CN110055252A

CN110055252A - One kind specifically expressed promoter PCHF45 and its application in paddy pollen

Info

Publication number: CN110055252A
Application number: CN201910343747.XA
Authority: CN
Inventors: 黄培劲; 龙湍; 吴春瑜; 张维; 刘昊; 吴永忠; 曾翔; 李新鹏; 安保光
Original assignee: Hainan Bolian Rice Gene Science & Technology Co Ltd
Current assignee: Hainan Bolian Rice Gene Science & Technology Co Ltd
Priority date: 2019-04-26
Filing date: 2019-04-26
Publication date: 2019-07-26
Anticipated expiration: 2039-04-26
Also published as: CN110055252B

Abstract

Specifically expressed promoter PCHF45 and its application in paddy pollen that the invention discloses one kind, are related to genetic engineering and molecular biology field.Promoter PCHF45 of the present invention has nucleotide sequence shown in SEQ ID NO.1, and the present invention further provides the expression vector containing promoter PCHF45, expression casette, engineering bacteria or cell lines.The present invention also provides the primer pairs of amplification pollen specific promoter.Pollen-specific promoter of the invention is rice endogenous gene, highly beneficial to paddy gene engineering, can drive that foreign gene is specific expressed in pollen and expression is accurate, provides driving foreign gene new method specific expressed in paddy pollen.

Description

One kind specifically expressed promoter PCHF45 and its application in paddy pollen

Technical field

The present invention relates to genetic engineering fields, specifically, being related to paddy pollen specificity promoter PCHF45 and its answering With.

Background technique

Transcriptional control is one of principal mode of control of plant gene expression, by cis-acting elements and trans-acting factor Coordinate to complete.Promoter is one of most important cis element in plant gene transcription regulation, is normally at gene 5 ' and holds upstream, It is identification and the binding site of RNA polymerase and some trans-acting factors.Promoter mainly includes core promoter Qu Yuzhuan Record two functional areas of control region.Core promoter area is the most short promoter fragment of starting transcription, and generally 40nt is one section of quilt DNA sequence dna RNA polymerase family I, II and III identification and combined.This region includes some important function element, Neng Gouzhun True positioning transcriptional start point and direction are the bases of gene expression regulation.Transcription regulating region is located at the upstream of core promoter (or downstream), can be combined with special transcription factor to the space-time of transcription and it is strong and weak play regulating and controlling effect, such as enhancer and Silencer etc..The expression pattern of further investigation promoter not only improves the expression and regulation mechanism and biological function for understanding gene, Facilitate the expression of control foreign gene again.

Promoter can be divided into constitutive promoter, inducible promoter and space-time specific promoter etc. by its expression way Three classes.Constitutive promoter promotor gene can transcribe in all or most of tissues, and gene expression is made to have space-time duration With expression quantity constancy.The 35S promoter of tobacco mosaic virus (TMV), the Actin promoter of rice and the Ubiquitin of corn are opened Mover belongs to constitutive promoter.Constitutive promoter is widely used in the genetic engineering research of plant, is used for target base Cause, such as pest-resistant and anti-herbicide gene overexpression.Inducible promoter can open under the stimulation of certain physically or chemically signals Move or greatly improve gene expression.They have the sequential structure of enhancer, silencer or similar functions, and have apparent single-minded Property.Inducible promoter can be divided into photoinduction promoter, heat shock promoter, low temperature induction according to the difference of inducement signal and open Mover, drought-induced promoter, wound-induced promoter, hormone induction promoter etc..Space-time specific promoter is only specific Growth phase or position in promotor gene expression.Tissue-specific promoter is one kind of space-time specific promoter, is only existed Start expression in specific cells, tissue or organ.It is controlled in the genetic transformation of plant with the promoter of organizing specific expression The expression of target gene can more effectively avoid such as can be reduced composing type table using constitutive promoter bring potential side effect Up to increased metabolic burden, reduction Safety of GM Food risk and to the adverse effect with environment, and reuse identical The gene silencing etc. that promoter causes.The type of current developed rice tissue specific promoter is varied, root, stem, Leaf, seed and fruit etc. have almost had been found that organized specific expressed promoter in various tissues.

Male sterility is the basis of heterosis utilization, is the core technology of hybrid rice industry, Study On Rice fertility tune The mechanism of control has great theoretical and practical significance.Anther is that rice generates mature male gametophyte (i.e. pollen or microspore) Organ.In the early stage of anther development, there are a kind of primary sporogenous cell, which passes through a series of divisions and differentiation, produces Raw tapetum and pollen mother cell.Tapetum provides nutrition, including various enzymes and non-enzymatic albuminoid for the development of male gametophyte Matter, and participate in the synthesis of exposore.Pollen mother cell then carries out meiosis, generates tetrad.It is unicellular in tetrad Effect through tapetum secretion callosity enzyme separates Haploid production microsporinite.Monoploid microsporinite is further developed, to evening The big vacuole in center is formed when the phase.Central big vacuole squeezes nucleus to cell edges (referred to as mid-late uninucleate stage), forms cell Polarity.This cell polarity promotes microsporinite to carry out primary unequal mitosis, generates a big vegetative cell and one A small reproduction cell.Small reproduction cell is completely contained in big vegetative cell, and cell is special existing in formation cell As.Reproduction cell then carries out second of mitosis, generates two spermatids, forms mature pollen grain.Therefore, screening and Paddy pollen specific promoter is determined by for the genetic engineering of rice, especially in the side such as sterility changing and transgenosis drift prevention and control Face provides new selection.

Summary of the invention

The object of the present invention is to provide a kind of plant pollen specificity promoter and its applications.

A kind of plant pollen specificity promoter PCHF45 provided by the invention, to include

1) nucleotide sequence shown in SEQ ID No:1,

Or 2) in the nucleotide sequence shown in SEQ ID No:1 it is substituted, lacks or adds one or several nucleotide And the nucleotide sequence as derived from 1) with same pollen specific startup function；

Or 3) the sequence complementary with nucleotide sequence shown in SEQ ID No:1.

Wherein, the nucleotide sequence as derived from 1) described in 2), the nucleotides sequence described in 1) is classified as to be had compared with 1) 70% or more homology, 80% or more homology, 85% or more homology, 90% or more homology, 95% or more homology, 98% or more homology or 99% or more homology, and the nucleotide sequence with same pollen specific promoter function.

Those skilled in the art can easily identify and utilize and plant pollen specificity promoter according to identical purpose Therefore the DNA molecular of PCHF45 nucleotide sequence complementation has promoter activity and can start under strict conditions with the present invention The DNA sequence dna of subsequence or the hybridization of its segment is included in the invention.Wherein, the nucleotide sequence is complementary, refers to stringent Under the conditions of can hybridize with PCHF45.

It is more than to hybridize with other sequences (as at least that stringent condition, which refers to that probe will be hybridized to detectable degree with its target sequence, 2 times of backgrounds) condition.Stringent condition has sequence dependent, and different because of the difference of the other conditions of experiment.Pass through control The stringency of hybridization and/or wash conditions, can identify the target sequence complementary with probe 100% (same to source detection).Selectively, Adjustable stringent condition is to allow some sequence mismatch, so that detecting the similitude (heterologous detection) of lower degree.In general, Probe length is no more than 1000 nucleotide, is preferably shorter than 500 nucleotide.

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

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

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

The present invention provides the expression casette containing plant pollen specificity promoter PCHF45 of the present invention, expression carries Body and host cell containing the expression vector.

The expression casette has structural gene for the downstream connection in pollen specific promoter PCHF45, adjusts base Because of the antisense gene of, structural gene, the table of MicroRNA gene that adjusts the antisense gene of gene or can interfere with endogenous gene expression Up to box.

The present invention further provides the expression vectors of the expression casette containing above-mentioned pollen specific promoter PCHF45.

The present invention provides above-mentioned paddy pollen specificity promoter PCHF45 or the expression cassette containing it, expression vector or The host cell application specific expressed in plant pollen in driving foreign gene.

The present invention provides paddy pollen specificity promoter PCHF45 or contain its expression cassette, expression vector or host Application of the cell in prepare transgenosis plant.

The genetically modified plants are foreign gene specifically expressed genetically modified plants in pollen, preferably pollinate, are fertilized Genetically modified plants ability enhancing or weakened, more preferably male sterility genetically modified plants.

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

The present invention also provides the primer pair for expanding the pollen specific promoter PCHF45, the nucleosides of the primer pair Acid sequence is SEQ ID NO:2-3 or SEQ ID NO:6-7.

The present invention provides a kind of method for separating pollen specific promoter PCHF45, to use SEQ ID NO:2-3 Or the nucleotide sequence of SEQ ID NO:6-7 primer pair PCR amplification pollen specific promoter PCHF45.

The present invention also provides driving foreign gene specifically expressed methods in pollen, include the following steps:

Paddy pollen specificity promoter PCHF45 and purpose exogenous gene cloning of the invention are contained into carrier There is the recombinant expression carrier of the expression cassette of PCHF45 and purpose foreign gene, and be introduced into Plant Genome, obtains external source base Because of the specifically expressed genetically modified plants in pollen.

Pollen-specific promoter PCHF45 provided by the invention has the advantages that

1) PCHF45 is rice DNA sequence endogeneous, and Transgene-safty risk is extremely low.

2) PCHF45 can drive foreign gene specific expressed in pollen, and expression is accurate.

3) the present invention provides a kind of new methods that driving foreign gene is specific expressed in pollen.

Detailed description of the invention

Fig. 1 is the recombinant expression carrier 1300gus-PCHF45 carrier of pollen specific promoter PCHF45 in embodiment 2 Map.

Fig. 2 is the recombinant expression carrier DX2182-PCHF45 map of pollen specific promoter PCHF45 in embodiment 3.

Fig. 3 is that 11 Rice Anthers and pollen GUS stained photographs are spent in non-transgenic, is dyed to blue without pollen.

Fig. 4 is 1300gus-PCHF45 transgenic paddy rice anther and pollen GUS stained photographs in embodiment 6.

Fig. 5 is 1300gus-PCHF45 transgenic paddy rice gynoecium GUS stained photographs in embodiment 6.

Fig. 6 is 1300gus-PCHF45 transgenic paddy rice glume GUS stained photographs in embodiment 6.

Fig. 7 is 1300gus-PCHF45 transgenic paddy rice root GUS stained photographs in embodiment 6.

Fig. 8 is 1300gus-PCHF45 transgenic paddy rice stem GUS stained photographs in embodiment 6.

Fig. 9 is 1300gus-PCHF45 transgenic paddy rice leaf GUS stained photographs in embodiment 6.

Figure 10 is that 11 paddy pollens photo under green fluorescence microscope is spent in non-transgenic, is issued without pollen glimmering Light.

Figure 11 is Actin promoter transgenic paddy rice pollen photo under green fluorescence microscope in embodiment 7, wherein greatly Most pollen can issue fluorescence.

Figure 12 is photo of the DX2182-PCHF45 transgenic paddy rice pollen under green fluorescence microscope in embodiment 7, In have half pollen that can issue fluorescence.

Figure 13 is photo of the DX2182-PCHF45 transgenic paddy rice anther under green fluorescence microscope in embodiment 7.

Figure 14 is photo of the DX2182-PCHF45 transgenic paddy rice glume under green fluorescence microscope in embodiment 7.

Figure 15 is photo of the DX2182-PCHF45 transgenic paddy rice blade under green fluorescence microscope in embodiment 7.

Figure 16 is photo of the DX2182-PCHF45 transgenic paddy rice gynoecium under green fluorescence microscope in embodiment 7.

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Without departing substantially from spirit of that invention In the case where essence, to modifications or substitutions made by the method for the present invention, step or condition, all belong to the scope of the present invention.

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

The acquisition of 1 paddy pollen specificity promoter PCHF45 of embodiment

1. the extraction of oryza sativa genomic dna

Oryza sativa genomic dna is extracted using plant DNA Isolation kit (Chengdu Fu Ji Bioisystech Co., Ltd).Gene Group derives from the Fresh leaves of rice varieties OryzasativaLcv.Nipponbare.It is saved backup after the genomic DNA packing of extraction in -20 DEG C.

2.PCHF45 PCR primer design and amplification

Design of primers uses Gibson Assembly method, and amplified production is inserted into 1300GUSplus carrier (will GUSplus element insertion pC1300 multiple cloning sites obtain) I and Hind the III digestion site Nco and be inserted into DX2182 load Xma I, the Sal I restriction enzyme site of body.Expand the primer sequence such as SEQ ID NO:2-3 and SEQ ID NO:6-7 institute of PCHF45 Show.There are 15 nucleotide sequences (cgacggccagtgcca) at the end of primer sequence SEQ ID NO:2-3 upstream and downstream primer 5 ' and carry The corresponding link position overlapping of body, so as to Gibson Assembly connection.Primer sequence SEQ ID NO:6-7 upstream primer 5 ' is held Containing Xma I restriction enzyme site sequence cccggg, Sal I restriction enzyme site sequence gtcgac is contained at the end of downstream primer 5 '.

PCR reaction system (100 μ L): oryza sativa genomic dna DNA profiling: 3 μ L (50ng), KOD polymerase (are purchased from Japan Mill company): 2 μ L, 10 × buffer:10 μ L, 10 μM of forward primers: 3 μ L, 10 μM of reverse primers: 3 μ L, 10 μM of dNTP:10 μ L, MgSO₄: 4 μ L, 1/10 DMSO:20 μ L, ddH₂O:45 μ L.

PCR program: 95 DEG C of initial denaturation, 4min.94 DEG C of denaturation, 30s；50 DEG C of annealing, 30s；Extend 68 DEG C, 2min；35 Circulation.Extend 68 DEG C, 10min.

Amplified production includes the pollen specific promoter PCHF45 (sequence such as SEQ ID NO.1) of 2051bp.

The recombinant expression carrier p1300gusplus-PCHF45 of the building of embodiment 2 promoter PCHF45

The PCR product obtained in embodiment 1 is subjected to electrophoresis, recycling 2051bp or so size in 1% Ago-Gel Band.With Nco I and Hind III double digestion carrier p1300gusplus, linear digestion carrier is recycled.

With Lightening Cloning Kit (Jin Fusai (Beijing) Biotechnology Co., Ltd) to PCR recovery product and Linearisation p1300gusplus empty carrier is attached, and 10 μ L systems are as follows: 2.5 μ L recovery products (50ng/ μ L), 0.5 μ L enzyme It cuts carrier (100ng/ μ L), 2.5 μ L Ligation Mix.Linker: 50 DEG C, 60min.

Take the above-mentioned 5 electroporated competent escherichia coli cell of μ L of connection product.Use primer SEQ ID NO:4 and SEQ ID NO:5 carries out bacterium colony PCR, selects positive colony sequencing verifying.Correct carrier is sequenced and is named as p1300gusplus- PCHF45, map are shown in Fig. 1.P1300gusplus carrier contains gus gene.Indigo plant is presented in the tissue of expression gus gene after dyeing Color may be used to indicate the expressive site and intensity of promoter.

Embodiment 3 constructs the recombinant expression carrier of promoter sequence containing PCHF45 DX2182-PCHF45

1. constructing the recombinant cloning vector pGEM-PCHF45 of the promoter sequence containing PCHF45

Referring to the method for embodiment 1, replaced with sequence primer pair as shown in SEQ ID NO:6 and SEQ ID NO:7 real It applies primer pair shown in sequence SEQ ID NO:2 and SEQ ID NO:3 in example 1 to be expanded, obtained amplified production carries out plus A Tail (3 μ L dNTP is added into non-purified product (50 μ L), 0.5 μ L Taq enzyme continues 72 DEG C of reaction 20min).Add A tail product It being attached after purification and recovery with pGEM-T carrier, operating procedure is carried out by Promega Products pGEM-T carrier specification, Obtain recombinant cloning vector pGEM-PCHF45.Competent escherichia coli cell is converted, positive colony is further selected, is sequenced.It will Correctly clone extracts plasmid for sequencing.

2. constructing the recombinant expression carrier DX2182-PCHF45 of the promoter sequence containing PCHF45

With the resulting recombinant cloning vector pGEM-PCHF45 of restriction enzyme XmaI, SalI double digestion embodiment 3 and table Up to carrier DX2182, the 2051bp of the promoter containing PCHF45 cut or so size segment is inserted into linear digestion carrier Xma I, the Sal I restriction enzyme site of DX2182.

PCR recovery product and linearisation DX2182 empty carrier are attached with T4 ligase, 10 μ L systems are as follows: 4 μ L Recovery product (50ng/ μ L), 4 μ L digestion carriers (100ng/ μ L), 1 μ L T4 ligase.Linker: 22 DEG C, 2 hours.

Take the above-mentioned 5 electroporated competent escherichia coli cell of μ L of connection product.Use primer SEQ ID NO:8 and SEQ ID NO:9 carries out bacterium colony PCR, selects positive colony sequencing verifying.Correct carrier is sequenced and is named as DX2182-PCHF45, schemes Spectrum is shown in Fig. 2.DX2182 carrier contains EGFP gene (a kind of enhanced GFP gene).It is glimmering that the group of expression EGFP gene is woven in green Fluorescence is issued under light microscope, may be used to indicate the expressive site and intensity of promoter.

The acquisition of 4 p1300gusplus-PCHF45 transgenic paddy rice of embodiment

Take the Agrobacterium EHA105 of -70 DEG C of preservations in containing 50 μ g/mL rifampin plate streakings, 28 DEG C of cultures.Picking single bacterium It falls and is inoculated in 50mL YEB fluid nutrient medium, 220rpm28 DEG C of shaken cultivation 12-16hr.Take the switching of 2mL bacterium solution in 100mL In (containing antibiotic) YEB fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5.It is pre-chilled 10 minutes on ice, 5000rpm is centrifuged 10min (refrigerated centrifuge is pre-chilled to 4 DEG C).Aseptic deionized water is washed 2 times (each 10mL), and 10% glycerol washes 1 It is secondary to be dissolved in 3mL10% glycerol.Respectively into two pipes, 100 μ L competent cell plus obtained in 1 μ L embodiment 2 and embodiment 3 P1300gusplus-PCHF45 plasmid, 2.5 KV are electroporated.28 on YEB culture plate containing kanamycin and rifampin DEG C culture, select positive colony, carry out PCR verifying with p1300gusplus vector-specific primers SEQ ID NO:4-5.

Verifying is correctly cloned, and is infected by Agrobacterium-mediated genetic transformation method and is spent 11 (Hiei Y Ohta in rice S,Komari T,Kumashiro T(1994)Efficient transformation of rice(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T- DNA.The Plant Journal6:271-282).Through co-cultivation, the links such as screening, breaks up, takes root and obtain T₀For transgenosis children Seedling.The blade total DNA for extracting transformed plant carries out PCR positive detection using primer SEQ ID NO:10 and SEQ ID NO:11, The positive plant cultivation verified through PCR is chosen, self-fertility obtains T₁Generation.Take T₀Or T₁Subsequent analysis is carried out for plant.

The acquisition of 5 DX2182-PCHF45 transgenic paddy rice of embodiment

Take the Agrobacterium EHA105 of -70 DEG C of preservations in containing 50 μ g/mL rifampin plate streakings, 28 DEG C of cultures.Picking single bacterium It falls and is inoculated in 50mL YEB fluid nutrient medium, 220rpm28 DEG C of shaken cultivation 12-16hr.Take the switching of 2mL bacterium solution in 100mL In (containing antibiotic) YEB fluid nutrient medium, 28 DEG C of 220rpm shaken cultivations to OD600=0.5.It is pre-chilled 10 minutes on ice, 5000rpm is centrifuged 10min (refrigerated centrifuge is pre-chilled to 4 DEG C).Aseptic deionized water is washed 2 times (each 10mL), and 10% glycerol washes 1 It is secondary to be dissolved in 3mL10% glycerol.Add DX2182-PCHF45 plasmid obtained in 1 μ L embodiment 3 into 100 μ L competent cells, 2.5 KV are electroporated.28 DEG C of cultures, select positive colony on YEB culture plate containing kanamycin and rifampin, use DX2182 vector-specific primers SEQ ID NO:8-9 carries out PCR verifying.

6 transgenic paddy rice GUS staining analysis of embodiment

Prepare GUS dyeing liquor X-Gluc reaction solution (50mM phosphoric acid receives buffer, pH value 7.0, the 0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 0.5mg/ml X-Gluc, 20% methanol of volumn concentration, 0.1%Triton X-100), at random Choose 5 or more the 1300gus-PCHF45 transgenic positive strains that obtain in example 4 acquire anther, gynoecium, glume, The tissue samples such as root, leaf, stem are immersed in 37 DEG C of 2h in X-Gluc reaction solution or stay overnight, then use 75% second of volumn concentration Alcohol observes photograph after sloughing the chloroplaset color of tissue.The results show that 11 pollen is spent in wild type cannot dye (Fig. 3), and The pollen of transgenic paddy rice has half to be dyed to blue (Fig. 4), other gynoeciums (Fig. 5), glume (Fig. 6), root (Fig. 7), stem (figure 8), the tissues such as leaf (Fig. 9) cannot be colored, and show that PCHF45 promoter can drive gus gene specific in paddy pollen Expression, and the expression of foreign gene cannot be driven in tissues such as Rice Anther, glume, root, stem, blade, gynoeciums.

The analysis of 7 transgenic paddy rice pollen EGFP green fluorescence of embodiment

Randomly select 5 or more DX2182 transgenic positive strains obtained in embodiment 5 acquisition maturity period anther and Pollen film-making is placed under green fluorescence microscope and analyzes result.The results show that 11 pollen is spent not fluoresce in wild type (Figure 10), and the transgenosis of positive control Actin promoter (also constructing in the upstream of DX2182 carrier EGFP reporter gene) flower Powder can fluoresce (Figure 11).Referring to positive control as a result, the transgenic pollen of PCHF45 promoter can issue obvious fluorescence (figure 12), the tissues such as other anther (Figure 13), glume (Figure 14), blade (Figure 15), gynoecium (Figure 16) cannot fluoresce, and show this The PCHF45 promoter of invention can drive EGFP gene specific expressed in paddy pollen, and in Rice Anther, glume, leaf The tissues such as piece, gynoecium cannot drive the expression of foreign gene.

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

Sequence table

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gaaatttacc ctcagatcta ccatctccat ggctggaggt gg 42

<210> 4

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gatcagttta aagaaagatc aaagctc 27

<210> 5

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ctgcaaggcg attaagttgg gtaac 25

<210> 6

<211> 33

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

cccgggccta gctctatgta cttcccataa gcc 33

<210> 7

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gtcgacggtg cctgcctgcc tatatg 26

<210> 8

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

aactgcctgg cacagcaatt g 21

<210> 9

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gacgttgtgg ctgttgtagt tgtac 25

<210> 10

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cttagccaga cgagcgggtt c 21

<210> 11

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gcttctgcgg gcgatttgt 19

Claims

1. paddy pollen specificity promoter PCHF45, which is characterized in that it is with following any nucleotide sequence:

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

2) one or several nucleotide are substituted, lack or added in the nucleotide sequence shown in SEQ ID NO.1 and have The function of same pollen specific promoter, the nucleotide sequence as derived from 1)；

3) nucleotide sequence complementary with nucleotide sequence shown in SEQ ID NO.1.

2. the expression casette containing paddy pollen specificity promoter PCHF45 described in claim 1.

3. the expression vector containing paddy pollen specificity promoter PCHF45 described in claim 1.

4. the host cell containing expression vector described in expression cassette described in claim 2 or claim 3.

5. paddy pollen specificity promoter PCHF45 described in claim 1 or expression cassette, expression vector or place containing it The chief cell application specific expressed in plant pollen in driving foreign gene.

6. paddy pollen specificity promoter PCHF45 described in claim 1 or expression cassette, expression vector or place containing it Application of the chief cell in prepare transgenosis plant.

7. paddy pollen specificity promoter PCHF45 described in claim 1 or expression cassette, expression vector or place containing it Application of the chief cell in prepare transgenosis rice.

8. expanding the primer pair of paddy pollen specificity promoter PCHF45 described in claim 1, which is characterized in that the primer Pair nucleotides sequence be classified as nucleotide sequence shown in SEQ ID NO:2-3 or SEQ ID NO:6-7.

9. paddy pollen specificity promoter PCHF45 described in claim 1 or expression cassette, expression vector or place containing it Chief cell or primer pair according to any one of claims 8 are preparing foreign gene in the specifically expressed transgenic paddy rice of paddy pollen Using.

10. the method for driving foreign gene specific expressed in plant pollen, which comprises the steps of: by water Rice pollen specific promoter PCHF45 and purpose foreign gene, which are imported into carrier, to be obtained containing PCHF45 and purpose external source base The recombinant expression carrier of the expression cassette of cause, and it is introduced into Plant Genome, it is specifically expressed in pollen to obtain foreign gene Genetically modified plants.