CN102965374A - Preparation method and applications of rape BnRabGDI3 promoter - Google Patents

Abstract

The invention discloses a preparation method and applications of a cabbage type rape BnRabGDI3 promoter. The preparation method comprises the steps of: firstly, obtaining a primer sequence amplified by the PBnRabGDI3 promoter: designing a primer according to an upstream 2kb sequence of a PBnRabGDI3 gene obtained from rape whole genome sequencing for PCR (Polymerase Chain Reaction) amplification; and secondly, extracting rape leaf genome DNA (Deoxyribose Nucleic Acid) by use of an SDS (Sodium Dodecyl Sulfate) cracking method, carrying out PCR amplification, connecting to a pMD18-T vector after carrying out purification and recycling of a gel extraction kit, converting the competent cell of a gold strain by a heat shock method, picking positive clones to obtain the upstream 2kb flanking sequence of the target gene, namely PBnRabGDI3. A GUS (glucosiduronide) staining result shows that PBnRabGDI3 is efficiently expressed in rape anthers and pollen grains and not expressed in the tissues of root, stem, leaf, silique, seed and the like. The promoter has good application potentiality in the aspects of improving crop quality, manually establishing germplasm resources, and the like.

Description

Preparation method and the application thereof of rape BnRabGDI3 promotor

Technical field

The present invention relates to plant genetic engineering and biological technical field, be specifically related to a kind of swede type rape BnRabGDI3 gene promoter (called after P _BnRabGDI3, below identical), also relate to simultaneously a kind of preparation method of swede type rape BnRabGDI3 promotor.The invention still further relates to the carrier that contains this promotor or its essence homologous nucleotide sequence and utilize the application of this promotor in rape and other plant genetic engineering with relating to.

Background technology

Plant gene promoter plays keying action in the expression regulation of gene.Regulation of Gene expression is the result of many factors comprehensive action.Generally according to the sequencing of an event, the regulation and control of gene are divided into the regulation and control of the regulation and control of transcriptional level, translation skill and the regulation and control of protein level of processing etc.The protein of genes encoding and RNA and secondary metabolite thereof are of crucial importance for the whole vital movement of keeping organism.The mistake of any gene expression regulation all can cause serious consequence to life.Therefore, the mechanism research for gene expression regulation is the focus of molecular biology research always.The regulation and control of transcriptional level are the most important.Promotor is the critical elements of transcriptional level control, also is an important component part of gene engineering expression carrier.

Plant gene promoter is the section of DNA sequence that is positioned at structure gene 5 ' end upstream, contains cis-acting elements, is determining specificity, direction and efficient that downstream gene is transcribed, is the factor of most critical in gene transcription regulation mechanism and the expression pattern.In addition, promotor is making up and can play a key effect in the process of high level expression heterogenous expression carrier, it determined the genetic expression of foreign gene sequence of time and space, expression intensity, transcribe the expression level of efficient and gene.So plant genetic engineering machine-processed and day by day maturation has very important scientific meaning to the functional sequence of research promotor for gene expression regulation.

Find that by the analysis to the various plants gene promoter area promotor of most functional protein genes all has common structural pattern, generally is comprised of core promoter element and upstream element.The TATA box that is positioned at transcription initiation site upstream-20--30bp place is core promoter element, it is the conserved sequence district of being rich in AT, relevant with unwinding of dna double chain, and the selection of decision transcripting start point, be that most plant promoter corrections are necessary.The conserved sequence of TATA box upstream is called the promotor upstream element, comprise upstream-75bp place CAAT box and-near the 80--110bp general upstream promoter element and other special upstream elements such as GC box, such as element (Zhang Chunxiao etc., Review on Plant Gene Promoters such as jasmonate response element (JRE), ethylene response elements (ERE).Acta Genetica Sinica, 2004,31(12): 1455-1464; Wait quietly on the road, Plant Promoter and applied research progress thereof.The natural science progress, 2004,14(8): 856862).CAAT box is the sequence of relatively guarding, and with the identification of RNA polymerase with in conjunction with relevant, genetic transcription is had stronger activation.Yet some gene is without this frame, as being replaced by the CATC frame without CAAT box in the storage protein gene of cereal crop.The conserved sequence of GC box is 5 ' GGGCGG3 ', and a plurality of copies can be arranged, and can exist and do not affect its function (road is quiet, Zhao Huayan, He Yikun, Song Yanru, Plant Promoter and applied research thereof progress with any direction.The natural science progress, 2004,14(8): 856862; The summer east of a river, Chen Zaiquan, Wu Yusheng, Ji Pengzhang, Plant Promoter function and structure progress.Yunnan Prov Agriculture University's journal, 2006,21(1): 714).As long as had these conservative structural frames, so just can have the function of corresponding startup downstream gene expression.

Transcriptional profile according to plant promoter can be divided into it constitutive promoter, organizing specific type promotor and inducible promoter.The foreign gene that constitutive promoter drives is stably express in all developmental stages of transfer-gen plant and tissue.Conversion to many dicotyledonss, usually all use from the 35S promoter of cauliflower mosaic virus (CaMV) or from the plasmid vector of the nopaline synthetic enzyme no promotor of bacterium, and the most frequently used in monocotyledons transforms be to contain rice actin Act promotor, the plasmid vector of corn ubiquitin Ubi promotor and 35S promoter (Guan Liying etc., the effective expression of foreign gene and safety evaluation thereof in the transgenic plant.Capital Normal University's journal, 2002,23(2): 52-56).But many times foreign gene continuing in recipient plant efficiently expresses the waste that not only causes the energy in the organism, and the expression in a organized way might have toxic action to plant itself, even cause Transgene-safty problem (Jia S-R.Environment and food biosafty assessment of transgenic plants.Advanced of Bioengineer.1997,17:37-42; Moris S H, Adley C.C.Irish public perceptions and attitudes to modern biotechnology:an overview with a focus on GM foods.Trends in Biotechnology.2001,19:43-48).Therefore, the research of inducible promoter and tissue-specific promoter and application are subject to breeder's attention day by day.The inducible promoter of identifying in transgenic plant mainly comprises the abiotic stress inducible promoter, (Nie Lina etc., the clone of plant gene promoter and the Research progress on Function thereof such as biological stress induced promoter and hormone induction type promotor.The plant genetic resources journal, 2008,9 (3): 385-391).But the application of inducible promoter also has certain limitation, and the external condition that recipient plant is carried out is processed, and such as heat shock, HORMONE TREATMENT etc. may cause a series of biochemical reactions in the organism and be unfavorable for the normal growth of plant.In addition, the Methylprednisone acetate (dex, dexamethasone), estradiol (estradiol) and the tsiklomitsin (tetracycline) that are used as inductor in the chemical regulation system are all harmful to ecotope, should not be for the production of practice.Use that the tissue-specific promoter of itself just can avoid this problem in the plant materials, obviously, the biological safety that the tissue specific expression of foreign gene will the Effective Raise genetically modified crops.(Song Yang etc., the research of plant tissue specificity promoter.The biotechnology circular, 2007, (4): 21-24).

In the last few years, made significant headway about tissue-specific promoter's research, these tissue-specific promoters mainly comprise reproductive organ specific expression promoter (Song Yang etc., the researchs of plant tissue specificity promoter such as the organ specific expression promoter such as blade, phloem, vascular bundle and root and pollen, floral organ, fruit, seed.The biotechnology circular, 2007, (4): 21-24).Separated LTP12 such as Ariizumi etc., the promotor of XTH3 and PGA4 and arabidopsis thaliana transformation, GUS dyeing shows that these 3 promotors are anther specific expression promoter, and there are differences (Ariizumi et al in different expression in period, Comparative study of promoter activity of three anther-specific genes encoding lipidtransfer protein, xyloglucan endotransglucosylase/hydrolase and polygalacturonase in transgenic Arabidopsis thaliana.Plant Cell Report.2002,21:90 – 96).The clone obtains the promotor of microsome oleic acid dehydrogenase gene (FAD2) in sesame, and forecast analysis shows that the E-box in this promotor is relevant with the biosynthesizing of triacylglycerol with the G-box element.Express by detecting gus gene behind the arabidopsis thaliana transformation, the result shows this promotor specifically expressing (Mi Jung Kim etal.Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2promoter and enhancers in the5'-UTR intron.Molecular Genetics and Genomics.2006,276:351-368) in seed.The strange grade of Geng An separated 4 promotors and transformation of tobacco from turnip type rape, swede type rape, tender flower stalk, wild cabbage, the GUS staining analysis shows that it is promotor (Geng etal.Expression analysis of four flower-specific promoters of Brassica spp.in the heterogeneous host tobacco.African Journal of Biotechnology.2009,8 (20): 51935200) of floral organ specifically expressing.In addition, Mariani etc. merge tobacco anther tapetum specific expression gene promotor TA29 and nuclease gene Barnase, RnaseT1 with rear conversion of plant, nuclease gene is specifically expressing in flower pesticide, and destruction tapetum, obtain male sterile tobacco and rape (Mariani C.etal.Induction of male sterility in plants by a chimaeric ribonuclease gene.Nature, 1990,347:737741).At present the TA29 promotor is in the plants such as tobacco, corn, rape, Arabidopis thaliana, paddy rice use and succeed (Song Yang etc., the research of plant tissue specificity promoter.The biotechnology circular, 2007, (4): 2124).This shows, even exist between the internal milieu difference between the different plants and gene and make mutually difference, even the plant larger with the Arabidopis thaliana nature difference, start zone and corresponding conservative controlling element as long as have conservative core, just can in other different plants, bring into play the biological function that starts downstream gene expression.

In recent years, functional study to promotor, the method that the lot of documents report adopts is normally first with after the information biology tentative prediction and analyzing promoter sequence, again promoter fragment and reporter gene are constructed expression vector, Cells In Vitro or the plant materials of transformation mode plant Arabidopis thaliana, tobacco, paddy rice etc. are analyzed the function of promotor by the expression of examining report gene in the transgenosis individuality again and are used.The promotor of a large amount of bibliographical information demonstration other plants is transformed in the above-mentioned plant all can exercise its biological function normally, as: the promotor of the microsome oleic acid dehydrogenase gene (FAD2) of cloning in sesame, the E-box in this promotor is relevant with the biosynthesizing of triacylglycerol with the G-box element.In transgenic arabidopsis and other transgenic plant, also demonstrated expression (the Mi Jung Kim of seed-specific, Heeja Kim, Mi Chung Suh.Seed-specific expression of sesame microsomal oleic acid desaturase is controlledby combinatorial properties between negative cis-regulatory elements in the SeFAD2promoter and enhancers in the5 '-UTR intron.Molecular Genetics and Genomics.2006,276 (4): 351-368).The ZmGLU1 gene promoter of from corn, cloning, be converted in the tobacco after connecting gus reporter gene, detect the analytical results corn the ZmGLU1 promoters driven gus gene efficiently express (Riliang Gu at the tobacco root, Li Zhao, Guoying Wang.Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco.Plant Cell Reports.2006,25 (11): 1157-1165).From potato, separate one promotor with closely similar TDF511 (tran-script derived fragment) the gene Stgan of ethanol dehydrogenase.Make up Stgan promotor-GUS fusion expression vector transformation of tobacco, the GUS histochemical stain shows that this promoters driven gene is at tobacco stem tubercle place specifically expressing, may participate in stem tuber forming process (Trindade L M, et al.Isolation and functional characterization of a stolon specific promoter from potato (Solanum tuberosum L.) .Gene, 2003,303:77-84.).More than these reports all be to utilize transgenic technology between different plants, to carry out the example that promoter function is analyzed, these examples show and utilize model plant Arabidopis thaliana, tobacco etc. as carrier, and the expression analysis by transfer-gen plant confirms to come from the function of promotor of other plant by extensively recognition and acceptance.

Rape is extensively planted in the Yangtze valley as the main oil crops of China, and national economy is had material impact.Along with the maturation of transgenic technology, transgene rape will inevitably face the public opinion of Transgene-safty risk.Being used in the expression that the promotor of not expressing fully in the Semen Brassicae campestris starts goal gene is a feasible method that addresses this problem, and has both reached and has both improved rape each side quality, does not cause again the purpose of edible oil security risk.

Therefore, the present invention has developed a swede type rape endogenesis promoter.The gene of the expression characteristic proof promoters driven by examining report gene GUS is not expressed in root, stem, blade, angle fruit, seed, and a large amount is expressed in flower pesticide and pollen granule.Our result is indicating that the promotor of this gene has a good application prospect in transgenic plant.

Summary of the invention

First purpose of the present invention is to be to provide a kind of swede type rape P _BnRabGDI3Promotor, its advantage is two aspects: at first, derive from the endogenous tissue-specific promoter of rape and can accurately locate the gene regulated and control, drive goal gene and express in particular organization or the period of transgene rape, avoid causing the waste of plant self energy and material.Secondly, improve the expression efficiency of foreign gene in transgenic plant, limit its expressive site.Therefore, this promotor can be applicable to the genetically engineered research of plant and seed with the safe transgenic research of rape.

Second purpose of the present invention is to be to provide a kind of swede type rape P _BnRabGDI3The preparation method of promotor.The method is carried out pcr amplification take the swede type rape genomic dna as template, obtains swede type rape P _BnRabGDI3Sequence.Its advantage is simple to operate, and the result is reliable and stable.

The 3rd purpose of the present invention is to be to provide a kind of recombinant vectors that plant efficient is expressed promotor that contains, and it contains described promotor nucleotide sequence.This carrier size is fit to, in plant easily with transform, institute with marker gene GUS expression intensity high, easily detection.Can obtain the Arabidopis thaliana transfer-gen plant that gus gene efficiently expresses by this carrier arabidopsis thaliana transformation in plant.

The 4th purpose of the present invention is to be to provide a kind of swede type rape P _BnRabGDI3The application of promotor in flower pesticide, pollen granule.Under the driving of this promotor, mainly meticulous expression in the flower pesticide of plant of goal gene is not expressed at other position.This promotor with tissue specific expression has good using value in creating the genetically engineered such as sterile line, manual creation germ plasm resource and Transgene-safty (edible, pollen drift).

In order to finish above-mentioned purpose, the present invention adopts following technical scheme:

In order to obtain the present invention, the contriver has carried out deeply and comprehensively research the genes involved in the Rape Development process, found that a kind of new promotor.The native gene of this promoters driven efficiently expresses in the flower pesticide of Arabidopis thaliana and pollen granule.

According to an aspect of the present invention, above-mentioned purpose can realize by a kind of promotor is provided, described promotor can specificity drives downstream gene and efficiently expresses in Arabidopis thaliana, described promotor contain SEQ ID No.1 nucleotide sequence or with the SEQ ID No.1 nucleotide sequence of homology in fact.

According to another aspect of the present invention, above-mentioned purpose can realize by the recombinant vectors that the nucleotide sequence that contains described promotor is provided.

According to another aspect of the present invention, above-mentioned purpose can realize by the microorganism that transforms with described recombinant vectors is provided.

According to another aspect of the present invention, above-mentioned purpose can realize by providing with the transgenic arabidopsis of described microbial transformation.

1 one kinds of swede type rape promotor P _BnRabGDI3The preparation method, the steps include:

1.1 homologous sequence method clone P _BnRabGDI3Sequence:

1.1.1 rape promotor P _BnRabGDI3Primer sequence:

BnRabGDI3 gene order information according to the acquisition of rape genome sequencing, carry out pcr amplification at its upstream, ATG initiator codon place 2kb position sequence design pair of primers, this sequence comprises the initiator codon of swede type rape BnRabGDI3 gene and the promoter sequence of this gene.The primer that adopts is PBnRabGDI3S:5 '-(Kpn I) CAGaagcttGAGTAAATACAGAACAGTGTTAC-3 ' and PBnRabGDI3A:5 '-(Xba I) GACAtctagaTGTTGCAACCAATCTCTATTATC-3 '.Used forward primer PBnRabGDI3S contains Kpn I restriction enzyme site, and reverse primer PBnRabGDI3A contains Xba I restriction enzyme site.

1.2.2 rape promotor P _BnRabGDI3Preparation:

The used rape of the present invention is two No. nine (the Wang Xinfa assistant researcher of oil crops institute provides, below identical) in the swede type rape (Brassica napus L.).In be seeded in the land for growing field crops, normal field management two No. nine.Utilize SDS cracking process (J. Pehanorm Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, below identical) extract in two No. nine rape leaf genomic dnas, carry out pcr amplification as template, step is: extract genomic dna 25 μ l, increase as template, reaction system is 50 μ l, add respectively 10 * Ex Taq buffer5 μ l, dNTP4 μ l, 5 ' primer, 1 μ l, 3 ' primer, 1 μ l, ExTaq0.5 μ l, the about 50ng of DNA masterplate 1 μ l, ddH ₂O37.5 μ l.Primer is PBnRabGDI3S and the PBnRabGDI3A (front is stated) according to the BnRabGDI3 upstream region of gene 2kb sequences Design of rape genome sequencing acquisition.The amplified production size is 2064bp, the PCR response procedures be 94 ℃ 5 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 2 minutes, 33 circulations, 72 ℃ 10 minutes, 16 ℃ 3 hours, the PCR product is through 1.0%(sepharose/TE solution quality volume ratio, below identical) agarose gel electrophoresis detects, gel reclaims test kit (available from root biochemical technology Beijing, sky company limited, below identical) after purifying reclaims, be connected to the pMD18-T carrier (available from the precious biotechnology in Dalian company limited, below identical), heat shock method (J. Pehanorm Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, below identical) transform the competent cell of gold bacterial strain (available from the precious biotechnology in Dalian company limited, below identical), coat and contain penbritin 50 μ g/mL(mass volume ratios, below identical) the LB solid medium (it is as follows to fill a prescription: take by weighing respectively 10 the gram Tryptoness, 5 the gram yeast extracts and 10 the gram sodium-chlor, 8 gram agar are dissolved in the distilled water successively, and constant volume is in 1000 milliliters.Be sub-packed in 500 milliliters of triangular flasks, 121 ℃, autoclave sterilization is 20 minutes under 6.859 * 104Pa.Minute install in the culture dish, 4 ℃ of refrigerations are for subsequent use, below identical) on the flat board, 37 ℃ of overnight incubation are selected 6 of hickies.Adopt the M13 primer: 5 '-TGTAAAACGACGGCCAGT-3 ' and 5 '-CAGGAAACAGCTATGACC-3 ', be bacterium colony PCR and detect.Bacterium colony PCR concrete grammar (following identical) is to do masterplate with a small amount of bacterial plaque of toothpick picking of sterilization, and reaction system is that 10 μ L include: 10 * Taq buffer(contains MgCl ₂) 1 μ l, 1.5mmol/L dNTP (10mmol/L) 1 μ l, 5 ' primer (10 μ mol/L), 0.5 μ l, 3 ' primer (10 μ mol/L), 0.5 μ l, Taq (5U/ μ l) 0.5 μ l, ddH ₂O6.5 μ l.Reaction conditions be 94 ℃ 5 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 2 minutes 30 seconds, 33 circulations, 72 ℃ 10 minutes, amplification size is 2064bp, 1.0%(states the front) the agarose gel electrophoresis detected magnitude correctly after.The bacterial plaque that the PCR detected magnitude is correct is inoculated into the liquid LB substratum that contains penbritin 50 μ g/mL, and (it is as follows to fill a prescription: take by weighing respectively 10 gram Tryptoness, 5 gram yeast extracts and 10 gram sodium-chlor, be dissolved in the distilled water, constant volume is in 1000 milliliters, 121 ℃, 6.859 autoclave sterilization is 20 minutes under the * 104Pa, below identical), 37 ℃ of lower 200r/min shaking culture are spent the night, (J. Pehanorm Brooker .D.W. Russell is outstanding for alkaline process in a small amount, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, below identical) extract plasmid, 1.0%(states the front) agarose gel electrophoresis detect the plasmid DNA size correct after (for 2064bp), I/(2 kinds of enzymes are available from TaKaRa company for Xba I enzyme to adopt Kpn, below identical) cut plasmid carried out double digestion (following identical), it is 10 μ l that enzyme is cut system (following identical), comprises plasmid DNA 5 μ l, Kpn I 0.5 μ l, Xba I0.5 μ l, ddH ₂O4 μ l, 37 ℃ are spent the night, 1.0%(states the front) the agarose gel electrophoresis detection.Correct positive recombinant clone called after pMD18-P will be detected _BnRabGDI3(with purpose fragment P _BnRabGDI3Be connected into the pMD18-T vector construction and form, below identical), in order to ensure the sequence information of promotor in the carrier, with pMD18-P _BnRabGDI3Serve Hai Yingjun company and check order, analytical results shows, has obtained a kind of rape BnRabGDI3 full length gene promotor of separation, and its sequence is nucleotide sequence shown in the SEQ ID NO:1.Called after P _BnRabGDI3

With the BnRabGDI3 upstream sequence P that clones and check order and obtain _BnRabGDI3With Signal Scan Search (the Higo et al. among core promoter element and the Upstream cis acting usefulness forecasting software PLACE, Plant cis-acting regulatoryDNA elements (PLACE) database.Nucleic Acids Research.1999,27:297～300.http: //www.dna.affrc.go.jp/PLACE/signalscan.html), PlantCARE(Magali Lescot, Patrice DAehais, Gert Thijs, et al.PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools in silico analysis of promoter sequences.Nucleic Acids Res, 2002,30 (1): 325-327.http: //bioinformatics.psb.ugent.be/webtools/plantcare/html/) and PlantPromDB (the IIham A.Shahmuradov of Softberry, Alex J.Gammerman, John M.Hancock, et al.PlantProm:a database of plant promoter sequences.Nucleic Acids Res., 2003,31 (1): 114-117.http: //linux1.softberry.com/berry.phtml topic=plantprom﹠amp; Group=data﹠amp; Subgroup=plantprom) these online softwares carry out the on-line prediction analysis of BnRabGDI3 core promoter element and Upstream cis acting.

The result shows, BnRabGDI3 promoter sequence P _BnRabGDI3Contain promoter in eukaryote necessary core parts TATA-box and CAAT-box, with TATA-box at a distance of 30bp.With CAAT-box be transcription initiation site at a distance of the A at 60bp place, mostly to be this rule of A consistent with Eukaryotic transcription initiation site for this.Further analyzing this promoter sequence finds, except necessary core parts, also have multiple known promoter function element: (1) POLLENILELAT52 (AGAAA), LAT enhancer element (TGTGA), GTGA-motif (GTGA) and TTTCT element in the BnRabGDI3 promoter sequence, these are functional element of pollen-specific promotor; (2) CCGTCC-box(CCGTCC), be meristem specifically expressing element; (3) Skn-1_motif(GTCAT), be the cis-regulating element of endosperm high level expression; (4) MBS(TAACTG), for responding the MYB protein binding site of drought stress; (5) TCA-element(GAGAAGAATT), be the Whitfield's ointment cis-regulating element; (6) CGTCA-motif(CGTCA), be the methyl jasmonate response element; (7) O2-site(GATGATGTAG), be the metabolic regulation site; (8) DOFCOREZM(AAAG), action site (the Bate N of Dof transcription factor, Twell D.Functional architecture of a late pollen promoter:pollen-specific transcription is developmentally regulated by multiple stage-specific and codependent activator elements.Plant Molecular Biology.1998,37,859 – 869; Park, J.I., Hakozaki, H., Endo, M., et al.Molecular characterization of mature pollen-specific genes encoding novelsmall cysteine-rich proteins in rice (Oryza sativa L.) .Plant Cell Reports, 2006,25 (5): 466-474; Rogers, H.J., Bate, N., Combe, J., et al.Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10[J] .Plant MolecularBiology, 2001,45 (5): 577 – 585.) (Fig. 2).The applicant predicts that the promotor of BnRabGDI3 may be an anther-specificpromoter thus, and because the existence of the special element of a plurality of mature pollens and enhanser, it may be the highest in the mature pollen cells amount of flower pesticide.The present invention will be by turning P _BnRabGDI3Connect the plant expression vector pBI-P that reporter gene GUS makes up _BnRabGDI3Arabidopsis thaliana transformation carries out the active result who detects to the transfer-gen plant that obtains and show that the gus gene of this promoters driven only efficiently expresses, and does not all express in the flower pesticide of Arabidopis thaliana in seed, blade, root and stem.Therefore, P _{BnRabG DI3}Have the downstream gene of driving and in Arabidopis thaliana flower pesticide, efficiently express, and the function of not expressing at seed and other positions.This promotor with tissue specific expression has good using value in plant genetic engineering and Transgene-safty (eating).

2, a kind of swede type rape promotor P _BnRabGDI3Application in the flower pesticide of transfer-gen plant the steps include:

2.1P _BnRabGDI3The structure of plant expression vector and agrobacterium tumefaciens bacterial strain EHA105(are purchased from still bio tech ltd of Shanghai, Shanghai, below identical) conversion:

The recombinant vectors that makes up is that the 35S promoter on the plasmid pBI121 (available from TaKaRa company, below identical) is obtained P with cloning in the technical scheme 1 _BnRabGDI3Fragment is replaced.For finishing this purpose, at first use Xba I/Kpn I (Chen, P.Y., Wang, C.K., Soong, S.C.To, K.Y.Complete sequence of the binary vector pBI121and itsapplication in cloning T-DNA insertion from transgenic plants.Mol.Breed.11,287-293) double digestion cloning vector pMD18-P _BnRabGDI3Promoter fragment, use simultaneously Xba I/Kpn I (front is stated) enzyme to cut the pBI121 plasmid.It is 10 μ l that enzyme is cut system (front is stated), and endonuclease reaction carries out in 37 degree incubators, after about 4-6 hour, states with the 1.0%(front) the agarose gel electrophoresis detection.With cloning vector pMD18-P _BnRabGDI3The small segment (about 1400bp) that large fragment (size is 2064bp, and the pBI121(front the is stated) enzyme that enzyme downcuts downcuts reclaims test kit (front is stated) with dna gel and reclaims.Press pMD18-P _BnRabGDI3Large fragment and pBI121(front that enzyme downcuts are stated) and the small segment that downcuts of enzyme (the 150ng large fragment: the 50ng small segment)=ratio (molar concentration rate) biased sample of 3:1, add T ₄Dna ligase 1 μ l, 10 * reaction buffer, 1 μ l, aseptic ddH ₂O replenishes volume to 10 μ L, and 16 ℃ of connections are spent the night.After heat shock method (front is stated) transforms the competent cell (front is stated) of gold bacterial strain, in the dull and stereotyped screening of the solid LB substratum (front is stated) that contains kantlex (50 μ g/mL), picking white bacterial plaque is done bacterium colony PCR(front and is stated), extract plasmid (front is stated), cut the recombinant plasmid called after pBI-P of checking size correct (endonuclease bamhi is 2064bp) through enzyme _BnRabGDI3(Fig. 3).Utilize freeze-thaw method (J. Pehanorm Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press, below identical) transform Agrobacterium EHA105(front and state): step is as follows:

1: get 0.2ml Agrobacterium competence, in ice, slowly melt.

2: add about 2 μ g recombinant plasmid dnas, mixing behind the ice bath 30min, drops into liquid nitrogen flash freezer 1min gently, and then 37 ℃ of water-bath 5min melt cell.

3: add 800 μ l and do not contain microbiotic LB liquid nutrient medium (front is stated), 28 ℃ of jogs are cultivated 4-5h.

4:12000rpm, 30s removes supernatant, and cell is resuspended in 0.2ml LB liquid nutrient medium (front is stated) substratum.

5: culture is uniformly coated on contain Rif(50mg/L), Str(50mgL) and on the two anti-agar plates of LB solid medium (front is stated) Kan(50mgL), cultivated 2 days for 28 ℃, picking list bacterium colony carries out bacterium colony PCR detection validation (front is stated) after transformant occurring on the flat board.

2.2P _BnRabGDI3Functional analysis:

2.2.1P _BnRabGDI3Plant expression vector pBI-P _BnRabGDI3Genetic transformation in Arabidopis thaliana and transfer-gen plant screening:

Adopt inflorescence infestation method (Zhang X.R.et al.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method.Nature, 2006,1:1-6, below identical) carry out transformation of Arabidopsis thaliana.Preparation contains recombinant vectors pBI-P _BnRabGDI3Agrobacterium tumefaciens EHA105(front state) bacterium liquid, change in the LB liquid nutrient medium (front is stated) that contains kantlex 50 μ g/ml, Rifampin 50 μ g/ml 28 ℃ of incubated overnight over to the day before yesterday transforming.Second day uses ultraviolet spectrophotometer (SPEKOL1300) to detect the light absorption value of bacterium liquid under the 276nm nano wave length, takes out when the light absorption value of bacterium liquid reaches between 1.6-2.0.With the centrifugal 10min of 4000g, abandon supernatant under the room temperature (20-25 ℃, below identical), precipitation is suspended in isopyknic 5% sucrose solution (mass volume ratio of sucrose and distilled water, below identical).The sucrose solution of muddiness is poured in the large culture dish, and adding final concentration before transforming is the 0.02%(volume ratio) Silwet l-77(available from the Five continents, Beijing unit industry science and trade center, below identical).The whole inflorescence of Arabidopis thaliana to be transformed being immersed in the sucrose gently, silent several 15 seconds, take out plant behind the mixing.Plant after the conversion is wrapped with a black plastic bag, is placed on the growth case and cultivates.Second day is opened plastics bag, and cultivate in the place that is placed on light intensity.Every the conversion that tries again in a week.Cultivate and gathered in the crops seed in about one month, seed under incubator or daylight dry 3-5 days.To transform the T0 of results for seed 70%(volume ratio) alcohol and 0.1%(mass ratio) mercuric chloride distinguish surface sterilization 3 minutes and 10 minutes, then with distilled water wash several (5～7 times), then blow and beat to containing MS solid screening culture medium (the MS macroelement mother liquor 100ml that concentration is the 50mg/L kantlex equably; MS trace element mother liquor 10ml; The organic mother liquor 10ml of MS; MS molysite 10ml; Inositol 10ml; Sucrose 30g; Transfer PH to 5.8 with 1M NaOH, the 12g agar powder is settled to 1L, and is for subsequent use after the high pressure 121 degree sterilizations.Add the 8g agar powder and can be configured to solid medium.Various mother liquor prescriptions see Table 1) surface.4 ℃ vernalization 4-6 days, it is dark to put into constant incubator (photoperiod 16(daytime)/8() hour, temperature: 22 ℃ of daytimes, 20 ℃ of dark cycles) cultivate, the transformed plant that screening obtains claims T1 for transformed plant (following identical).According to distinctive kalamycin resistance screening positive plant on the expression vector.Blade is long gets a little green seedling leaf during to enough sizes (3-4 leaf phase), and extraction DNA carries out the PCR positive detection (following identical) of converting material.Primer is NPT II F:5 '-GATGGATTGCACGCAGGT-3 ' and NPT II R:5 '-TCAGAAGAACTCGTCAAG-3 ', and reaction system is that 10 μ l include: template DNA template 1 μ L (about 50ng), 10 * Taq buffer(contains MgCl ₂) 1 μ l, 1.5mmol/L dNTP (10mmol/L) 1 μ l, 5 ' primer (10 μ mol/L), 0.5 μ l, 3 ' primer (10 μ mol/L), 0.5 μ l, Taq (5U/ μ l) 0.5 μ l, ddH ₂O5.5 μ l.Reaction conditions be 94 ℃ 5 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 1 minute, 32 circulations, 72 ℃ 10 minutes, the amplification size be 800bp(Fig. 4).The result shows and has obtained transgenic positive plant (plant that contains the testing goal fragment claims positive plant, below identical).

Table 1MS substratum mother liquor prescription

2.2.2P _BnRabGDI3Functional analysis:

Use P _BnRabGDI3Replacing the pBI121(front states) 35S promoter sequence on the plasmid, form pBI-P _BnRabGDI3The recombinant plant expression vector, reporter gene GUS wherein is subjected to P _BnRabGDI3Regulation and control.Utilize agrobacterium tumefaciens EHA105(front to state) mediation inflorescence infestation method (front is stated) arabidopsis thaliana transformation.Results T0 for seed after, detect through kalamycin resistance screening and PCR and to obtain T1 for conversion positive plant (front is stated).

Positive T1 for the transfer-gen plant seed in that to contain concentration be that the MS(front of 50mg/L kantlex is stated with PCR checking) the substratum upper seeding wheel, in the growth case, sprout after 4 ℃ of vernalization, sprouting condition: light application time 16h, temperature: 22 ± 2 ° of C began sampling dyeing after emerging 5 days.Dyeing course is as follows: sample is immersed in GUS dye liquor (X-gluc0.5mg/mL, phosphoric acid buffer 50mmol/L, each 0.5mmol/L of the Tripotassium iron hexacyanide and yellow prussiate of potash, EDTA10mmol/L, the Triton-x-1000.001%(volume ratio), methyl alcohol 20%(volume ratio) in, vacuum suction 5 minutes, 37 ℃ are spent the night.Second day decolours with alcohol-acetic acid (volume ratio is 1:1), until blade bleaches, uses afterwards distilled water rinsing for several times (5～7 times), and Stereo microscope (OLYMPUS SZX16) is taken pictures.Got respectively seedling stage 5 days, 10 days, 15 days whole plant; Reproductive stage, blade are got tender leaf and mature leaf; Flower rounds the flower that inflorescence contained out and the bud of not opening; After fertilization was really got 5 days in the angle, and 10 days, the angle fruit of 15 days and 20 days.It is exactly the position that gus gene is expressed that plant is dyed to blue position.Explore P by the expressive site and the expression intensity that detect gus gene under the different space-times _BnRabGDI3The spatial and temporal expression pattern.

The result shows that the gus gene of this promoters driven only efficiently expresses, and does not all express (Fig. 5) in seed, blade, root and stem in the flower pesticide of Arabidopis thaliana.Therefore, this promotor has the downstream gene of driving and efficiently expresses in plant anther, and does not express at seed and other positions.This promotor with tissue specific expression has good using value in plant genetic engineering and Transgene-safty (eating).As containing the carrier that this promoters driven causes the gene of Anther Abortion by structure, the transformation receptor plant, the artificial male sterile material of creating, be applied in the production of cross-breeding, or the control transgenic plant are by the elegant genetically modified escape that causes of pollen, or some improves the gene of pollens nutrition composition by promoters driven, improves pollens nutrition etc.Simultaneously because P _BnRabGDI3The gene that drives is not expressed in seed, therefore can not occur the protein product of external source quiding gene in seed, has important using value in transgenosis food safety field.

The description of a kind of swede type rape BnRabGDI3 promotor full length sequence SEQ ID NO:1 functional expression in model plant Arabidopis thaliana different tissues.Utilize homologous sequence method clone to obtain 5 ' upstream promoter sequence of swede type rape BnRabGDI3 gene.State with Signal Scan Search (front is stated), PlantCARE(front among the forecasting software PLACE with core promoter element and Upstream cis acting) and the PlantPromDB (front is stated) of Softberry core parts and the Upstream cis acting of BnRabGDI3 promotor have been carried out the on-line prediction analysis.The result shows that the promoter sequence of cloning contains TATA box core promoter sequence, (front has been stated, Fig. 2) for the upstream promoter elements such as CAAT and pollen-specific Expression element such as POLLENILELAT52 (AGAAA), LAT enhancer element (TGTGA), GTGA-motif (GTGA) and TTTCT element.Make up on this basis by the plant expression vector pBI-PBnRabGDI3(front of the reporter gene GUS of this promoter regulation and state).Adopt agriculture bacillus mediated florescence infestation method (front is stated) arabidopsis thaliana transformation, in T1 generation, stated in the MS(front that is added with kantlex) preliminary screening transfer-gen plant on the substratum, after Arabidopis thaliana grows two true leaves, be transplanted to and continue plantation on the vermiculite, after inflorescence appears in plant, carry out PCR and detect, utilize Molecular tools to identify the transgenic positive strain.T2 is for selecting 10 transgenic lines to carry out GUS dyeing.The dyeing of GUS histochemical method shows that the gus gene of this promoters driven is only expressed in the flower pesticide of Arabidopis thaliana.This shows that the gus gene of this promoters driven has certain spatial and temporal expression specificity, have the downstream gene of driving and in flower pesticide, express, do not express the function of (Fig. 5) in the seed fully.This promotor with tissue specific expression has using value in manual creation germ plasm resource, plant genetic engineering and Transgene-safty (eating).As utilize the goal gene of this promoters driven pollen granule related to development, at first make up P _BnRabGDI3Drive the over-express vector of goal gene, the transformation receptor plant, then goal gene is at P _BnRabGDI3Under the driving of promotor, can improve the expression amount of goal gene in above-mentioned tissue, in seed, not express fully simultaneously, can not cause food-safety problem.

Advantage of the present invention:

1, the promotor that the promotor cloning process that provides is cloned into can by regulation and control and the histochemical analysis to gus gene, obtain to have the promotor of tissue specific expression function.

2, P _BnRabGDI3Promotor is in the security of rape edible oil and utilize the transgene improvement crop quality, and the aspects such as manual creation germ plasm resource have good application potential.P with the rape source _BnRabGDI3Replacing the pBI121(front states) 35S promoter sequence on the plasmid, form pBI-P _BnRabGDI3The recombinant plant expression vector, gus gene wherein is subjected to P _BnRabGDI3Regulation and control.Prove P by transgenic experiments _BnRabGDI3In rape flower pesticide, pollen granule, efficiently express, in the tissues such as root, stem, leaf, angle fruit and seed, do not express.Characteristic applies people according to this promoter expression can replace the CaMV35S strong promoter to drive the gene relevant with pollen development with this promotor, so that pollen development genes involved overexpression in pollen, artificial sterile line, the restorer created enriched germ plasm resource; Or the diffusion of inhibition foreign gene, prevent that gene from drifting about, and improves the biological safety of transgenic plant.Therefore, this promotor improves the aspects such as crop quality aspect and manual creation germ plasm resource in the security of rape transgenosis edible oil, has good application potential.

Description of drawings

Fig. 1 is a kind of swede type rape BnRabGDI3 of a kind of amplification promoter gene fragment electrophoresis synoptic diagram.

Swimming lane 1 is the nucleic acid Marker of DL2000; Swimming lane 2 is the promoter fragment result of the pcr amplification take genomic dna as template.

Fig. 2 is a kind of P _BnRabGDI3Promoter sequence analytical results synoptic diagram.

Shaded boxes represents the primary element element relevant with anther-specific expression. TGTGGWith TGTGAIt is LAT enhanser original paper;

It is the POLLENLELAT52 element;

It is the reverse complementary sequence of POLLENLELAT52 element;

It is the GTGA element.

Fig. 3 is a kind of pBI121-BnRabGDI3 carrier synoptic diagram of structure.

Fig. 4 is that the transfer-gen plant PCR of a kind of pBI121-PBnRabGDI3 identifies synoptic diagram.

Swimming lane 1 is the nucleic acid Marker of DL2000; Swimming lane 2 is pBI121-P BnRabGDI3 positive plasmid; Swimming lane 3 is the wild-type Arabidopis thaliana; Swimming lane 4-22 is the transgenic positive plant.

Fig. 5 is that a kind of flower pesticide efficiently expresses P _BnRabGDI3The histochemical stain result schematic diagram of the gus gene of promoters driven.

A is cotyledon period seedling (colourless); B is seedling (colourless) on the 20th; C is the whole inflorescence (11-14 phase flower pesticide is blue) at florescence; D is initial stage angle fruit (colourless); E is ripening stage angle fruit (colourless); F is 13 phase buds (flower pesticide is blue); G is the stamen (blueness) in the 13 phase buds, and the upper right corner is pollen granule (blueness); H is 9 phase anther tissue sections (colourless); I is 10 phase anther tissue sections (blueness); J is 13 phase anther tissue sections (blueness); K is 14 phase anther tissue sections (blueness).

Embodiment

According to following examples, can better understand the present invention, but described embodiment is in order better to explain the present invention rather than limitation of the present invention.

Embodiment 1.:

A kind of swede type rape promotor P _BnRabGDI3The preparation method of promotor the steps include:

1, rape promotor P _BnRabGDI3Primer sequence:

The 1 pair of primer of BnRabGDI3 upstream region of gene 2kb sequences Design that obtains according to the rape genome sequencing carries out pcr amplification, and the primer of employing is PBnRabGDI3S:5 '-(Kpn I) CAGaagcttGAGTAAATACAGAACAGTGTTAC-3 ' and PBnRabGDI3A:5 '-(Xba I) GACAtctagaTGTTGCAACCAATCTCTATTATC-3 '.Used forward primer PBnRabGDI3S contains Kpn I restriction enzyme site, and reverse primer PBnRabGDI3A contains Xba I restriction enzyme site.

2, rape promotor P _BnRabGDI3Preparation:

The used rape of the present invention is two No. nine (front is stated) in the swede type rape (Brassica napus L.).In be seeded in the land for growing field crops, normal field management two No. nine.Utilize SDS cracking process (front is stated) to extract the rape leaf genomic dna, carry out pcr amplification as template, step is: extract genomic dna 25 μ l, increase as template, reaction system is 50 μ l, add respectively 10 * Ex Taq buffer5 μ l, dNTP4 μ l, 5 ' primer, 1 μ l, 3 ' primer, 1 μ l, ExTaq0.5 μ l, the about 50ng of DNA masterplate 1 μ l, ddH ₂O37.5 μ l.The BnRabGDI3 upstream region of gene 2kb sequences Design PCR the primer that obtains according to the rape genome sequencing is that the PBnRabGDI3S(front is stated) and the PBnRabGDI3A(front state).The amplified production size is 2064bp, the PCR response procedures be 94 ℃ 5 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 2 minutes, 33 circulations, 72 ℃ 10 minutes, 16 ℃ 3 hours, the PCR product is stated through the 1.0%(front) the agarose gel electrophoresis detection, gel is connected to pMD18-T carrier (front is stated) after reclaiming the recovery of test kit (front is stated) purifying, heat shock method (front is stated) transforms the competent cell (front is stated) of gold bacterial strain, coat and contain penbritin 50 μ g/mL((fronts and state) LB solid medium (front is stated) flat board on, 37 ℃ of overnight incubation are selected 6 of hickies, adopt M13 primer (front is stated) to be bacterium colony PCR and detect (front is stated), 1.0%(states the front) after the agarose gel electrophoresis detected magnitude is correct.The bacterial plaque that the PCR detected magnitude is correct is inoculated in the liquid LB substratum that contains penbritin 50 μ g/mL (front is stated), 37 ℃ of lower 200r/min shaking culture are spent the night, alkaline process (front is stated) extracts plasmid in a small amount, 1.0%(states the front) agarose gel electrophoresis detection plasmid DNA size correct rear (for 2064bp), I/Xba I(front is stated to adopt Kpn) enzyme carries out double digestion (front is stated) to plasmid, it is 10 μ l that enzyme is cut system (front is stated), 37 ℃ are spent the night, and 1.0%(states the front) the agarose gel electrophoresis detection.Detect correct positive recombinant clone, called after pMD18-P _BnRabGDI3(front is stated) is with pMD18-P _BnRabGDI3Serving Hai Yingjun company checks order.Analytical results shows, obtains BnRabGDI3 full length gene promoter sequence, called after P _BnRabGDI3A kind of promotor of separation, its series be the nucleotide sequence shown in the SEQ ID No.1 or with the SEQ ID No.1 nucleotide sequence of homology in fact.

3, P _BnRabGDI3The structure of recombinant expression vector and agrobacterium tumefaciens transform:

With Xba I/Kpn I (front is stated) respectively double digestion be connected with P _BnRabGDI3PMD18-P _BnRabGDI3(front is stated) and pBI121(front are stated) plasmid.Gel reclaims the P that enzyme scales off _BnRabGDI3Fragment and pBI121(front are stated) fragment, connect, heat shock method (front is stated) transforms intestinal bacteria, is built into plant expression vector pBI-P _BnRabGDI3(front is stated) (Fig. 3).Using at last freeze-thaw method (front is stated) that this carrier is imported agrobacterium tumefaciens EHA105(front states), select positive colony containing on kantlex (50mg/L) and the two resistance LB flat boards of Rifampin (50mg/L) (front is stated), state with bacterium colony PCR(front) whether checking contain target fragment.

Embodiment 2:

PBI-P _BnRabGDI3Transformation of Arabidopsis thaliana and PCR detect:

Adopting inflorescence infestation method (front is stated) arabidopsis thaliana transformation, according to the peculiar kalamycin resistance of transfer-gen plant, in that to contain concentration be that the MS(front of 50mg/L kantlex is stated) substratum grows, and the green seedling of acquisition is tentatively thought transformed plant.Plant to be transformed is transplanted to it in vermiculite after growing two true leaves, after inflorescence appears in plant to be planted, gets a slice true leaf and extracts genomic dna (front is stated) with the SDS method, carries out PCR and identifies.Primer sequence is that NPT II F and NPT II R(front are stated), the PCR reaction system is as follows: template DNA 1 μ L (about 50ng), 10 * Taq buffer(contain MgCl ₂) 1 μ l, 1.5mmol/L dNTP (10mmol/L) 1 μ l, 5 ' primer (10 μ mol/L), 0.5 μ l, 3 ' primer (10 μ mol/L), 0.5 μ l, Taq (5U/ μ l) 0.5 μ l, ddH ₂O5.5 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min32 circulations, 72 ℃ are extended 5min.With 1% sepharose (front is stated) electrophoresis detection PCR reaction product, size is 800bp.The result shows P _BnRabGDI3Expression vector pBI-P _BnRabGDI3Successfully change Arabidopis thaliana (Fig. 4) over to, obtained altogether the positive seedling of 19 strains.

Embodiment 3:

Swede type rape P _BnRabGDI3The functional analysis of promotor:

The present invention clones first and obtains P _BnRabGDI3Sequence, and it has been carried out functional analysis.From embodiment 2, detect (front is stated) screening by model plant transformation of Arabidopsis thaliana (front is stated) and PCR and obtain transgenic positive seedling T1 generation, selfing results seed (being T2 generation).At different times, the different tissues of getting T2 generation 10 strain transformed plants carries out GUS dyeing.

T2 is as follows for the GUS dyeing course of transfer-gen plant and tissue: sample is immersed in the middle vacuum suction of GUS dye liquor (front is stated) 5 minutes, and 37 ℃ are spent the night.Second day decolours with alcohol-acetic acid (volume ratio is 1:1), until blade bleaches, uses afterwards distilled water rinsing 3-5 time, takes pictures under Stereo microscope (OLYMPUS SZX16).Get the whole plant of different time sections seedling stage; Reproductive stage, blade are got tender leaf and mature leaf; The inflorescence of the flower that flower took away and the bud of not opening; The angle fruit of different times is really got at the angle.It is exactly the position that gus gene is expressed that plant is dyed to blue position.

Om observation result according to the section of the cytology of flower pesticide, Arabidopis thaliana can be occurred to anther dehiscence from the former base of stamen comes off and at length is divided into (Sanders et al.Anther developmental defects in Arabidopsis thaliana male-sterile mutants.Sex Plant Repro in 14 periods, 1999,11:297-322), coloration result is found (table 3, Fig. 5), early development flower pesticide (1-9 phase) does not occur blue, development later stage flower pesticide (10-14 phase) and pollen granule are dyed to blueness, and exothecium also has a small amount of dyeing; In the fruit of angle, blueness does not appear in seed, does not occur blue in other tissue.This shows that the gus gene of this promoters driven mainly in development later stage flower pesticide (10-14 phase) and pollen granule and middle expression, has a small amount of expression in the exothecium.In other tissue, do not express.

Experimental result shows, rape P _BnRabGDI3Have following biological function: the gus gene of this promoters driven is expressed in the flower pesticide of Arabidopis thaliana and pollen granule, and a small amount of expression is arranged in the exothecium, does not express in other tissue.P _BnRabGDI3Gus gene under the regulation and control has certain spatial and temporal expression characteristic, and this illustrates P _BnRabGDI3Have and drive the function that downstream reporter gene GUS expresses in recipient plant.Under the regulation and control of this promotor, mainly meticulous expression in the later stage of transfer-gen plant flower pesticide (10-14 phase) and pollen granule thereof of gus gene, and in other tissue, do not express (table 3, Fig. 5) fully.This promotor with tissue specific expression has using value in plant genetic engineering and Transgene-safty (eating).

A kind of swede type rape P _BnRabGDI3Promotor is in the flower pesticide of model plant Arabidopis thaliana and the application in the pollen granule.Utilize homologous sequence method clone to obtain 5 ' upstream promoter sequence of rape BnRabGDI3 gene.This promoter sequence of process bioinformatic analysis has core parts and the upstream element of promoter expression, makes up the plant expression vector pBI-P by the reporter gene GUS of this promoter regulation _BnRabGDI3(front is stated).Adopt agriculture bacillus mediated florescence infestation method arabidopsis thaliana transformation, in T1 generation, stated in the MS(front that is added with kantlex) preliminary screening transfer-gen plant on the substratum, after Arabidopis thaliana grows two true leaves, be transplanted to and continue plantation on the vermiculite, after inflorescence appears in plant, carry out PCR and detect, utilize Molecular tools to identify positive strain.T2 is for selecting 10 transgenic lines to carry out GUS dyeing.The dyeing of GUS histochemical method shows that the gus gene of this promoters driven is expressed in the later stage of Arabidopis thaliana flower pesticide (10-14 phase) and pollen granule thereof.This shows that the gus gene of this promoters driven has certain spatial and temporal expression specificity, have the downstream gene of driving and in later stage flower pesticide (10-14 phase) and pollen granule thereof, express, do not express the function of (Fig. 5) in other tissue fully.This promotor with tissue specific expression has good using value in plant genetic engineering and Transgene-safty (eating), as utilizes this promoters driven pollen fertility or the goal gene relevant with oleaginousness, at first makes up P _BnRabGDI3Drive the over-express vector of goal gene, the transformation receptor plant, then goal gene is at P _BnRabGDIUnder the driving of 3 promotors, in later stage flower pesticide (10-14 phase) and pollen granule thereof, express, can improve the expression amount of goal gene in above-mentioned tissue, do not express fully in the seed, can not cause food-safety problem.

Table 3P _BnRabGDI3Transgenic arabidopsis T2 is for the GUS dyeing cartogram of strain

SEQUENCE LISTING

＜110〉Inst. of Oil Crops, Chinese Academy of Agriculture

＜120〉preparation method of rape BnRabGDI3 promotor and application thereof

＜130〉preparation method of rape BnRabGDI3 promotor and application thereof

<160> 1

<170> PatentIn version 3.1

<210> 1

<211> 2064

<212> DNA

＜213〉rape BnRabGDI3 promotor total length

<400> 1

cagttgtgtt gattctgacg aatcaaaatt tcaagatgaa tattttttgc acataaattt 60

ggtaaatttg caagtacgaa acgaaaagca gcttcagatt aaagaatgaa agcaaacaac 120

gtttaggttg tggtcggtaa tctacacaac gactgtagat ccttttgcct tgtgtggtat 180

gttatgctat ttgtcatcaa gtcattgaag tacaatatat tgtttgatat atttgatctt 240

cagacatatt ttacaaaaga aagttataga attgggtata acttcagccg gtaacaactt 300

tctttcaata aaatgtataa gccaaaatga aacaagtctg atcagaagct tgctacctct 360

aatctgagaa aacaagtgtt ggtgccttac ttccatcgca actatatcat ccgaaaccag 420

ttcttggata tgatacataa aggttataca taagattaca ttacattgat tccacaaacg 480

aatcaaagct acatcatttt caactactaa caatacatag gcccgagaac atgcaaccac 540

aaaacaaaca aggacataat aaaggtctta ctttcactaa gtagccttga ccttatcagc 600

tacaacaaca caaaagtatt aacatatttt tcaagaactc atcaagttcc tcaaaaaact 660

ctatcctttt gatatcatcc tcctttctta ttcttctttg catgatctct ctcctcatca 720

tcatcagata tgttaatata taactcgtca ccaccatcct ccacgtcgtg atagtttctt 780

ggagctggtg ttttaacatt tgcctgatga tgatcagcgc ttagcacagg gcagtggaag 840

agatgtgaaa ccggcgacca accagaacca gaggttttag catcacctga aaaccctacc 900

ggaaacatag cccacgaaca gaaaaagtaa acgccctggc cattactctc caccggcggc 960

gtctccatct taaccgcttc aaacgctctc ccacactgct gacacctcaa cgcgcattcc 1020

tcgtagccct tagggtactc gaaaagcgcg taacagtacg gacacgccgt ccaaaagctc 1080

gcagcattag gactctccga ggtctcagct aactgctccc gttggctttg aggttgaccg 1140

agttggtggt cgtagattga ttttctcgga ggatcggaga ggacgcacca agcctcagct 1200

tgagcgcttg gtcggcgaaa gggagacggt tcacagtggg gttgaggaga agagcgagtc 1260

tacggtactg aaccgcgatg cgttcgaggc tttgggttag tttttcgagt cggagcaccg 1320

cgtaccagtc aggtagttta gaatctccga cggtggattc tccggcgagg agagtgtcgg 1380

cgatggcgat gatgtagtct gctgcatgtg ttcgggacga gtcgtcttcg catgcgcgga 1440

tggctaatgc ctttgctcct ttgaagtcac ttgaggctag aagctccttc gatgtgacta 1500

gccaccgatc cgcctccgct ccgttatcgt cgccgctgac catatctttg tttctcaacc 1560

tttgtttttg agtaaataca gaacagtgtt actttgctag tggaccaaca agacactgtc 1620

ggactataca caatatctat gggtagtttt gtcatttcca ctaggcctct ctaaaccggc 1680

cggtatatag cataccaatg attgttattg aactgatctc aagttttttt ttttgtcttg 1740

gtcaaccatt gttattagga caattttgct ttcccacatc tttgtctgaa tatagtaaat 1800

tttgggagat ttctttaaca atttctttac atttgaagtt atgaaccaat ataactcctg 1860

acaatgcggt tttgattatt aacgtggaac attctcatga ggtgaaacgg taagatcgga 1920

ggtggtggta aagactgtaa taacagccac acatcatcct ctatttcttt ttcttcccac 1980

gcattttgtt tctctgatat aacaaaaact attagagatt agagaagaat taaagacgat 2040

cgataataga gattggttgc aaca

Claims (5)

1. the promotor PBnRabGDI3 of a separation, its sequence is the nucleotide sequence shown in the SEQ ID No.1.

2. the promotor of a kind of separation according to claim 1, it is characterized in that: described promotor contains recombinant vectors pBI-PBnRabGDI3.

3. the preparation method of a kind of swede type rape promotor PBnRabGDI3 claimed in claim 1 the steps include:

(1) primer sequence of rape promotor PBnRabGDI3:

The Bn RabGDI3 upstream region of gene 2kb sequences Design pair of primers that obtains according to the rape genome sequencing carries out pcr amplification, amplification obtains 5 ' the upstream promoter sequence of rape BnRabGDI3, and the primer is PBnRabGDI3S:5 ' CAGaagcttGAGTAAATACAGAACAGTGTTAC-3 ' and PBnRabGDI3A:5 '-GACAtctagaTGTTGCAACCAATCTCTATTATC-3 ';

(2) preparation of rape promotor PBnRabGDI3:

The used primer of 5 ' upstream 2kb sequences Design of the BnRabGDI3 that obtains according to the rape genome sequencing: PBnRabGDI3S and PBnRabGDI3A, utilize the SDS cracking process to extract the rape leaf genomic dna, carry out pcr amplification as template, step is: extract rape genomic dna 25 μ l, increase as template, reaction system is 50 μ l, add respectively 10 * Ex Taq buffer, 5 μ l, dNTP 4 μ l, 5 ' primer, 1 μ l, 3 ' primer, 1 μ l, ExTaq 0.5 μ l, DNA masterplate 1 μ l50ng, ddH2O 37.5 μ l, the amplified production size is 2064 bp, the PCR response procedures be 94 ℃ 5 minutes, 94 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 2 minutes, 33 circulations, 72 ℃ 10 minutes, 16 ℃ 3 hours, the PCR product detects through 1.0% mass volume ratio agarose gel electrophoresis, after gel reclaims the recovery of test kit purifying, be connected to the pMD18-T carrier, the heat shock method transforms the competent cell of gold bacterial strain, picking positive colony, PCR detects positive and enzyme is cut the rear order-checking of checking, obtaining goal gene upstream 2kb flanking sequence, is the promotor full length sequence of BnRabGDI3 through this sequence of bioinformatic analysis, called after PBnRabGDI3.

4. the application of the promotor PBnRabGDI3 of a kind of separation described in the claim 1 in Arabidopis thaliana flower pesticide.

5. the application of the promotor PBnRabGDI3 of a kind of separation described in the claim 1 in the thaliana flower powder.

Images