CN102115751A - Rape BnPABP 5 gene and application of promoter thereof - Google Patents

Abstract

The invention discloses a rape BnPABP 5 gene and the application of a promoter thereof. The sequence of a segregation gene is nucleotide sequence indicated by SEQ ID No. 1, SEQ ID No.2. The promoter comprises a recombinant vector. The invention also discloses the application of the rape BnPABP 5 gene the promoter thereof in controlling pollen fertility and anther. The gene and the promoter thereof are predominantly expressed in the rape anther. The invention further relates to the isolation, cloning, analysis of expression and application of gene BnPABP5 and the promoter thereof for controlling microspore development and the anther fertility. The BnPABP 5 gene has the function of controlling the anther development of rape as well as the pollen fertility. The expression of the BnPABP 5 gene can be weakened through the technique of gene engineering so that the pollen fertility can be controlled and male sterile line materials as well as restorer line materials can be created, therefore, the rape BnPABP 5 gene can be applied to the development and research and the quality improvement of rape andro gamete. The promoter of the BnPABP 5 gene is only predominantly expressed in the anther, and the promoter with tissue preference has higher potential of edible safety on transgenic oil crops, thus having certain development and application value.

Description

The application of rape BnPABP5 gene and promotor thereof

Technical field

The present invention relates to plant genetic engineering and biological technical field.Be specifically related to a kind of gene relevant and control the promotor that this gene efficiently expresses in pollen or flower pesticide with pollen development, and the preparation method of this gene and promotor.The invention still further relates to the carrier that contains this gene and its promotor or its homologous nucleotide sequence and utilize this gene or the application of its promotor in the rape genetically engineered with relating to.

Background technology

The ripe mRNA molecule of most of eukaryotes all has one section poly (A) tail, and it is relevant with the initial adjusting stable and translation of mRNA.Conjugated protein (poly (A) the binding protein of Poly (A), PABP) extensively be present in the dissimilar cell of eukaryote, with 25bp or longer poly (A) very strong avidity (Sachs A B is arranged, Davis R W and Kornberg R.D.A single domain ofyeastpoly (A)-binding protein is necessary and sufficient for RNA binding and cell viability.Mol Cell Biol., 1987,7 (9): 3268-3276.).PABP albumen can with the shortest polymer combination that comprises 12 VITAMIN B4, form a complex body, this complex body can cover 27 VITAMIN B4 fragment (BaerW at most, Kornberg D.Repeating structure of cytoplasmic poly (A)-ribonucleoprotein.ProcNatl Acad Sci USA 1980,77:1890-1892.), PABP albumen mainly is to discern Poly (A) sequence (Burd G by the RRM in the tertiary structure (RNA recognition motif), Dreyfuss G.Conservedstructures and diversity of functions of RNA-binding proteins.Science 1994,265:615-621.).

The research of pair cell nuclear PABP at present falls behind than tenuigenin PABP research, mainly is because their crystal and NMR (nuclear magnetic resonance) structure are not also determined.However, existing at present determining at its N-terminal has a RRM structural domain, there is one to be rich in arginic structural domain (Wahle E at C-terminal, Ruegsegger U:3-end processing of pre-mRNA in eukaryotes.FEMS Microbiol Rev 1999,23:277-295.).The zymic survival must have the existence of nucleus PABP, it is by gene NAP2 coding (David A Mangus, Matthew C Evans, Allan Jacobson.Poly (A)-binding proteins:multifunctional scaffolds for the posttranscriptional controlof gene expression Genome Biol.2003; 4 (7): 223.).

PABP albumen plays an important role in the genetic expression process.As the cis acting factor be combined in new synthetic or sophisticated mRNA on, it is special to play a part in the process such as initial of the polyadenous purineization of transcript, transportation, translation.It by with the biosynthesizing of transcribing post-treatment: mRNA that combines at least regulating mRNA aspect following three of poly (A) tail, initial (the Dmitry A.Belostotsky.Unexpected Complexity of Poly (A)-Binding Protein Gene Families inFlowering Plants:Three Conserved Lineages That Are at Least 200Million Years Oldand Possible Auto-and Cross-Regulation Genetics.163 (1): 311-319) of mRNA degraded and protein synthesis.To Poly (A) protein-bonded studies show that they all exist four placed in-line RNA recognition structure territories (RNA recognitionmotif, RRM).Although these four RRM there are differences, but its three-dimensional structure is similar, all comprise and to discern and in conjunction with β 1-α 1-β 2-β 3-α 2-β 4 structures (the Dreyfuss G of Poly (A), M J Matunis, SPinol-Roma, and C G Burd.hnRNP Proteins and the Biogenesis of mRNA.AnnualReview of Biochemistry.1993,62:289-321.).Research to the PABP gene mainly concentrates in yeast and animal, and their primary structure and gene structure all have conservative property, and less to the research of higher plant.The deletion mutant of PABP gene is the type that causes death in yeast cell, this shows that this gene plays an important role in vital process, and Arabidopis thaliana PABP5 gene is an anther-specific expression, and it can complementary zymic deletion mutant, recover activity (the Belostotsky D A of yeast cell, Meagher R B.Apollen-, ovule-, and early embryo-specific poly (A) binding protein from Arabidopsiscomplements essential functions in yeast.Plant Cell, 1996,8 (8): 1261-75.).Infer that thus this gene may also play important effect in the microspore development process, its disappearance may cause the abortion of plant.At present from yeast (Sachs A.B, Bond M.W.and Kongherg R.D.A singlegene from yeast for both nuclear and cytoplasmic polyadenylate-binding proteins:domain structure and expression.Cell, 1986,20; 45 (6): 827-35.), the pawl frog (Lefrere V, Vincent A, Amalric F.Drosophila melanogaster poly (A)-binding protein:cDNAcloning reveals an unusually long 3 '-untranslated region of the mRNA, also present inother eukaryotic species.Gene, 1990,15; 96 (2): 219-25.), people (Grange T, de Sa CM, Oddos J, Pictet Ral.Human mRNA polyadenylate binding protein:evolutionaryconservation of a nucleic acid binding motif.Nucleic Acids Res.1987June, 15 (12): 4771-4787.), Arabidopis thaliana (Belostotsky D A, Meagher R B.Differential organ-specificexpression of three poly (A)-binding-protein genes from Arabidopsis thaliana.ProcNatlAcad Sci U S A.1993,90 (14): 6686-6690.), Radix Dauci Sativae (Lin Huixin, Zhang Lei, Yang Zhi climbs, Huang Meijuan, Wu Naihu. the separation of Radix Dauci Sativae DcPAB gene and structure and functional analysis. the Biochemistry and Molecular Biology newspaper, 2004,20 (3): 319-324.), wheat (Hanh.Le, Su-chih.Chang, Tanguay R.L, Gallie D.R.The wheat poly (A)-binding protein functionally complements pab l inyeast.Eur.J.Biochem, 1997,243 (1-2): 350-7) be separated to the PABP gene in the species such as grade, but still do not had the correlative study report of rape PABP gene.

Plant gene promoter plays keying action in the expression of gene regulation and control.The regulation and control of genetic expression are result of various factor comprehensive action.Generally according to the sequencing of an incident, 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.To a certain extent, promotor has determined the space-time order and the expression intensity of genetic expression.So the function sequence of research promotor has very important significance for gene expression regulation mechanism and day by day sophisticated plant genetic engineering.

Promotor rises and is making up and can play a key effect in the process of high level expression heterogenous expression carrier, and what it had determined foreign gene transcribes efficient and expression of gene level.The used promotor of plant transgene breeding can be divided into composing type and Idiotype two classes.The foreign gene that constitutive promoter drives stable expression in all developmental stages of transfer-gen plant and tissue.Conversion to many dicotyledonss, usually all use the 35S promoter that contains cauliflower mosaic virus (CaMV) or the plasmid vector of nopaline synthetic enzyme no promotor, 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 transgenosis safety problem (Jia S-R.Environment and food biosafty assessment of transgenic plants.Advanced ofBioengineer.1997,17 (6): 37-42; Morris S H, Adley C.C.Irish public perceptions andattitudes to modern biotechnology:an overview with a focus on GM foods.Trends inBiotechnology, 2001,19 (2): 43-48).Therefore, the research of inducible promoter and tissue-specific promoter and use the person's that is subjected to the breeding work day by day attention.The inducible promoter of identifying in transgenic plant comprises heat-inducible promoter, derives from inducible promoter of spinach nitrate reductase or the like.(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 the application of inducible promoter also has certain limitation, and the external condition that recipient plant is carried out is handled, and 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.And be used as in the chemical regulation system inductor Methylprednisone acetate (dex, dexamethasone), estradiol (estradiol) and tsiklomitsin (tetracycline) be all harmful to ecotope, should not be used for production practice.And the tissue-specific promoter of itself just can avoid this problem in the use plant materials.Obviously, the tissue specific expression of foreign gene will effectively improve the biological safety of genetically modified crops.Obtained great success in recent years in this respect.Specific expressed all kinds of promotors constantly obtain research (Song Yang etc., the research of plant tissue specificity promoter in different tissues.The biotechnology circular.2007，(4)：21-24)。

Rape is extensively planted in the Yangtze valley as the main oil crops of China, and national economy is had material impact.Rape heterosis is obvious, and utilizing the combination of nuclear male sterility hybridization advantage is the important channel of heterosis utilization.Found polytype nuclear male sterility material at present both at home and abroad, and obtained impressive progress in theoretical and application facet, and the discovery of novel sterile source and research are the bases of Study on Heterosis always, and create more natural and artificial sterile material with this, by breeding and production are used.

Along with the maturation of transgenic technology, rape will inevitably face the public opinion of transgenosis security risk.The expression that starts goal gene with the organizing specific type promotor of not expressing fully in the Semen Brassicae campestris is the feasible method that addresses this problem.Both improve rape each side quality to reach, do not cause the purpose of edible oil security risk again.

Therefore, the present invention has developed a gene relevant with the Pollen Brassicae campestris growth and the endogenesis promoter of rape.Express by this gene of fluorescence quantitative PCR detection a large amount in the flower pesticide of fertile plant.Suppress this expression of gene by the transgenic experiments proof and can cause plant anther heteroplasia, cause male sterile.Prove the spatial and temporal expression pattern of promotor by the expression characteristic of examining report gene GUS.Our result is indicating that this gene and promotor thereof have suitable application prospect in transgene rape.

Summary of the invention

The objective of the invention is to be to provide a kind of rape BnPABP5 gene, this gene efficiently expresses ， Tong Over and suppresses the fertility that this expression of gene can reduce plant anther in the flower pesticide of rape, thereby obtains male sterile plants, is applied to cross-breeding.And this gene do not express in other most of plant tissue, and therefore reticent this expression of gene can not cause the variation in vegetation growth of plant stage in plant genetic engineering.

Another object of the present invention is the preparation method who has been to provide a kind of rape BnPABP5 gene, by the est database of BLAST comparison rape, behind the electronic cloning of acquisition rape full length sequence, uses the sequence that simple PCR method promptly can obtain this gene.

A further object of the present invention is to be to provide a kind of recombinant vectors that contains the plant anther high efficient expression starter, and it contains described promotor nucleotide sequence.This carrier size is fit to, easy and conversion in plant, and the marker gene GUS expression intensity height that is had detects easily.Can obtain the Arabidopis thaliana transfer-gen plant that gus gene efficiently expresses by this carrier arabidopsis thaliana transformation in flower pesticide.

A further object of the invention is to be to provide the application of a kind of rape BnPABP5 gene in the control pollen fertility.By reticent this expression of gene, the contriver can manually create male sterile material, uses in cross-breeding is produced.

A further object of the invention is to be to provide the application of a kind of rape BnPABP5 gene promoter in flower pesticide.Under the driving of this promotor, mainly meticulous expression in the flower pesticide of transfer-gen plant or pollen, and do not express fully in the seed.This promotor with tissue specific expression has using value in plant genetic engineering and transgenosis safety (eating).

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 the genes involved in the Pollen Brassicae campestris growth course and comprehensively research, found that a new gene, and this gene is under a kind of control of new promotor, in the flower pesticide of fertile plant, efficiently express, and in sterile plant, do not express.Therefore new gene can be with the fertility of eco-friendly mode controlling plant pollen together with its promotor.Transgenic experiments has also confirmed this point: suppress this expression of gene and can cause plant anther heteroplasia, cause male sterile.

According to an aspect of the present invention, above-mentioned purpose can realize by providing plant anther to efficiently express gene, described gene contain SEQ ID No.1 nucleotide sequence or with SEQ ID No.1 homologous nucleotide sequence in fact.

According to another aspect of the present invention, above-mentioned purpose can realize by a kind of promotor is provided, described promotor can specificity drives gene and efficiently expresses in plant anther, described promotor contain SEQ IDNo.2 nucleotide sequence or with SEQ ID No.2 homologous nucleotide sequence 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 providing with described recombinant vectors microorganism transformed.

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

A kind of rape BnPABP5 gene the steps include:

1, Pollen Brassicae campestris efficiently expresses the clone of gene:

In liquid nitrogen plant young leaflet tablet sample is ground to powdery, the extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit (available from Invitrogen company, below identical).The total RNA that extracts is dissolved in the distilled water of no RNase.DNase I (available from Promega company, below identical) remove may be residual DNA.(DU 650BECKMAN USA) detects RNA respectively at 260 nanometers and 280 nanometer absorbance values, identifies purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis with the Protein Detection instrument.RNA with acquisition is that template is carried out reverse transcription, and is standby in-20 ℃ of preservations after the cDNA packing that obtains.

Compare with BLASTN (http://www.ncbi.nlm.nih.gov/BLAST) on the NCBI website according to Arabidopis thaliana PABP5 gene order among the GenBank, obtain respectively with Arabidopis thaliana PABP5 gene order 5 ' end homologous rape est sequence and and 3 ' hold homologous rape est sequence, then according to the rape est sequence and Arabidopis thaliana PABP5 gene start codon and the terminator codon homology zone design primer that obtain, thereby guarantee that amplified production is a full length sequence, root, stem, leaf, flower, angle cDNA really with swede type rape are masterplate, carry out pcr amplification.Reclaim and the purifying amplification PCR products, be connected on the pMD18-T carrier (available from Promega company, below identical), transformed into escherichia coli is selected positive colony, shakes bacterium and extracts plasmid, order-checking.

According to Arabidopis thaliana PABP5 gene order, on the NCBI website, compare with BLASTN (http://www.ncbi.nlm.nih.gov/BLAST), obtain many effective rape est sequences, extend the cDNA sequence of the PABP5 gene that has obtained total length 2196bp based on the electronics in rape EST library.CDNA obtains band (Fig. 1) about a treaty 2.0kb with rape PABP5 amplimer amplification rape bud, and above-mentioned product is checked order.Obtain a kind of isolating gene, its sequence is a nucleotide sequence shown in the SEQ ID No.1.

Utilize the open reading frame of NCBI, finder (ORF founder) is determined the open reading frame of BnPABP5 gene, derive the protein sequence amino acids coding, proteinic molecular weight and theoretical iso-electric point are carried out at line computation (http://us.expasyorg/tools/pi tool.htm).Show that with the online conservative region analytical results that carries out of the Conserved Domains instrument (http://wwwncb.inlm.nih.gov/Structure/cdd/wrpsb.cgi) of NCBI this gene belongs to the conjugated protein superfamily of Poly (A); By the Blastx program NR database of NCBI is carried out the homology comparison, and the PABP albumen with homology, different plant species source is carried out the multisequencing comparison with ClustalX1.83; With this proteinic basic structure territory of Prosite software on-line analysis.According to the aforesaid method analytical results, be BnPABP5 with the rape PABP5 unnamed gene that obtains.

2, rape BnPABP5 expression of gene pattern:

The used rape of the present invention is swede type rape (Brassicanapus L.) dominant genic male sterile heterozygosis sterile line J03AB (Msmsrfrf*msmsrfrf).System backcrosses through for many years and hands over and breed with sister.Sterile strain called after A can educate strain called after B, and the two can be considered a pair of near isogenic line, only exist sterile site difference (Hu Shengwu, Yu Chengyu, Zhao Huixian, the road is bright, the seed selection of hybrid rape dominant nuclear sterile material Shaan-GMS homozygous two-type line 803AB.Northwest agricultural journal, 2002, ii (4): 25-2).Be seeded in the land for growing field crops for the examination material, normal field management.

Get root, stem, leaf, bud, angle fruit from the identical fertile plant of same grown in field condition, anatomical isolation gynoecium and flower pesticide from bud, every kind of material is got three repetitions at least, each repeats at least one strain, the sampling back is positioned in the liquid nitrogen-80 ℃ of preservations rapidly with the masking foil parcel.The extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit (front is stated).Get bud from rape fertile plant and sterile plant respectively in the land for growing field crops, after taking back the laboratory, flower pesticide and gynoecium on ice chest in difference picking fertile plant and the sterile plant bud, every kind of material is got three repetitions at least, each repeats at least one strain, the sampling back is positioned in the liquid nitrogen-80 ℃ of preservations rapidly with the masking foil parcel.The extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit (front is stated).

Quantitative real time PCR Instrument is RT ^TM-Cycler (Bo Ao) adopts rape Actin as internal standard gene (accession number AF111812), and the Actin gene primer is 5 '-CTGGAATTGCTGACCGTATGAG-3 ' and 5 '-ATCTGTTGGAAAGTGCTGAGGG-3 '.The BnPABP5 gene primer is 5 '-TGGACCAAACTGTCAACGAAG-3 ' and 5 '-CCAAGTGAACGACGAGTCAAG-3 '.

Quantitative fluorescent PCR reacts every group of experiment and all finishes three biology repetitions, and each biology repeats to do at least three technology and repeats.Detect the expression of BnPABP5 in rape tissue (root, stem, leaf, flower, angle fruit, flower pesticide, gynoecium) respectively.

Calculate the relative expression quantity of gene with comparing Ct method (Δ Δ Ct).By 2 ^{-Δ Δ Ct}Relative expression quantity and systematic error (Kenneth J Livak.Thomas D Schmittgen.2001, the Analysis ofRelative Gene Expression Data Using Real-Time Quantitative PCR and the 2 of estimation goal gene ^{-Δ Δ C}TMethod.METHODS 25,402-408.).When calculating relative expression quantity, be reference with the sample of root, be about to its value and convert 1 (standard value) to, other sample again with its relatively, obtain relative expression's value.

3, the structure of genomic walking cloning promoter sequence and pMD18-BnPABP5 (order-checking) carrier: utilize SDS cracking process (J. Sa nurse Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press) the extraction genomic dna, according to genomic walking test kit (available from Clontech company) schedule of operation, carry out genomic walking.Concrete steps are: extract genomic dna 25 μ L (0.1 μ g/ μ L), cut with endonuclease Dra I, EcoR I, Pvu II and Stu I enzyme respectively, add joint behind the purifying and form four different genomic dna storehouses, be used as genomic walking first round pcr template first, increase according to upstream joints primer AP1 (table 1) in goal gene conserved sequence design Auele Specific Primer GSP1 (table 1) and the genomic walking test kit (purchasing company) in Clontech.Second to take turns PCR be template with 50 times of diluents (water diluent) of first round PCR product, increases with the Auele Specific Primer GSP2 and the joint primer AP2 (table 1) of quadrat method design, carries out second subsequently and take turns genomic walking.All PCR reactions are 50 μ L amplification systems: template 1 μ L, and dNTPs 0.2mM, primer 0.5mM, LATaq enzyme 1.25 units, 10 * LA-PCR buffer (contains MgCl ₂) 5 μ L, moisturizing to 50 μ L.Reaction conditions is: 94 ℃ of pre-sex change 1min; 98 ℃ of 10s, 72 ℃ 6min7 circulation; 98 ℃ of 10s, 33 circulations of 67 ℃ of 6min; 72 ℃ are extended 10min.The PCR product detects through 1.0% (mass volume ratio) agarose gel electrophoresis, 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 TaKaRa company, below identical), transform the gold bacterial strain (available from the precious biotechnology in Dalian company limited, below identical) competent cell, the picking positive colony, enzyme is cut the order-checking of checking back, obtain the flanking sequence of goal gene upstream, this sequence comprises the promoter sequence of the initiator codon of swede type rape BnPABP5 gene to the section between the GSP2 and this gene.According to the sequence that obtains, design amplification segmental primer PBnPABP5S:CAGAAGCTTAAAAGACTTACCGAGTTGAACC of promoter region and PBnPABP5A:GACATCTAGACGCTGTCGTTGGGGCAATTC, with the genomic dna is template, carries out pcr amplification.5 of all primers ' end contains Hind III restriction enzyme site, and 3 ' end contains Xba I restriction enzyme site.Reaction system is that 25 μ L include: 1 * PCR buffer., and MgCI 1.5mmol/L, dNTP 0.2mmol/L (every liter of mmole), primer concentration are 0.5mol/L, Pfu enzyme 1.5 units, the about 100ng of template is provided with loop parameter as the case may be.PCR product 1.0% (mass volume ratio, below identical) sepharose detects, and gel reclaims test kit and reclaims each purpose deletion fragment.By the deletion fragment of each recovery: carrier (the 0.3pmol deletion fragment: the 0.1pmol carrier segments)=3: 1 mixed sample, add T4DNA ligase enzyme 5 units, 1 * reaction buffer, sterilized water replenish volume to 25ml, 16 ℃ of ligations of spending the night.(J. Sa nurse Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press to freeze-thaw method transformed into escherichia coli gold bacterium, 96-99) in the competent cell.(it is as follows to fill a prescription: take by weighing 10 gram Tryptoness respectively, 5 gram yeast extracts and 10 restrain sodium-chlor, and 8 gram agar are dissolved in the distilled water successively, and constant volume is in 1000 milliliters to coat the LB solid that contains penbritin 50 μ g/mL.Be sub-packed in 500 milliliters of triangular flasks, 121 ℃, autoclave sterilization is 15 minutes under 6.859 * 104Pa.4 ℃ of refrigerations are standby) on the flat board, 37 ℃ of overnight incubation, respectively select three of hickies, be bacterium colony PCR and detect, positive recombinant is inoculated in the liquid LB substratum that contains penbritin 50 μ g/mL, 37 ℃ of 200r/min shaking culture are spent the night, (J. Sa nurse 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) extract plasmid, Hind III/Xba I digested plasmid 1 μ g, 0.8% (mass volume ratio) sepharose detects.Detect correct positive recombinant clone, (the purpose fragment is connected into the pMD18-T vector construction to form called after pMD18-BnPABP5 respectively, below identical), in order to ensure the sequence information of promotor in the carrier, with M13F/M13R be primer (M13F5 '-AGCGGATAACAATTTCACACAGGA-3 '; M13R5 '-GTAAAACGACGGCCAGT-3 '), pMD18-BnPABP5 is carried out sequencing, analytical results has obtained a kind of isolating rape BnPABP5 full length gene promotor, and its sequence is a nucleotide sequence shown in the SEQ ID NO:2.

4, the conversion of the structure of BnPABP5 full length gene promotor plant expression vector and agrobacterium tumefaciens bacterial strain EHA105 (purchasing still identical below the bio tech ltd) in Shanghai, Shanghai:

The recombinant vectors that makes up is that the 35S promoter on the plasmid pBI121 (available from TaKaRa company) is replaced with the goal gene promoter fragment that early stage, the clone obtained.For finishing this purpose, at first use the promoter fragment of Xba I/HindIII double digestion cloning vector pMD18-BnPABP5, cut pBI121 (Chen with Xba I/Hind III enzyme simultaneously, P.Y., Wang, C.K., Soong, S.C.To, K.Y.Complete sequence ofthe binaryvector pBI 121and its application in cloning T-DNA insertion from transgenic plants.Mol.Breed.11,287-293) plasmid.Endonuclease reaction carries out in 37 degree incubators, after about 4-6 hour, detects with 1% agarose gel electrophoresis.Small segment that cloning vector pMD18-BnPABP5 go up is downcut and pBI121 (front is stated) go up the big fragment of downcutting and reclaim test kit (front is stated) with dna gel and reclaim.In the promoter fragment on the pMD18-BnPABP5 50ng carrier segments)=3: 1 ratio (molar concentration rate) biased sample than pBI121 (front is stated) carrier (150ng deletion fragment:, add T4DNA ligase enzyme 5 units, 10 * reaction buffer, sterilized water replenishes volume to 20 μ L, and 16 ℃ of connections are spent the night.After the conversion, screen containing on kantlex (50 μ g/mL) the solid LB flat board, choose spot and be bacterium colony PCR, and the upgrading grain, enzyme is cut the correct recombinant plasmid called after pBI121-BnPABP5 of checking.Utilize freeze-thaw method to change pBI121-BnPABP5 over to agrobacterium tumefaciens EHA105 (available from the precious biotinylated biomolecule in Dalian Products Co., Ltd then, below identical), the two resistant panel screenings of kantlex (50 μ g/mL) and Rifampin (50 μ g/mL) are chosen spot, bacterium colony PCR detection validation.

5, the functional analysis of total length promotor

5.1BnPABP5 genetic transformation and the transfer-gen plant screening of full length gene promoter vector pBI121-BnPABP5 in Arabidopis thaliana:

Method in the reference literature carry out Arabidopis thaliana transform (Zhang X.R.et al.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dipmethod.Nature, 2006,1:1-6).Preparation contains agrobacterium tumefaciens EHA105 (front is stated) the bacterium liquid that builds carrier, changes in the LB liquid nutrient medium that contains kantlex 50 μ g/ml, Rifampin 50 μ g/ml 28 ℃ of incubated overnight over to the day before yesterday in conversion.Second day, in the light absorption value that the 276nm nano wave length detects down, when reaching between 1.6-2.0, takes out the light absorption value of bacterium liquid with ultraviolet spectrophotometer (SPEKOL 1300).Room temperature (20-25 ℃, below identical) with the centrifugal 10min of 4000g, is abandoned supernatant, and precipitation is suspended in isopyknic 5% sucrose (mass volume ratio).The sucrose solution of muddiness is poured in the big culture dish, added the Silwet 1-77 that final concentration is 0.02% (volume ratio) (available from the Five continents, Beijing unit industry science and trade center, below identical) before transforming.The whole inflorescence of Arabidopis thaliana to be transformed being immersed in the sucrose gently, silent number 15s 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.Plastics bag was opened in second day, 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.With the T0 that transforms results for seed with the mercuric chloride surface sterilization of 70% (volume ratio) alcohol and 0.01% (volume ratio) after 10 minutes with distilled water wash several (5～7 times), (it is as follows to fill a prescription: ammonium nitrate 1.65g to blow and beat 1/2MS then equably, saltpetre 1.9g, potassium primary phosphate 0.17g, sal epsom 0.37g, calcium chloride 0.44g, sucrose 30g, agar 8g, adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8, boils (100 ℃) and is settled to 1000ml, divide to install to 500ml triangular flask (250ml/ bottle), seal sterilization.) solid screening and culturing primary surface.4 ℃ vernalization 4-6 days, put into constant incubator and cultivate.Screen positive seedling according to peculiar kalamycin resistance on the expression vector.Blade is long when enough big or small (3-4 leaf phase), gets a little green seedling leaf and carries out the PCR positive detection.Obtained the transgenic positive seedling.

5.2BnPABP5 the full length gene promoter function is analyzed:

35S promoter sequence with on BnPABP5 gene promoter replacement pBI121 (front the is stated) plasmid forms pBI121-BnPABP5 recombinant plant expression vector, and gus gene wherein is subjected to the regulation and control of BnPABP5 promotor.Utilize the inflorescence infestation method arabidopsis thaliana transformation of agrobacterium tumefaciens EHA105 (front is stated) mediation.T1 obtains positive plant for utilizing kalamycin resistance and PCR to detect screening.

With PCR checking be male T1 for the transfer-gen plant seed at 1/2MS (front is stated) substratum upper seeding wheel, in the growth case, sprout after 4 ℃ of vernalization, the back of emerging began sampling dyeing in 5-7 days.Dyeing course is as follows: sample is immersed in GUS dye liquor (X-gluc 0.5mg/mL, phosphoric acid buffer 50mmol/L, each 0.5mmol/L of the Tripotassium iron hexacyanide and yellow prussiate of potash, EDTA 10mmol/L, Triton-x-1000.001% (volume ratio), in the methyl alcohol 20% (volume ratio), vacuum suction 5 minutes, 37 ℃ are spent the night.Decoloured with alcohol-acetate (volume ratio is 1: 1) in second day, and bleached until blade, use distilled water rinsing (5～7 times) for several times afterwards, Stereo microscope (OLYMPUS SZX16) is taken pictures.Get the whole plant of different time sections seedling stage; Reproductive stage, blade are got tender leaf and mature leaf; The inflorescence of 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 blue position.The spatial and temporal expression pattern that expressive site by detecting gus gene under the different space-times and expression intensity are explored the purpose promotor.

The pBI121-BnPABP5 transgenic arabidopsis that utilizes the same manner to choose T2 generation carries out the analysis of GUS tissue staining, gets the seedling of different developmental phases and different plant tissues respectively and dyes.This promoter-driven GUS gene is all expressed in the tip of a root of Arabidopis thaliana, and the expression of very short time is arranged at the vegetative point place of the rough leaf that has just grown, and along with the growth of plant, gus gene only efficiently expresses in flower pesticide.Illustrate that this promotor has the downstream gene of driving and efficiently expresses in the flower pesticide of plant, the tip of a root is expressed basic, and the function of not expressing fully in seed.This promotor with tissue specific expression has using value in plant genetic engineering and transgenosis safety (eating).Cause the carrier of the gene of flower pesticide abortion as some that can contain this promoters driven 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.

6, the functional verification of BnPABP5 gene:

6.1BnPABP5 the structure of gene RNAi plant expression vector pGKannibal-1-2 and the conversion of Arabidopis thaliana

In order to study the function that pollen among the present invention efficiently expresses gene BnPABP5, adopted RNAi (RNAinterference) technology.Because the PABP5 of rape BnPABP5 and Arabidopis thaliana has similar expression pattern (Belostotsky A, Meagher B.A pollen-, ovule-, and early embryo-specific poly (A) binding protein from Arabidopsis complements essential functions in yeast.Plant Cell, 1996,8 (8): 1261-75.), gene structure (stone is of heap of stone. rape PABP gene family member's clone, expression pattern is analyzed and the PABP5 functional study. ([doctorate paper]. Beijing: the Chinese Academy of Agricultural Sciences, 2009), 76% albumen homology, infer that both have identical functions, so the function of rape PABP5 is to obtain by the function of studying this gene of Arabidopis thaliana.Increase on Arabidopis thaliana PABP5 gene order by PCR method and to obtain disturbing segment.The pulsating connection primer of forward is PABP-Ri-1-1:5 '-GACATCTAGA (XbaI) GAGTCGTGCGATGGAAAG-3 ' and PABP-Ri-1-2:5 '-CGCGGATCC (BamHI) CTTGCAGGAAGTCACATTC-3 '.The PCR reaction system is that 25 μ L include: 1 * PCR buffer., and MgCI 1.5mmol/L, dNTP 0.2mmol/L (every liter of mmole), primer concentration are 0.5mol/L, Pfu enzyme 1.5 units, the about 100ng of template.PCR response procedures: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 8min.PCR product 1.0% (mass volume ratio, below identical) sepharose detects, and reclaims test kit and reclaims each purpose deletion fragment.Reclaim product and intermediate carrier pKannibal (Wesley S V with Xbal I+BamH I difference double digestion, Helliwell C A, Smith N A, et al.Construct design for efficient, effective and high-throughput gene silencing in plants.Plant J, 2001,27 (6): 581～590), each reclaims the purpose fragment, by 3: 1 mixed sample, adds T4DNA ligase enzyme 5 units, 1 * reaction buffer, sterilized water replenishes volume to 25ml, and 16 ℃ of ligations of spending the night make up intermediate carrier pKannibal-1.Freeze-thaw method is transformed in the intestinal bacteria gold bacterial strain competent cell.The pulsating connection primer of forward is PABP-Ri-2-1:5 '-CCGGAATTC (EcoRI) CCATGG (Nco I) GAGTCGTGCGATGGAAAG-3 ' and PABP-Ri-2-2:5 '-CGGGGTACC (KpnI) CTTGCAGGAAGTCACATTC3-', the PCR reaction conditions, the vector construction condition is the same, makes up intermediate carrier pKannibal-1-2.With SpeI and NotI double digestion intermediate carrier pKannibal-1-2, reclaim big segment, and with SpeI and NotI double digestion pGreen0229, reclaim the double digestion product,, add T4DNA ligase enzyme 5 units by 3: 1 mixed sample, 1 * reaction buffer, sterilized water replenishes volume to 25ml, and 16 ℃ of ligations of spending the night make up plant expression vector pGKannibal-1-2.Transform Agrobacterium, arabidopsis thaliana transformation (front is stated, 5.1).

6.2BnPABP5 the functional analysis of gene

Verify jamming effectiveness and observe transgenic arabidopsis T1 representative type by quantitative fluorescent PCR.Observations shows that each RNAi transfer-gen plant is at vegetative growth phase and wild-type Arabidopis thaliana and no significant difference, and notable difference is arranged in reproductive growth period and wild-type Arabidopis thaliana: in 10 transformants that obtain altogether, there are 3 strain transgenic arabidopsis shaky fully, 6 strain Arabidopis thalianas fruit pod shortens, seed tails off, and variation is not observed in 1 strain.Compare with the wild-type Arabidopis thaliana, the flower pesticide of RNAi transfer-gen plant shortens, and diminishes, and flower pesticide can not touch column cap, and column cap had not begun to expose when bud was bloomed, and shows the common trait (Figure 10) of typical male sterile plants.These results show and suppress or reduce the BnPABP5 expression of gene to cause the plant pollen abortion, make the solid ability drop of plant or lose fully, obtain male sterile plants.The BnPABP5 gene has the function of control pollen fertility, has using value for utilizing genetic engineering technique to create male sterile material.

A kind of rape flower pesticide efficiently expresses the description (1, technical scheme 1 Pollen Brassicae campestris efficiently express among the clone of gene and obtain) of gene BnPABP5 full length sequence SEQ ID NO:1.The flower pesticide that utilizes the homologous clone method to obtain rape efficiently expresses gene BnPABP5, the cDNA sequence total length 1998bp of coding region, with the homology of Arabidopis thaliana PABP5 gene coding region be 79.6%, 651 amino acid of encoding.Be approximately 71598.4Da at the proteinic molecular weight of line computation, theoretical iso-electric point is about 8.38.BLASTp on-line analysis software carries out homology relatively with the proteins encoded and the sequence among the Genebank of deriving, and finds that the protein amino acid sequence homology of this sequence and Arabidopis thaliana AtPABP5, tobacco NtPABP, rice Os PABP, Radix Dauci Sativae DcPABP, corn ZmPABP, cucumber CsPABP, wheat TaPABP reaches 76%, 52%, 52%, 53%, 51%, 52%, 53% respectively.The online conservative region analytical results that carries out shows that this gene belongs to the conjugated protein superfamily of Poly (A), identical with above-mentioned several PABP albumen, all contain 4 RNA recognition sites (RNA recognition motif, RRM), each RRM comprises two more conservative RNP I and RNP II sequential element.The expression amount of this gene in root is minimum, expression amount is root＜stem＜leaf＜angle fruit＜bud from low to high, expression amount in the root is made as 1, relative expression quantity in stem, leaf, angle fruit, the bud is respectively 1.98,4.34,6.99,70.36, and the expression amount in the bud is significantly higher than other tissue (Fig. 2 A).In bud, the expression amount of flower pesticide is higher than the expression amount in the gynoecium, is 4.46 times (Fig. 2 B) of gynoecium.This gene is not expressed in flower pesticide in sterile material, and expression intensity in gynoecium and fertile plant suitable (Fig. 3).

The description () of a kind of rape BnPABP5 gene promoter full length sequence SEQ ID NO:2 functional expression in plant tissue.Utilize genomic walking method clone to obtain 5 ' upstream sequence of rape BnPABP5 gene.Structure is by the plant expression vector pBI121-BnPABP5 (front is stated) of the reporter gene GUS of this promoter regulation.Adopt agriculture bacillus mediated florescence infestation method arabidopsis thaliana transformation, T1 is for being added with preliminary screening transfer-gen plant on the 1/2MS of kantlex (front the is stated) 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 the molecule means to identify positive strain.T2 is for selecting a plurality of transgenic lines to carry out GUS dyeing.The dyeing of GUS histochemical method shows that this promoter-driven GUS gene is expressed in the tip of a root of Arabidopis thaliana, the strongest in dyeing seedling stage on the 5th, still along with the growth of plant, staining power has a declining tendency; The expression of very short time is arranged at the vegetative point place of the rough leaf that has just grown, and along with the growth of plant, gus gene is not just expressed in true leaf; At the whole reproductive stage of Arabidopis thaliana, gus gene is specific expressed in flower pesticide.Not observing in whole dyeing course has gus gene to express in other tissue.This shows that this promoter-driven GUS gene has must the spatial and temporal expression specificity, have the downstream gene of driving and in flower pesticide, efficiently express, express on a small quantity, do not express the function of (Fig. 8) in the seed fully in the leaf primordium of the tip of a root, rough leaf.This promotor with tissue specific expression has using value in plant genetic engineering and transgenosis safety (eating).

The description of a kind of rape BnPABP5 full length gene sequence SEQ ID NO:1 functional expression in plant tissue (technical scheme 1 Pollen Brassicae campestris efficiently expresses among the clone of gene and obtains).Utilize the RNAi technology in Arabidopis thaliana, to suppress the function that this expression of gene has been studied this rape gene.The silence of BnPABP5 gene can't cause that the phenotype of vegetation growth of plant phase changes in transgenic arabidopsis, but can cause the pollen abortion of Arabidopis thaliana at reproductive stage, be embodied in: compare with the wild-type Arabidopis thaliana, setting percentage reduces or is shaky, the flower pesticide smaller volume of RNAi transgenic arabidopsis, pollen obviously reduces, the abortion level of pollen obviously raise (Figure 10 B).Select the higher transgenic line of abortion degree, make the observation that frozen section is observed the pollen abortion of transfer-gen plant, find pollen granule crumple folding, atrophy (Figure 10 F).Show and suppress or reduce the BnPABP5 expression of gene to cause the plant pollen abortion, make the solid ability drop of plant or lose fully, obtain male sterile plants.The BnPABP5 gene has the function of control pollen fertility, has using value for utilizing genetic engineering technique to create male sterile material.

Advantage of the present invention:

1 by this gene of clone, studies this gene in the developmental effect of rape flower pesticide, and clear and definite this gene has the function of control anther development.By disturbing this expression of gene, the applicant can manually create male sterile material, provides new sterile gene for realizing manually operated pollination system.

2 are cloned into the promotor of the BnPABP5 gene in rape source.Consider that from security of rape edible oil and transgenosis environmental organism secure context this promotor has application potential.The expression intensity enlightenment applicant of this promotor in flower pesticide can come replaced C aMV35S strong promoter to start the expression of gene relevant with anther development with this promotor, and do not cause the edible and environmental safety problem of rape transgenosis.

The present invention efficiently expresses in the flower pesticide of fertile plant, and does not express in sterile plant under a kind of control of new promotor.Therefore new gene can be with the fertility of eco-friendly mode controlling plant pollen together with its promotor.Transgenic experiments has also confirmed this point: suppress this expression of gene and can cause plant anther heteroplasia, cause male sterile.

Description of drawings

Fig. 1 efficiently expresses the clone's of gene electrophorogram for a kind of Pollen Brassicae campestris.

Swimming lane 1 is for being the pcr amplification result of template with the genomic dna; Swimming lane 2 is the pcr amplification result of template for the cDNA with bud; Swimming lane 3 is the nucleic acid Marker of DL2000.

Fig. 2 efficiently expresses expression of gene pattern analysis figure for a kind of rape flower pesticide.

A figure shows that by quantitative fluorescent PCR this gene efficiently expresses in bud; B figure shows that by quantitative fluorescent PCR this gene efficiently expresses at the flower pesticide of bud.

Fig. 3 for a kind of rape flower pesticide efficiently express gene can educate with sterile plant in expression pattern figure.

J03BP, J03BS represent the gynoecium and the flower pesticide of fertile plant; J03AP, J03AS represent gynoecium and the flower pesticide of sterile plant; This figure shows that a kind of flower pesticide efficiently expresses gene and efficiently expresses in the flower pesticide of fertile plant, and does not express in sterile plant.

Fig. 4 efficiently expresses gene BnPABP5 promoter fragment electrophorogram for a kind of genomic walking a kind of rape flower pesticide that increases. Swimming lane 1,2,3,4 is represented the pcr amplification result in four genomic dnas " storehouse " that form respectively after Dra I, EcoR I, Pvu II and Stu I enzyme are cut; Swimming lane M represents the nucleic acid Marker of DL2000.

A figure moves electrophorogram in the genomic step first round; B figure genomic second takes turns the step and moves electrophorogram.

The pBI121-BnPABP5 carrier synoptic diagram of Fig. 5 for making up.

The transfer-gen plant PCR of Fig. 6 conversion carrier pBI121-BnPABP5 identifies figure

M, DNA marker; Lane 1, the HindIII/XbalI double digestion; The 1:pBI121-BnPABP5 positive

Plasmid; 2,3,4,5,6 positive strains: 7 wild-type Arabidopis thalianas;

Fig. 7 transforms the transfer-gen plant PCR evaluation figure of RNAi carrier pGKannibal-1-2

M, DNA marker DL2000; Lane 1,2,3,4,5,6,7,8,9,10 positive strains:

Lane 11 wild-type Arabidopis thalianas;

Fig. 8 efficiently expresses the histochemical stain result of the promoter-driven GUS gene of gene for a kind of flower pesticide.

A is seedling on the 5th; B is seedling on the 7th; C is seedling on the 15th; D is from leave; E is stem, lateral bud, stem leaf; F is a seed; G is an inflorescence; H flower pesticide.

The Arabidopis thaliana interference carrier pGKannibal-1-2 synoptic diagram of Fig. 9 for making up.

Figure 10 is the phenotypic map of transgenic arabidopsis.

A is the Arabidopis thaliana of vegetative growth phase; B is the bud of Arabidopis thaliana; C is the Alexandria dyeing of the flower pesticide of Arabidopis thaliana; D is the inflorescence of Arabidopis thaliana; E is the angle fruit of Arabidopis thaliana; F is Arabidopis thaliana frozen section figure.

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

Embodiment 1.:

A kind of rape flower pesticide efficiently expresses clone and the expression pattern analysis of gene BnPABP5:

1, a kind of rape flower pesticide efficiently expresses the clone of gene BnPABP5

Extract the requirement of test kit (front is stated) according to Trirol and carry out the extraction of RNA, concrete grammar is as follows: root, stem, leaf, flower, the angle fruit sample of getting 0.05-0.1g respectively, in liquid nitrogen, be ground to powdery, extract the requirement of test kit according to Trirol and carry out the extraction of RNA.The total RNA that extracts is dissolved in the distilled water of no RNase of 60uL.DNase I removes the residual DNA of possibility.(DU 650BECKMAN USA) detects the absorbance value of RNA under 260 nanometers and 280 nanometers respectively, identifies purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis with the Protein Detection instrument.RNA with above-mentioned acquisition is that template is carried out reverse transcription by following scheme: add 1 μ L Oligo (dT) among the 2 μ g RNA, 70 ℃ of incubation 5min, place 5min on ice immediately, of short duration centrifugal, add 5 * M-MLV Buffer, 4 μ L, dNTP (10mmol/L) 1 μ L, RNase Inhibitor 20 units, M-MLV reversed transcriptive enzyme (available from Promega company) 200 units, mend to cumulative volume 20 μ L, mixing, 42 ℃ of incubation 1h with the sterilized water that DEPC handled, 70 ℃ of water-bath 15min, standby after the cDNA packing that obtains in-20 ℃ of preservations.

With rape root, stem, leaf, angle fruit, bud cDNA is the full-length clone that template is carried out the BnPABP5 gene.According to Arabidopis thaliana PABP5 gene order among the GenBank ( NM 105835.) on the NCBI website, compare with BLASTN (http://www.ncbi.nlm.nih.gov/BLAST), extract E-value≤e ^-5Est sequence, obtain respectively with Arabidopis thaliana PABP5 gene order 5 ' end homologous rape est sequence ( CD837708.) and and 3 ' end homologous est sequence ( EV150118.), then according to the rape est sequence and Arabidopis thaliana PABP5 gene start codon and the terminator codon homology zone design primer that obtain, thereby guarantee that amplified production is a full length sequence.Rape PABP5 amplimer sequence is respectively: 5 '-GCGATGGTTGATCAAGTCAT-3 ' (5 ' end primer); 5 '-TCACTCCGTTGAGGAGGG-3 ' (3 ' end primer).The PCR program is: 94 ℃, and 5min; 94 ℃, 45s, 57 ℃, 45s, 72 ℃, 2min, 33 circulations; 72 ℃, 10min.The result as shown in Figure 1.The PCR reaction product is electrophoresis on (mass volume ratio) low melting-point agarose of 1%, the amplified production band is put into 1.5ml Eppendoff centrifuge tube from the glue cutting-out, 65 ℃ of water-bath 15min, add equal-volume phenol (PH7.9), put upside down and shake up 5min, 13000 rev/mins centrifugal 8 minutes, get supernatant, add the equal-volume chloroform: primary isoamyl alcohol (volume ratio 24: 1) is put upside down and is shaken up 5min, 13000 rev/mins centrifugal 8 minutes, get supernatant, 3 mol sodium-acetate (PH5.2) solution that add 1/10 volume, 95% (mass volume ratio, below identical) ethanol of 2 times of volume precoolings, in the mixing postposition-20 ℃ refrigerator more than the 20min, 13000 rev/mins centrifugal 15 minutes, outwell and use 75% behind 95% (front is stated) ethanol again (mass volume ratio, below identical) ethanol and embathe precipitation, natural air drying, the DNA precipitation is dissolved in 20 μ l aseptic deionized waters.Press pMD18-T (available from TaKaRa company) carrier specification sheets, be connected on the pMD18-T carrier, (J. Sa nurse Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press to transformed into escherichia coli, 96-99).Method is as follows: PCR selects positive colony by bacterium colony, and primer sequence is: M13F 5 '-AGCGGATAACAATTTCACACAGGA-3; M13R5 '-GTAAAACGACGGCCAGT-3), reaction system is: 10 * Taq enzyme reaction buffer solution 2ul, 25mMMgCL ₂1.2ul, 2mM dNTP 1.5ul, 10uM primer 0.2ul, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min.Each PCR product is selected 3 clones respectively and is shaken bacterium and extract plasmid, and order-checking obtains BnPABP5 full length gene sequence, a kind of isolating gene, its sequence be nucleotide sequence shown in the SEQ ID No.1 or with SEQ ID No.1 homologous nucleotide sequence in fact.

Utilize the open reading frame of NCBI, finder (ORF founder) is determined the open reading frame of nucleotide sequence shown in the SEQ ID No.1, derive the protein sequence amino acids coding, proteinic molecular weight and theoretical iso-electric point are carried out at line computation (http://us.expasyorg/tools/pi tool.htm).Show that with the online conservative region analytical results that carries out of the Conserved Domains instrument (http://wwwncb.inlm.nih.gov/Structure/cdd/wrpsb.cgi) of NCBI this gene belongs to the conjugated protein superfamily of Poly (A); By the Blastx program NR database of NCBI is carried out the homology comparison, and the PABP albumen with homology, different plant species source is carried out the multisequencing comparison with ClustalX1.83; With this proteinic basic structure territory of Prosite software on-line analysis.Show according to the aforesaid method analytical results: 651 amino acid of protein sequence coding that the opening code-reading frame of this gene is derived.Be approximately 71598.4Da at the proteinic molecular weight of line computation, theoretical iso-electric point is about 8.38.SignalP and Tmpred analytical results show: BnPABP is a non-secretory albumen, does not have the signal peptide section, does not also have membrane spaning domain.BLASTp on-line analysis software carries out homology relatively with the proteins encoded and the sequence among the Genebank of deriving, and finds that the protein amino acid sequence homology of this sequence and Arabidopis thaliana AtPABP5, tobacco NtPABP, rice Os PABP, Radix Dauci Sativae DcPABP, corn ZmPABP, cucumber CsPABP, wheat TaPABP reaches 76%, 52%, 52%, 53%, 51%, 52%, 53% respectively.The online conservative region analytical results that carries out shows that this gene belongs to the conjugated protein superfamily of Poly (A), identical with above-mentioned several PABP albumen, all contain 4 RNA recognition sites (RNA recognition motif, RRM), each RRM comprises two more conservative RNP I and RNP II sequential element.The PABP albumen of having cloned is carried out genealogical tree analysis revealed BnPABP5 and Arabidopis thaliana AtPABP5 sibship is nearest.The homology of the cDNA sequence of coding region and Arabidopis thaliana PABP5 gene coding region is 79.6%.Therefore the gene of inferring amplification really is the PABP5 full length gene sequence of rape, is BnPABP5 with this unnamed gene, and BnPABP5 has similar function with At PABP5.

2, a kind of rape flower pesticide efficiently expresses the expression pattern analysis of gene BnPABP5:

The used rape of the present invention (Brassica napus L.) is dominant genic male sterile heterozygosis sterile line J03AB (Msmsrfrf*msmsrfrf) (material is that oil crops functional genome of institute of the Chinese Academy of Agricultural Sciences and Molecular Biology Research Lab preserve, and the front is stated).System is seeded in the land for growing field crops for the examination material, normal field management through backcrossing with sister's friendship for many years and breeding (sterile strain called after A can educate strain called after B, and the two can be considered a pair of near isogenic line, only has sterile site difference).

Get root, stem, leaf, bud, angle fruit from the identical fertile plant of same grown in field condition, anatomical isolation gynoecium and flower pesticide from bud, every kind of material is got three repetitions at least, each repeats at least one strain, the sampling back is positioned in the liquid nitrogen-80 ℃ of preservations rapidly with the masking foil parcel.Extract RNA (the method front is stated).Get respectively flowering period can educate with sterile plant on get bud, go back to the laboratory after, on ice chest, take out flower pesticide and gynoecium respectively, every kind of material is got three repetitions at least, and each repeats at least one strain, and wrap up with masking foil the sampling back, be positioned in the liquid nitrogen-80 ℃ of preservations rapidly.Extract RNA (the method front is stated).

Quantitative real time PCR Instrument is RT ^TM-Cycler (Bo Ao) adopts rape Actin (accession number AF111812) as internal standard gene, the Actin gene primer be forward primer 5 '-CTGGAATTGCTGACCGTATGAG-3 ' and reverse primer 5 '-ATCTGTTGGAAAGTGCTGAGGG-3 '.The BnPABP5 gene primer be forward primer 5 '-TGGACCAACTGTCAACGAAG-3 ' and reverse primer 5 '-CCAAGTGAACGACGAGTCAAG-3 '.

The quantitative fluorescent PCR reaction system is 20 μ L: contain SYBR Mix 10 μ L, and each 0.8 μ L of forward and reverse primer (10 μ mol/L), template 2 μ L, the sterilized water that DEPC handled complements to 20 μ L.Amplification condition is: 94 ℃, and 5min:94 ℃, 15s, 60 ℃, 20s, 72 ℃, 30s, 40 circulations; Each is circulated in 72 ℃ of renaturation ends and carries out fluoroscopic examination.Reaction is heated to 95 ℃ earlier after finishing, and reduces to 72 ℃ then, slowly is warming up to 95 ℃ again, writes down the variation of fluorescent signal, draws the melting curve of amplified production.Every group of experiment all finished three biology and repeated, and each biology repeats to do at least three technology and repeats.Detect the expression of BnPABP5 in rape tissue (root, stem, leaf, flower, angle fruit, flower pesticide, gynoecium, stamen) respectively.

Calculate the relative expression quantity of gene with comparing Ct method (Δ Δ Ct).Utilize software that instrument is with, at first optimize internal standard gene and goal gene amplification condition, measure the Ct value of Actin and goal gene respectively, choose that the most approaching three results (systematic error of Ct value is less than 0.3) average in nine measured value of experiment, by internal standard gene goal gene is proofreaied and correct then and obtained Δ Δ Ct, at last by 2 ^{-Δ Δ Ct}The relative expression quantity and systematic error (the Kenneth J Livak.Thomas D Schmittgen Analysis of Relative GeneExpression Data Using Real-Time Quantitative PCR and the 2 of estimation goal gene ^{-Δ Δ C}T MethodMETHODS 25,402-408 (2001)).When calculating relative expression quantity, be reference with the sample of root, be about to its value and convert 1 to, other sample again with its relatively, obtain relative expression's value.

The result shows: in fertile plant, the expression amount of BnPABP5 in root is minimum, expression amount is root＜stem＜leaf＜angle fruit＜bud from low to high, expression amount in the root is made as 1, relative expression quantity in stem, leaf, angle fruit, the bud is respectively 1.98,4.34,6.99,70.36, and the expression amount in the bud is significantly higher than other tissue (Fig. 2 A).In bud, the expression amount of flower pesticide is higher than the expression amount in the gynoecium, is 4.46 times (Fig. 2 B) of gynoecium.This gene is expressed at flower pesticide middle part in sterile plant, and in the expression intensity of gynoecium and fertile plant expression intensity quite (Fig. 3), this explanation BnPABP5 is a main gene that efficiently expresses in flower pesticide.

Embodiment 2.:

A kind of preparation of rape BnPABP5 promotor:

1. the clone of rape BnPABP5 gene promoter:

Utilize the SDS cracking process to extract rape DNA (J. Sa nurse Brooker .D.W. Russell work, Huang Peitang etc. translate molecular cloning test guide (third edition) Science Press), according to genomic walking test kit (purchasing company) schedule of operation in Clontech, carry out genomic walking twice, the capable two-wheeled pcr amplification of each step shift-in, clone's rape BnPABP5 gene promoter (3688bp).Two hyposynchronization move the primer such as following table.

Table 1 genomic walking clone rape BnPABP5 promotor the primer

It is as follows that step is moved concrete steps:

(purchase company according to the genomic walking test kit in Clontech, Universal.GenomeWalker) explanation, respectively with producing flat terminal restriction enzyme DraI, EcoRV, PvuII and StuI cuts about 2.5 μ g at 100 μ l system endoenzymes genomic dna, setting up four through restriction enzyme " storehouse " that handle, that be connected with the DNA of specificity joint AD, is that template is carried out two-wheeled PCR reaction with it.Get 1 μ lDNA (1 μ g/ μ L) and cut, detect its purity with Dra I enzyme.System is as follows: DNA 5 μ l, Dra I (10 units/μ l) 1.6 μ l, 10 * buffer, 2 μ l, sterilized water polishing to 20 μ l.37 ℃, spend the night, detect enzyme with 1% agarose gel electrophoresis and cut effect.Cut DNA with DraI, EcoRV, PvuII and StuI enzyme respectively, reaction system is as follows: DNA (0.1 μ g/ μ l) 25 μ l, and EcoRV (10 units/μ l) 8 μ l, 10 * buffer, 10 μ l, sterilized water polishing to 100 μ l, 37 ℃ are spent the night.Cut the 3M NaOAc of adding 10 μ l in the liquid and 95% ethanol of 50 μ l to enzyme ,-20 ℃ precipitation is more than 3 hours down, and 12000rpm is centrifugal 5 minutes then, twice of 70% washing with alcohol, centrifugal 5 minutes of 12000rpm, (20-25 ℃) dries under the room temperature, with 20 μ l sterilized waters dissolving air dried DNA.The connection of specificity joint, reaction system is as follows: 10 * T4ligase buffer, 2.0 μ l, enzyme cut the DNA8.0 μ l of processing, T4ligase (1 unit/μ l) 1 μ l, AD1 (joint primer, test kit carries) (25 μ M) 4.0 μ l, sterilized water is mended to 20 μ l, and 16 ℃ are spent the night.To connect liquid with 10 times of distilled water dilutions, be stored in-20 ℃.Carry out first round PCR reaction with genomic walking Auele Specific Primer GSP1 (table 1), system is as follows: 10 * buffer (MgCl ₂Free) 5 μ l, dNTPs mixture (10mM) 1.0 μ l, GSP1 (10 μ M) 2.5 μ l, AP1 (10 μ M) 2.5 μ l, Taq polymerase (5 units/μ l) 0.2 μ l connects liquid 0.1 μ l, and sterilized water is mended to 50 μ l.Response procedures is as follows: 94 ℃ of 1min; 98 ℃ of 10s, 72 ℃ of 2min, totally 7 circulations; 98 ℃ of 10s, 68 ℃ of 2min, totally 33 circulations; 72 ℃ of 10min., carry out second with genomic walking Auele Specific Primer GSP2 (table 1) and take turns the PCR reaction as second template of taking turns the PCR reaction with 50 times of distilled water dilution first round PCR reaction product, reaction system is as follows: 10 * buffer (no MgCl ₂) 5 μ l, dNTPs mixture (10mM) 1.0 μ l, GSP2 (10 μ M) 2.5 μ l, AD2 (joint primer, test kit carries) (10 μ M) 2.5 μ l, Taq polysaccharase (5 units/μ l) 0.2 μ l, template solution 1.0 μ l, sterilized water is mended to 50 μ l.Response procedures is with first round PCR reaction, pcr amplification result such as Fig. 4.Reclaim purifying second and take turns the PCR product, and connect, transformed into escherichia coli competence (front is stated), order-checking in order-checking T carrier (front is stated).Obtain BnPABP5 gene promoter full length sequence, a kind of isolating promotor, its series be the nucleotide sequence shown in the SEQ ID No.2 or with SEQ ID No.2 homologous nucleotide sequence in fact.

2, the structure of rape BnPABP5 gene promoter recombinant expression vector and agrobacterium tumefaciens transform: the pMD18-BnPABP5 (front is stated) and pBI121 (front the is stated) plasmid that are connected with the BnPABP5 promotor with Xba I/Hind III double digestion.Gel reclaims each promoter fragment and pBI121 (front the is stated) fragment that enzyme scales off, and connects, and transformed into escherichia coli (the method front is stated) is built into plant expression vector pBI121-BnPABP5 (front is stated) (Fig. 5).Change this carrier over to agrobacterium tumefaciens EHA105 (front is stated) with freeze-thaw method at last, contain select positive colony on kantlex (50mg/L) and the two resistant panel of Rifampin (50mg/L) after, whether contain target fragment with PCR checking.Described BnPABP5 gene promoter full length sequence contains the recombinant vectors of the nucleotide sequence of promotor, and recombinant vectors is: pBI121-BnPABP5.Recombinant vectors, it contains a kind of isolating promotor, and its sequence is the nucleotide sequence shown in the SEQ ID No.2.Microorganism, it contains recombinant vectors and transforms (freeze-thaw method transforms) transgenic plant, and it contains described microorganism and transforms (inflorescence infestation method).

Embodiment 3:

The Arabidopis thaliana of pBI121-BnPABP5 transforms and PCR detects:

According to above-mentioned document (Zhang X R.et al.Agrobacterium-mediated transformation ofArabidopsis thaliana using the floral dip method.Nature, 2006,1:1-6) middle method for transformation arabidopsis thaliana transformation.Grow containing on the 1/2MS that concentration is the 50mg/L kantlex (front the is stated) substratum according to the peculiar kalamycin resistance of transfer-gen plant, the green seedling of acquisition is tentatively thought positive seedling.After treating that green seedling grows two true leaves, it is transplanted in the vermiculite, treat that inflorescence appears in plant after, get a slice true leaf and extract genomic dna with the SDS method, do the PCR positive identification.The primer sequence sequence is, 5 '-AAAAGACTTACCGAGTTGAACC-3 ' and 5 '-CGCTGTCGTTGGGGCAATTC-3 '; The PCR reaction system is as follows: genomic dna template 1 μ L (about 50ng), 10 * Taq enzyme reaction buffer solution 2ul, 25mM MgCL ₂1.2ul, 2mM dNTP 1.5ul, each 0.2ul of 10uM primer, 0.3 Taq of unit enzyme, add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 5min, and 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 2min 32 circulations, 72 ℃ are extended 5min.Detect the PCR reaction product with 1% agarose gel electrophoresis, the result shows that this BnPABP5 full length gene promoter expression vector pBI121-BnPABP5 has successfully changed Arabidopis thaliana (Fig. 6) over to.

Embodiment 4:

The functional analysis of BnPABP5 gene:

1.BnPABP5 the structure of gene RNAi plant expression vector:

Sequences Design RNAi interference fragment according to second exon of Arabidopis thaliana.Primer such as table 2.

Table 2 makes up RNAi interference carrier primer

The PCR reaction system is 25 μ, includes: 1 * PCR buffer., and MgCI 1.5mmol/L, dNTP 0.2mmol/L (every liter of mmole), primer concentration are 0.5mol/L, Pfu enzyme 1.5 units, the about 100ng of template adds sterilized water to 25 μ l.Response procedures is: 94 ℃ of sex change 3min, and 94 ℃ of 50s, 55 ℃ of 80s, 72 ℃ of 90s35cycles, 72 ℃ are extended 5min, 2 fragments, 10 pipes that respectively increase, purifying (method is stated) PCR product.2 dna fragmentations of purifying are connected respectively to RNAi carrier pKannibal go up (front is stated).Concrete grammar is that increasing on Arabidopis thaliana PABP5 gene order by PCR method obtains disturbing segment.It is PABP-Ri-1-1 and PABP-Ri-1-2 that the forward segment connects primer.Reaction system is that 25 μ L include: 1 * PCR buffer., and MgCI 1.5mmol/L, dNTP 0.2mmol/L (every liter of mmole), primer concentration are 0.5mol/L, Pfu enzyme 1.5 units, the about 100ng of template.The PCR response procedures is as follows: 94 ℃ of 5min; 94 ℃ of 30sec, 55 ℃ of 45sec, 72 ℃ of 1min, 32 circulations; 72 ℃ are extended 8min.(mass volume ratio, below identical) sepharose detects the PCR product with 1.0%, reclaims PCR product (method is stated).With Xbal I+BamH I respectively the product that reclaims of double digestion and intermediate carrier pKannibal (front is stated) (enzyme is available from Takara company, the enzyme blanking method is pressed the operation of Takara specification sheets), reclaim enzyme respectively and cut product, molar ratio biased sample by 3: 1, add T4DNA ligase enzyme 5 units, 1 * reaction buffer, sterilized water replenish volume to 25ml, 16 ℃ of ligations of spending the night make up intermediate carrier pKannibal-1.In freeze-thaw method transformed into escherichia coli gold bacterium (front the is stated) competent cell.Reverse pulsating connection primer is PABP-Ri-2-1 and PABP-Ri-2-2, the PCR reaction conditions, and the vector construction condition is the same, makes up intermediate carrier pKannibal-1-2.With SpeI and NotI double digestion pKannibal-1-2, reclaim big segment, and be inserted into the SpeI/NotI site (method is the same) of plant expression vector pGreen0229 (http://www.pgreen.ac.uk/jit/pG0229.htm).Make up BnPABP5 gene RNAi plant expression vector pGKannibal-1-2 (Fig. 9), the carrier freeze-thaw method that builds is changed over to Agrobacterium EHA105 (front is stated).

2., the evaluation of the conversion of Arabidopis thaliana and positive plant: the method for having stated document according to the front is with BnPABP5 gene RNAi plant expression vector pGKannibal-1-2 arabidopsis thaliana transformation and gather in the crops seed.The Arabidopis thaliana planting seed on vermiculite and 1: 1 composite soil of nutrition soil, 4 ℃ vernalization 2-5 days, transfer to temperature 20-25 ℃, intensity of illumination 5000-10000 lumen, photoperiod is 16 hours illumination/8 hour dark, and atmospheric moisture is greater than between the cultivation more than 80%, normal management.After Arabidopis thaliana grows a slice true leaf, spray the weedicide Basta (available from Bayer AG) of 30ppm.After one week, spray the Basta of 50ppm, obtain the positive seedling of 10 strains.After treating that plant grows inflorescence, get a slice true leaf, extract DNA (the method front is stated), do the PCR positive identification.Concrete grammar is as follows: primer sequence is 5 '-TTTCGGTGACGGGCAGGAC-3 ' and 5 '-CTGCACCATCGTCAACCAC-3 '; The cumulative volume of PCR reaction system is 20 μ l, genomic dna template 1ul (about 50ng), 1 * Taq enzyme reaction buffer solution, 25mM MgCL ₂1.2ul, 2mM dNTP1.5ul, each 0.2ul of 10uM primer, 50% glycerine 2ul, 0.3u Taq enzyme (Takara company), add sterilized water to 20 μ l.Response procedures is: 94 ℃ of sex change 3min, and 94 ℃ of 50s, 55 ℃ of 80s, 72 ℃ of 90s 35 circulations, 72 ℃ are extended 5min.Agarose gel electrophoresis with 1% detects, and the result shows that BnPABP5 gene RNAi plant expression vector pGKannibal-1-2 has successfully changed Arabidopis thaliana (Fig. 7) over to.

3, the phenotypic evaluation of Arabidopis thaliana positive plant: the solid situation of the transgenic arabidopsis that detects by an unaided eye.Check the growth of flower pesticide respectively with Alexandria staining and frozen section.Alexandria staining concrete grammar is as follows: prepare slide glass, tweezers, staining fluid, pencil, pipettor and rifle head, gloves, spirit lamp, filter paper etc.; Get required fresh sample; Sample is put into the 1.5ml centrifuge tube, (to prepare about 100 milliliters of staining fluids is example: ethanol 10ml to add the Alexandria dye liquor, 1% malachite green spirituous solution 1ml, distilled water 50ml, glycerine 25ml, 1% C.I. 42685 aqueous solution 5ml, 1% orange G aqueous solution 0.5ml, Glacial acetic acid 1-4ml, phenol 5 grams, Chloral Hydrate 5 grams), 50 ℃ of dyeing 24h in flower pesticide immersion and the staining fluid; Take out sample, blot moisture on the sample with filter paper, microscopically is observed.Frozen section method concrete grammar is as follows:

1, preparation work: slide glass, PBS damping fluid (80g NaCl, 2g KCl, 6.1g, anhydrous Na 2HPO ₄, 2g, anhydrous K H2PO4 add water to 1000ml, stock solution can be used distilled water diluting as required).50% glycerine, dissecting needle, tweezers, embedding medium, pipettor and rifle head, gloves, spirit lamp, filter paper etc.;

2, get fresh bud sample and at room temperature put into the 2ml centrifuge tube, (take by weighing 2 gram phenodins and be dissolved in the propionic acid of 100ml45%, obtain the phenodin stock solution with propionic acid-Chloral Hydrate-phenodin dye liquor; Take by weighing 0.5 gram siderotil and be dissolved in the propionic acid of 100ml45%, obtain the siderotil stock solution.Get phenodin stock solution and siderotil stock solution balanced mix, add chloral hydrate again, deposit and to use later in 1 day to saturated.) dyeing 5min, take out and blot dyestuff with filter paper; PBS phosphoric acid buffer cleaning in the 2ml centrifuge tube under the room temperature.Carry out corresponding mark;

3, sample preparation: blot the PBS phosphoric acid buffer of sample with filter paper, put it into (available from Ling Fei biological products company limited) in the OCT embedding liquid, carefully put into the liquid nitrogen quick-frozen, take out-20 ℃ of preservations in back.；

4, section: the section temperature is-20 ℃, cuts up to seeing sample with 20 μ m left and right thicknesses earlier, repaiies the slice, thin piece that sheet cuts with the fine cutting of 10 μ m left and right thicknesses again, will be attached on the slide glass, carries out mark.

5, dyeing: drip the several propionic acid-Chloral Hydrate-phenodin dye liquors 5min that on sample, (covers sample), with filter paper just dye liquor blot, drip 50% glycerine and on sample, can observe photograph.

Get the Arabidopis thaliana bud that has just showed money or valuables one carries unintentionally for each strain transgenic arabidopsis, with the strip off gently of petal, sepal, avoid hurting flower pesticide with needle point, carry out Alexandria dyeing, microscopically pollen is observed, result such as Figure 10 and table 2.Compare with the wild-type Arabidopis thaliana, the flower pesticide smaller volume of the transgenic arabidopsis that setting percentage is low, pollen obviously reduces, and the abortion level of pollen obviously raises.Select the higher transgenic line of abortion degree, make the observation that frozen section is observed the pollen abortion of transfer-gen plant, find pollen granule crumple folding, atrophy (Figure 10 F).The silence that PABP5 gene in the Arabidopis thaliana is described can cause the abortion of plant.The PABP5 gene has the function of control pollen fertility and the solid ability of plant.

The solid cartogram of table 2RNAi transformed plant

Plant number	Positive	Solid situation	The fertility observations
				Transformant
1	YES	Normally	Normally
				Transformant
2	YES	The knot small quantities of seed	Half is sterile
				Transformant
3	YES	Not solid	Sterile fully
				Transformant 4	YES	Not solid	Sterile

Transformant 5	YES	The knot small quantities of seed	Half is sterile
				Transformant
6	YES	The knot small quantities of seed	Half is sterile
				Transformant
7	YES	The knot small quantities of seed	Half is sterile
				Transformant
8	YES	Not solid	Sterile
				Transformant
9	YES	The knot small quantities of seed	Half is sterile
				Transformant
10	YES	The knot small quantities of seed	Half is sterile

4.BnPABP5 the detection of gene RNAi plant expression vector transgenic arabidopsis jamming effectiveness: get PCR and detect the inflorescence of the T2 of back acquisition for positive plant, extract RNA, method is as follows, in liquid nitrogen plant young leaflet tablet sample is ground to powdery, the extraction of RNA is carried out in the requirement that utilizes Trirol to extract test kit.The total RNA that extracts is dissolved in the distilled water of no RNase.DNase I removes the residual DNA of possibility.(DU 650BECKMAN USA) detects RNA respectively at 260 nanometers and 280 nanometer absorbance values, identifies purity and the concentration of RNA in conjunction with 1% (mass volume ratio) agarose gel electrophoresis with the Protein Detection instrument.RNA with acquisition is that template is carried out reverse transcription (front is stated), and is standby in-20 ℃ of preservations after the cDNA packing that obtains.

Quantitative real time PCR Instrument is RT ^TM-Cycler (Bo Ao) adopts Arabidopis thaliana Actin as internal standard gene.Arabidopis thaliana PABP primer is 5 '-TCAAGTTGCTCCAGTTCACA-3 ' and 5 '-TGCCTTTCCCACTCAATCTA-3 '.The quantitative fluorescent PCR reaction system is 20 μ L: contain SYBRMix 10 μ L, and each 0.8 μ L of forward and reverse primer (10 μ mol/L), template 2 μ L, the sterilized water that DEPC handled complements to 20 μ L.Amplification condition is: 94 ℃, and 5min; 94 ℃ of 15s, 60 ℃ of 20s, 72 ℃ of 30s, 40 circulations; Each is circulated in 72 ℃ of renaturation ends and carries out fluoroscopic examination.Reaction is heated to 95 ℃ earlier after finishing, and reduces to 72 ℃ then, slowly is warming up to 95 ℃ again, writes down the variation of fluorescent signal, draws the melting curve of amplified production.Every group of experiment all finished three biology and repeated, and each biology repeats to do at least three technology and repeats.

Calculate the relative expression quantity of gene with comparing Ct method (Δ Δ Ct).Utilize software that instrument is with, at first optimize internal standard gene and goal gene amplification condition, measure the Ct value of Actin and goal gene respectively, choose that the most approaching three results (Ct valve system error is less than 0.3) average in nine measured value of experiment, by internal standard gene goal gene is proofreaied and correct then and obtained Δ Δ Ct, at last by 2 ^{-Δ Δ Ct}The relative expression quantity and the systematic error (front is stated) of estimation goal gene.When calculating relative expression quantity, be reference with the sample of wild-type Arabidopis thaliana inflorescence, be about to its value and convert 1 to, other sample again with its relatively, obtain relative expression's value.The result shows that the genetically modified jamming effectiveness of RNAI is very obvious: the expression efficiency of transfer-gen plant AtPABP5 only is the 2.7-15% of wild-type, and is suitable with the expression level of blade.Although offspring's expression level of homophyletic system is not quite similar, its difference amplitude is also little.Show that in conjunction with the result of case study on implementation 4.3 suppressing the PABP5 expression of gene can cause the plant pollen abortion, make the solid ability drop of plant or lose fully, obtain male sterile plants.The BnPABP5 gene has the function of control pollen fertility, has using value for utilizing genetic engineering technique to create male sterile material.

Embodiment 5:

The functional analysis of rape BnPABP5 gene promoter:

The present invention clones first and obtains rape BnPABP5 gene promoter sequence, and it has been carried out functional analysis.From embodiment 3, Arabidopis thaliana transforms and PCR detects that screening obtains in the step positive seedling T1 generation, selfing results seed (being T2 generation).The different times tissue of getting T2 generation 9 strain systems carries out GUS dyeing.

T2 is as follows for getting dyeing course: sample is immersed in GUS dye liquor (X-gluc 0.5mg/mL, phosphoric acid buffer 50mmol/L, each 0.5mmol/L of the Tripotassium iron hexacyanide and yellow prussiate of potash, EDTA 10mmol/L, Triton-x-1000.001%, methyl alcohol 20% (volume ratio) vacuum suction 5 minutes, 37 ℃ are spent the night.Decoloured with alcohol-acetate (volume ratio is 1: 1) in second day, and bleached until blade, use distilled water rinsing 3-5 time afterwards, Stereo microscope (OLYMPUS SZX16) is taken pictures.Get the whole plant of different time sections seedling stage; Reproductive stage, blade are got tender leaf and mature leaf; The inflorescence of flower that flower took away and the bud of not opening; The angle fruit of different times is really got at the angle; Seed removes back about the 10 days seed of blooming.It is exactly the position that gus gene is expressed that plant is dyed blue position.

Coloration result finds that this promoter-driven GUS gene is all expressed at the tip of a root in the whole growth process of (Fig. 8) Arabidopis thaliana, and the strongest in dyeing seedling stage on the 5th, still along with the growth of plant, staining power has a declining tendency; The expression of very short time is arranged at the vegetative point place of the rough leaf that has just grown, and along with the growth of plant, gus gene is not just expressed in true leaf; At the whole reproductive stage of Arabidopis thaliana, gus gene is specific expressed in flower pesticide.Not observing in whole dyeing course has gus gene to express in other tissue.This shows that this promoter-driven GUS gene is mainly expressed in flower pesticide, a small amount of expression is arranged, do not express in the seed in the leaf primordium of the tip of a root, rough leaf.

Experimental result shows, rape BnPABP5 gene promoter has following biological function: this promoter-driven GUS gene is all expressed in the tip of a root of Arabidopis thaliana, the expression of very short time is arranged at the vegetative point place of the rough leaf that has just grown, along with the growth of plant, gus gene efficiently expresses in flower pesticide.Gus gene under the BnPABP5 promoter regulation has certain spatial and temporal expression characteristic, and this explanation BnPABP5 promotor has the downstream reporter gene GUS of driving and express the function of (Fig. 8) in recipient plant.Under the regulation and control of this promotor, gus gene can be mainly meticulous expression in the flower pesticide of transfer-gen plant or pollen, and do not express fully in the seed.

This promotor with tissue specific expression has using value in plant genetic engineering and transgenosis safety (eating).

SEQUENCE?LISTING

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

＜120〉application of rape BnPABP5 gene and promotor thereof

＜130〉application of rape BnPABP5 gene and promotor thereof

<160>2

<170>Patentln?version?3.1

<210>1

<211>1998

<212>DNA

＜213〉rape

<400>1

atggcggcgg?cggttgcatc?tggaattgcc?ccgacgacag?cgatggttga?tcaagtcatc 60

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gcggctgcgg?cggccgaagt?gttacaaacg?caccctaact?cgtctctcta?tgtcggagat 180

ttggaccaaa?ctgtcaacga?agcacatctg?ttggatctct?tcaaccaagt?ggctcctgtc 240

caaacagtta?gggtttgtcg?cgacttgact?cgtcgttcac?ttggatacgc?ttacgtcaac 300

ttcgctaatc?ccgatgatgc?catgcgagca?atggatattc?tcaactacac?tccgatcaaa 360

gacagaccca?taaggatcat?gcgttcgaac?cgtgacccga?gcacaagact?tagcggaaaa 420

ggcaatgttt?tcatcaaaaa?cctggacctt?accattgata?acaaagcctt?gtacgacacc 480

ttctcgagtt?tcgggaccat?actctcatgc?aaggtagcca?tggacggtac?gggaaagtca 540

agaggctacg?gcttcgttca?gttcgagaag?gaagaaaccg?ctcaagccgc?tatcgacaag 600

ttgaacggga?tgcttctcaa?cgacaagcaa?gtctttgtgg?gaccctttgt?ccgtcgtcag 660

gacaggactc?gcgagagcgg?tgctgtcccg?cgtttcacaa?atgtctacgt?gaagaatctg 720

cctaaggaga?ttacagatga?tgagcttaag?aagacgtttg?ggaagtacgg?agagatctct 780

agtgcggttg?tggtgaaaga?cgagagtggg?aactcgaggt?gtttcgggtt?tgtgaacttc 840

gagagccctg?aggcggctgc?ggttgcggtt?gagaagatga?atgggattag?tcttggtgag 900

gatgtgttgt?acgttgggag?ggcgcagaag?aaggctgaga?ggggtgagga?gctgaggagg 960

aagtatgagc?aagagaggtt?ggagaagtcg?aacggaacga?atttgtatgt?gaagaatctt 1020

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tcaagcaagg?ttatgacgaa?cccagaaggt?ttgagcagag?ggtttggttt?tgttgcgtac 1140

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ccagctgcag?ggccacacca?tcaaatgtac?atgggccaga?acggtcaagg?tctggtgccg 1380

tcacagccta?tggggtacgg?gtaccaactt?cagttcatgc?ctggggtgcg?tccgggtgcc 1440

ggcccggcta?acttcatgat?gctttaccct?ctccagaggc?caaatcaacc?tggtcctcgt 1500

tttgggttca?ggcgtggtgc?tcctaacatg?cagcacaact?ttcagcagca?acaacaaatg 1560

atgcagcaca?atgtaaactc?tggaatgaga?tacatgggag?gtccgggtaa?caggatgaac 1620

ggagtggaag?cagctgcccc?acaaggcatt?gtggatgcat?ctgcaatctc?tcacaatgct 1680

tctcaaaacc?cacagaggcc?acctctccta?cccatttcta?agctcacttc?tgccttggca 1740

ctggctagcc?cttccaatca?ctcgcagatt?cttggagagc?agctttaccc?acttgtggaa 1800

aagcaggagc?cagttcatgt?ggccaaagtt?acagggatgc?tgctagagat?ggaccaagcg 1860

gagatcttgc?accttcttga?gtcacctgaa?gctcttaagg?ccaaagtgtc?tatggctttg 1920

gatgtcctca?gactctctgc?taatccatca?gctgtgtcat?ccgttgatga?ccagtttgct 1980

ccctcctcaa?cggagtga 1998

<210>2

<211>1936

<212>DNA

＜213〉rape

<400>2

aaaagactta?ccgagttgaa?ccgaattatt?tttaagattt?gtctaattga?acggaattta 60

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tttggtcaag?taaatagttt?cttggaatca?cacctctgga?tctcaaaggt?aaacaaagac 180

aaatatagaa?accacaaagg?aagccacagt?tggctcaata?tctttggata?ctattgttac 240

aagaaattaa?gaagaaaaaa?aacgtataaa?agttgcaata?tttcttgtct?tttccaggat 300

tctctgctca?aacccattac?aagaatcgtg?cttggagtcg?cgattgttat?gctgctaata 360

acgtttctat?tgaaagaccc?tccagcgtgg?atcaagaaca?ctatctccat?ggggaacttt 420

cctccgtggg?tgctcgcgtg?catagcaata?gtaatcaccc?gagcgaggaa?gaggaccaga 480

gacttcttca?agaagtgtgg?ctggtgatat?cacagtccct?aacatcttgt?ctacttgtaa 540

atgtgctgga?aattgtaatc?cgtcatcatg?taatatagag?attctcttat?cttaaaccca 600

acaagttccc?ttccttggta?aacacttgtt?gtctttgtta?agccttgagg?cgccaaaaat 660

tgcttgcttt?cagaaccaag?agggcaaatc?attttctcca?tttgcatttg?ttgaacttta 720

taggtttatg?cattttcatg?tgtatgcgag?tggaaattga?taatttaatc?tctcgaaacc 780

caaaagtttg?gtcaaagtaa?caagcctagc?catgttatat?tttcttttgt?attattcctg 840

atggaaaatc?ttacagttag?aaaaaataaa?aatctacaac?cggtcactag?tttatgttat 900

gcacaatttt?ttaaaaagtt?ggtagacaat?gattaggaaa?tatctaaaat?tatttgactt 960

tatgtatgat?ttttttcaca?cgaactttgt?attttgaatg?cttattaacc?ttttgaacaa 1020

attgatagag?tatataagtg?aaagtcaaca?tgtttttaac?caaataatct?tccccagaaa 1080

aaaaagttat?taagaaacta?ttgaatgaat?tcatctacaa?aagttggtat?ggatgtacaa 1140

actgaaatag?caaaaaaaaa?tgtaaaacac?aatttgactt?gaatttatca?gatagttcca 1200

aagtacccat?caacattgta?acaaatataa?gtaaatagtt?gcctgatact?ttttcacata 1260

ttttggaatc?atatccacat?tggatgttag?aagaagttat?taactcacgt?tgaagaactc 1320

atcacagaga?gcatcttcga?tttttaaaac?ataaacaatt?gaacacaata?ttatacgatt 1380

taattttgac?ctaatttttc?ttatgtacta?tcctcttcta?tctgtacttt?cccgcatgta 1440

aagtggtcag?gtcaaatcat?aagaaacgca?gaagtagaag?atatatggtt?aaattgaata 1500

tccacgttac?tcttgtagat?gcacaataaa?aggcgacaaa?aaacaaaaga?aagacggaca 1560

cgtgtcacca?tgttatacat?tatttgttaa?gttatgggcc?cacaaaactc?gatcaatctg 1620

aaaagacaca?tcagttcgaa?ctcagatccg?tacggaccga?ttcgttagtt?agcgaatgta 1680

taaatacaca?acagcacatc?taccacactc?ttatcgtttc?tccgaagtta?tcaaaaaagg 1740

aaaaaaatca?aaatcaaaac?caaaatatca?aataaataaa?aatctcgcaa?aatattctct 1800

gaaagattta?aaagatcgca?aaacacacaa?acttaatctc?tctcgctgtc?ttgcacacat 1860

acgaaacaaa?aaccctaaat?atcagagcgg?tatggcggcg?gcggttgcat?ctggaattgc 1920

cccaacgaca?gcgatg 1936

Claims (5)

1. isolating gene, its sequence is a nucleotide sequence shown in the SEQ ID No.1.

2. isolating promotor, its sequence is the nucleotide sequence shown in the SEQ ID No.2.

3. a kind of isolating promotor according to claim 2, base is characterised in that: described promotor contains recombinant vectors pBI121-BnPABP5.

4. the application of the described a kind of rape BnPABP5 gene of claim 1 in the control pollen fertility.

5. the application of the described a kind of rape BnPABP5 gene promoter of claim 2 in flower pesticide.

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