNA.Then be connected to pMD18-T (purchased from TaKaRa company) carrier and be used for order-checking, the sequence of this fragment and gene order are spliced, obtain the fragment that AaTPS7 upstream region of gene is about 1.1kb.
PCR reaction system taken turns by table 3 second
The sequence of the AaTPS7 gene promoter obtained is as follows:
Wherein, the positive-sense strand of shown sequence is namely consistent with the sequence shown in SEQIDNo:1.
Embodiment 2
Analyze the cis-acting elements of the AaTPS7 gene promoter obtained, determine the type of AaTPS7 gene promoter
The AaTPS7 gene promoter sequence length obtained in the present invention is 1167bp.In order to find the cis-acting elements in promotor, with Plantcare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), the promotor of AaTPS7 gene is analyzed.Analyze find polyclone to promotor on have except TATAbox and CAATbox, also there is a lot of cis-acting elements: CGTCA-motif, HSE, MBS, MRE, TC-richrepeats, WUN-motif etc.
In promoter sequence, cis-acting elements is in table 4:
Table 4: controlling element analysis in promoter sequence
In table 4, the position of-980 ,-907 ,-678 and-130 is see the corresponding position in the sequence listed in embodiment 1.
CGTCA-motif is methyl jasmonate response element; HES responds heat stress; MBS, MRE are MYB binding sites; TC-richrepeats is that plant is on the defensive, the element of response drought stress; WUN-motif receives physical abuse induction.Above interpretation of result shows, AaTPS7 gene promoter is the promotor of an induction type, can induce by many factors.
Embodiment 3
The promotor obtained is connected into pCAMBIA-1391z carrier, merges gus reporter gene
In order to study the expression of gene promoter in plant different tissues position, the promotor proTPS7 of AaTPS7 gene is connected pCAMBIA-1391z carrier and merges gus reporter gene, in order to realize the structure to expression vector pCAMBIA1391z-proTPS7, PstI restriction enzyme site is introduced in forward primer, introduce NcoI restriction enzyme site in reverse primer, primer sequence is as shown in the table:
Table 5pCAMBIA1391z-proTPS7 vector construction PCR primer
Embodiment 4
The pCAMBIA1391z-proTPS7 vector agrobacterium tumefaciens built is detected
Proceed to agrobacterium tumefaciens (EHA105) by containing the plant expression vector built, performing PCR of going forward side by side is verified.Result shows: the plant binary expression vector containing promoter gene fragment is successfully building up in Agrobacterium tumefaciens strain, thus obtains the Agrobacterium tumefaciens strain of the plant expression vector pCAMBIA1391z-proTPS7 merged containing gene promoter and gus gene.
Embodiment 5
By the Agrobacterium tumefaciens transformation sweet wormwood with pCAMBIA1391z-proTPS7 carrier
1) preculture of explant
Seeds of southernwood volume fraction is the alcohol immersion 1min of 75%; The NaClO of 20% (w/v) is used to soak 20min again; Aseptic water washing 3 ~ 4 times; Surface-moisture is blotted with aseptic thieving paper; Be inoculated in the MS substratum without hormone, this MS substratum is the solid medium of Murashige and Skoog invention in 1962, and this solid medium can be obtained by commercial channel; The daylight of 25 DEG C, 16 hours and the night of 8 hours cultivate, and can obtain sweet wormwood aseptic seedling, grow to after about 5cm until seedling, and clip tests for sterility explant is used for transforming;
2) Dual culture of Agrobacterium and explant
By described leaf explant, forward in the Dual culture substratum that 1/2MS and 100 μm ol/LAS (Syringylethanone) forms, drip the 1/2MS suspension of the agrobacterium tumefaciens engineering bacteria containing the above-mentioned plant expression vector activated, explant is fully contacted with bacterium liquid, 28 DEG C of light culture 3 days, to drip leaf explant at the 1/2MS liquid nutrient medium suspension of the agrobacterium tumefaciens without goal gene for contrast;
3) screening of resistance regeneration plant
The described Dual culture sweet wormwood explant of 3 days is transferred in germination screening culture medium that MS, 0.5mg/L6-BA (6-benzyl purine), 0.05mg/LNAA (naphthylacetic acid), 50mg/LKan (kantlex) and 500mg/LCb (Pyocianil) form, cultivate in the dark (dark) of the daylight (light) of 25 DEG C, 16 hours and 8 hours, every two weeks succeeding transfer culture once, Kan resistance Multiple Buds can be obtained after 2-3 subculture, well-grown resistance Multiple Buds is cut and proceeds to 1/2MS
0the root media of (the MS substratum without hormone) and 125mg/LCb composition is cultured to and takes root, thus obtain Kan resistance regeneration sweet wormwood plant.
Embodiment 6
1, PCR detects transfer-gen plant
Design forward primer (proTPS7:5 '-GGTAATTTGCCCTTATTAATTCGCA-3 ') and reverse primer (GUSR:5 '-GACATCGGCTTCAAATGGCGTA-3 ') respectively according to promoter and the GUS sequence in expression cassette promoter-GUS to detect gus gene; Result shows, the PCR special primer designed by utilization, can amplify specific DNA fragment, and with non-transformed sweet wormwood genomic dna for template time, do not amplify any fragment, result is as shown in Figure 1.This shows, contains expression cassette promoter-GUS in this transfer-gen plant.
2, the determination of the expressive site of gus reporter gene in plant that guides of promotor
To the sweet wormwood plant of PCR test positive, get its blade and carry out GUS tissue staining, result as shown in Figure 2, result shows, dyeing part is specific expressed in the nonsecreting type glandular hairs of sweet wormwood, illustrate that AaTPS7 gene promoter can guide foreign gene specifically expressing in glandular hairs in transgene abrotanum, as can be seen here, the proTPS7 gene promoter that the present invention clones can be used for utilizing plant glandular hairs tissue expression with in the genetic engineering breeding producing meta-bolites and industrialization.
More than describe preferred embodiment of the present invention in detail.Should be appreciated that the ordinary skill of this area just design according to the present invention can make many modifications and variations without the need to creative work.Therefore, all technician in the art, all should by the determined protection domain of claims under this invention's idea on the basis of existing technology by the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (10)
1. the promotor expressed in nonsecreting type glandular hairs of regulatory gene, it is characterized in that, the nucleotide sequence of described promotor is as shown in SEQIDNo:1.
2. the promotor expressed in nonsecreting type glandular hairs of regulatory gene as claimed in claim 1, it is characterized in that, the cis-acting elements in described promotor comprises: CGTCA-motif, HSE, MBS, MRE, TC-richrepeats, WUN-motif.
3. prepare a method for the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs, it is characterized in that, comprise the following steps:
Step one, from sweet wormwood genome, clone the genome sequence of described promotor;
Step 2, the cis-acting elements analyzed in described promotor, determine described promoter and enhancer;
Step 3, the sequence of described promotor obtained in step one to be connected with carrier;
Step 4, the vector agrobacterium tumefaciens will obtained in step 3;
Step 5, the described agrobacterium tumefaciens stable conversion plant with described carrier will obtained in step 4;
Step 6, detect proceeding to of described promotor, and determine the expressive site of gene in plant that described promotor guides.
4. the method preparing the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs as claimed in claim 3, it is characterized in that, it is template that described step one comprises with genomic dna, adopts two-wheeled nested PCR method to increase described promoter sequence.
5. the method preparing the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs as claimed in claim 3, it is characterized in that, carrier in described step 3 is pCAMBIA1391z, utilize primer pair 1391z-proTPS7-F and 1391z-proTPS7-R, in the described promoter sequence obtained, restriction enzyme site PstI and NcoI is introduced by PCR, thus be connected on carrier pCAMBIA1391z, wherein primer sequence is as follows:
1391z-proTPS7-F：5’-AACTGCAGAAGAGAAAGCTACATACAATGGGAA-3’；
1391z-proTPS7-R：5’-CATGCCATGGGGAGAAAGATCGGTTATGC-3’。
6. the method preparing the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs as claimed in claim 3, it is characterized in that, described step 5 comprises:
1) preculture of explant;
2) Dual culture of Agrobacterium and explant;
3) screening of resistance regeneration plant.
7. a carrier, is characterized in that, described carrier is connected with the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs.
8. an expression cassette, is characterized in that, described expression cassette comprises the promotor that regulatory gene as claimed in claim 1 is expressed in nonsecreting type glandular hairs.
9. the promotor expressed in nonsecreting type glandular hairs of a regulatory gene as claimed in claim 1 is for utilizing plant glandular hairs tissue expression and the application in the genetic engineering breeding of plant production meta-bolites.
10. the application of the promotor expressed in nonsecreting type glandular hairs of a regulatory gene as claimed in claim 1 in the initiation and development of nonsecreting type glandular hairs and the metabolic regulation research of secondary metabolite wherein.
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| CN105925577A (en) * | 2016-05-06 | 2016-09-07 | 上海交通大学 | Promoter for regulating and controlling predominant expression of gene in glandular secretory trichome based cells and application of promoter |
| CN105924510A (en) * | 2016-05-06 | 2016-09-07 | 上海交通大学 | Artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1 and application |
| CN108070596A (en) * | 2018-01-31 | 2018-05-25 | 上海交通大学 | Sweet wormwood glandular hairs predominant expression AaTCP14 gene promoters and its preparation method and application |
| CN109852614A (en) * | 2018-12-06 | 2019-06-07 | 上海交通大学 | A kind of promoter and its application that controlling gene is expressed in nonsecreting type glandular hairs |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925577A (en) * | 2016-05-06 | 2016-09-07 | 上海交通大学 | Promoter for regulating and controlling predominant expression of gene in glandular secretory trichome based cells and application of promoter |
| CN105924510A (en) * | 2016-05-06 | 2016-09-07 | 上海交通大学 | Artemisia apiacea MYB type transcription factor coding sequence AaMIXTA1 and application |
| CN105925577B (en) * | 2016-05-06 | 2019-06-07 | 上海交通大学 | The promoter and application of controlling gene predominant expression in secreting type glandular hairs basal cell |
| CN105924510B (en) * | 2016-05-06 | 2019-09-10 | 上海交通大学 | Sweet wormwood MYB class transcription factor coded sequence AaMIXTA1 and application |
| CN108070596A (en) * | 2018-01-31 | 2018-05-25 | 上海交通大学 | Sweet wormwood glandular hairs predominant expression AaTCP14 gene promoters and its preparation method and application |
| CN109852614A (en) * | 2018-12-06 | 2019-06-07 | 上海交通大学 | A kind of promoter and its application that controlling gene is expressed in nonsecreting type glandular hairs |
| CN109852614B (en) * | 2018-12-06 | 2022-11-22 | 上海交通大学 | Promoter for regulating expression of gene in non-secretory glandular hair and application thereof |
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