CN101921764B - Chemically Induced promoter induced by antibiotics and application thereof - Google Patents

Abstract

The invention belongs to the biotechnological field, particularly relating to a chemically Induced promoter. The chemically Induced promoter comprises the clone method of a chemically Induced promoter induced by mitomycin and bleomycin, a building method of a plant transgenic carrier by using the promoter, the analysis and analysis of a reporter gene expression mode driven by the promoter and a method for inducing the promoter. The GUS reporter gene driven by the promoter has no expression or has low-level expression in most parts of tissue in the plant; after being processed by antibiotics, the promoter can drive the reporter gene to have strong expression in the whole seedling stage and spica. In addition the antibiotics, the promoter is hardly induced by other factors and has strong specificity, and thus the promoter can be used for building the plant transgenosis expression carrier induced by the antibiotics and has an important application value on scientific research and agricultural production.

Description

A kind ofly be subjected to microbiotic inductive chemical induced promotor and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of new chemical induced promotor, the activation analysis of the structure, this promotor that comprises clone, the plant transgene carrier of arabidopsis thaliana promoter subsequence in different tissues, this promotor are to the growth effect analysis to plant of the ageing analysis of microbiotic mitomycin and bleomycin induced response and used microbiotic.

Background technology

Promotor is meant the section of DNA sequence that RNA polymerase identification, combination and initial gene are transcribed, and is usually located at upstream region of gene.A typical promotor comprises CAAT-box and TATA-box, and they are respectively identification and the binding sites that depends on the RNA polymerase of DNA, generally is positioned at tens base places, transcription initiation site upstream.Traditionally, the promotor with plant gene is divided into constitutive promoter (constitutive promoter), inducible promoter (inducible promoter) and tissue-specific promoter (tissue-specific promoter) by its expression of gene mode.But in some cases, one type promotor often has the characteristic of other type promotor concurrently.

Constitutive promoter is meant that the genetic expression of gene in different tissues organ and etap that promotor drives does not have notable difference, can both promotor gene in a organized way expression, have persistence, do not show the space-time specificity.Cauliflower dish mosaic virus CaMV35S promotor (Odell etc., 1985) for example.

Tissue-specific promoter, be meant the only expression in some specific organ and tissue of gene that promotor drives, and often the characteristic of regulating, for example flower specific promoter (Vantunen etc. are grown in performance, 1988), blade specific promoter (Taylor etc., 2001).Research to these promotors, not only help to illustrate the effect of gene in phytomorph, growth, pathways metabolism etc., and can be by these promotors, the driving purposes gene is expressed in specific site, thereby reaches the specific purpose that sets in production practice or the scientific research.

Inducible promoter is meant that for example physics, chemistry, bio signal (being referred to as " inducible factor ") are when existing at some specified conditions, and the gene that promotor can drive the downstream strengthens expression, and transcriptional level improves greatly.It is characterized by, when not having inducible factor to exist, in a single day the gene of the type promotor control is not expressed or is had only low-down expression (being also referred to as " background expression "), but is subjected to inducing of inducible factor, and gene expression amount increases considerably rapidly.Inducible promoter all has significant application value in basic scientific research and production practice.What it can be used to study gene expresses influence for plant phenotype at regular time and quantity, thus function in the body of research gene; Also can be used in the production practice, make transgenosis high expression level in needs of some external source, do not express when not required or low the expression, thereby reach certain specific purpose in the production.

At present, according to the token stimulus kind that plant is experienced, inducible promoter mainly can be divided into the promotor of physical factor (as temperature, light, drought etc.), chemical factor (ion, organism, hormone etc.) and biotic factor (germ, histoorgan, etap etc.) abduction delivering.Such promotor is often named with inducement signal, for example (Wu Xuefeng etc., 2004) such as photoinduction expressing gene promotor, thermal induction expressing gene promotor, wound-induced expressing gene promotor, hormone induction expressing gene promotor, fungal induction expressing gene promotor and symbiotic bacterium abduction delivering gene promoters.Often because inductor is an external source, there is not induction factor in chemically inducible promoter in the organism or in the environment, be compared to environmental factors inductive promotors such as biotic factor or temperature, specificity is stronger, and the switch of gene more easy to control is so use more extensive in production or scientific research.

The activation analysis of promotor is commonly used to GUS (β-glucuronidase, coding beta-glucuronic acid Glycosylase) as reporter gene, it can be used as the downstream that foreign gene is inserted into promotor, analyzes according to histochemical stain and just can obtain the expression space time information of gene at the different sites of plant intuitively.In the present patent application, adopt gus gene to report the activity of promotor equally.

Summary of the invention

The purpose of this invention is to provide a kind of microbiotic inductive chemical induced promotor that is subjected to, it also is a kind of tissue-specific promoter simultaneously.

Promotor provided by the invention is one section order of being cloned into from the upstream of the PARP1 gene of Arabidopis thaliana.Its nucleotides sequence is classified as shown in the SEQ ID NO.1, is designated as the PARP1 promotor.PARP1 albumen is responsible for the poly ADP ribose modification of plant protein, plays an important role in the DNA of plant repairs.Itself can be subjected to the induced strong of mitomycin and bleomycin the PARP1 gene.

PARP1 promotor provided by the invention is a kind of mitomycin and bleomycin height inductive chemical induced promotor of being subjected to, except mitomycin and bleomycin, it is subjected to inducing of other endogenous or extrinsic factor hardly, so inducing specific is strong especially.Such inducible promoter, for making up the plant transgene carrier, it is all significant to be used for scientific research or production practice.

Tissue-specific promoter provided by the invention has activity in seedling stage base root, the early stage flower pesticide of plant in flowering period and filigree.

This promotor is cloned in the reporter gene carrier, find that by transgenic technology its institute's driven GUS reporter gene is only at the basic root position transient expressions of 3-5 days seedling; And in maturation plant, only there is expression at flower pesticide before the 8th phase and filigree top near the flower pesticide place.Therefore, the PARP1 promotor can drive foreign gene specifically expressing in these tissues, thereby reaches directionally genetically modified purpose.Foreign gene localization and expression in transgenic plant can be improved the expression level of foreign gene at privileged site, increase genetically modified purpose.

Important, promotor provided by the invention is a kind of chemical induced promotor that is subjected to microbiotic mitomycin and bleomycin induced.

Promotor feature provided by the invention be do not induce before, gene expression amount is very low, behind bleomycin induced 30h with 22 μ g/ml mitomycin+1.5 μ g/ml, can make its great expression, the multiple that improves in different tissues is different, this experiment with the quantitative PCR means detected gus reporter gene in spending (induce intensity strong) and lotus throne leaf (inducing weak strength), induce before and after expression amount change, find in flower the abduction delivering amount up to 32 times, and in the lotus throne leaf, improved 7 times.This experiment also utilizes the seedling of Arabidopis thaliana to attempt inducing effect with what mitomycin and bleomycin were handled separately, finds when with the effect of the bleomycin induced 50h of 1.0 μ g/ml and suitable with the effect of the bleomycin induced 30h of 22 μ g/ml mitomycin+1.5 μ g/ml.Compare with bleomycin, a little less than mitomycin induces effect, so with the bleomycin induced of lower concentration external source goal gene high strength in seedling is expressed separately.

The present invention has also detected the influence of the bleomycin of 1.0 μ g/ml to plant-growth, and the seedling of Arabidopis thaliana is grown on the substratum of the bleomycin that contains 1.0 μ g/ml, found that this concentration does not have the significant adverse influence to the growth of plantling.These presentation of results, this promotor can become a safety, inducible promoter effective, easy to use, thereby the structure that can be used for transgene carrier is realized the artificial regulatory to genetic expression, make goal gene regularly, quantitatively, ground, location expresses.

The present invention also provides the method for utilizing the PARP1 promotor to make up the plant transgene carrier.Concrete steps are as follows:

Arabidopis thaliana PARP1 promotor can be connected to the upstream of the foreign gene initiation codon of any transgene carrier by restriction enzyme site, to drive the expression of downstream gene.The application is an example with transgene carrier pAKK687, and the PARP1 promotor of being cloned into is inserted into the upstream of the carrier gus reporter gene of pAKK687 by the two enzyme sites of EcoR I/Sma I, and having constituted is the plant transgene carrier of reporter gene with GUS.By inflorescence infusion method transformation mode plant Arabidopis thaliana, successfully obtain transfer-gen plant.Can detect the activity of this promotor under different tissues and extraneous factor stimulation by the GUS histochemical stain.Foreign gene can substitute the downstream that gus reporter gene is inserted in the PARP1 promotor.

The present invention also provides the detection method of method, antibiotic administration concentration and expression level that microbiotic induces the PARP1 promotor.

Concrete grammar: spray antibiotic solution till drip in seedling stage or flowering period, can induce with the bleomycin of 22 μ g/ml mitomycin+1.5 μ g/ml, the time is 30 hours, but this concentration may impact plant-growth.If induce with the bleomycin that is low to moderate 1.0 μ g/ml, time is 50 hours, also can reach the almost same effect of inducing, and under this concentration, microbiotic does not have disadvantageous effect (plant grows a week among Fig. 7, and the growth of plant is not subjected to obviously coercing) to plant-growth on the substratum of the bleomycin that contains 1.0 μ g/ml.

Can be after the sprinkling according to the foreign gene design gene-specific primer that inserts, extracted total RNA, with quantitative PCR detect induce before and after the expression of gene level change.

Description of drawings

The amplification of Fig. 1 PARP1 promoter fragment.Arrow is depicted as the promotor segment of being cloned into.

Fig. 2 recombinant plasmid synoptic diagram is inserted into the PARP1 promotor upstream of gus reporter gene by restriction enzyme site.

The tissue specificity activation analysis of Fig. 3 PARP1 promotor.

PARP1 promotor institute driven GUS reporter gene is only at the basic root position transient expressions of 3-5 days seedling; And in maturation plant, only flower pesticide before the 8th phase and filigree have expression near the flower pesticide place.A: 3-5 days seedling grow; B: the lotus throne leaf of flowering plant; C: stem leaf; D: Hua Sui; E: the flower of early stage (before the 8th phase); F: open flower; G: stem; H: young tender fruit pod (before embryo's cotyledon period); I: mellow fruit pod (behind the cotyledon period).

Fig. 4 PARP1 promotor is expressed before and after the bleomycin induced in different tissues and is changed.0h/30h represents the bleomycin induced 0h/30h with 22 μ g/ml mitomycin+1.5 μ g/ml, and the level of gus reporter gene changed before and after diagram flower fringe, seedling and developmental fruit pod were induced.The very low or not expression of GUS expression level in these tissues induces the back in whole colored fringe before inducing, and in the whole plants in seedling stage and the whole fruit pod strong expression is arranged all, shows the intensive blue signal.

Fig. 5 grows the seedling in a week with the bleomycin induced of 1.0 μ g/ml.A. contrast; B.0.5 μ g/ml bleomycin induced is 5 hours; C.0.5 μ g/ml bleomycin induced is 50 hours; D.1.0 μ g/ml bleomycin induced is 5 hours; E.1.0 μ g/ml bleomycin induced is 50 hours; F.35S promotor is in the activity in seedling stage.

Fig. 6 utilizes quantitative PCR analysis to induce the activity change of front and back PARP1 promotor.The abduction delivering amount is up to 32 times (stronger) in flower, and expression amount is about 7 times (weak) in the lotus throne leaf.

Fig. 7 is the grow seedling in a week of the 1/2MS substratum that contains 1.0 μ g/ml bleomycin.1.0 μ g/ml bleomycin does not cause obvious injury to plant, the seedling in one week of growth is crooked slightly except root on the substratum of this concentration, and cotyledon size and root are long approaching with contrast.

Fig. 8 PARP1 promotor is subjected to the situation of other external environment factor affecting.As can be seen from the figure in seedling except mitomycin and bleomycin, PARP1 is subjected to inducing of other external environment factor hardly.

Embodiment

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Following is the experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, and Sambrook equimolecular clone for example: the condition of being narrated in the laboratory manual, or the condition of advising according to manufacturer.

The segmental clone of embodiment 1PARP1 gene promoter

1.DNA extraction get the blade of the environmental Col-0 plant of Arabidopis thaliana, liquid nitrogen grinding adds 0.4ml 2%CTAB (20mM EDTApH8.0,1.4M NaCl faces with before adding mercaptoethanol to 0.2% for 2%CTAB, 100mM Tris-Cl pH8.0), mixing; 65 ℃ of water-bath 30min; Add the 0.4ml chloroform, mixing; 4000rpm, centrifugal 10min shifts supernatant to new centrifuge tube; Add 400 μ l Virahols, mixing, room temperature leaves standstill 10min; 12000rpm, centrifugal 10min; Abandon supernatant, 70% washing with alcohol; 37 ℃ of oven dryings add 20 μ l water dissolution.Detect the quality of DNA with 0.8% sepharose.

2.PARP1 the segmental amplification of gene promoter is according to two pairs of primers of PARP1 gene 5 ' upstream sequence design, a pair of primer that does not contain restriction enzyme site, upstream primer: 5 '-ATT TTG AGG CGG TGG AGT TTC-3 '; Downstream primer: 5 '-CGT CGA CTT TGA GCT TGT TCG-3 '.The a pair of restriction enzyme site that contains, upstream primer: 5 '-GAA TTC TTTTGA GGC GGT GGA GTT TC-3 '; Downstream primer: 5 '-CCC GGG TTT CGT CTT CTT CTT CAGGAG AAT AG-3 '.See SEQ ID NO.2.At first increase, utilize the LATaq enzyme, press following condition: 94 ℃ of 2min with a pair of primer that does not contain restriction enzyme site; 94 ℃ of 30s, 53 ℃/51 ℃/49 ℃/47 ℃/45 ℃ 30s, 72 ℃ of 2min30s totally 32 circulations.Recovery is greater than the band of 2kb, as template.Carry out secondary PCR with a pair of primer that has restriction enzyme site again, utilize high-fidelity pfu enzyme, press following condition: 94 ℃ of 2min; 94 ℃ of 30s, 50 ℃/49 ℃/48 ℃ 30s, 72 ℃ of 2min30s totally 32 circulations amplify the fragment (see figure 1) of a single 2179bp.

The structure of the transgene carrier of embodiment 2PARP1 promoters driven

After the recovery of PCR product electrophoresis, use T ₄Dna ligase is connected among the intermediate carrier PCR Blunt, transformed into escherichia coli DH5 α, and selected clone shakes bacterium and extracting plasmid.The clone who chooses insertion after electrophoresis is identified carries out enzyme and cuts evaluation, order-checking then, the correct clone that checks order can utilize restriction enzyme to downcut the purpose fragment, is inserted into (being pAKK687 in the present patent application) (see figure 2) in the final transgene carrier, transformed into escherichia coli.The clone who obtains carries out plasmid amplification after enzyme is cut evaluation correctly, in order to transforming Agrobacterium.

The activation analysis of embodiment 3PARP1 promotor in different tissues

Method for transformation changes above-mentioned expression vector over to Agrobacterium GV3101 routinely, and the laggard performing PCR of resistance screening is identified.Be accredited as the male transformant by inflorescence infusion method arabidopsis thaliana transformation, collect T ₀Carry out the Basta resistance screening for seed.Get T ¹Carry out the GUS histochemical stain for the seed separation than the seedling that is 3: 1, observe the expression of gus gene, take pictures at each plant organ of transgenic arabidopsis.The GUS discovery of dyeing, promotor in 3-5 days seedling stages base root place and early stage flower pesticide (before the 8th phase) have of short duration activity, but show active (see figure 3) in stamen filigree top always, do not observe obvious activity in other tissue.

The activation analysis of embodiment 4PARP1 promotor under different antibiotic concentrations are induced

Get the different tissues of transgenic positive plant, place the centrifuge tube that contains 22 μ g/ml mitomycin+1.5 μ g/ml bleomycin to handle 5h, 30h respectively.The expression of gus reporter gene that the microbiotic induced strong has been found in the GUS staining analysis.At Hua Suizhong, except the filigree top, the GUS expression level is all very low in the whole colored fringe before inducing, induce the back in anthocaulus, whole filigree, stem and gynoecium GUS all by induced strong, but a little less than in flower pesticide, inducing degree relatively; The seedling that on substratum, has grown 7 days, before inducing, microbiotic do not detect the GUS signal, signal begins to occur after inducing 5h, induce after the 30h, all there is blue the appearance positions such as whole, vein, stem apex, signal strong (Fig. 4 only shows the situation of 30h), induce the intensity that reached can with present widely used high expression level promotor Caulimovirus 35S promoter almost near (Fig. 6).In the tender fruit pod of children, do not detect the GUS signal before not inducing, at first blue signal occurs after inducing 5h at fruit pod two ends, induced 30h after, blue signal spreads all over whole fruit pod (Fig. 4).Other tissue such as stem, stem leaf, lotus throne leaf are after having induced 30h, vein at the incision of stem, lotus throne leaf, stem leaf can both detect blue signal, but signal is not as strong (not shown) in the seedling, thus this promotor is more suitable in the whole plants in seedling stage or the spending of flowering plant in the high expression level of induction exogenous gene product.

The active level analysis of embodiment 5PARP1 promotor under microbiotic is induced

This experiment changes with the expression amount that the quantitative PCR means have detected with GUS in flower and the lotus throne leaf before and after the bleomycin induced 30h of 22 μ g/ml mitomycin+1.5 μ g/ml, and primer sequence is as follows: F-primer:5 '-AGT GGC AGT GAAGGG CGA ACA GT-3 '; R-primer:5 '-TCA GCG TAA GGG TAA TGC GAG GT-3 '.See SEQ IDNO.3.95 ℃ of 3min; 95 ℃ of 5s, 60 ℃ of 30s, 40 circulations.Found that induce intensity higher spend in induce and improved 32 times, in inducing more weak lotus throne leaf, induce and improved 7 times of (see figure 5)s.

Embodiment 6 antibiotic concentrations are to the impact analysis of plant-growth

Test lower antibiotic concentration, and utilize bleomycin and mitomycin to induce respectively, found that with respect to bleomycin, mitomycin induce effect a little less than, when with the effect of the bleomycin induced 50h of 1.0 μ g/ml and effect suitable (Fig. 6), so can adopt the bleomycin of 1.0 μ g/ml to induce during practical application with the bleomycin induced 30h of 22 μ g/ml mitomycin+1.5 μ g/ml.

To evenly sow seeds after the Arabidopis thaliana seed disinfection to the 1/2MS substratum of the bleomycin that contains 1.0 μ g/ml, put 4 ℃ of vernalization and move to illumination box two days later, the growth of one week back discovery Arabidopis thaliana is not subjected to significantly influencing (see figure 7), illustrate that this induced concentration does not have the significant adverse influence to the most responsive plant-growth in seedling stage, can be used for inducing of promotor.

The response condition analysis of other external environmental condition of embodiment 7PARP1 gene pairs and endogenous biotic factor

Arabidopis thaliana information site (www.arabidopsis.org) almost combines all about some information of arabidopsis gene group level at present, it is used to the gene chip result from the different experiments chamber, provides Arabidopis thaliana to transcribe the changing conditions of group under different extraneous factor influences.We can inquire about the changing conditions of a certain gene under the varying environment factor affecting information that provides according to this website.We find that in the Arabidopis thaliana seedling (gas first portion and root) PARP1 expression of gene (Fig. 8) is subjected to other common external world to coerce the influence of factor such as hot and cold, arid, salt, oxidation, injury etc. hardly, only induces to increase substantially when handling with Genotoxin (mitomycin+bleomycin).This illustrates that from another point of view the PARP1 promotor is subjected to the inductive specificity very strong, is not subject to the interference of other factors during application.

Sequence table

1.SEQ the information of ID NO.1

(i) sequence signature: long 2179bp, linear kernel thuja acid

(ii) molecule type: nucleic acid

(iii) sequence and sequence mark: red (5 '-UTR); Blue ATG (PARP1 gene start codon)

1-50 TTTTGAGGCG?GTGGAGTTTC?TATTGATTGG?TTATCGATGA?CCACTTGCAT

→

F-primer

51-100 CGTCAAATGG?TTCTTTGATT?CGTGGTTTCG?GCCCCAGCTT?CATACTATCA

101-150 CGAGACTCGA?CCATCTTGTT?TTTTTCTTCT?TCTTCTTGGT?AAATGGTTTC

151-200 ACAACTTGGT?TCCTCTTTCT?TTCATTTTAT?TTTATATACT?ATCATAGCAT

201-250 TTTGGTTGGA?TTTCTCGAGA?TAGTATATTT?TTTAGTTACT?ATCATTACAT

251-300 AAGTATATTT?TAAAAAACTA?ATTATATGAA?TTATGTAGCT?AACTAGATAG

301-350 ATAATCGTAT?AACCAATTCA?TGTTAGTATA?GTATAGTTTA?AGTATGTATT

351-400 TTGGGATTAC?AAGTGTGGTT?GGCATCAAGA?CAAGGATGGT?GATAGCCTTT

401-450 CTCTGTAATT?TGGTTTAAGA?AAAGTTTTTG?CATTTTATGT?ATAAACGTGT

451-500 TTTTTTTTTA?TAATTTAAAA?TTTCAACAAA?AAACAATTTT?TTTTAATAAT

501-550 GATTGACCAC?TATAGACAAT?TTAAATGATA?AAAAAAACCC?CCAATTTTTC

551-600 ACAATGTTTT?GGAGATTAGT?CTAGATTTTT?TGTCCAAATT?TTCCGATTGT

601-650 AAGAATTAAG?AAGCAATGAA?CATTTGTGTT?AAGCTTAATG?ATTTGTACTC

651-700 ACAATATCTT?TTAAATTTAA?AATTGTTAAC?CAAAATATCC?TATATATTGT

701-750 ACTTGTAATA?GAAATATAAA?CTATTAAAAA?CAACACTTTA?TTCATATAAT

751-800 ATAAGTTAAA?ACATATGTTT?TTTTTAGTAT?GTTCTAATCA?CACCTATTAA

801-850 AAAAAGTTGA?AGCTAAATGA?GCCAAAAAGA?AAAATAAAGA?TAGGGGATGG

851-900 GGACAGGCTG?TAATGTTAGG?CGGTTGGTAT?ATGAACTGAG?AACATGTCTG

901-950 TTGGTTCGGT?CCATCTACAC?CACTCAACCA?TTTGGCTATG?TTTTCTTTTT

951-1000 GGCTTTTGCA?TGTTCTCTCT?ACTTTTCTTC?TTTGGTCAAA?ATCTCTATCT

1001-1050?CGTCTTTTAC?ATGGCTTACC?CGAATGTTAG?TTGTCATGTA?AATTTGGTTA

1051-1100?TGAAAAGATA?TTTTATATAA?ACTTTATCGT?ATATTAATAT?CGTTATCATC

1101-1150?TAACCATTTT?TTAAAACTAA?ACTAGAACCA?TCCAGTTTTA?CAAGAGTTTT

1151-1200?TTTTTTTTTT?TTTCTAACTA?AATAATATTT?GAAGTGTACA?ATATTAACAA

1201-1250?TATATGGGCC?AAATAATAGT?GGAAACCAAA?TCGTTAGTCC?CACTTTATGA

1251-1300?TGGGCCTGTT?GATTCTTATG?TCTTCTTCGT?AAGTTGTGAT?TATGCAGATT

1301-1350?ACGGGCTAAT?AAACATGCAT?GTTTAGTTTT?TACTGTCCAA?GTAACGAAAT

1351-1400?TTTATCTTTT?GGGTTGTTGG?CCCATTTCAT?ATATTCCAAA?TGCCAAATCC

1401-1450?AGCCCGGCTC?GACACAGCAC?TGCTCGGCTC?AACACTCGTA?TGCGGTTGGT

1451-1500?AGCCACTTAA?GACCTTGGTT?TGATTAACAT?GTTACGAATA?ATTTGTGTCC

1501-1550?CTTTTTCTTC?AAGGAGACTA?ATCTCTTTTA?ATAAAAAAGA?ATTGTGTCAT

1551-1600?TAGTCAACAC?AAGTCCTATA?ATCCGTTTAC?GGTAATTTGT?ATGCACGTCC

1601-1650?TTGGAAAAGT?GAGTAGTGGC?GTAGCATTAC?AGCCAAAAAC?TATTTGTATA

1651-1700?TTTTTCTTTT?CGTTAAACAA?CCAGCAAAAT?TTTCAGAAAA?ATGTTCTTAA

1701-1750?ATTATAAATT?AGTAGTACAT?TTTAAAACAT?AGAGATTTTT?TGTTTCTTTT

1751-1800?AATAGAAGAG?TTAAACCTAT?GTACAAAATT?TCAACTCCTT?TTCAAAGTAT

1801-1850?TTGCCTGTTA?CTAGATTTTT?AACCTTTTTT?TTTTTATCTT?TCATGATTTT

1851-1900?CTATTGCTTG?CCATCATCAA?TGGTAGGAAA?TAAATACTAT?TTTAAAAAGG

1901-1950?TCAGGGGTGG?ATTTAAGAAT?CAATCCAAAA?GTTTGGGGTC?TTTTGGAGAT

1951-2000?TAAAAAGTTA?TATGGGAAAT?ATCCACAAAT?ATGAACGAGA?ACTTTTGTCA

2001-2050?AAAAAATTTA?AAATAATTTT?TCAAAAAGCC?CTAAAGCTTT?CAAGGGAAGC

2051-2100?CATCGATGAA?GAAGAAAACG?AAGAAGAAGA?CTCTTCAAAT?GCTCGCGCGA

2101-2150?ACTCACTTCT?GACGAAAACC?ATACTTCCTC?AGTCTCATTC?CCTTTCCGAC

2151-2200?GAA CTATTCT?CCTGAAGAAG?AAGACGAAAA?TG

←

R-primer

2.SEQ?ID?NO.2

The primer of PARP1 promotor is used to increase:

Do not contain restriction enzyme site:

Upstream primer: 5 '-ATT TTG AGG CGG TGG AGT TTC-3 '

Downstream primer: 5 '-CGT CGA CTT TGA GCT TGT TCG-3 '

Contain restriction enzyme site:

Upstream primer: 5 '-GAA TTC TTT TGA GGC GGT GGA GTT TC-3 '

Downstream primer: 5 '-CCC GGG TTT CGT CTT CTT CTT CAG GAG AAT AG-3 '

3.SEQ?ID?NO.3

The primer that is used for quantitative PCR:

Upstream primer: 5 '-AGT GGC AGT GAA GGG CGA ACA GT-3 '

Downstream primer: 5 '-TCA GCG TAA GGG TAA TGC GAG GT-3 '.

Claims (4)

1. a chemically inducible promoter is characterized in that this promotor is one section dna sequence dna of being cloned into from Arabidopis thaliana, and its nucleotides sequence is classified as shown in the SEQ ID NO.1.

2. the application of chemically inducible promoter in tissue specific expression according to claim 1 is characterized in that being used for foreign gene at the transgenic plant localization and expression, to improve the expression level of foreign gene at privileged site, increases genetically modified purpose.

3. the application of chemically inducible promoter in making up transgene carrier according to claim 1 is characterized in that being used for the structure of transgene carrier, realizes the artificial regulatory to genetic expression, make goal gene regularly, quantitatively, ground, location expresses.

4. recombinant vectors that comprises the described chemical promotor of claim 1.

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