CN106434673B

CN106434673B - A kind of plants flower pesticide specificity promoter PCHF15 and its application

Info

Publication number: CN106434673B
Application number: CN201611113767.0A
Authority: CN
Inventors: 黄培劲; 张维; 吴春瑜; 陈思兰; 吴永忠
Original assignee: Hainan Bolian Rice Gene Science & Technology Co Ltd
Current assignee: Hainan Bolian Rice Gene Science & Technology Co Ltd
Priority date: 2016-12-06
Filing date: 2016-12-06
Publication date: 2017-11-03
Anticipated expiration: 2036-12-06
Also published as: CN106434673A

Abstract

The present invention relates to genetic engineering and molecular biology, a kind of plant pollen specificity promoter PCHF15 and its application are specifically disclosed.The present invention also provides the expression vector containing plant pollen specificity promoter PCHF15, expression casette, and the carrier containing the expression casette.Present invention also offers amplification pollen specific promoter PCHF15 primer pair.The pollen-specific promoter PCHF15 of the present invention is paddy rice endogenous gene, highly beneficial to paddy gene engineering, and driving foreign gene expression specific expressed in pollen is accurate, and there is provided a kind of new method for driving foreign gene specific expressed in pollen.

Description

A kind of plants flower pesticide specificity promoter PCHF15 and its application

Technical field

The present invention relates to genetic engineering and biology field, specifically, it is related to a kind of plants flower pesticide specificity and opens Mover PCHF15 and its application.

Background technology

Plant gene is mainly regulated and controled on transcriptional level, is in a variety of cis-acting elements and trans-acting factor Between mutually coordinated effect.Promoter is important regulating and controlling element in plant gene transcription, is normally at the end of structural gene 5 ' upstream, It is RNA polymerase and the identification of some trans-acting factors and binding site.Promoter is the regulation and control center of genetic transcription, is deeply ground Study carefully promoter and not only improve grasp control gene transcript pattern and regulatory mechanism, contribute to the regulation and control of exogenous gene expression again. Promoter mainly has two regions, respectively core promoter area and transcription regulatory region.Core promoter area is transcription initiation Most short promoter fragment, generally 40nt, comprising core promoter element, is one section and known by RNA polymerase family I, II and III DNA sequence dna that is other and combining.This region includes some important function element, can accurately position transcriptional start point and direction, It is the key foundation of gene regulation.Transcription regulating region is located at the upstream (or downstream) of core promoter, can be with special transcription The factor is combined so as to the space-time to transcription, strong and weak regulating and controlling effect, such as enhancer and silencer.

Core promoter classics conserved sequence has following four：1) transcription initiation site (initiator, Inr), usual position Locate in translation initiation codon (AUG) upstream -40 to -70bp.Transcription initiation site has no very strict DNA sequence homologous, common Conserved sequence be in+1 A and+3 T.The transcription factors such as TFIID and TFII-I can recognize that transcription initiation site and start Transcription.2) TATA box, are core parts, are the recognition sites of rna plymerase ii, are also to be combined with some trans-acting factors One of site.The TATA box of plant promoter are normally at 32 ± 7bp of transcripting start point upstream, and its conserved sequence is 5 ' T₃₇C₄₂A₃₈C₄₇T₉₆A₉₇T₉₀A₉₄T₅₃A₉₅T₆₃A₇₁G₄₁3’.TATA box direction does not determine the direction of transcription.TATA box work( Can rely on position of its position relative to initiation site, the displacement of base can inactivate minimal promoter.Therefore, TATA The required dependenc RNA polymerase II transcriptions of box, are combined by the identification with transcription factor TFIID and determine RNA polymerase starting and turn The site of record, forms preceding transcription complex and starts transcription, and regulate and control upstream activat albumen.3) CAAT box, are a kind of enhancings The element of transcription.Near-the 75bp for being normally at transcripting start point, its conserved sequence is GGC/TCAATCT.CAAT box with it is suitable Formula controlling element has synergy, there is stronger activation to genetic transcription, controls the frequency of transcription initiation.4)GC Near the box, -90bp for being usually located at transcripting start point, its conserved sequence be GGGCGG, can have multiple copies, must with transcription because Sub- SP1 interactions could promote genetic transcription.

Promoter has following feature：1) sequence-specific.Promoter contains base in conservative sequence frame, sequence frame Change can influence transcriptional level.2) direction specificity.Promoter is a kind of directive cis regulatory factor, point unilateral initiative Son and the class of bidirectional promoter two.3) location specific.Promoter is normally at the upstream of gene coding region or functional areas.4) kind Specificity.Different plant species have different types of promoter.5) specific Function.Different tissues and different biochemical routes have not The promoter of same type.

Promoter can be divided into three classes by its expression way：Constitutive promoter, inducible promoter and tissue specificity are opened Mover.Constitutive promoter be can in a organized way in can promotor gene expression, the gene started have space-time continue Property and expression quantity constancy, but Space-time speciality is not showed.There are six aggressiveness TGACTG in most of constitutive promoters, be located at Transcription initiation site upstream 100bp or so, and often repeat.Constitutive promoter is in plant genetic engineering frequently as table Up to carrier.Inducible promoter promotor gene can be expressed under the induction of some physically or chemically signals, or can be significantly Improve the transcriptional level of gene.They have the sequential structure of enhancer, silencer or similar functions, and have obvious selectivity. A part of inducible promoter has tissue specificity simultaneously, and such promoter is often named with inducement signal, such as photoinduction promoter, Heat shock promoter, low temperature induction promoter, drought-induced promoter, wound-induced promoter, hormone induction promoter etc..Group Knitting specific expressing promoter, only promotor gene is expressed in certain cell, tissue or organ, and tissue specific expression starts Son can not only make target gene accumulate expression product in certain cell, tissue or organ, increase local expression amount, while may be used also To avoid the unnecessary waste of plant nutrient.Tissue-specific promoter not only has the structure of general promoter, also exists several Plant and generally there is tissue-specific enhancer and silencer in the element of control tissue specific expression, its structure, thus regulation and control Transcriptional level of the gene in specific tissue or organ.These promoters also tend to show Chemical Physics signal induction, Also influenceed by histocyte physiological status and stage of development.Such promoter has root-specific promoter, dimension Restrain specificity promoter, cane and blade specific promoter, flower specific promoter, seed and fruit-specific promoter Deng.

Paddy rice is one of most important cereal crops in the world, is also the main cereal crops of China, therefore, Study On Rice Flowering mechanism is significant.The pollen formation of paddy rice occurs in the flower pesticide in stamen, and flower pesticide contains reproduction cell, these reproductions Cell formation tapetum and pollen mother cell.Tapetum provides nutrition, including various enzymes and non-enzymatic for the development of male gametophyte Proteinoid, and participate in the synthesis of exposore.Pollen mother cell then carries out meiosis, produces tetrad.In tetrad The unicellular effect for secreting callosity enzyme through tapetum separates Haploid production microsporinite.Monoploid microsporinite is further sent out Educate, central big vacuole is formed during to late period.Nucleus exclusion is arrived cell edges (being referred to as mid-late uninucleate stage), shape by central big vacuole Into cell polarity.The promotion microsporinite of this cell polarity carries out not waiting mitosis once, produces a big trophonucleus With a small reproduction cell.Small reproduction cell is completely contained in big vegetative cell, forms the spy of cell in cell Different phenomenon.Reproduction cell will carry out second of mitosis, produce two spermatids, form ripe pollen grain.Pollen-specific Property promoter can drive foreign gene specifically expressing in pollen, so as to control pollen fertility, these promoters be water The genetic engineering of rice, particularly provides new selection in terms of control fertility and transgenosis drift.But at present in plant The Pollen Anthers specificity promoter of identification is less, it is necessary to further identify and find pollen specific promoter.

The content of the invention

It is an object of the invention to provide a kind of plants flower pesticide specificity promoter and its application.

A kind of plants flower pesticide specificity promoter PCHF15 that the present invention is provided, it is with nucleotides sequence 1) or 2) Row：

1) nucleotide sequence shown in SEQ ID No.1；

2) be substituted, lack or add in the nucleotide sequence shown in SEQ ID No.1 one or several nucleotides and The nucleotide sequence as derived from 1) with equal anther-specific startup function.

Wherein, the nucleotide sequence as derived from 1) described in 2), the nucleotides sequence described in 1) is classified as to be had compared with 1) More than 70% homology, and the nucleotide sequence with equal anther specific promoter function.2) described in by 1) derivative Nucleotide sequence.

The present invention provides the recombinant expression carrier containing plants flower pesticide specificity promoter PCHF15 of the present invention.This hair It is bright to provide the host cell containing above-mentioned recombinant expression carrier.

The present invention also provides the DNA molecular complementary with plants flower pesticide specificity promoter PCHF15 nucleotide sequences.Ability Field technique personnel can easily identify according to identical purpose and utilize and plants flower pesticide specificity promoter PCHF15 nucleotides The complementary DNA molecular of sequence, therefore, with promoter activity and under strict conditions with promoter sequence of the present invention or its fragment The separation sequence of hybridization is included in the invention.

Wherein, the nucleotide sequence is complementary, refers to PCHF15 to hybridize under strict conditions.

Stringent condition refers to that probe will be hybridized to detectable degree with its target sequence and exceed with other sequence hybridizations (as at least 2 times of backgrounds) condition.Stringent condition has a sequence dependent, and different and different because of environment.By control hybridization and/ Or the stringency of wash conditions, the target sequence (same to source detection) complementary with probe 100% can be identified.Selectively, it can adjust Save stringent condition to allow some sequence mismatch so that detect the similitude (heterologous detection) of lower degree.Generally, probe is long Degree is shorter than about 1000 nucleotides, is preferably shorter than 500 nucleotides.

Typically, stringent condition is that salinity is less than about 1.5M Na ions under pH value 7.0-8.3, typically about 0.01-1.0M Na ion concentrations (or other salts), temperature is right at least about 30 DEG C of short probe (such as 10-50 nucleotides) At least about 60 DEG C of long probe (such as more than 50 nucleotides).Strict bar can be also obtained by adding destabilizing agent such as formamide Part.Low stringency condition, it may for example comprise molten in the buffering of 30-35% formamides, 1M NaCl, l%SDS (dodecyl sodium sulfate) 37 DEG C of hybridization in liquid, 1 × the 50-55 DEG C of washing into 2 × SSC (20 × SSC=3.0M NaCl/0.3M trisodium citrates).In Spend stringent condition, it may for example comprise 37 DEG C of hybridization in 40-45% formamides, 1.0M NaCl, l%SDS cushioning liquid, 0.5 × 55-60 DEG C of the washing into 1 × SSC.High stringency, it may for example comprise in 50% formamide, 1M NaCl, l%SDS Cushioning liquid in 37 DEG C hybridization, in 0.1 × SSC 60-65 DEG C washing.Optionally, lavation buffer solution can be containing about 0.1%-1% SDS.Hybridization time is generally less than about 24 hours, generally about 4-12 hours.

Especially it is typically the function of post-hybridization washing, key factor is the ionic strength and temperature of final wash solution. For DNA-DNA crossbreds, Tm can be from Meinkoth and Wahl (1984) Anal.Biochem.138：267-284 equation Estimation：Tm=81.5 DEG C+16.6 (logM)+0.41 (%GC) -0.61 (%form) -500/L；Wherein M is univalent cation Molar concentration, %GC is the percentage of guanylic acid and cytidylic acid in DNA, and %form is formamide in hybridization Percentage in solution, L is length of the crossbred in base-pair.Tm is 50% complementary target sequence and pairing probe hybridization completely Temperature (under defined ionic strength and pH).Every 1% mispairing needs Tm to reduce about l DEG C；Therefore, Tm hybridizes and/or washed The condition of washing can be conditioned with the sequence hybridization with required homogeneity.If for example, the sequence sought have >=90% homogeneity, Tm can reduce by 10 DEG C.Usually, the stringent condition of selection is less than about 5 DEG C of the thermal melting point (Tm) of particular sequence, and It is complementary under defined ionic strength and pH.But, high stringency, which can be applied, is less than thermal melting point (Tm) 1,2,3 Or 4 DEG C of hybridization and/or washing；Moderate stringency can be applied less than thermal melting point (Tm) 6,7,8,9 or 10 DEG C miscellaneous Hand over and/or wash；Low stringency conditions, which can be applied, is less than thermal melting point (Tm) 11,12,13,14,15 or 20 DEG C of hybridization And/or washing.Using this equation, hybridization and cleaning compositions and required Tm, those of ordinary skill in the art can understand hybridization And/or the condition of wash solution changes with the change of stringency.If required extent of mismatch makes Tm (water-soluble less than 45 DEG C Liquid) or 32 DEG C (formamide solution), preferably increase SSC concentration to use higher temperature.The guide of nucleic acid hybridization is seen Tijssen (1993) biochemistries and the nucleic acid probe hybridization of Molecular Biology Lab's technology one, part i, the 2nd chapter (Elsevier, New York)；With Ausubel et al. editor's (1995) Current Protocols method the 2nd chapter (Greene Publishing and Wiley-Interscience,New York).See Sambrook et al. (1989) molecular cloning：Experiment Room handbook (second edition, Cold Spring Harbor Laboratory Press, Plainview, NewYork).

The stringent condition is preferably in 6 × SSC (sodium citrate), 0.5%SDS (dodecyl sodium sulfate) solution In, hybridize at 65 DEG C, film is then respectively washed with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS 1 time.

The present invention provides complementary containing of the present invention and plants flower pesticide specificity promoter PCHF15 nucleotide sequences The recombinant expression carrier of DNA molecular.The invention provides the host cell containing above-mentioned recombinant expression carrier.

The present invention further provides the expression casette containing the sub- anther specific promoter PCHF15.

The expression casette be anther specific promoter PCHF15 downstream connection have structural gene, regulation base Cause, the antisense gene of structural gene, the antisense gene of regulatory gene or the gene table that can interfere with the tiny RNA that endogenous gene is expressed Up to box.

The invention provides the expression cassette recombinant expression carrier containing said gene expression cassette.The invention provides containing upper State the host cell of expression cassette recombinant expression carrier.

The present invention further provides the plant anther of the expression casette containing above-mentioned anther specific promoter PCHF15.

Present invention also offers the primer pair for expanding the anther specific promoter PCHF15, the nucleosides of the primer pair Acid sequence is shown in SEQ ID NO.2-3 or SEQ ID NO.5-6.

A kind of method for expanding anther specific promoter PCHF15, it is to pass through SEQ ID NO.2-3 primer pairs or SEQ ID NO.5-6 primer pairs PCR amplification anther specific promoters PCHF15 nucleotide sequence.

The present invention also provides driving foreign gene method specific expressed in flower pesticide, comprises the following steps：

Anther-specificpromoter PCHF15 and purpose foreign gene are imported into carrier and obtain opening containing anther-specific Mover PCHF15 and purpose foreign gene expression cassette recombinant expression carrier, and Plant Genome is introduced into, obtain external source base Because of the genetically modified plants of the specifically expressing in flower pesticide.

The invention provides plants flower pesticide specificity promoter PCHF15, promoter PCHF15 complementary DNA molecule, contain Application of the expression casette of the anther specific promoter PCHF15 in prepare transgenosis plant.

The genetically modified plants are the genetically modified plants of foreign gene specifically expressing in flower pesticide, preferably pollination/fertilization The genetically modified plants of ability enhancing/weakening, more preferably male sterility genetically modified plants.

The plant includes but not limited to paddy rice, corn, jowar, barley, oat, wheat, grain, sugarcane, soybean, Btassica Species, cotton, safflower, tobacco, clover and sunflower.

The anther-specificpromoter PCHF15 advantages that the present invention is provided are as follows：

1) anther-specificpromoter PCHF15 is paddy rice endogenous gene, highly beneficial to paddy gene engineering.

2) anther-specificpromoter PCHF15 drives the specific expressed experiment of gus gene to show, anther-specificpromoter PCHF15 driving foreign genes expression specific expressed in flower pesticide is accurate.

3) the invention provides a kind of new method for driving foreign gene specific expressed in flower pesticide.

Brief description of the drawings

The recombinant expression carrier 1300gus-PCHF15 that Fig. 1 is anther specific promoter PCHF15 in embodiment 2 is built Flow chart.

The recombinant expression carrier DX2181-PCHF15 that Fig. 2 is anther specific promoter PCHF15 in embodiment 3 builds stream Cheng Tu.

Fig. 3 is that 1300gus-PCHF15 chooses monoclonal bacterium colony PCR agarose gel figures in embodiment 5；It is respectively from left to right Challenge clone bacterium 1-7 promoter PCHF15PCR bands by oneself.

Fig. 4 is to spend 11 paddy rice GUS stained photographs in transgenosis in embodiment 5；A. flower pesticide, b. gynoeciums, c. roots, d. blades.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention In the case of essence, the modifications or substitutions made to the inventive method, step or condition belong to the scope of the present invention.

Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.

Embodiment 1：Rice Anther specificity promoter PCHF15 is obtained

1. the extraction of oryza sativa genomic dna

Rice genome is extracted according to plant DNA Isolation kit (Chengdu Fu Ji Bioisystech Co., Ltd) method explanation DNA.Genomic source is in paddy rice Nipponbare kind Fresh leaves.- 20 DEG C of the genomic DNA packing preservation of extraction is standby.

2. design PCR PCHF15 primers

Design of primers use Gibson Assembly methods, amplified production be inserted into 1300GUSplus carriers NcoI and HindIII restriction enzyme sites.PCHF15 gene primers are expanded as shown in sequence SEQ ID NO.2 and SEQ ID NO.3.Above and below wherein Trip primer 5 ' end has 15 or so nucleotide sequences link position corresponding to carrier to repeat, so that Gibson Assembly connect Connect.

PCR reaction systems (100 μ L)：

DNA profiling：3μL(50ng)

KOD polymerases (spin company) purchased from Japan：2μL

10X buffer：10μL

Forward primer：3μL(10μM)

Reverse primer：3μL(10μM)

dNTP：10μL(10mM)

MgSO₄：4μL

1/10DMSO：20μL

H₂O：45μL

PCR programs：95 DEG C of 4min of pre-degeneration, are denatured 94 DEG C of 30s, and anneal 50 DEG C of 30s, extends 68 DEG C of 2min 30s, 35 circulations, extend 68 DEG C of 10min.

Amplified production about 2000bp, for restriction endonuclease sites and protection group as shown in SEQ IE NO.1 Promoter.

Embodiment 2：Build promoter PCHF15 recombinant expression carrier 1300gus-PCHF15

Build flow and see Fig. 1, the amplified production of embodiment 1 is inserted into 1300GUSplus carrier NcoI and HindIII digestions Site.

It is left that primer SEQ ID NO.2 and SEQ ID NO.3 amplification PCR primer 1% agarose gel electrophoresis reclaims 2000bp Right product.NcoI and HindIII digestion carrier 1300GUSplus, reclaim linear digestion carrier.

2X connections kit connects promoter PCHF15 to 1300GUSplus, and 10 μ L systems are as follows：

2.5 μ L PCHF15PCR products (50ng)

2.5 μ L digestions carriers (100ng)

5μL Ligation Mix

Linker：50℃ 60min.

Conversion：Take the above-mentioned electroporated competent escherichia coli cells of μ L of connection product 5.Positive colony sequencing is selected, is tied Fruit is as shown in Figure 3.It is named as 1300gus-PCHF15.

1300GUSplus carriers contain gus gene, and gus gene sequence is as shown in SEQ ID NO.4, its dyed presentation Blueness, for detecting the specific expressed of promoter.

Embodiment 3：Build the recombinant expression carrier of promoter sequence containing PCHF15 DX2182-PCHF15

The structure flow of reference picture 2, first builds the recombinant cloning vector pGEM-PCHF15 of the promoter sequence containing PCHF15, The recombinant expression carrier DX2182-PCHF15 of the promoter sequence containing PCHF15 is built it based on again.

1. build the recombinant cloning vector pGEM-PCHF15 of the promoter sequence containing PCHF15

With reference to the method for embodiment 1, replace real with primer pair of the sequence as shown in SEQ ID NO.5 and SEQ ID NO.6 Apply the primer pair in example 1 shown in sequence SEQ ID NO.2 and SEQ ID NO.3 to be expanded, obtained amplified production and pGEM- Carrier T is attached, and operating procedure is carried out by Promega Products pGEM-T carriers specification, obtains recombinant cloning vector pGEM-PCHF15.Competent escherichia coli cell is converted, positive colony is further selected, is sequenced.The correct clone of sequencing is carried Take plasmid.

2. the recombinant expression carrier DX2182-PCHF15 of the promoter sequence containing PCHF15 is built with restriction enzyme Recombinant cloning vector pGEM-PCHF15 and expression vector DX2182 obtained by HindII digestions step 1, contain PCHF15 by what is cut The fragment of promoter is inserted into the HindIII restriction enzyme sites of DX2182 carriers, builds recombinant vector DX2182-PCHF15.

T4 connections kit connects promoter PCHF15 to DX2182, and 10 μ L systems are as follows：

4 μ L PCHF15 endonuclease bamhis (50ng)

4 μ L digestions carriers (100ng)

1 μ L T4 ligases

1μL 10×Buffer

Linker：22 DEG C 2 hours.

Electroporated competence Escherichia coli, select positive colony sequencing.DX2182-PCHF15 is included in promoter downstream Green fluorescence protein gene GFP genes, GFP gene orders, can be for checking PCHF15 promoters as shown in SEQ ID NO.7 Expression specificity and intensity.

Embodiment 4：Turn the acquisition of 1300gus-PCHF15 paddy rice

The Agrobacterium EHA105 of -70 DEG C of preservations is taken in containing 50 μ g/mL rifampin plate streakings, 28 DEG C of cultures.Picking single bacterium Fall to be inoculated in 50mL YEB fluid nutrient mediums, 28 DEG C of shaken cultivation 12-16hr of 220rpm.2mL bacterium solutions are taken to transfer in 100mL In (containing antibiotic) YEB fluid nutrient mediums, 28 DEG C of 220rpm shaken cultivations to OD600=0.5.Precooling on ice 10 minutes, 5000rpm 10min (refrigerated centrifuge is pre-chilled to 4 DEG C).Aseptic deionized water washes 2 times (each 10mL), and 10% glycerine is washed 1 time It is dissolved in the glycerine of 3mL 10%.The 1300gus-PCHF15 plasmids obtained in 100 μ L competent cells plus 1 μ L embodiments 1 are taken, 2.5KV it is electroporated.28 DEG C of cultures, select positive colony on the YEB culture plates containing kanamycins and rifampin, use PCAMBIA1300NH-GUSplus vector-specific primers SEQ ID NO.8 and SEQ ID NO.9 enter performing PCR checking.

The correct clone of checking, is infected by Agrobacterium-mediated genetic transformation method and 11 (Hiei Y Ohta is spent in paddy rice S,Komari T,Kumashiro T(1994)Efficient transformation of rice(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA.The Plant Journal 6：271-282).T0 is obtained for transgenosis through the link such as co-culturing, screening, break up, take root Seedling, extracts DNA, and the transgenic positive plant verified through PCR, self-fertility obtains T1 generations.After T0 or T1 are taken for plant progress Continuous analysis.

Embodiment 5：Transgenic paddy rice GUS staining analysis

Prepare GUS dyeing liquor X-Gluc reaction solutions (50mM phosphoric acid receives buffer solution, pH value 7.0, the 0.5mM potassium ferricyanides, 0.5mM potassium ferrocyanides, 0.5mg/mL X-Gluc, the methanol of volumn concentration 20%, 0.1%Triton X-100), it will adopt Each organization material of paddy rice of collection is immersed in X-Gluc reaction solutions 37 DEG C of 2h or overnight, then with the second of volumn concentration 70% Alcohol observes photograph after sloughing the chloroplaset color of tissue.As a result as shown in figure 4, showing blueness only in the flower pesticide of transgenic paddy rice, Blueness is had no in the other floral organs tissue (gynoecium, ovary) and nutritive issue (root, leaf) of paddy rice, shows that PCHF15 promoters can Drive gus gene specific expressed in Rice Anther.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Hainan Bo Lian paddy genes Science and Technology Ltd.

<120>A kind of plants flower pesticide specificity promoter PCHF15 and its application

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caccgtcgcc gtggacaaca tcctcgacga tagcaccctc ccggtggggc tgtacagcga 660

gcgccacgaa gagggcctcg gaaaagtcat tcgtaacaag ccgaacttcg acttcttcaa 720

ctatgcaggc ctgcaccgtc cggtgaaaat ctacacgacc ccgtttacgt acgtcgagga 780

catctcggtt gtgaccgact tcaatggccc aaccgggact gtgacctata cggtggactt 840

tcaaggcaaa gccgagaccg tgaaagtgtc ggtcgtggat gaggaaggca aagtggtcgc 900

aagcaccgag ggcctgagcg gtaacgtgga gattccgaat gtcatcctct gggaaccact 960

gaacacgtat ctctaccaga tcaaagtgga actggtgaac gacggactga ccatcgatgt 1020

ctatgaagag ccgttcggcg tgcggaccgt ggaagtcaac gacggcaagt tcctcatcaa 1080

caacaaaccg ttctacttca agggctttgg caaacatgag gacactccta tcaacggccg 1140

tggctttaac gaagcgagca atgtgatgga tttcaatatc ctcaaatgga tcggcgccaa 1200

cagcttccgg accgcacact atccgtactc tgaagagttg atgcgtcttg cggatcgcga 1260

gggtctggtc gtgatcgacg agactccggc agttggcgtg cacctcaact tcatggccac 1320

cacgggactc ggcgaaggca gcgagcgcgt cagtacctgg gagaagattc ggacgtttga 1380

gcaccatcaa gacgttctcc gtgaactggt gtctcgtgac aagaaccatc caagcgtcgt 1440

gatgtggagc atcgccaacg aggcggcgac tgaggaagag ggcgcgtacg agtacttcaa 1500

gccgttggtg gagctgacca aggaactcga cccacagaag cgtccggtca cgatcgtgct 1560

gtttgtgatg gctaccccgg agacggacaa agtcgccgaa ctgattgacg tcatcgcgct 1620

caatcgctat aacggatggt acttcgatgg cggtgatctc gaagcggcca aagtccatct 1680

ccgccaggaa tttcacgcgt ggaacaagcg ttgcccagga aagccgatca tgatcactga 1740

gtacggcgca gacaccgttg cgggctttca cgacattgat ccagtgatgt tcaccgagga 1800

atatcaagtc gagtactacc aggcgaacca cgtcgtgttc gatgagtttg agaacttcgt 1860

gggtgagcaa gcgtggaact tcgcggactt cgcgacctct cagggcgtga tgcgcgtcca 1920

aggaaacaag aagggcgtgt tcactcgtga ccgcaagccg aagctcgccg cgcacgtctt 1980

tcgcgagcgc tggaccaaca ttccagattt cggctacaag aacgctagcc atcaccatca 2040

ccatcacgtg tga 2053

<210> 5

<211> 33

<212> DNA

<213>Artificial sequence

<400> 5

cgtttaagct tacgtacgac gaggatgata tcc 33

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence

<400> 6

ttcctaagct tggatcgagt tcttggcgc 29

<210> 7

<211> 720

<212> DNA

<213> GFP

<400> 7

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<400> 8

cacgggtctc ggtgttgatc 20

<210> 9

<211> 25

<212> DNA

<213>Artificial sequence

<400> 9

ggaaacgaca atctgatcca agctc 25

Claims

1. a kind of plants flower pesticide specificity promoter PCHF15, it is characterised in that its nucleotide sequence such as SEQ ID No.1 institutes Show.

2. the complementary DNA molecular with plants flower pesticide specificity promoter PCHF15 described in claim 1.

3. the restructuring containing DNA molecular described in plants flower pesticide specificity promoter PCHF15 described in claim 1 or claim 2 Expression vector.

4. the expression casette containing claim 1 anther specific promoter PCHF15.

5. the expression cassette recombinant expression carrier containing expression casette described in claim 4.

6. expand the primer pair of anther specific promoter PCHF15 described in claim 1, it is characterised in that the primer pair Nucleotides sequence is classified as the nucleotide sequence shown in SEQ ID NO.2-3 or SEQ ID NO.5-6.

7. the method for driving foreign gene specific expressed in flower pesticide, it is characterised in that methods described comprises the following steps：

Anther-specificpromoter PCHF15 and purpose foreign gene described in claim 1 is imported into carrier and obtained containing flower Medicine specificity promoter PCHF15 and purpose foreign gene expression cassette recombinant expression carrier, and it is introduced into Plant Genome, Obtain the genetically modified plants of foreign gene specifically expressing in flower pesticide.

8. applications of the plants flower pesticide specificity promoter PCHF15 in prepare transgenosis plant described in claim 1.

9. the expression casette containing anther specific promoter PCHF15 described in claim 4 is in prepare transgenosis plant Using.