CN105316334B - The identification and application of plant anther specific expression promoter pTaASG019 - Google Patents

Abstract

The invention belongs to plant biotechnology fields, and in particular to the separation of plant anther specific expression promoter and Function Identification and application.Promoter disclosed in this invention specifically expressing in the anther of plant has a good application prospect in plant transgene field.

Description

The identification and application of plant anther specific expression promoter pTaASG019

Technical field

The invention belongs to plant biotechnology fields, specifically, can instruct can the present invention relates to isolated DNA It is operatively connected to nucleic acid downstream specific transcription and/or expression in plant anther.In addition, the invention further relates to include this The expression cassette of DNA and plant etc., and it is related to the application of the DNA.

Background technique

Plant gene regulatory is mainly to carry out on transcriptional level, by a variety of cis-acting elements and trans-acting factor It is mutually coordinated.Promoter is important cis-acting elements, it is to be located at structural gene 5 ' upstream region controlling gene is held to turn The section of DNA sequence of record can activate RNA polymerase, be allowed to accurately be combined with template DNA, it is ensured that transcription is accurately and effectively Starting, plays a crucial role in transcriptional control.The different characteristics of gene expression is driven according to it, promoter is divided into composing type starting Son and specificity promoter.Constitutive promoter can be in all cell or tissues, regardless of time and spatially starting transcription；It is special Specific Promoters can be divided into tissue-specific promoter and inducible promoter again, and wherein inducible promoter, which does not start usually, turns Record or transcriptional activity are very low, but under the stimulation of certain specific adverse circumstance signals, transcriptional activity can improve significantly.

Exogenous DNA array starts subclass by being connected to expression of the specific promoter to starting in plant host The selection of type determines expression time and the position of gene.At present widely applied mainly some groups in agricultural biological technical field Molding strong promoter, such as CaMV 35S promoter and corn Ubiquitin-1 promoter, however utilizing these promoters When inducing quality of the crops such as target gene rice transformation to Crop Improvement, often due to the time of destination gene expression (stage of development specificity) or space (tissue and organ specificity) cannot control well and cause improved effect unobvious, or The growth and development of plant is impacted since these constitutive promoter inducible gene expression amounts are too high, these are all current The obstacle encountered when crop improvement quality using composing type strong promoter binding function gene.

In addition, when studying certain metabolic processes or adjusting approach, it is often necessary to will be more than two in same approach Genetic transformation converts another base into the same strain, using converting after one of gene obtains transgenic plant again Cause or two genes are hybridized again after the completion of converting respectively, are required to wait longer time, be shortened to improve efficiency The time of multiple genetic transformation has been reported that the conversion that can use new carrier while carrying out multiple genes recently, but more If reusing the same promoter when genetic transformation, sink also due to the high homology of promoter sequence may cause gene It is silent.

In recent years, existed by genetic engineering regulation plant pollen fertility, creation male sterility line of plants and its restorer It succeeds on some crops, has started new prospect for the utilization of crop heterosis.It is created currently with genetic engineering male Property infertility strategy mainly utilize the specific promoter of pollen development chimeric with foreign gene, construction of expression vector, convert plant Object blocks the process of pollen development to reach male sterile purpose.Mariani etc. is special using tobacco anther tapetum Promoter TA29 connect with RNase T1 or Barnase and is built into mosaic gene, is transferred to tobacco and rape by mediated by agriculture bacillus, Obtain stable male sterile transformant (Mariani C etc., Induction of male sterility in Plants by a chimaeric ribonuclease gene, Nature, 1990,347:737-741).Then, Ta Menyou Barstar gene is driven with TA29, creates restorer (Mariani C etc., the A chimaeric of above-mentioned male sterile line ribonuclease-inhibitor gene restores fertility to male sterile plants,Nature, 1992,357:384-387).In addition, other research groups are synthesized using anther-specificpromoter starting antisense chalkone The specifically expressing in anther such as enzyme gene, antisense actin gene, β -1,3- glucanase gene also successfully obtains respectively Male sterile plants (Meer etc., Promoter analysis of the chalcone synthase gene of petunia hybrid:a 67bp promoter region directs flower-specific expression, Plant Biol.,1990,15:95-109；New is appointed red male sterility gene to import Wheat cultivar by Li Yanhong etc. First Report of Studies, Journal of Agricultural Biotechnology, 1999,7:255-258；Curtis etc., Genomic male sterility in lettuce,a base line for the production of F₁hybrid,Plant Sci.,1996,113:113- 119)。

The driving activity and specificity of plant pollen or anther promoter, which are determined, regulates and controls pollen by genetic engineering means Fertility, the success or failure for creating plant sterile line and restorer.The driving activity height and the good plant pollen of specificity being currently known Or anther-specificpromoter is also relatively fewer, and wheat is because of the reasons such as its genome is larger and structure is complicated, in pollen or anther Research in terms of development molecular mechanism is deficienter, therefore, clone and functional analysis to Wheat Pollen specific expression promoter For utilizing genetic engineering regulation pollen fertility in wheat, creating male sterility line of plants, to be provided for wheat heterosis Source making full use of in wheat breeding lays the foundation.

Summary of the invention

The object of the present invention is to provide a kind of specifically expressed promoter sequence of Plant Pollen Development advanced stage anther and clones And using the method for the promoter.

The present invention takes pollen to be in meiophase, monokaryotic stage, the wheat anther of dicaryotic phase and three core phases, uses Trizol (Invitrogen) total serum IgE is extracted, and carries out DNaseI (Promega) processing, and then purify mRNA (Ambion).By purifying MRNA carries out reverse transcription (Invitrogen), ultrasound interrupts (Fisher), preparation library (illumina) and expands (illumina), sequencing reaction is finally carried out on illumina machine.

The result of wheat transcript profile high-flux sequence passes through Trinity software first and carries out sequence assembly, obtained splicing Sequence further removes redundancy and similitude cluster.For the expression mutation analysis for the transcript contig that splicing obtains, respectively The sequence of high-flux sequence passes through TopHat (http://tophat.cbcb.umd.edu/) software and transcript first in sample The result of splicing is compared.Then Cufflink software being capable of homogenization expression of the calculating ratio to upper transcript contigs Amount, with " Kilobases (the fragments per kilobase of exon model per of every million aligned fragment of exon Million mapped fragments, FPKM) " it indicates.

By the full-length genome expression pattern analysis to different development stage wheat anther, finds pollen and be in the subtrahend separation phase Anther in do not express and expressed in the anther that pollen is in monokaryon, double-core and three core phases transcript contig 7187. As shown in Figure 1, comp106333_c0_seq1 (sequence is as shown in SEQ ID NO:1) pollen is in subtrahend separation phase and monokaryotic stage Little Hua in do not express and be in double-core and the anther of three core phases and express in pollen.It will be corresponding to comp106333_c0_seq1 Unnamed gene be TaASG019 (AntherSpecific Gene 019).

Due to the allohexaploid that wheat is made of tri- sets of genomes of A, B, D, the average copy number of gene is 2.8, Wherein there is 3-4 copy close to the gene of half (46%), 12% gene has 1-2 copy, 42% gene copy number >=5 It is a.From the sequence of comp106333_c0_seq1, () as shown in SEQ ID NO:1, utilizes CerealsDB and IWGSC The sequencing information for the common wheat that (International Wheat Genome Sequencing Consortium) is announced, with And the wheat ancestors Uralensis Fisch (Triticum urartu, A Genome donor) delivered on Nature for 2013 and thick mountain The sequencing information of sheep's hay (Aegilops tauschii, D Genome donor) carries out electronic cloning, obtains 3 TaASG019 bases Cause is respectively designated as TaASG019-1, TaASG019-2 and TaASG019-3, and wherein comp106333_c0_seq1 corresponds to TaASG019-1.The cDNA sequence of 3 TaASG019 genes is respectively such as SEQ ID NO:2, SEQID NO:3 and SEQ ID NO:4 Shown, the homology between three is about 95%.It separately designs for TaASG019-1, TaASG019-2 and TaASG019- The specific primer of 3cDNA, using RT-PCR method, to these three genes in wheat root, stem, leaf, different development stage In the Various Tissues material such as anther and other floral organs in addition to anther carry out expression specificity analysis, as a result as shown in Fig. 2, The expression of TaASG019-1 gene does not have specificity, and two genes of TaASG019-2 and TaASG019-3 are only in pollen It is specific expressed in the anther of dicaryotic phase and three core phases, other floral organs of phase and root, stem, leaf and meiophase at the same time It is not expressed in the histoorgans such as tassel, the anther of monokaryotic stage and other floral organs, illustrates TaASG019-2 and TaASG019-3 Two genes are anther specifically expressing and the only specifically expressed gene in the anther in pollen development advanced stage.

The present invention also provides two specifically expressed promoters of anther, the promoter is prepared by the following: from The cDNA sequence of TaASG019-2 and TaASG019-3 gene is set out, and CerealsDB and IWGSC (International are utilized Wheat Genome Sequencing Consortium) announce common wheat sequencing information and Nature in 2013 On the wheat ancestors Uralensis Fisch (Triticum urartu, A Genome donor) and aegilops tauschii (Aegilops delivered Tauschii, D Genome donor) sequencing information carry out electronic cloning, obtain TaASG019-2 and TaASG019-3 gene Promoter, be respectively designated as TaASG019-2 promoter and TaASG019-3 promoter, in the present invention the promoter It can be referred to as pTaASG019-2 and pTaASG019-3, length is respectively 1918bp and 2064bp, nucleotide sequence point Not as shown in SEQ ID NO:5 and SEQ ID NO:6.

Using PlantCARE database and PLACE database, to TaASG019-2 promoter and TaASG019-3 promoter Carry out cis element analysis.As shown in figure 3, translation initiation site ATG is indicated with runic underscore, with translation initiation site ATG A be defined as "+1 ", GTGA motif is indicated with box.There are 7 GTGA motifs in TaASG019-2 gene promoter, There are 12 GTGA motifs in TaASG019-3 gene promoter.GTGA motif is relevant cis- to pollen/anther specifically expressing Controlling element, the presence of multiple GTGA motifs shows that the promoter can in the promoter of TaASG019-2 and TaASG019-3 gene It can be promoter related with pollen/anther specifically expressing.

In order to further verify the function of the two promoters, by one of promoter SEQ ID NO:5 and report base Because GUS is connected, plant is converted, all can't detect the table of gus gene in the nutrition organs such as the root, stem and leaf of transgenic paddy rice It reaches, is in the tassel of meiophase, the anther of monokaryotic stage and other floral organs of the same period, pollen in pollen and is in dicaryotic phase With the expression that also can't detect gus gene in other floral organs in addition to anther of three core phases, TaASG019-2 promoter is only Gus gene can be started to express in the anther that pollen is in dicaryotic phase and three core phases, illustrate that promoter provided by the present invention is One specifically expressed promoter of pollen development advanced stage anther.

Plant anther specific expression promoter provided by the present invention, containing shown in SEQ ID NO:5 or 6 in ordered list Nucleotide sequence, or the nucleotides sequence comprising having 90% or more similitude with nucleotide sequence listed by SEQ ID NO:5 or 6 Column, or comprising 100 and 100 or more the continuous nucleotide fragments in SEQ ID NO:5 or 6 sequences, and can be with Driving is listed in the expression in plant anther with the nucleotides sequence that the promoter is operatively connected.Expression containing above-mentioned sequence carries Body, transgenic cell line and host strain etc. all belong to the scope of protection of the present invention.Expand SEQ ID disclosed in this invention The primer pair of NO:5 or any nucleotide fragments of 6 promoters is also within protection scope of the present invention.

Promoter nucleotide sequence provided by the present invention can also be used to separate from other plants other than wheat corresponding Sequence especially carries out homologous clone from other monocotyledons.According to promoter sequence listed by these corresponding sequences and this paper Sequence homology between column, or the homology with this promoter gene are identified using the technologies such as such as PCR, hybridization and separate these Corresponding sequence.Therefore, the sequence according to listed by them and the present invention between SEQ ID NO:5 or 6 promoter sequences (or its segment) Column similitude and isolated respective segments, are also included in embodiment.

" promoter " of the present invention refers to a kind of DNA regulatory region, generally comprises energy guide RNA polymerase II and exists The TATA box of the suitable transcription initiation site starting RNA synthesis of specific coding sequence.Promoter also may include other identification sequences, These identification sequences are usually located at the upstream or 5 ' ends of TATA box, commonly known as upstream promoter element, play regulatory transcription effect The effect of rate.Those skilled in the art should know, although having identified the core for promoter region disclosed by the invention Nucleotide sequence, but separate and identify other tune of the TATA box upstream region for the specific promoter region identified in the present invention It is also within the scope of the invention to control element.Therefore, promoter region disclosed herein is usually further defined as comprising upstream Controlling element, such as those of tissue expression for regulating and controlling coded sequence and temporal expressions function element, enhancer etc..With Identical mode can be identified, isolate the promoter member for making it possible to be expressed in destination organization (such as male tissue) It is used together by part with other core promoters, to verify the preferential expression of male tissue.Core promoter refers to starting transcription Required minimal sequence, such as the sequence of referred to as TATA box, this is that the promoter of the gene of coding protein is usual All have.Therefore, optionally, SEQ ID NO:5 of the present invention or 6 promoters can with its own or from it is other come The core promoter in source, which is associated with, to be used.

Core promoter can be core promoter known to any one, such as cauliflower mosaic virus 35S or 19S are opened Mover (United States Patent (USP) No.5,352,605), ubiquitin promoter (United States Patent (USP) No.5,510,474), IN2 core promoter (beauty State patent No.5,364,780) or figwort mosaic virus promoter.

The function of the gene promoter can be analyzed by the following method: can by promoter sequence and reporter gene It is operatively connected, forms transformable construct, then the construct is transferred in plant, in obtaining transgenic progeny, pass through Expression of the visual report gene in each histoorgan of plant confirms its expression characterization；Or it is above-mentioned construct is sub- It clones into the expression vector for transient expression experiment, promoter or the function of its control region is detected by transient expression experiment

Host will be depended on and by the table for the selection of test starting or the appropriate expression vector of regulatory region function The method for introducing host up to carrier, such methods are well known to those of ordinary skill in the art.For eucaryote, in carrier In region include control transcription initiation and control processing region.These regions are operably connected to reporter gene, institute Stating reporter gene includes YFP, UidA, gus gene or luciferase.Table comprising the presumption control region being located in genomic fragment It can be introduced into complete tissue, such as interim anther up to carrier, or introduce callus, to carry out functional verification.

The activity and intensity of promoter can be surveyed according to the expression quantity of the mRNA of the reporter gene of its driving or protein It is fixed.Reporter gene (reporter gene) is a kind of gene of protein or enzyme that coding can be detected, that is to say, that is one Its a expression product is very easy to certified gene.Its coded sequence and Gene expression and regulation sequence are blended to be formed it is embedding Close gene, or blended with other target gene, expressed under regulating and controlling sequence control, thus using its expression product come Determine the expression regulation characteristic of target gene.Common reporter gene has beta-glucosiduronatase gene GUS and green fluorescence egg White gene GFP.

The present invention detects the activity and expression characterization of promoter by gus reporter gene.Used in being detected according to gus gene Substrate it is different, there are three types of detection methods: (sensitivity is spectrophotomelric assay for histochemical method, spectrophotometry and fluorescence method Method highest), wherein the most commonly used is histochemical method.Histochemical method is detected with the chloro- 3- indoles-β-glucose of the bromo- 4- of 5- Thuja acid (X-Gluc) is used as reaction substrate.Buffer of the tested material containing substrate is impregnated, if histocyte has been transferred to GUS Gene, and GUS zymoprotein has been given expression to, under appropriate conditions, which can hydrolyze X-Gluc and generate blue product, this is The bipseudoindoxyl dye formed is acted on through oxidative dimerization by its initial product, it makes the position for having GUS expression activity in each histocyte Or blue is presented in site, with the naked eye or under the microscope can be seen, and can reflect GUS according to the dyeing depth under to a certain degree Active power.Therefore expression of the foreign gene in certain organs, tissue or even individual cells can be observed using this method Situation.

In addition, promoter of the invention can be with the nucleotide sequence phase of not TaASG019-2 or TaASG019-3 gene Even, to express other heterologous nucleotide sequences.Promoter nucleotide sequence of the invention and its segment and variant can be with heterologous cores Nucleotide sequence is assembled in together in an expression cassette, for expressing in purpose plant, more specifically, in the spermogonium of the plant It is expressed in official.The expression cassette has suitable restriction enzyme site, for being inserted into the promoter and heterologous nucleotide sequence. These expression cassettes can be used for carrying out genetic manipulation to any plant, to obtain desired corresponding phenotype.

TaASG019-2 promoter and TaASG019-3 promoter disclosed in this invention can be used for driving following heterologous core The expression of nucleotide sequence, so that the plant of conversion obtains male sterile phenotype.The heterologous nucleotide sequence codified promotes The enzyme or modification enzyme of carbohydrate degradation, amylase, debranching enzyme and pectase, more specifically such as a amylase gene, auxin (auxin), rot B, cytotoxin gene, diphtheria toxin, DAM methylase, Avidin, or can be selected from protokaryon regulation system System, can also be dominant male sterility gene.

In some embodiments, the core mentioned in the present invention for being operatively coupled on promoter downstream of the present invention Acid, wherein " nucleic acid " can be the structural gene being operatively connected on promoter disclosed herein, adjust base Because of the antisense gene of, structural gene, the tiny RNA that adjusts the antisense gene of gene or can interfere with endogenous gene expression.

Promoter sequence provided by the present invention is isolated from any plant, including but not limited to Btassica, corn, small Wheat, sorghum, two section shepherd's purse categories, sinapsis alba, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus, peanut, clover, Oat, rapeseed, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheat (Spelt), Emmer, flax, gramagrass (Gramma grass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sugarcane, the red certain kind of berries Tongue fur, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, dendrobium nobile, gladiolus, chrysanthemum, Liliaceae, cotton, eucalyptus, Xiang Certain herbaceous plants with big flowers, rape, beet, coffee, ornamental plant and conifer etc..Preferably, plant include corn and soybean, it is safflower, leaf mustard, wheat, big Wheat, rye, rice, cotton and sorghum.

The invention also includes the construct containing TaASG019-2 promoter or TaASG019-3 promoter, the construct Including usually said carrier or expression cassette.It may also include other components in above-mentioned construct, this depends primarily on vector construction Purpose and purposes, such as can further comprise selectable marker gene, targeting or regulating and controlling sequence, critical sequences or boot sequence, Introne etc..Expression cassette will also be included in plant at 3 ' ends of desired heterologous nucleotide sequence has functional transcription and translation Terminator.Terminator can be the terminator of gene provided by the present invention, be also possible to the terminator from external source.More specifically Ground, above-mentioned terminator can be nopaline synthase or octopine synthase termination area.

It is desirable that guiding the expression product of heterologous nucleotide sequence into specific cells device, such as plastid, amyloplast, Huo Zheyin To endoplasmic reticulum, or in the case where cell surface or cell exocrine, expression cassette also may include the nucleosides for encoding transit peptides Acid sequence.Such transit peptides be it is known in the art, its include but is not limited to the small subunit of Rubisco, plant EPSP synthase, Corn Brittle-1 chloroplast transit peptides etc..

During preparing expression cassette, a variety of DNA fragmentations can be operated, be in proper orientation to provide, or DNA sequence dna in correct reading frame.To reach this purpose, adapter or connector can be used, DNA fragmentation is linked up, or Person further comprises other operations, to provide convenient restriction enzyme site etc..

Further, it may also include selectable marker gene in construct provided by the present invention, it is transformed for selecting Cell or tissue.The selectable marker gene includes assigning antibiotic resistance or the gene to Herbicid resistant.Suitable selection Marker gene includes but is not limited to: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistance Gene, sulfamido resistant gene, glyphosate gene, the careless bony resistant gene of fourth.The selectable marker gene can also be red Color fluorogene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene, flower The genes such as green glucoside p1.

Expression cassette or carrier provided by the present invention can be inserted into plasmid, clay, yeast artificial chromosome, bacteria artificial dye Colour solid or other be suitble to be transformed into any carrier in host cell.Preferred host cell is bacterial cell, is especially used In cloning or storage polynucleotides or bacterial cell for converting plant cell, for example, Escherichia coli, Agrobacterium tumdfaciens and Agrobacterium rhizogenes.When host cell is plant cell, expression cassette or carrier can be inserted into the base for the plant cell being converted Because in group.Insertion can be positioning or random insertion.Preferably, such as homologous recombination is inserted through to realize.In addition, table It is positively retained at outside chromosome up to box or carrier.Expression cassette or carrier of the invention may be present in the core, chloroplaset, line of plant cell In plastochondria and/or plastid.Preferably, expression cassette of the invention or carrier are inserted into the chromosomal DNA of plant nucleolus.

The invention also includes the applications of disclosed TaASG019-2 promoter and/or TaASG019-3 promoter, at certain In the embodiment applied a bit, it can start using TaASG019-2 promoter provided by the present invention and/or TaASG019-3 Son realizes that some fertility-related genes are mutated the breeding and holding of male sterile line obtained, the fertility-related gene packet Include but be not limited to Ms26, Ms45, MSCA1 etc..

It is specific expressed in anther that provided anther specific expression promoter of the invention can be used for foreign gene, To avoid foreign gene continuous expression adverse effect in its hetero-organization of plant, plant anther can be also used for The functional analysis and identification of growth and development related gene；It can be used for the creation of male sterile line and restorer；And it can be applied to spend In powder abortion experiment, so that brought bio-safety problem of being escaped by plant transgene drift or pollen is avoided, to plant hero The creation of property sterile line and restorer is of great significance.

Genetically modified plants of the invention are prepared using method for transformation known to plant biotechnology field technical staff.It is any Method can be used for for recombinant expression carrier being transformed into plant cell, to generate genetically modified plants of the invention.Method for transformation It may include method for transformation directly or indirectly.Suitable direct method includes that the DNA of polyethylene glycol induction takes in, is liposome-mediated Conversion, imported using particle gun, electroporation and microinjection, etc..In a specific embodiment of the invention, of the invention Use the transformation technology based on agrobacterium (reference can be made to Horsch RB etc. (1985) Science 225:1229；White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.15-38；The .Techniques such as Jenes B For Gene Transfer, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Agrobacterium bacterial strain (such as Agrobacterium tumdfaciens or hair root soil bar Bacterium) it include plasmid (Ti or Ri plasmid) and T-DNA element, the plasmid and element are transferred to plant after with Agrobacterium transfection Object, and T-DNA is integrated into the genome of plant cell.T-DNA can be located on Ri- plasmid or Ti- plasmid, or independently wrap It is contained in so-called binary vector.Agrobacterium-mediated method for transformation is described in for example.Agrobacterium-mediated conversion is most It is suitble to dicotyledon, but is also suitble to monocotyledon.Agrobacterium is described in for example the conversion of plant.Conversion can be led Cause instantaneous or stable conversion and expression.Although nucleotide sequence of the invention, which can be inserted into, falls into appointing in these broad varieties In what plant and plant cell, but it is particularly suitable for crop plants cell.

Below by specific embodiment, in conjunction with attached drawing, the invention will be described in further detail, but not in any way It limits the scope of the invention.

Detailed description of the invention

Fig. 1 is that comp106333_c0_seq1 in pollen is in meiophase (WT-0), monokaryotic stage (WT-1), dicaryotic phase (WT-2) the expression analysis in the anther of He Sanhe phase (WT-3), abscissa is pollen different development stage, and ordinate is FPKM, the expression of response gene.

Fig. 2 is 3 homologous genes of TaASG019 in the anther of wheat different tissues organ and different development stage RT-PCR analysis.1 indicates root, and 2 indicate stem, and 3 indicate that blade, 4 expression pollen are in the tassel of meiophase, and 5 indicate pollen Anther in monokaryotic stage, 6 expression pollen are in the anther of dicaryotic phase, and 7 expression pollen are in the anther of three core phases, and 8 indicate flower Powder is in the spending of monokaryotic stage other floral organs in addition to anther, and 9 expression pollen are in the spending of dicaryotic phase in addition to anther Other floral organs, 10 expression pollen be in the spending of three core phases the others floral organs in addition to anther.

Fig. 3 indicates TaASG019-2 promoter sequence (A) and TaASG019-3 promoter sequence (B).Translation initiation site ATG indicates that the A of translation initiation site ATG is defined as "+1 " with runic underscore, and GTGA motif is indicated with box.GTGA is indicated Conserved motifs relevant to pollen/anther specific expression promoter.

Fig. 4 is the area the T-DNA map of expression vector p166.LB and RB is respectively the left margin and right margin of T-DNA； NPTII indicates neomycin phosphotransferase II gene；The promoter of P35S expression CaMV35S gene；T35S indicates CaMV35S base The terminator of cause；GUS indicates beta-glucosiduronatase gene；The terminator of Tnos expression rouge alkali synthetase (no) gene； HindIII, BamHI, BglII, SacI and EcoRI respectively indicate the restriction enzyme site of restriction enzyme；TaASG019-2 starting Son is exactly the separated specifically expressed promoter of wheat anther of the present invention.

Fig. 5 is the histoorgan GUS dyeing of p166 transgenic wheat.A is root；B leaf；C stem；D is pollen in subtrahend point Split the flower of phase；E is the flower that pollen is in monokaryotic stage；F is the flower that pollen is in dicaryotic phase；G is the flower that pollen was in for three core phases；H The anther of three core phases is in for pollen；I is the pollen of three core phases, and the upper right corner is the pollen of DAPI dyeing.

Specific embodiment

Method therefor is conventional method unless otherwise instructed in following embodiments, and the primer is by Shanghai English fine horse biology Technology company's synthesis, sequencing are won Radix Polygalae biotechnology Co., Ltd by Beijing three and are completed, PCR kit, vector construction mistake Endonuclease and pMD20-T carrier in journey are purchased from precious bioengineering Co., Ltd, TransStart FastPfu DNA Polymerase is purchased from Beijing Quan Shijin biotech company, and T4DNA ligase is purchased from NEB company, and method is referring to kit The method of offer carries out.

The full-length genome expression pattern analysis and pollen development later period anther table of 1. different development stage wheat anther of embodiment Up to the acquisition of contig

It takes pollen to be in meiophase, monokaryotic stage, the wheat anther of dicaryotic phase and three core phases, uses Trizol (Invitrogen) total serum IgE is extracted, and carries out DNaseI (Promega) processing, and then purify mRNA (Ambion).By purifying MRNA carries out reverse transcription (Invitrogen), ultrasound interrupts (Fisher), preparation library (illumina) and expands (illumina), sequencing reaction is finally carried out on illumina machine.

The result of wheat transcript profile high-flux sequence passes through Trinity software first and carries out sequence assembly, obtained splicing Sequence further removes redundancy and similitude cluster.For the expression mutation analysis for the transcript contig that splicing obtains, respectively The sequence of high-flux sequence passes through TopHat (http://tophat.cbcb.umd.edu/) software and transcript first in sample The result of splicing is compared.Then Cufflink software being capable of homogenization expression of the calculating ratio to upper transcript contigs Amount, with " Kilobases (the fragments per kilobase of exon model per of every million aligned fragment of exon Million mapped fragments, FPKM) " it indicates.

By the full-length genome expression pattern analysis to different development stage wheat anther, finds pollen and be in the subtrahend separation phase Anther in do not express and expressed in the anther that pollen is in monokaryon, double-core and three core phases transcript contig 7187. As shown in Figure 1, comp106333_c0_seq1 (sequence is as shown in SEQ ID NO:1) pollen is in subtrahend separation phase and monokaryotic stage Little Hua in do not express and be in double-core and the anther of three core phases and express in pollen.It will be corresponding to comp106333_c0_seq1 Unnamed gene be TaASG019 (Anther Specific Gene 019).

The tissue expression specificity of embodiment 2.RT-PCR verifying TaASG019 gene

Due to the allohexaploid that wheat is made of tri- sets of genomes of A, B, D, the average copy number of gene is 2.8, Wherein there is 3-4 copy close to the gene of half (46%), 12% gene has 1-2 copy, 42% gene copy number >=5 It is a.From the sequence of comp106333_c0_seq1, () as shown in SEQ ID NO:1, utilizes CerealsDB and IWGSC The sequencing information for the common wheat that (International Wheat Genome Sequencing Consortium) is announced, with And the wheat ancestors Uralensis Fisch (Triticum urartu, A Genome donor) delivered on Nature for 2013 and thick mountain The sequencing information of sheep's hay (Aegilops tauschii, D Genome donor) carries out electronic cloning, obtains 3 TaASG019 bases Cause is respectively designated as TaASG019-1, TaASG019-2 and TaASG019-3, and wherein comp106333_c0_seq1 corresponds to TaASG019-1.The cDNA sequence of 3 TaASG019 genes is respectively such as SEQ ID NO:2, SEQID NO:3 and SEQ ID NO:4 Shown, the homology between three is about 95%.It separately designs for TaASG019-1, TaASG019-2 and TaASG019- The specific primer of 3cDNA, using RT-PCR method, to these three genes in wheat root, stem, leaf, different development stage In the Various Tissues material such as anther and other floral organs in addition to anther carry out expression specificity analysis, as a result as shown in Fig. 2, The expression of TaASG019-1 gene does not have specificity, and two genes of TaASG019-2 and TaASG019-3 are only in pollen It is specific expressed in the anther of dicaryotic phase and three core phases, other floral organs of phase and root, stem, leaf and meiophase at the same time Do not expressed in the histoorgans such as other floral organs of the anther and monokaryotic stage of tassel and monokaryotic stage, illustrate TaASG019-2 and Two genes of TaASG019-3 are anther specifically expressing and the only specifically expressed gene in the anther in pollen development advanced stage.

The RT-PCR primer of TaASG019-1 gene are as follows:

Primer 1:5'-CTGCCATTTCTCCTTTCTCATCTAA-3'(SEQ ID NO:7)

Primer 2: 5'-TGGGCTGCTTTCCGTCGGT-3'(SEQ ID NO:8)

The RT-PCR primer of TaASG019-2 gene are as follows:

Primer 3:5'-TCCATTCTCCTCTCTCGTCTAGAT-3'(SEQ ID NO:9)

Primer 4:5'-CTGCTTTCCGTCAGACCACTT-3'(SEQ ID NO:10)

The RT-PCR primer of TaASG019-3 gene are as follows:

Primer 5:5'-GATGAGTACGTAGGTGTCCACTGATT-3'(SEQ ID NO:11)

Primer 6:5'-GGGAGTAGTGGACCTTCTCGACT-3'(SEQ ID NO:12)

The acquisition of embodiment 3.TaASG019-2 and TaASG019-3 gene promoter sequence and cis element analysis

From the cDNA sequence of TaASG019-2 and TaASG019-3 gene, CerealsDB and IWGSC are utilized The sequencing information for the common wheat that (International Wheat Genome Sequencing Consortium) is announced, with And the wheat ancestors Uralensis Fisch (Triticum urartu, A Genome donor) delivered on Nature for 2013 and thick mountain The sequencing information of sheep's hay (Aegilops tauschii, D Genome donor) carries out electronic cloning, obtain TaASG019-2 and The promoter of TaASG019-3 gene is respectively designated as TaASG019-2 promoter and TaASG019-3 promoter, length point Not Wei 1918bp and 2064bp, nucleotide sequence is respectively as shown in SEQ ID NO:5 and SEQ ID NO:6.

Using PlantCARE database and PLACE database, to TaASG019-2 promoter and TaASG019-3 promoter Carry out cis element analysis.As shown in figure 3, translation initiation site ATG is indicated with runic underscore, with translation initiation site ATG A be defined as "+1 ", GTGA motif is indicated with box.There are 7 GTGA motifs in TaASG019-2 gene promoter, There are 12 GTGA motifs in TaASG019-3 gene promoter.GTGA motif is relevant cis- to pollen/anther specifically expressing Controlling element, the presence of multiple GTGA motifs shows that the promoter can in the promoter of TaASG019-2 and TaASG019-3 gene It can be promoter related with pollen/anther specifically expressing.

The clone of embodiment 4.TaASG019-2 promoter and the building of plant expression vector

By plant expression vector pBI121 with restriction enzyme HindIII and EcoRI double digestion, obtained 35S:GUS Segment is carried with the pCAMBIA2300 of the T4DNA ligase CAMBIA company for being connected into same HindIII and EcoRI double digestion Body, new carrier are named as p2300 35S:GUS.

It is held and ATG upstream design primer from the 5 ' of TaASG019-2 promoter:

Primer 7:5 '-aagctt CATAATGATACCAAGCCACACTATCAAT-3 ' (SEQ ID NO:13)

Primer 8:5 '-agatct CGTCCTCGCCTTTCCTCTG-3 ' (SEQ ID NO:14)

Sequence aagctt is the restriction enzyme site of HindIII in primer 7, and sequence agatct is the digestion position of BglII in primer 8 Point.

It using the genomic DNA of wheat as template, is expanded with primer 7 and primer 8, reaction condition is: 94 DEG C of initial denaturations 5 Minute；94 DEG C are denaturalized 30 seconds；60 DEG C are annealed 30 seconds；72 DEG C extend 2 points 30 seconds；35 circulations；72 DEG C extend 10 minutes.Reaction knot Shu Hou, PCR product are detected through 1% agarose gel electrophoresis and are recycled, and product is connected into pMD20-T carrier, and screening positive clone is simultaneously Sequence verification is carried out, for sequence as shown in SEQ ID NO:5, which is known as p159.

With restriction enzyme HindIII and BglII double digestion p159, obtained TaASG019-2 promoter T4DNA Ligase is connected into the p2300 35S:GUS carrier with HindIII and BamHI double digestion, obtains plant expression vector p166, the matter The structure of grain is as shown in Figure 4.

The rice transformation of 5. mediated by agriculture bacillus of embodiment and the Molecular Identification of transgenic plant

Plant expression vector p166 is transferred to Agrobacterium AGL0 bacterial strain using heat shock method.

Rice embryo callus subculture is infected with Agrobacterium, is co-cultured in the dark 2-3 days, then break up by two step resistance screenings, in advance, Differentiation and culture of rootage, it is final obtain have kalamycin resistance, turn p166 rice T0 for plant.

Design primer carries out PCR identification to transgenic rice plant.

Primer 9:5 '-GTCCCCTGATCGATCTGTCCA-3 ' (SEQ ID NO:15)

Primer 10:5 '-GCCGTCGAGTTTTTTGATTTCAC-3 ' (SEQ ID NO:16)

Reaction condition are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds；55 DEG C are annealed 30 seconds；72 DEG C extend 30 seconds；30 A circulation；72 DEG C extend 10 minutes.Amplification is TaASG019-2 promoter and the Partial Fragment of GUS, length 200bp.Mirror It the result shows that, is calmly the positive plant for turning p166 gene using the resistance regeneration plant that the rice conversion of mediated by agriculture bacillus obtains.

Histochemistry's detection of 6. transgenic rice plant different tissues organ gus gene of embodiment expression

X-Gluc mother liquor: 100mg X-Gluc is dissolved in 5ml DMF.

X-Gluc base fluid: 50mM PBS pH7.0,10mM EDTA2Na, 0.1%Triton X-100,5mM iron hydrogenation Potassium, 0.5mM ferrous iron hydrofining.

X-Gluc uses liquid :+950 μ l base fluid of 50 μ l mother liquor.

It selects in the transgenic seedlings or specific organization's immersion GUS dye liquor with gus reporter gene of suitable size, 37 DEG C Stained over night sucks reaction solution, ethanol gradient decoloration, micro- sem observation photograph.

To GUS coloration result such as Fig. 5 of each histoorgan of p166 transgenic rice plant, root, stem in transgenic paddy rice With the expression (Fig. 5 A-C) that all can't detect gus gene in the nutrition organs such as leaf, pollen be in meiophase, monokaryotic stage, The flower and pollen of dicaryotic phase is in other floral organs in addition to anther of three core phases the table that also can't detect gus gene It reaches, TaASG019-2 promoter can only start gus gene and express (Fig. 5 D-G) in the anther that pollen was in for three core phases, explanation TaASG019-2 promoter is the specifically expressed promoter of pollen development advanced stage anther.Further, pollen was in for three core phases Anther wall in can't detect the expression (Fig. 5 H) of GUS, and the expression (Fig. 5 I) of GUS is able to detect that in the pollen of three core phases, Therefore, TaASG019-2 promoter is a Wheat Pollen development advanced stage specifically expressed promoter.

SEQUENCE LISTING

<110>Unnamed Xingwang System Crop Design Front Laboratory (Beijing) Co., Ltd.

Hebei Bo Nong agricultural technology development corporation, Ltd.

Xingwang Investment Pty Ltd.

<120>identification and application of plant anther specific expression promoter pTaASG019

<130>

<150> 201410342557.3

<151> 2014-07-18

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 215

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 1

gaaggaatca ctgatcggtt agcatttgtc tatatataac ccgtgatgac tgatgagtac 60

tacgtaggtg gtcactgatc gatctgccat ttctcctttc tcatctaaat cggcgccggc 120

ggctcgatcg agcagaggaa aggcgacgac gatgtcggcc acgacggggg cggtgtggcg 180

aacggcgggg gtgacctaca tcggctactc caacg 215

<210> 2

<211> 443

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 2

ctgccatttc tcctttctca tctaaatcgg cgccggcggc tcgatcgagc agaggaaagg 60

cgacgacgat gtcggccacg acgggggcgg tgtggcgaac ggcggggatg acctacatcg 120

gctactccag gctctgcgcg tccattgtgc ggagctgcct caaggagccc tacaagtccg 180

aggccgccga agtcgagaag gtccactact cccgctccaa gtggaccgac ggaaagcagc 240

ccaagcccag ctactgcctc tgtccggtgg gcctagctgt cgggtcgtcg gttgaccagc 300

atggtttatt cggtgaggtt gaaccgttcc tttccttgcc atctgcgaag atgataaatg 360

aaagatccgg tgccatggtg acagagtcct tcctacggac gttttcaaaa gctactgtgt 420

ttcgtgctgg ccattctacc taa 443

<210> 3

<211> 534

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 3

tgaagactga tgagtacgta ggtgtcccct gatcgatctg tccattctcc tctctcgtct 60

agatcggcgt cggcggctcg atcgagcaga ggaaaggcga ggacgatgtc ggccacgacg 120

ggggcggtgc cgttctggcg aacggcgggg atgacctaca tcggctactc caggctctgc 180

gcgtccatgg tgcggagctg cctcaaggag ccctacaagt ccgaggccgc cgaggtcgag 240

aaggtccact actcccgctc caagtggtct gacggaaagc agcccaagcc cagaactgca 300

aaagtagatg tagttgaatt ttgggatgct caagctactg cctctgtccg gtgggcctag 360

ctgtcgggtc gtgggttgac cagcatgggt tattcggtga ggttgaaccg ttcctttcct 420

ttgtctatgc catctgcgaa gatgataaat gaaagatccg gtgccatggt gacacagtcc 480

ttcctacgga cgttttgaaa agctattgtg tttcgtgccg gccattctac ctaa 534

<210> 4

<211> 526

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 4

gatgagtacg taggtgtcca ctgattgatc tgtccattct actctctcgt ctagatcggc 60

gtcggcggct cgatcgagca gaggaaaggg gacggcgatg tcggccacga cggcggcggt 120

gcccttctgg cgagcggcgg ggatgaccta cgtcggctac tccaggctct gcgcgtccat 180

ggtgcggagc tgcctcaagg agccctacaa gtccgaggcc gccgaagtcg agaaggtcca 240

ctactcccgc tccaagtggt cggacggaaa gcaggccaag cccagaactg caaaagtaga 300

tgtagttgaa ttttgggatg ctcaagctac tgcctctgtc cggtgggcct acctgtcggg 360

tcgtgggttg accagcatgg tttattccgt gaggttgaac cgttcctttc ctttggctat 420

gccatctgcg aagatgataa atgaaagatc tggtgccatg gtgacacagt ccttcctacg 480

gacgttttca aaagctactg tgtttcgtgc tggccattct acctaa 526

<210> 5

<211> 1918

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 5

cataatgata ccaagccaca ctatcaatgg catattttct aatctttcta atcttcaacc 60

tcattttctc catcttgatc ttgtgatcat cgacgacatc ggcaacatgc aactccaatt 120

ccatcttctc cctcacaatt cttttcaatt tttctttcaa atactcgttt tctctttcaa 180

ctaaatttaa cctctcgaca atagggtcgg ttggaatttc cggttcacat acctcctaga 240

taaaaatatc tatgtcaact tgatgggcat aatttgtcat aagaacgaaa tgcaacaact 300

agttttaaaa gagaatatac cacatccgaa tcataacaag gacgagggcc gacggggacg 360

gatatcaaaa ccatggcact atgtataaca aacaacgtac gggtaagata attatacgag 420

taactatata tccaaatcac ataaacatca atttagtaat gtaaaacatt catgaacaag 480

agcctcacca gaaggtggtg ccggcgacgg gacggtgcgg gcgatcggcg gtggttacga 540

cggagattta gaaggcacta agtaaaccac acctacatat gcaaactaag tgttattttt 600

atgctcaaat tgcatataaa tcaaatacta gcaaatataa ttattcctcc caaattaatc 660

actatacaaa gcattgcaag agctaatcta gcaatgagag ttgaaaggac aaagttgcta 720

acctttgtga tcatttgaat ggatgggggc cttcaaatct tgacaaattt tgggcaaaat 780

ttgtgaggag cttgaggaga gagggggaag aacagaggaa gtgaggggaa agggggagaa 840

cagagtggct caggggggac gaagggttta tgtacgtcga cctttagtac cggttcgtgc 900

cacgaaccgg tactaaaggt gctggacggg ccccagactg acaacaccct gccaccaccc 960

tctttagtac cggttcgtgg cacgaaccgg tactataggt ttgccacgaa ccgatactaa 1020

tgagaacggc cggctagccg ttggaaccgg cactattgga cacattagtg ccggctcaaa 1080

accaaaccgg cactaatgtg tctcatacta ggaccttttt ctactagtgt ctcttgctat 1140

taagtattat tctcagttga aagagaataa gttaaatttt atagtgttta tgtttttttt 1200

tgcgggatag tggcctttta tgttgtgtag tggcctatct tgtttgaaaa gttgccttag 1260

attattaatt aagatcaaaa ctagctcgag aacaactcga gatcgataca agcagagcat 1320

gagtcacttt atacatctcg accttgagat tcactccgtt tgacctcgct caaattttaa 1380

tacaagtcga agtgagccta gtgcaactcg ctcgaactcg attcgtttgc agccctacgc 1440

ccctaccttt gatgagttca tttttgcatc actttttatt ctttcttttg cattggtcgt 1500

tgcctttttc ttttcttttt tctgagagga aaaggaaaaa agaatacgtt agtttgttgg 1560

atccgaggtg cacgttcgtt cgttaaccgc gtttaacagt gttctggcgg aacgcaagac 1620

acggtcgtcg atccaagttc tcgtgaagga gttcgttcct ctcgcaaaaa aaggaggtgg 1680

aattaatccg atccgtaatt gcaggcccgg ccgtaggtac atccactcaa tgcagtgcga 1740

tccatgcaag cgcatgcagc ccgccctctg accgggatca ctgatcggtt tgcatttttt 1800

ctatatataa ccgtgaagac tgatgagtac gtaggtgtcc cctgatcgat ctgtccattc 1860

tcctctctcg tctagatcgg cgtcggcggc tcgatcgagc agaggaaagg cgaggacg 1918

<210> 6

<211> 2064

<212> DNA

<213>wheat (Triticum aestivum L.)

<400> 6

gtcctacaac tatggaaaac gggtagtttg attgaatcat gcttggctat atccatatac 60

tatttgccta aaaagatttt ttactttacc caactctgga tgtatcatga atcatgctct 120

atcaacatga ttgttgtgtg tgtttctgtt attgcttaca aagctatttc ttaaatgttt 180

aagttacaca tgaagttctc aacttcacaa catgtagctc tacaatgttg tcttcccaag 240

ttcatgtgtt ctgactagct ccattcttat tatgtcctgc cgcccaacaa gcagatgttg 300

tagtttgcat ctaagtgttg tattgaatct tgcctaatgt accatgtcgg tcctcctgga 360

agatgctgtc agcgaatgcg caagtatata tgcaatactt gtggttaatt agttgtttca 420

atggtgcaat atatatatcc acaccttttg attttgctat gcaaaagtat atttcttgtg 480

tattatagtg ctttctttgt tatcgggttg gatatgattg gaccagatga ggcatttgag 540

actcaggctc tgaggcacta gacatgataa acaagcttgc cagtggttac ttgtatgttg 600

tgagatacat gagctccttc tcagatcagc ttcataatgt catagcacat ggctttcctc 660

ctataggaca aaagcaataa tctgaagaag aggttgcatg ccataaaaat acagtctcag 720

gcacaagagc atgaggtgtt tgcaccatct ggagttgctt tcttcacact gaggccatgg 780

cgttgatcgt cgatggatgt caccggtcac tctgataagt attcggtgat gtcatgccac 840

cagcatgtga tcgtcgtgta aactaccatg tgatctccct ctccttttgt gaacatgacc 900

tctttgttga gtctatccat tcttttgtac tcaatatccc gcacttaatg tcaagtgata 960

tacttgctct ctactgttca cacttttagt actaaactta atgtcaagtg gttacctttt 1020

gtagtctcat gaacacactg aactcgatgc tgcctataac ctcatgacga tgttgaactc 1080

aatgatgtct tcattatatt ataatttaat gttctctctt tcttttattt tgttgtcctg 1140

cctgtataca tatgctaaat caagatcccc tactaacttg agcattactt gcttgactat 1200

atcgttagga ccggcaccct cgcgccaagg cgcgttaccg atctagttaa gtattattgt 1260

cagttgaaag agaataagtt aaattttaca gtgacctttt atgttgtgta gtggcctaac 1320

ttgtttgaaa aattgcctta gattattaag atcagaacta cctcgagatc aactcgagat 1380

cgatacaaac tgagcatgag tcactttcta cagctcgacc ttgagcttca ctcagtttga 1440

gctcactcaa attttaatat aagtagaggt gagcctagtg catctcgctc gaaatcgact 1500

cgtttgcagc cgtatgcccc tacctttgat gagttcattt ttgcatcact ttttattctt 1560

tgttttgcat tggtcataag atttccgtga ttttagtgcg actcaagggt gcgtgtgtga 1620

atggtgtacc agcgacttcg ttggttggtt ttttcatttt ttctgaaaag aagaaggaaa 1680

aagaatacgt taattggttg gatccgaact acacgttcgt tcgttaaccg cgtttcacac 1740

tgttctggcc gaacgcaaga cacggtcgtc gatccaagtt ctcgtgaagg agttcctctc 1800

gcaaaagaag gaggtggaat taatctgatc cgtaactaca ggcccggccg taggtacacc 1860

aagtcaccaa tccaatgcaa tgcgatccat gcacgggcat gcagcccgcc ctctgaccgg 1920

aatcactgat cggtttgcat tttcttctat atataaccgt gaagactgat gagtacgtag 1980

gtgtccactg attgatctgt ccattctact ctctcgtcta gatcggcgtc ggcggctcga 2040

tcgagcagag gaaaggggac ggcg 2064

<210> 7

<211> 25

<212> DNA

<213>artificial synthesized

<400> 7

ctgccatttc tcctttctca tctaa 25

<210> 8

<211> 19

<212> DNA

<213>artificial synthesized

<400> 8

tgggctgctt tccgtcggt 19

<210> 9

<211> 24

<212> DNA

<213>artificial synthesized

<400> 9

tccattctcc tctctcgtct agat 24

<210> 10

<211> 21

<212> DNA

<213>artificial synthesized

<400> 10

ctgctttccg tcagaccact t 21

<210> 11

<211> 26

<212> DNA

<213>artificial synthesized

<400> 11

gatgagtacg taggtgtcca ctgatt 26

<210> 12

<211> 23

<212> DNA

<213>artificial synthesized

<400> 12

gggagtagtg gaccttctcg act 23

<210> 13

<211> 34

<212> DNA

<213>artificial synthesized

<400> 13

aagcttcata atgataccaa gccacactat caat 34

<210> 14

<211> 25

<212> DNA

<213>artificial synthesized

<400> 14

agatctcgtc ctcgcctttc ctctg 25

<210> 15

<211> 21

<212> DNA

<213>artificial synthesized

<400> 15

gtcccctgat cgatctgtcc a 21

<210> 16

<211> 23

<212> DNA

<213>artificial synthesized

<400> 16

gccgtcgagt tttttgattt cac 23

Claims (17)

1. a kind of promoter has the specifically expressed characteristic of anther, it is characterised in that the nucleotide sequence of the promoter such as SEQ Sequence shown in ID NO:5.

2. a kind of expression cassette has the specifically expressed characteristic in anther, it is characterised in that the expression cassette includes claim 1 The promoter sequence.

3. a kind of expression vector, it is characterised in that the expression vector includes expression cassette as claimed in claim 2.

4. a kind of engineering bacteria, it is characterised in that the engineering bacteria contains expression vector as claimed in claim 3.

5. a kind of method that purpose nucleotide sequence is expressed in plant, the method includes importing DNA construct to plant, The DNA construct contains promoter and the purpose nucleotide sequence for being operatively connected to the promoter, wherein the starting Nucleotide sequence sequence as shown in SEQ ID NO:5 of son.

6. method described in claim 5, wherein the plant is monocotyledon.

7. method described in claim 5, wherein the plant is gramineae plant.

8. method described in claim 5, wherein the plant is rice or wheat.

9. method described in claim 5, wherein the purpose nucleotide sequence can be structural gene, adjust gene, knot The antisense gene of structure gene, the antisense gene for adjusting gene or the tiny RNA that can interfere with endogenous gene expression are sent out in pollen Educate the fertility and pollen germination of the specific expressed adjustable pollen in advanced stage.

10. method described in claim 5, wherein the purpose nucleotide sequence can be coding and carbohydrate is promoted to drop The enzyme of solution.

11. method described in claim 5, wherein the purpose nucleotide sequence can be modification enzyme, amylase, debranching enzyme And pectase.

12. method described in claim 5, wherein the purpose nucleotide sequence is selected from corn a amylase gene, growth The group that element, rotB, cytotoxin gene, diphtheria toxin, DAM methylase, Avidin or dominant male sterility gene are constituted One of.

13. promoter described in claim 1 is cultivating the application in plant variety or strain.

14. application described in claim 13, wherein the cultivation plant variety or strain, which can be, cultivates Pollination Fertilization ability Enhancing plant variety or strain cultivate plant variety or strain and cultivate male sterile plants product that Pollination Fertilization ability slackens Kind or strain.

15. application described in claim 13, which is characterized in that the plant is monocotyledon.

16. application described in claim 13, which is characterized in that the plant is gramineae plant.

17. application described in claim 13, which is characterized in that the plant is rice or wheat.