CN104975022A - Identification and applications of plant anther-specific expression promoter pTaASG036 - Google Patents

Abstract

The present invention belongs to the field of plant biotechnology, and particularly relates to separation, functional identification and applications of a plant anther-specific expression promoter. According to the present invention, the promoter is specifically expressed in the plant anther, and provides good application prospects in the plant transgene field.

Description

The qualification of plant anther specific expression promoter pTaASG036 and application

Technical field

The invention belongs to plant biotechnology field, specifically, the present invention relates to the DNA of separation, it can instruct the nucleic acid specific transcription and/or the expression in plant anther that are operatively coupled on its downstream.In addition, the invention still further relates to the expression cassette and plant etc. that comprise this DNA, and relate to the application of this DNA.

Background technology

Plant gene regulatory mainly carries out on transcriptional level, by the mutual coordination of multiple cis-acting elements and trans-acting factor.Promotor is important cis-acting elements, it is positioned at the section of DNA sequence that structure gene 5 ' holds upstream region regulatory gene to transcribe, and can activate RNA polymerase, make it to be combined exactly with template DNA, guarantee to transcribe accurately and effectively initial, play a crucial role in transcriptional control.Drive the different characteristics of genetic expression according to it, promotor is divided into constitutive promoter and specificity promoter.Constitutive promoter can all cells or tissue in, regardless of time and spatially startup transcribe; Specificity promoter can be divided into tissue-specific promoter and inducible promoter again, wherein inducible promoter do not start at ordinary times transcribe or transcriptional activity very low, but under the stimulation of some specific adverse circumstance signal, transcriptional activity can improve significantly.

Exogenous DNA array is by being connected to specific promotor thus being enabled in the expression in plant host, and the selection of promoter and enhancer determines expression time and the position of gene.At present at the strong promoter of mainly some composing types of agricultural biological technical field widespread use, such as CaMV 35S promoter and corn Ubiquitin-1 promotor, but when the quality utilizing these promotors to induce the crops such as goal gene rice transformation to Crop Improvement, often because time (etap specificity) of destination gene expression or space (tissue and organ specificity) can not control well and cause improved effect not obvious, or because these constitutive promoter inducible gene expression amounts are too high, growing of plant is impacted, these obstacles run into when being all and utilizing composing type strong promoter combined function gene to carry out Crop Improvement quality at present.

In addition, when studying some metabolic process or the approach of adjustment, usually need the plural gene transformation in same approach in same strain, adopt to transform after one of them gene obtains transfer-gen plant and transform another one gene again, or hybridize again after two genes have transformed respectively, all need to wait for the longer time, in order to raise the efficiency the time shortening multiple gene transformation, there is report that new carrier can be utilized to carry out the conversion of multiple gene recently simultaneously, if but reuse same promotor when polygene transforms, also gene silencing may can be caused due to the high homology of promoter sequence.

In recent years, succeeded on some crops by genetic engineering regulation plant pollen fertility, creation male sterility line of plants and restorer thereof, new prospect has been started in the utilization for crop heterosis.Utilizing genetically engineered to create male sterile strategy at present mainly utilizes the specific promoter of pollen development chimeric with foreign gene, and construction of expression vector, conversion of plant blocks the process of pollen development thus reaches male sterile object.Mariani etc. utilize tobacco anther tapetum specific promoter TA29 to be connected with RNase T1 or Barnase and are built into mosaic gene, tobacco and rape is proceeded to by agriculture bacillus mediated, obtain stable male sterile transformant (Mariani C etc., Induction of male sterility in plants by a chimaeric ribonucleasegene, Nature, 1990,347:737-741).Subsequently, they drive Barstar gene with TA29 again, create restorer (the Mariani C etc. of above-mentioned male sterile line, A chimaeric ribonuclease-inhibitorgene restores fertility to male sterile plants, Nature, 1992,357:384-387).In addition, other research groups utilize anther-specificpromoter to start the specifically expressing in flower pesticide such as antisense chalkone synthase gene, antisense actin gene, β-1,3-glucanase gene and also successfully obtain male sterile plants (Meer etc. respectively, Promoter analysis of the chalcone synthase gene of petunia hybrid:a 67bp promoter region directs flower-specific expression, Plant Biol., 1990,15:95-109; Li Yanhong etc., import the First Report of Studies of Wheat cultivar, Journal of Agricultural Biotechnology, 1999,7:255-258 by new red male sterility gene of appointing; Curtis etc., Genomic male sterility in lettuce, a base line for the production of F ₁hybrid, Plant Sci., 1996,113:113-119).

The driving activity of plant pollen or anther promoter and specificity determine and are regulated and controled pollen fertility by genetic engineering means, created the success or failure of plant sterile line and restorer.Driving activity known is at present high and the plant pollen that specificity is good or anther-specificpromoter are also relatively less, and wheat is because of the comparatively large and reasons such as complex structure of its genome, research in pollen or anther development molecular mechanism is deficienter, therefore, to the clone of Wheat Pollen specific expression promoter and functional analysis for utilizing genetic engineering regulation pollen fertility in wheat, creating male sterility line of plants, thus lay the first stone for wheat heterosis resource making full use of in wheat breeding.

Summary of the invention

The object of this invention is to provide a kind of promoter sequence of Plant Pollen Development flower pesticide in late period specifically expressing and clone and apply the method for this promotor.

Get the wheat anther that pollen is in meiophase, monokaryotic stage, dikaryophase and three core phases, total serum IgE is extracted with Trizol (Invitrogen), and carry out DNaseI (Promega) process, and then purified mRNA (Ambion).The mRNA of purifying carried out reverse transcription (Invitrogen), ultrasonicly interrupt (Fisher), preparation library (illumina) increase (illumina), finally on illumina machine, carry out sequencing reaction.

First the result of wheat transcript profile high-flux sequence carries out sequence assembly by Trinity software, and the splicing sequence obtained removes redundancy and similarity cluster further.For the expression mutation analysis splicing the transcript contig obtained, the result that in each sample, first the sequence of high-flux sequence is spliced by TopHat (http://tophat.cbcb.umd.edu/) software and transcript is compared.Then Cufflink software can calculate the homogenization expression amount of the transcript contigs in comparison, represent with " the every megabit of exon is to the Kb (fragments per kilobase of exon model per million mapped fragments, FPKM) of fragment ".

By the full-length genome expression pattern analysis to different development stage wheat anther, find pollen to be in and do not express in the flower pesticide of subtrahend separation period and be in the transcript contig 7187 expressed in monokaryon, double-core and the flower pesticide of three core phases at pollen.As shown in Figure 1, comp170457_c0_seq1 (sequence is as shown in SEQ ID NO:1) pollen is in not express in the flower pesticide of subtrahend separation period and be in monokaryon, double-core and the flower pesticide of three core phases at pollen and expresses.Be TaASG036 (Anther SpecificGene 036) by the unnamed gene corresponding to comp170457_c0_seq1.

Wheat overlaps by A, B, D tri-allohexaploid that genome forms, and the average copy number of gene is 2.8, and the gene (46%) wherein close to half has 3-4 copy, and the gene of 12% has 1-2 copy, gene copy number >=5 of 42%.From the sequence of comp170457_c0_seq1 (as shown in SEQ ID NO:1), the order-checking information of the common wheat utilizing CerealsDB and IWGSC (International Wheat Genome SequencingConsortium) to announce, and wheat ancestors Uralensis Fisch (Triticum urartu Nature delivered in 2013, A Genome donor) and aegilops tauschii (Aegilops tauschii, D Genome donor) order-checking information carry out electronic cloning, obtain 3 TaASG036 genes, called after TaASG036-1 respectively, TaASG036-2 and TaASG036-3, wherein comp170457_c0_seq1 corresponds to TaASG036-1.The cDNA sequence of 3 TaASG036 genes is respectively as shown in SEQ ID NO:2, SEQID NO:3 and SEQ ID NO:4, and the homology between three is about 93%.Design respectively for TaASG036-1, the Auele Specific Primer of TaASG036-2 and TaASG036-3cDNA, utilize RT-PCR method, to these three genes at wheat root, stem, leaf, expression specificity analysis is carried out in the flower pesticide of different development stage and other floral organ official rank Various Tissues materials except flower pesticide, result as shown in Figure 2, TaASG036-1, TaASG036-2 and TaASG036-3 gene is all only in monokaryotic stage at pollen, specific expressed in the flower pesticide of dikaryophase and three core phases, other floral organs of phase and root at the same time, stem, do not express in the histoorgans such as the tassel of leaf and meiophase, TaASG036-1 is described, TaASG036-2 and TaASG036-3 gene is flower pesticide specifically expressing, and the only gene of specifically expressing in the flower pesticide in pollen development late period.

Present invention also offers the promotor of three flower pesticide specifically expressings, described promotor obtains by the following method: from TaASG036-1, the cDNA sequence of TaASG036-2 and TaASG036-3 gene is set out, the order-checking information of the common wheat utilizing CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium) to announce, and wheat ancestors Uralensis Fisch (Triticum urartu Nature delivered in 2013, A Genome donor) and aegilops tauschii (Aegilops tauschii, D Genome donor) order-checking information carry out electronic cloning, obtain TaASG036-1, the promotor of TaASG036-2 and TaASG036-3 gene, called after TaASG036-1 promotor respectively, TaASG036-2 promotor and TaASG036-3 promotor, its length is respectively 2267bp, 2065bp and 2049bp, sequence is respectively as SEQ ID NO:5, shown in SEQ ID NO:6 and SEQ ID NO:7.

Utilize PlantCARE database and PLACE database, cis element analysis is carried out to TaASG036-1 promotor, TaASG036-2 promotor and TaASG036-3 promotor.As shown in Figure 3, translation initiation site ATG runic underscore represents, is defined as "+1 " with the A of translation initiation site ATG, AGAAA motif shadow representation, the box indicating of GTGA motif.There are 21 AGAAA motifs and 11 GTGA motifs in TaASG036-1 promotor, in TaASG036-2 promotor, have 17 AGAAA motifs and 11 GTGA motifs, in TaASG036-3 promotor, have 1 AGAAA motif and 8 GTGA motifs.AGAAA motif and GTGA motif are and the cis-regulating element that pollen/flower pesticide specifically expressing is relevant that TaASG036-1, TaASG036-2 and multiple AGAAA motif in the promotor of TaASG036-3 gene and the existence of GTGA motif show that this promotor may be the promotor relevant with pollen/flower pesticide specifically expressing.

In order to verify the function of these two promotors further, one of them promotor SEQ ID NO:5 is connected with Reporter gene GUS, conversion of plant, at the root of transgenic paddy rice, the expression of gus gene is all can't detect in the vegetative organ such as stem and leaf, the tassel of meiophase is in and pollen is in monokaryotic stage at pollen, also the expression of gus gene is can't detect in other floral organ except flower pesticide of dikaryophase and three core phases, TaASG036-1 promotor can only start gus gene and be in monokaryotic stage at pollen, express in the flower pesticide of dikaryophase and three core phases, illustrate that promotor provided by the present invention is the promotor of pollen development flower pesticide in a late period specifically expressing.

Plant anther specific expression promoter provided by the present invention, containing the nucleotide sequence shown in SEQ ID NO:5,6 or 7 in ordered list, or comprise the nucleotide sequence with nucleotide sequence listed by SEQ ID NO:5,6 or 7 with more than 90% similarity, or comprise 100 and more than 100 continuous print nucleotide fragments deriving from SEQ ID NO:5,6 or 7 sequences, and the nucleotides sequence be connected with this promotor operability can be driven to be listed in expression in plant anther.Expression vector containing above-mentioned sequence, transgenic cell line and Host Strains etc. all belong to protection scope of the present invention.Increase SEQ ID NO:5 disclosed in this invention, 6 or 7 promotors the primer pair of arbitrary nucleotide fragments also within protection scope of the present invention.

Promotor nucleotide sequence provided by the present invention also can be used for being separated corresponding sequence from other plant beyond wheat, especially from other monocotyledonss, carries out homologous clone.According to these corresponding sequence and the sequence homology herein between listed promoter sequence, or with the homology of this promoter gene, use such as the technology such as PCR, hybridization is differentiated to be separated these corresponding sequence.Therefore, according to the respective segments that they are separated with the sequence similarity between the SEQ IDNO:5 listed by the present invention, 6 or 7 promoter sequences (or its fragment), be also included within embodiment.

" promotor " of the present invention refers to that a kind of DNA regulates and controls region, and it comprises the TATA box of energy guide RNA polymerase II in the initial RNA synthesis of the suitable transcription initiation site of specific coding sequence usually.Promotor also can comprise other recognition sequence, and these recognition sequences are usually located at upstream or the 5 ' end of TATA box, are commonly called upstream promoter element, play regulatory transcription efficiency.Those skilled in the art should know, although identified the nucleotide sequence for promoter region disclosed by the invention, separation andpreconcentration has been in other controlling element of the TATA box upstream region of the specific promoter region of the present invention's qualification also within the scope of the invention.Therefore, promoter region disclosed herein is defined as usually further comprises upstream regulatory elements, such as, for those element, enhansers etc. of the tissue expression and temporal expressions function that regulate and control encoding sequence.In an identical manner, can identify, isolate the promoter element making to carry out expressing in destination organization (such as male tissue), it be used together with other core promoter, to verify the expression that male tissue is preferential.Core promoter refers to the minimal sequence needed for initiation transcription, such as, be called as the sequence of TATA box, and this is that the promotor of the gene of coded protein all has usually.Therefore, alternatively, SEQ IDNO:5 of the present invention, 6 or 7 promotors can with himself or associate from other core promoter of originating and use.

Core promoter can be the known core promoter of any one, such as cauliflower mosaic virus 35S or 19S promotor (U.S. Patent No. 5,352,605), ubiquitin promoter (U.S. Patent No. 5,510,474), IN2 core promoter (U.S. Patent No. 5,364,780) or figwort mosaic virus promotor.

The function of described gene promoter can be analyzed by the following method: be connected with reporter gene operability by promoter sequence, form transformable construct, again this construct is proceeded in plant, in acquisition transgenic progeny, confirm its expression characterization by the expression of visual report gene in each histoorgan of plant; Or above-mentioned construct subclone is entered the expression vector for transient expression experiment, is detected the function of promotor or its control region by transient expression experiment

Being used for the selection of suitable expression vector of test starting or control region domain-functionalities will depend on host and this expression vector will be introduced the method for host, and these class methods are well known to those of ordinary skill in the art.For eukaryote, region in the carrier comprises the region controlling transcription initiation and controlled working.These regions are operably connected to reporter gene, and described reporter gene comprises YFP, UidA, gus gene or luciferase.The expression vector comprising the presumption control region being arranged in genomic fragment can be introduced into complete tissue, such as interim flower pesticide, or introduces callus, to carry out functional verification.

The mRNA of the reporter gene that the activity of promotor and intensity can drive according to it or protein expression amount measure.Reporter gene (reporter gene) is the gene of a kind of coding protein that can be detected or enzyme, that is, is that its expression product is very easy to certified gene.Its encoding sequence is merged formation mosaic gene mutually with Gene expression and regulation sequence, or merges mutually with other goal gene, express under regulating and controlling sequence controls, thus utilize its expression product to determine the expression regulation characteristic of goal gene.Conventional reporter gene has beta-glucosiduronatase gene GUS, and green fluorescence protein gene GFP.

The present invention detects activity and the expression characterization of promotor by gus reporter gene.Detect substrate used according to gus gene different, have three kinds of detection methods: histochemical method, spectrophotometry and fluorescent method (sensitivity is that Spectrophotometric Assays method is the highest), wherein the most conventional is histochemical method.Histochemical method detects using the bromo-4-of 5-chloro-3-indoles-beta-glucoside acid (X-Gluc) as reaction substrate.The damping fluid of tested material containing substrate is soaked, if histocyte has proceeded to gus gene, and given expression to GUS zymoprotein, under appropriate conditions, X-Gluc hydrolysis just can be generated blue product by this enzyme, and this is the bipseudoindoxyl dye formed through oxidative dimerization effect by its initial product, and it makes have the position of GUS expression activity or site to present blueness in each histocyte, with the naked eye or under the microscope can see, and the power of GUS activity can be reflected according to the dyeing depth under to a certain degree.Therefore the method is utilized to can be observed foreign gene at certain organs, tissue, the expression even in individual cells.

In addition, promotor of the present invention can be connected with the nucleotide sequence being not TaASG036-1, TaASG036-2 or TaASG036-3 gene, to express other heterologous nucleotide sequence.Promotor nucleotide sequence of the present invention and fragment thereof can be assembled in an expression cassette with variant together with heterologous nucleotide sequence, for expressing in object plant, more specifically, express in the male organs of this plant.Described expression cassette has suitable restriction enzyme site, for inserting described promotor and heterologous nucleotide sequence.These expression cassettes can be used for carrying out genetic manipulation to any plant, to obtain the corresponding phenotype wanted.

TaASG036-1 promotor disclosed in this invention, TaASG036-2 promotor and TaASG036-3 promotor, can be used for the expression driving following heterologous nucleotide sequence, obtains male sterile phenotype to make the plant of conversion.Described heterologous nucleotide sequence codified impels enzyme or modifying enzyme, amylase, debranching factor and the polygalacturonase of carbohydrate degradation, more specifically as a amylase gene, growth hormone (auxin), rot B, cytotoxin gene, diphtheria toxin, DAM methylase, avidin, or can be selected from protokaryon regulator control system, can also be dominant male sterility gene.

In some embodiments, the nucleic acid being operatively coupled on promotor downstream of the present invention mentioned in the present invention, wherein said " nucleic acid " can be the tiny RNA that operability is connected to structure gene on promotor disclosed herein, regulatory gene, the inverted defined gene of structure gene, the inverted defined gene of regulatory gene or native gene can be disturbed to express.

Promoter sequence provided by the present invention is separable from any plant, include but not limited to Btassica, corn, wheat, Chinese sorghum, two joint shepherd's purses belong to, sinapsis alba, Semen Ricini, sesame, cottonseed, Semen Lini, soybean, Arabidopsis, Phaseolus, peanut, clover, oat, Semen Brassicae campestris, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheat (Spelt), emmer, flax, gramagrass (Grammagrass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sugarcane, crowberry, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, the stem of noble dendrobium, gladiolus, chrysanthemum, Liliaceae, cotton, eucalyptus, Sunflower Receptacle, rape, beet, coffee, ornamental plant and conifer etc.Preferably, plant comprises corn, soybean, safflower, leaf mustard, wheat, barley, rye, rice, cotton and Chinese sorghum.

The present invention also comprises the construct containing TaASG036-1 promotor, TaASG036-2 promotor or TaASG036-3 promotor, and described construct comprises usually said carrier or expression cassette.Also can comprise other component in above-mentioned construct, this depends primarily on object and the purposes of vector construction, such as, can comprise selectable marker gene, target or regulating and controlling sequence, critical sequences or homing sequence, intron etc. further.3 ' the end at desired heterologous nucleotide sequence is also included in plant by expression cassette has transcribing and translation termination of function.Terminator can be the terminator of gene provided by the present invention, also can be the terminator from external source.More specifically, above-mentioned terminator can be nopaline synthase or octopine synthase termination area.

Guide the expression product of heterologous nucleotide sequence into specific cells device in hope, such as plastid, amyloplast, or guide endoplasmic reticulum into, or when cell surface or cell exocrine, expression cassette also can comprise the nucleotide sequence for encoding transit peptides.This type of transit peptides is known in the field, and it includes but not limited to the small subunit, plant EPSP synthase, corn Brittle-1 chloroplast transit peptides etc. of Rubisco.

In the process preparing expression cassette, can be operated multiple DNA fragmentation, be in proper orientation to provide, or be in the DNA sequence dna in correct reading frame.For reaching this object, adapter or joint can be used, DNA fragmentation is linked up, or comprise other operation further, with the restriction enzyme site etc. of providing convenience.

Further, in construct provided by the present invention, also selectable marker gene can be comprised, for selecting the cell or tissue through transforming.Described selectable marker gene comprises gives antibiotics resistance or the gene to Herbicid resistant.Suitable selectable marker gene includes but not limited to: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistant gene, sulfamido resistant gene, glyphosate gene, the bony resistant gene of careless fourth.Described selectable marker gene can also be the genes such as red fluorescent gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene, anthocyanin p1.

Expression cassette provided by the present invention or carrier can be inserted into plasmid, clay, yeast artificial chromosome, bacterial artificial chromosome or other be applicable to being transformed in any carrier in host cell.Preferred host cell is bacterial cell, such as, in particular for cloning or storing polynucleotide or the bacterial cell for transformed plant cells, intestinal bacteria, Agrobacterium tumefaciems and Agrobacterium rhizogenes.When host cell is vegetable cell, expression cassette or carrier can be inserted in the genome of the vegetable cell be converted.Insertion can be location or random insertion.Preferably, be inserted through such as homologous recombination to realize.In addition, expression cassette or carrier can remain on outside karyomit(e).Expression cassette of the present invention or carrier can be present in the core of vegetable cell, chloroplast(id), plastosome and/or plastid.Preferably, expression cassette of the present invention or carrier are inserted in the chromosomal DNA of plant nucleolus.

The present invention also comprises disclosed TaASG036-1 promotor and/or the application of TaASG036-2 promotor and/or TaASG036-3 promotor, in the embodiment of some application, can apply TaASG036-1 promotor provided by the present invention and/or TaASG036-2 and/or TaASG036-3 promotor to suddenly change the breeding of the male sterile line obtained and maintenance to realize some fertility-related genes, described fertility-related gene includes but not limited to Ms26, Ms45, MSCA1 etc.

Of the present invention provided anther specific expression promoter can be used for specific expressed in flower pesticide of foreign gene, thus avoid this foreign gene continuous expression adverse effect in its hetero-organization of plant, plant anther can also be used for and grow the functional analysis of genes involved and qualification; Can be used for the establishment of male sterile line and restorer; And can be applicable in pollen abortion experiment, thus avoid being drifted about by plant transgene or pollen is escaped the Biosafety problem brought, significant to the creation of male sterility line of plants and restorer.

Transgenic plant of the present invention use plant biotechnology field method for transformation preparation known to the skilled.Any method can be used to recombinant expression vector to be transformed in vegetable cell, to produce transgenic plant of the present invention.Method for transformation can comprise directly and indirectly method for transformation.Suitable direct method comprises the DNA absorption of polyoxyethylene glycol induction, liposome-mediated conversion, uses particle gun importing, electroporation and microinjection, etc.In the specific embodiment of the present invention, the transformation technology that present invention uses based on edaphic bacillus (can see (1985) Science 225:1229 such as Horsch RB; White FF, Vectors forGene Transfer in Higher Plants, Transgenic Plants, the 1st volume, Engineeringand Utilization, Academic Press, 1993, pp.15-38; .Techniques for Gene Transfer, the Transgenic Plants such as Jenes B, the 1st volume, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Edaphic bacillus bacterial strain (such as Agrobacterium tumefaciems or Agrobacterium rhizogenes) comprises plasmid (Ti or Ri plasmid) and T-DNA element, described plasmid and element are transferred to plant after with Agrobacterium transfection, and T-DNA is integrated in the genome of vegetable cell.T-DNA can be positioned on Ri-plasmid or Ti-plasmid, or is included in independently in so-called binary vector.Agrobacterium-mediated method for transformation is described in such as.The agrobacterium-mediated the most applicable dicotyledons of conversion, but be also applicable to monocotyledons.The conversion of edaphic bacillus to plant is described in such as.Conversion can cause instantaneous or stable conversion and expression.Although nucleotide sequence of the present invention can be inserted into any plant and vegetable cell that fall into these broad variety, it is particularly useful for crop plants cell.

Below by embodiment, by reference to the accompanying drawings the present invention is described in further detail, but the scope do not limited the present invention in any way.

Accompanying drawing explanation

Fig. 1 expression level analysis that to be comp170457_c0_seq1 at pollen be in the flower pesticide of meiophase (WT-0), monokaryotic stage (WT-1), dikaryophase (WT-2) and three core phases (WT-3), X-coordinate is pollen different development stage, ordinate zou is FPKM, the expression level of response gene.

Fig. 2 is that the RT-PCR of 3 homologous genes in the flower pesticide of wheat different tissues organ and different development stage of TaASG036 analyzes.1 represents root, 2 represent stem, 3 represent blade, 4 represent that pollen is in the tassel of meiophase, and 5 represent that pollen is in the flower pesticide of monokaryotic stage, and 6 represent that pollen is in the flower pesticide of dikaryophase, 7 represent that pollen is in the flower pesticide of three core phases, 8 represent that pollen to be in spending of monokaryotic stage except flower pesticide except other floral organ, and 9 represent that pollen to be in spending of dikaryophase except flower pesticide other floral organ, and 10 expression pollen to be in spending of three core phases except flower pesticide other floral organ.

Fig. 3 represents TaASG036-1 promoter sequence (A), TaASG036-2 promoter sequence (B) and TaASG036-3 promoter sequence (C).Translation initiation site ATG runic underscore represents, the A of translation initiation site ATG is defined as "+1 ", AGAAA motif shadow representation, the box indicating of GTGA motif.AGAAA with GTGA represents two conserved motifs relevant to pollen/anther specific expression promoter.

Fig. 4 is the T-DNA district collection of illustrative plates of expression vector p235.LB and RB is respectively left margin and the right margin of T-DNA; NPTII represents neomycin phosphotransferase II gene; P35S represents the promotor of CaMV35S gene; T35S represents the terminator of CaMV35S gene; GUS represents beta-glucosiduronatase gene; Tnos represents the terminator of rouge alkali synthetase (no) gene; HindIII, PstI, XbaI, SpeI, SacI and EcoRI represent the restriction enzyme site of restriction enzyme respectively; TaASG036-1 promotor is exactly the promotor of the wheat anther specifically expressing that the present invention is separated.

Fig. 5 is that the histoorgan GUS of p235 transgenic wheat dyes.A is root; B stem; C leaf; D is the flower that pollen is in meiophase; E is the flower that pollen is in monokaryotic stage; F is the flower that pollen is in dikaryophase; G is the flower that pollen was in for three core phases; H is the flower pesticide that pollen is in monokaryotic stage; I is the pollen of monokaryotic stage, and the upper right corner is the pollen of DAPI dyeing; J is the pollen of dikaryophase, and the upper right corner is the pollen of DAPI dyeing; K is the pollen of three core phases, and the upper right corner is the pollen of DAPI dyeing.

Embodiment

In following embodiment, method therefor is ordinary method if no special instructions, the primer synthesizes by Shanghai Ying Jun biotech company, order-checking is won polygala root biotechnology limited liability company by Beijing three and is completed, endonuclease in PCR kit, vector construction process is purchased from precious biotechnology company limited, pEASY-T1-simple connection test kit and TransStart FastPfu DNA Polymerase are purchased from Beijing Quan Shijin biotech company, T4DNA ligase enzyme is purchased from NEB company, and the method that the equal reference reagent box of method provides is carried out.

The acquisition of contig expressed by the full-length genome expression pattern analysis of embodiment 1. different development stage wheat anther and pollen development later stage flower pesticide

Get the wheat anther that pollen is in meiophase, monokaryotic stage, dikaryophase and three core phases, total serum IgE is extracted with Trizol (Invitrogen), and carry out DNaseI (Promega) process, and then purified mRNA (Ambion).The mRNA of purifying carried out reverse transcription (Invitrogen), ultrasonicly interrupt (Fisher), preparation library (illumina) increase (illumina), finally on illumina machine, carry out sequencing reaction.

First the result of wheat transcript profile high-flux sequence carries out sequence assembly by Trinity software, and the splicing sequence obtained removes redundancy and similarity cluster further.For the expression mutation analysis splicing the transcript contig obtained, the result that in each sample, first the sequence of high-flux sequence is spliced by TopHat (http://tophat.cbcb.umd.edu/) software and transcript is compared.Then Cufflink software can calculate the homogenization expression amount of the transcript contigs in comparison, represent with " the every megabit of exon is to the Kb (fragments per kilobase of exon model per million mapped fragments, FPKM) of fragment ".

By the full-length genome expression pattern analysis to different development stage wheat anther, find pollen to be in and do not express in the flower pesticide of subtrahend separation period and be in the transcript contig 7187 expressed in monokaryon, double-core and the flower pesticide of three core phases at pollen.As shown in Figure 1, comp170457_c0_seq1 (sequence is as shown in SEQ ID NO:1) pollen is in not express in the flower pesticide of subtrahend separation period and be in monokaryon, double-core and the flower pesticide of three core phases at pollen and expresses.Be TaASG036 (Anther specificgene 036) by the unnamed gene corresponding to comp170457_c0_seq1.

Embodiment 2.RT-PCR verifies the tissue expression specificity of TaASG036 gene

Wheat overlaps by A, B, D tri-allohexaploid that genome forms, and the average copy number of gene is 2.8, and the gene (46%) wherein close to half has 3-4 copy, and the gene of 12% has 1-2 copy, gene copy number >=5 of 42%.From the sequence of comp170457_c0_seq1 (as shown in SEQ ID NO:1), the order-checking information of the common wheat utilizing CerealsDB and IWGSC (International Wheat Genome SequencingConsortium) to announce, and wheat ancestors Uralensis Fisch (Triticum urartu Nature delivered in 2013, A Genome donor) and aegilops tauschii (Aegilops tauschii, D Genome donor) order-checking information carry out electronic cloning, obtain 3 TaASG036 genes, called after TaASG036-1 respectively, TaASG036-2 and TaASG036-3, wherein comp170457_c0_seq1 corresponds to TaASG036-1.The cDNA sequence of 3 TaASG036 genes is respectively as shown in SEQ ID NO:2, SEQID NO:3 and SEQ ID NO:4, and the homology between three is about 93%.Design respectively for TaASG036-1, the Auele Specific Primer of TaASG036-2 and TaASG036-3cDNA, utilize RT-PCR method, to these three genes at wheat root, stem, leaf, expression specificity analysis is carried out in the flower pesticide of different development stage and other floral organ official rank Various Tissues materials except flower pesticide, result as shown in Figure 2, TaASG036-1, TaASG036-2 and TaASG036-3 gene is all only in monokaryotic stage at pollen, specific expressed in the flower pesticide of dikaryophase and three core phases, other floral organs of phase and root at the same time, stem, do not express in the histoorgans such as the tassel of leaf and meiophase, TaASG036-1 is described, TaASG036-2 and TaASG036-3 gene is flower pesticide specifically expressing, and the only gene of specifically expressing in the flower pesticide in pollen development late period.

The RT-PCR primer of TaASG036-1 gene is:

Primer 1:5'-CAATGGAGGGGAGGAGAACC-3'(SEQ ID NO:8)

Primer 2: 5'-TAATAACAAGACCAGTCTACTCCTTGG-3'(SEQ ID NO:9)

The RT-PCR primer of TaASG036-2 gene is:

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

Primer 4:5'-AACTAGACGACCAGAGTAGACAGAGC-3'(SEQ ID NO:11)

The RT-PCR primer of TaASG036-3 gene is:

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

Primer 6:5'-GCCTCCCTAGCTAATAACAAAGACC-3'(SEQ ID NO:13).

The acquisition of embodiment 3.TaASG036-1, TaASG036-2 and TaASG036-3 gene promoter sequence and cis element analysis

From TaASG036-1, the cDNA sequence of TaASG036-2 and TaASG036-3 gene is set out, the order-checking information of the common wheat utilizing CerealsDB and IWGSC (International Wheat Genome Sequencing Consortium) to announce, and wheat ancestors Uralensis Fisch (Triticum urartu Nature delivered in 2013, A Genome donor) and aegilops tauschii (Aegilops tauschii, D Genome donor) order-checking information carry out electronic cloning, obtain TaASG036-1, the promotor of TaASG036-2 and TaASG036-3 gene, length is respectively 2267bp, 2065bp and 2049bp, sequence is respectively as SEQ ID NO:5, shown in SEQ ID NO:6 and SEQ ID NO:7.

Utilize PlantCARE database and PLACE database, cis element analysis is carried out to the promotor of TaASG036-1, TaASG036-2 and TaASG036-3 gene.As shown in Figure 3, translation initiation site ATG runic underscore represents, is defined as "+1 " with the A of translation initiation site ATG, AGAAA motif shadow representation, the box indicating of GTGA motif.There are 21 AGAAA motifs and 11 GTGA motifs in TaASG036-1 gene promoter, in TaASG036-2 gene promoter, have 17 AGAAA motifs and 11 GTGA motifs, in TaASG036-3 gene promoter, have 1 AGAAA motif and 8 GTGA motifs.AGAAA motif and GTGA motif are and the cis-regulating element that pollen/flower pesticide specifically expressing is relevant that TaASG036-1, TaASG036-2 and multiple AGAAA motif in the promotor of TaASG036-3 gene and the existence of GTGA motif show that this promotor may be the promotor relevant with pollen/flower pesticide specifically expressing.

The clone of embodiment 4.TaASG036-1 promotor and the structure of plant expression vector

By plant expression vector pBI121 restriction enzyme HindIII and EcoRI double digestion, the 35S:GUS fragment T4DNA ligase obtained is connected into the pCAMBIA2300 carrier of the CAMBIA company of same HindIII and EcoRI double digestion, and new carrier is named as p230035S:GUS.

5 ' end and the ATG upstream design primer from TaASG036-1 promotor:

Primer 7:5 '-ctgcag TGATTTATACAAAAAATGTACAGAGCATATG-3 ' (SEQID NO:14);

Primer 8:5 '-actagt TGGCGCAGCGCGCAGATGCAAAC-3 ' (SEQ ID NO:15);

In primer 7, sequence ctgcag is the restriction enzyme site of PstI, and in primer 8, sequence actagt is the restriction enzyme site of SpeI.

With the genomic dna of wheat for template, increase with primer 7 and primer 8, reaction conditions is: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds; Anneal 30 seconds for 60 DEG C; 72 DEG C extend 2 points 30 seconds; 35 circulations; 72 DEG C extend 10 minutes.After reaction terminates, PCR primer detects through 1% agarose gel electrophoresis and reclaims, and product is connected in pEASY-T1-simple carrier, and screening positive clone also carries out sequence verification, and sequence is as shown in SEQ ID NO:5, and this plasmid is called p231.

With restriction enzyme PstI and SpeI double digestion p231, the TaASG036-1 promotor T4DNA ligase obtained is connected into the p230035S:GUS carrier with PstI and XbaI double digestion, and obtain plant expression vector p235, the structure of this plasmid as shown in Figure 4.

The Molecular Identification of the rice transformation that embodiment 5. is agriculture bacillus mediated and transfer-gen plant

Utilize heat shock method that plant expression vector p235 is proceeded to Agrobacterium AGL0 bacterial strain.

Infect paddy rice embryo callus subculture with Agrobacterium, in the dark Dual culture 2-3 days, then through steps such as two step resistance screenings, pre-differentiation, differentiation and root culture, final obtain have kalamycin resistance, turn p235 paddy rice T0 for plant.

Design primer pair transgenic rice plant carries out PCR qualification.

Primer 9:5 '-CGGTATGAATATTGCAAAGCCAG-3 ' (SEQ ID NO:16)

Primer 10:5 '-GCCGTCGAGTTTTTTGATTTCAC-3 ' (SEQ ID NO:17)

Reaction conditions is: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds; Anneal 30 seconds for 55 DEG C; 72 DEG C extend 1 point 30 seconds; 30 circulations; 72 DEG C extend 10 minutes.Amplification be the Partial Fragment of TaASG036-1 promotor and GUS, length is 1304bp.Qualification result shows, the resistance regeneration plant utilizing agriculture bacillus mediated rice conversion to obtain is the positive plant turning p235 gene.

The histological chemistry that embodiment 6. transgenic rice plant different tissues organ gus gene is expressed is detected

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 potassium hydride KH, the ferrous potassium hydride KH of 0.5mM.

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

Select the transgenic seedlings with gus reporter gene of suitable size or particular organization to immerse in GUS dye liquor, 37 DEG C of stained over night, suck reaction solution, and ethanol gradient decolours, and microscopic examination is taken a picture.

To the GUS coloration result of each histoorgan of p235 transgenic rice plant as Fig. 5, at the root of transgenic paddy rice, the expression (Fig. 5 A-C) of gus gene is all can't detect in the vegetative organ such as stem and leaf, the flower of meiophase is in and pollen is in monokaryotic stage at pollen, also the expression of gus gene is can't detect in other floral organ except flower pesticide of dikaryophase and three core phases, TaASG036-1 promotor can only start gus gene and be in monokaryotic stage at pollen, (Fig. 5 D-G) is expressed in the flower pesticide of dikaryophase and three core phases, illustrate that TaASG036-1 promotor is the promotor of pollen development flower pesticide in a late period specifically expressing.Further, pollen is in the expression (Fig. 5 H) that can't detect GUS in the anther wall of monokaryotic stage, and the expression (Fig. 5 I-K) of GUS in the pollen of monokaryotic stage, dikaryophase and three core phases, can be detected, therefore, TaASG036-1 promotor is the promotor that a Wheat Pollen grows specifically expressing in late period.

SEQUENCE LISTING

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

Xingwang Investment Pty Ltd.

 

The qualification of <120> plant anther specific expression promoter pTaASG036 and application

<130>

<150> 201410137400.7

<151> 2014-04-08

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 433

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 1

tcatcaatct caatctcctt ccaagctcta ctcgcctcgc aaacgaaaga gcagatcaca 60

tcagatcaga gtgagtttgc atctgcgcgc tgcgccaatg gaggggagga gaaccgctct 120

ctgtttcctc gtcgtgctcg cgcttctcgc aagccctgct ctcgcagaac gcaagtgcta 180

caaggagcgc ttctactcca tgatatgcat gaagtggcac tgcgccatgg agtgcgtcaa 240

ccagagcccg gacctggtgc tcaagtcggc ccactgcacc accaagcacg tcatcatgcg 300

ctactgcaac tgcgagatgt gtaccaagga gtagactggt cttgttatta gctagggagg 360

cgtaccacca ataaatccac atgtagctag acaataatag ttattctagg agtatatatg 420

catgtcgtcg tcg 433

 

<210> 2

<211> 255

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 2

caatggaggg gaggagaacc gctctctgtt tcctcgtcgt gctcgcgctt ctcgcaagcc 60

ctactctcgc agaacgcaag tgctacaagg agcgcttcta ctccatgata tgcatgaagt 120

ggcactgcgc catggagtgc gtcaaccaga gcccggacct ggtgctcaag tcggcccact 180

gcaccaccaa gcacgtcatc atgcgctact gcaactgcga gatgtgtacc aaggagtaga 240

ctggtcttgt tatta 255

 

<210> 3

<211> 434

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 3

gccaatggag gggagaactg ctctctgttt cctcgtcgtg ctcgcgcttc tcgcaagccc 60

tgctctcgca ggtacgtgcg tgctcccctt ccatttcaca tcccctctct tttacaagaa 120

aagatctcct tctttccctt tacatctctg ttccacagaa aagatctcta atgacgatag 180

tgtttgtatg tttgttggct aattgcatct gtatatatat gtatctacat gcatgcagaa 240

cgcaagtgct acaaggagcg cttctactcc atgatatgca tgaagtggca ctgcgccatg 300

gagtgcgtca accagagccc ggacctggtg ctcaagtcgg cccactgcac caccaagcac 360

gtcatcatgc gctactgcaa ctgcgagatg tgtaccaagg agtagactgc tctgtctact 420

ctggtcgtct agtt 434

 

<210> 4

<211> 287

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 4

gagtctgcat ccgcgtgcta cgccaatgga gaggagaacc gctctctgtt tcctcgtcgt 60

gctcgccctt ctcgcaagcc ctgctctcgc agaacgcaag tgctacaagg agcgcttcta 120

ctccatgata tgcatgaagt ggcactgcgc catggagtgc gtcaaccaga gccccgacct 180

ggtgctcaag tcggcccact gcaccaccaa gcacgtcatc atgcgctact gcaactgcga 240

gatgtgtacc aaggagtagt ctggtctttg ttattagcta gggaggc 287

 

<210> 5

<211> 2267

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 5

tgatttatac aaaaaatgta cagagcatat ggacaaagta gacatagaaa tataatttaa 60

gatgtagtcc caatttgaaa aatgttaaac gtacatatat aaaatgttcc tgatgtatgc 120

aagaaatgca caatgtttat ggaaaaagta cacaacaaaa gtatatgttt actaaatgtt 180

aaatatgtat tttcaaaatg ctaaacgtgt atataaaaat attcctgatg tacaaaacaa 240

atgtacaata tttatggaca aaatagacat aacaaatata aacttcctaa taacatgtta 300

atcatgtatt tggaaaaata gccaaatgtg tatataaaaa atgttcctca tgcatacaga 360

aaatgaagta atccaataaa aagttaaaac tgaaaaagaa aaagaaataa acaaagagaa 420

ataggaaaaa acaaagaaaa gaaaaggtga aaaatgagaa ggaaagaaag gaaaacgaag 480

agaaccaatg gtaaaaagaa aagtaaaaaa accaataaaa atagagaaaa gaaacaaagg 540

aaccaaaaac aaatcacagt aaaaaaagga tagaaatgaa gagtaccgat aaaaaaagta 600

tagaaagaca gaaaccgaag aaaactatga agaagtgaag aaacccaata aaataaaata 660

aaataaataa aataaaaact ggaccaaaaa ccagtacaac ctacaaaaca gagagcttct 720

gagaaaaacg agtgacctat ctctctgatc ccgagcgtgc ggtcgaggag attaaatggg 780

ctggccaatt agacatgaca agaaaaaacc cttcacacaa gtgaaccgta gggcttgcta 840

taagcgagat atagtcgccg cggtgttaaa cgggtgtttc ctatttggag cttcagttgc 900

ccattagtat gcattttgca aacgggcata gacaccccct ccgctctggg ccgtcaatcc 960

accctttttc tgtcttttaa cccgggaaaa aagcatagtc atggtgaatc taaccagtga 1020

ccgcctgcat taaggtacgc ttcagcaacc acccatctac ctagcctgat tagatagcat 1080

gcatgttttg tatttttttt attttttcca tttttcaaat ccttgaaact tttttcaaat 1140

tgaggatttt ttttttcaaa ttgagtattt tttatgaaat gtttggacat tttttcgaat 1200

tccaggattt tttcaaatat ttttttagaa aattagacgt atttttttca aatttggcaa 1260

actatttttc aaaattgata tactttttta aaaatccaca attgtttttg tcaaattgat 1320

gaactttttt caaatccatg tcttttcttt caaaatctgt gcacatttgc gaattcatta 1380

atgtttgtct tttgcaaatg ttttttttat caaatgtgat cgagcgaacc agggggaaat 1440

aaaaatgcga tggagcgctc tcatctagat ggcccttagc tagatgagcc cgctgtccac 1500

tcatggcacc agcattttgt acgcttcact agaacaaaaa ggcaatttat atgctgattt 1560

tactgagaaa gcaccatgtt ttctcaaagt gccagctcca aaaatcctgg atagatcggg 1620

tgcagagcgg gatcccaaga attacacgtg catcaatctg catgaacacc tcctcaacgc 1680

gagccctgtc catgttggtg tagtatggtc ggatagacgc tttagctacg tgatgctgct 1740

actaggcgtt ttaattagtt ttattttttg aggattaggc attttaatta gtgcaaccat 1800

acacatgcag cccaaatgac gaggcccaat agcctattca ttagtgcaac cacatacatg 1860

cggcccaaat gtagagctac actacgacgg tgtttgcact aatgatgaat agtgtagctc 1920

tacatttgtg cggcccaaat gtatatctgg agtgaggcgc ggacaagcgg aacagccgag 1980

acggtatatt ctcctttttc ccgatcgcct cgcacgacag gcgtaacaac ggtctttgat 2040

tccttcaagc gctggatgcg gtcgcctcca cgggaagcgg gcggcgtcgt taggagattc 2100

cgtggatctg agcaatcctt cctccacctc tctccctcct atatatacct gtgccgcccg 2160

gcagagtgaa ccatcaatct caatctcctt ccaagctcta ctcgcctcgc aaacgaaaga 2220

gcagatcaca tcagatcaga gtgagtttgc atctgcgcgc tgcgcca 2267

 

<210> 6

<211> 2065

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 6

aatatgtatt tccaaaatgc taaacatgta tataaaaata ttcctgatgt acaaaacaaa 60

tgtacaatgt gtatagaaaa aataaacata acaaaatatt aaatttcaaa taatatgtta 120

atcatgcatt tgaaaaattg tcaaatgtgt atataaagaa tattcctcat gcatatggaa 180

aatgtacaaa ggaaaacgaa gaaaaccaat aaaatgttaa aaactgaaaa ggaaaaagga 240

ataaacaaag agaaatagga aaaacaaaca aagaaaagaa aaggtgaaaa tgagaaggaa 300

agaaaggaaa acgaagaaaa ctgatgaaaa taaagaaaat aaaagcaaat aaaaatagag 360

aaaagaaaaa aaggaaccaa aaagaactca aagaaaaaaa caatgataga aatgaagaat 420

gccaattaaa aaaagtatag aaagacagaa accgaagaaa actgtgaaga aacgcagaga 480

tccacaaaaa taaaataaaa taaaataaaa aaggaccaaa aactagtaca atccacaaaa 540

cagagagctt cgctatggaa aacgagcaag ctatctctct gaacccgagc gtgcgatcga 600

agagatgaaa tgggctggtc cattacaggt gacgtgaaaa acccctttag gtaagcgaac 660

cgtagggctt gctgtaagcg agatatagtc gttacggtgt taaacggtgt ttcctatttg 720

gagcttcagg cgcccgttag tatccatttt gcaaaggggt gcacacacct cctccattct 780

gggctggccg atccaccatt ttttctttct tttttccgtc ttttaaccca ggaaaaaagc 840

atactcgtgg tgaatctaac caatgacctc ttgcattgag gtacgcttca gtaaccagta 900

accacccctc cacccagcct gattagataa cgtacacgta ttttcatctt tttttcttca 960

tttctttttt ctttttttaa atcaattaac cttttttcaa attgatgaac tttttgcaaa 1020

ttttctggat attttttaaa atatatgaac ttcctttatg aatttttata aacttttttt 1080

caagttcgat gaacttttat aaaatttggc gaactatttt tcaactttga tgtacttttt 1140

ctctaaaaaa ttcacaattt tttgtcaaat tgatgagctt ttttcaaatc aatcatttgc 1200

ttttcgaaat ccatgcacat ttccgatttc cctaaatttt cgtcttttga gatttttttt 1260

tgttattttt tcaaatgtga tcgagcgaac caggggggaa taaaaaatgc gatggagcgc 1320

tcgcatctag atgggcccta ggctagatga gctatctcat ggacaccagc aatttatacg 1380

cttcgcttaa aaaggcaatt tatacgccca ttttactgag aaatcaccgt gttttctcga 1440

agtccaaaag tcctggatag atcgggtgca gagcgggatc ccaagaatta cacgtgcatc 1500

catgggcatg aacacctcct caacgcgagc cctgtcccgt gttgctgtag tatggccaat 1560

cagttcggat tagatggttt agctacgtga tgctgctact aggcctttta attagtgcaa 1620

ctgtatcctt ctcaaaaaaa taaatagtgc aactatatac atgcggccca aatgtagagc 1680

tacactagga cggtgcttgc ctctactgcg tgcgtatctg gagtgaggct tccctaaaat 1740

aaaataaaaa atctggagtg aggcgcgagc aagcggaaca gccgagacgg tatattctcc 1800

ttctcccgat cgcctcgcac gacaggctaa cagccttgat tccttcaagc gctgcatgcg 1860

gtcgcctcca cgggaagcgg gcggggtcgt taggagattc tgtggatctc accgatccat 1920

cctccacctc tctccctcct atatataggt gcgaggcccg gcagggtgaa tcatcaatca 1980

atctccttcc aagctctact cgtctcgcaa acgaaagagc agatcatatc agaccagagt 2040

gagtttgcat ctgcgagctg cgcca 2065

 

<210> 7

<211> 2049

<212> DNA

<213> wheat (Triticum aestivum L.)

<400> 7

cctcgttgat cagacaattt tggtggggga gcaagggagg aaaacgcaag ccgagttggg 60

tagcctggga cgagatgaca aagccaaagc atctaggcgg ccttggtttt agggatctgg 120

agatcttcaa tcttgcccta ctgtcgaagc aggcgtggag gatgctacaa aaccctacat 180

cgttgagcgc ccgcattctc aagggggttt acttccctga tgtatctctt tttgaagcaa 240

gcattgggaa ccatccatca caaatttggc gggcaatcct ggatgggcga gatatcatgg 300

tgcagggact tgtgagaaga attggaaacg gtgaaaccac ggacatctgg cacgataact 360

ggctacctcg cacacacatg aagcgaccat taacttctct ggttcaacat cctccgcaaa 420

aagtgtctaa actcatcaac aacaccactt gctcttggaa tgagcaacta gtccgggcta 480

cttttgttcc aatcgatgcg gaaactatca tgcagattcc tctctgcact agacagattg 540

aggatttttg ggtatggagc gaggacagac ggagtatttt ctctgtcaga actgcttacc 600

acatgatcca ccagaagaaa ctaagcagag aagcctggtt atatgagcaa gggggatcgt 660

ctcattcaca agctgatagt gagatctgga ctaagctatg gggttttaac gtaccatcga 720

agcttaaggt gttcctatgg agatttgaga agaacaccac accaaccgcg gcactcttgc 780

atcatcggaa catggcggac acgccagcgt gcgtcctttg cggagaggaa gacacttgga 840

gacacgcttt gctaaactgc acggtatccc ggagtacgtg ggctttgtca tctgagcata 900

tcatcgatgc actgagtaag aacgaagaag gtgacgcgaa gagatggctc tccgccatgc 960

acgaggcgct atcccaggag agttttacta cactcgtggt cacactctgg gccttgtggg 1020

gagcgcgccg aaaggcgatc catgagcaaa tataccaaac tccatttcag gtacatgcgt 1080

gtattcagtc ttacataaga gaactggaag ctatcaaaac tgtcaaaacg aggcgggatg 1140

gcaactctat tcctcgcccg acgagctaga ttcccccacc aacagggctg acaaaactga 1200

atgtagatgc tgcagtgggg aggggaagta gacacggctc tatagcggca atatcaagag 1260

actctactgg cctctttttg ggagcatcgg ctgtggtttt tgcaggcatt tccgatccgg 1320

ccactcttga gtgcatggcg accagggagg cgttgtccct tgcggatgac ctgaatgttt 1380

caactacaca agtggcgtcc gactcgaagg tggtggtcta ggacatccga gacaataatc 1440

ctacagcata tggtgcgatc atacatgaaa taatagagca tagatcaaat ttccttttgt 1500

gtaattttcg ccacgaattt aggagctcga atatcgaagc tcacaaactc acgaagcatg 1560

ctttatccct tccggctggc cgacatgttt ggttgggtca gccggacgga ctctctttcg 1620

tccatataaa cattgtgacg acgtaataaa agcttcggag tttgcctaaa aaaaaacatg 1680

cggtgcttgc ctttaccgcg tgcgtatctg gagtgaggcg cggccaagcg gaacagccga 1740

tcgagacggt gctcgaacat gcggtgcttc ccttctcctt ctcccgatcg cctcgcgcga 1800

caggcgtaac agccttgatt ccttcaagcg ctggatgcgg tcgcctccac gagaagcggg 1860

ctgggtcgtt aggagattcc gtggatctca ccgatccatt ctccacctct ctccctccta 1920

tatatacgtg ttccgcccgg cagagtgaat catcaatctc cctccttcca agctctactc 1980

ggctcacaaa cgaaagagca gagcagatca gatcagatca gcgtgagtct gcatccgcgt 2040

gctacgcca 2049

 

<210> 8

<211> 20

<212> DNA

<213> synthetic

<400> 8

caatggaggg gaggagaacc 20

 

<210> 9

<211> 27

<212> DNA

<213> synthetic

<400> 9

taataacaag accagtctac tccttgg 27

 

<210> 10

<211> 19

<212> DNA

<213> synthetic

<400> 10

gccaatggag gggagaact 19

 

<210> 11

<211> 26

<212> DNA

<213> synthetic

<400> 11

aactagacga ccagagtaga cagagc 26

 

<210> 12

<211> 20

<212> DNA

<213> synthetic

<400> 12

gagtctgcat ccgcgtgcta 20

 

<210> 13

<211> 25

<212> DNA

<213> synthetic

<400> 13

gcctccctag ctaataacaa agacc 25

 

<210> 14

<211> 37

<212> DNA

<213> synthetic

<400> 14

ctgcagtgat ttatacaaaa aatgtacaga gcatatg 37

 

<210> 15

<211> 29

<212> DNA

<213> synthetic

<400> 15

actagttggc gcagcgcgca gatgcaaac 29

 

<210> 16

<211> 23

<212> DNA

<213> synthetic

<400> 16

cggtatgaat attgcaaagc cag 23

 

<210> 17

<211> 23

<212> DNA

<213> synthetic

<400> 17

gccgtcgagt tttttgattt cac 23

 

 

Claims (10)

1. a promotor, has the characteristic of flower pesticide specifically expressing, it is characterized in that the nucleotide sequence of described promotor is selected from one of sequence of following group:

A () has SEQ ID NO:5, the sequence shown in 6 or 7;

(b) under strict conditions can with the DNA sequence dna of the DNA hybridization of sequence (a) described;

C () comprises the DNA sequence dna of at least 100 continuous nucleotides in SEQ ID NO:5,6 or 7; With

The DNA sequence dna of the arbitrary described complementary of (d) and (a)-(c).

2. an expression cassette, has the characteristic of specifically expressing in flower pesticide, it is characterized in that described expression cassette comprises promoter sequence according to claim 1.

3. an expression vector, is characterized in that described expression vector comprises expression cassette according to claim 2.

4. an engineering bacteria, is characterized in that described engineering bacteria contains expression vector according to claim 3.

5. in plant, express the method for object nucleotide sequence for one kind, described method comprises to plant materials importing DNA construct, described DNA construct contains the object nucleotide sequence that promotor and operability are connected to described promotor, and the nucleotide sequence of wherein said promotor is selected from one of sequence of following group:

A () has SEQ ID NO:5, the sequence shown in 6 or 7;

(b) under strict conditions can with the DNA sequence dna of the DNA hybridization of sequence (a) described;

C () comprises the DNA sequence dna of at least 100 continuous nucleotides in SEQ ID NO:5,6 or 7; With

The DNA sequence dna of the arbitrary described complementary of (d) and (a)-(c).

6. method according to claim 5, wherein said plant is monocotyledons, is preferably grass, is more preferably paddy rice or wheat.

7. method according to claim 5, wherein said object nucleotide sequence can be structure gene, regulatory gene, the inverted defined gene of structure gene, the inverted defined gene of regulatory gene or the tiny RNA that native gene can be disturbed to express, and it is at specific expressed fertility and the pollen germination that can regulate pollen in pollen development late period.

8. method according to claim 5, wherein said object nucleotide sequence can be enzyme or modifying enzyme, amylase, debranching factor and the polygalacturonase that coding impels carbohydrate degradation, more specifically as corn a amylase gene, growth hormone, rot B, cytotoxin gene, diphtheria toxin, DAM methylase, avidin, or can be selected from protokaryon regulator control system, can also be dominant male sterility gene.

9. the application of the promotor described in claim 1 in any one of following (a) to (d):

A () cultivates plants kind or strain;

B () is cultivated Pollination Fertilization ability and is strengthened plant variety or strain;

C plant variety that () cultivation Pollination Fertilization ability slackens or strain;

D () cultivates male sterile plants kind or strain.

10. application according to claim 9, is characterized in that, described plant is monocotyledons, and described monocotyledons is preferably grass, and more specifically, described grass is preferably paddy rice or wheat.