CN106834294A

CN106834294A - Rice Anther and seed efficient promoter POsAOM and its recombinant expression carrier and application

Info

Publication number: CN106834294A
Application number: CN201710209983.3A
Authority: CN
Inventors: 张毅; 吕俊; 杨昆; 黄远新; 管玉圣; 武丽娜
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2017-06-13
Anticipated expiration: 2037-03-31
Also published as: CN106834294B

Abstract

The invention discloses Rice Anther and seed efficient promoter POsAOM and its recombinant expression carrier and application, the promoter has the nucleotide sequence as shown in SEQ ID NO.23, promoter controllable genes of interest is high is expressed in flower pesticide and seed for this, pollen fertility and seed properties can be controlled, for producing new male sterible series of rice or fertile line, or for improved seed proterties, to realize rice fertility regulation and control or breed improvement.

Description

Rice Anther and seed efficient promoter POsAOM and its recombinant expression carrier and application

Technical field

The invention belongs to biological technical field, and in particular to Rice Anther and seed efficient promoter POsAOM, further relate to Recombinant expression carrier and application containing the promoter.

Background technology

Promoter is normally at the upstream of gene coding region, and cis-acting elements is contained in promoter, is that some are special DNA sequence dna, determines the combination of special transcription factor, so as to realize the transcriptional control of gene.Promoter also provides RNA polymerase and determines Position information, RNA polymerase is recognized and combines promoter relevant range, to start the transcription of RNA.Therefore, promoter is that gene turns Record institute is necessary, and special promoter determines the special expression pattern of the gene being attached thereto.Screening and separate some has tissue Or the promoter and the relevant important specific regulatory control element function of analysis of organ high expression level, in genetic engineering research There is extensive purposes.

Flower pesticide is that pollen is produced and ripe place, and normal pollen development needs many genes in flower pesticide or/and pollen Middle high efficient expression, using flower pesticide or pollen efficient promoter expression pollen development related gene or fragment, can regulate and control pollen and educate Property.Seed is the important results organ of cereal crops, and genes of interest can be expressed in seed using seed efficient promoter, with Obtain or improve the proterties such as yield, degeneration-resistant, quality and the mark of performance in seed.

Heterosis utilization has served very important improving rice yield, quality and resistance.At present, people are main The hybrid vigour of paddy rice is realized by Cytoplasm nuclear interaction male sterile line (three line method) and Temperature and light interaction (two line method).However, by Not free in Cytoplasm nuclear interaction male sterile line combo, available parent is limited, and seed produces, Temperature and light interaction more complicated than two line method Fertility influenceed by nature Thermo-Photo sensitive, the reason such as large area risk in hybrid seed production is big so that the hybrid vigour of paddy rice cannot be by this Two kinds of sterile lines find full expression.Flower pesticide and seed efficient promoter can regulate and control downstream gene specifically expressing in flower pesticide and kind Son, and pass through the expression that flower pesticide efficient promoter regulates and controls related gene, the development of flower pesticide and pollen can be influenceed to obtain male sterility System or male-fertile system, expressing related gene by seed efficient promoter can change seed correlated traits.Therefore, flower is separated Medicine and seed efficient promoter are significant to plant species improvement.

The content of the invention

In view of this, an object of the present invention is to provide flower pesticide and seed efficient promoter POsAOM, the promoter Regulation and control genes of interest high efficient expression controls the expression of the related genes such as microspore germination hole development in flower pesticide and seed, such as adjust The expression of control OsAOM genes (nucleotide sequence is as shown in SEQ ID NO.2), one GDSL lipase of the gene code, color The development of powder germinal aperature is relevant such that it is able to for producing new male sterible series of rice, or for expressing other mesh in seed Gene, played an important role in agricultural production, technical scheme is：

The nucleotide sequence of flower pesticide and seed efficient promoter POsAOM, the flower pesticide and seed efficient promoter POsAOM As shown in SEQ ID NO.23；Or sequence is added shown in SEQ ID NO.23, replace, insert or lack one or more Nucleotides generation with POsAOM promoter function identical nucleotide sequences；Or have with sequence shown in SEQ ID NO.23 The nucleotide sequence of 80% or more than 90% homology.

The second object of the present invention provides the recombinant expression carrier of flower pesticide and seed efficient promoter POsAOM, its technical side Case is：

Recombinant expression carrier containing the flower pesticide and seed efficient promoter POsAOM.

Preferably, the recombinant expression carrier is loading flower pesticide and seed efficient promoter POsAOM before genes of interest；More Preferably, the genes of interest is gus gene or gene or genetic fragment with regulation and control pollen fertility and seed properties.

The third object of the present invention is to provide the application of flower pesticide and seed efficient promoter POsAOM, and technical scheme is：

The flower pesticide and seed efficient promoter POsAOM answering in regulation and control plant pollen fertility and improved seed proterties With.

Preferably, the plant is cereal plantses.

It is furthermore preferred that the cereal plantses are the one kind or many in paddy rice, wheat, sorghum, barley, oat and rye Kind.

The beneficial effects of the present invention are：Flower pesticide disclosed by the invention and seed efficient promoter POsAOM, the promoter Regulate and control genes of interest high efficient expression in flower pesticide and seed, the adjusting and controlling rice pollen germination pore development related gene OsAOM in paddy rice Expression, influences the structure of pollen germination pore, and being obstructed when expression can produce male sterible series of rice；If be used in seed expression Related gene, can improve or increase seed properties.The invention also discloses flower pesticide and the weight of seed efficient promoter POsAOM Group expression vector, makes downstream gene high efficient expression in flower pesticide or seed under the regulation and control of POsAOM, is plant genetic engineering neck Domain studies gene and provides instrument in flower pesticide and seed high efficient expression, is with a wide range of applications.

Brief description of the drawings

In order that the purpose of the present invention, technical scheme and beneficial effect are clearer, the present invention provides drawings described below and carries out Explanation：

Figure l is OsAOM genes of the present invention normal bright extensive 63 and its mutant plants, morphology of the anther and pollen observation figure (A：Bright extensive 63 spike of rice；B：Osaom mutant spikes of rice；C：Bright extensive 63 flower pesticide；D：Osaom mutant flower pesticide；E：Bright extensive 63 pollen iodine Dye result；F：Osaom mutant pollen iodine contaminates result；G：Bright extensive 63 pollen scanning electron microscope；H：Osaom mutant pollen scannings Electron microscope).

Fig. 2 is the OsAOM assignments of genes gene mapping of the present invention and mutational site schematic diagram (A：OsAOM gene Primary Locations；B：OsAOM Gene finely positioning).

Fig. 3 is the expression point of flower pesticide of the present invention and seed efficient promoter POsAOM regulation and control OsAOM genes in Different Organs Analysis figure.

Fig. 4 is the tissue of flower pesticide of the present invention and seed efficient promoter POsAOM regulation and control OsAOM genes in each structure of flower pesticide Specifically expressing analysis chart.

Specific embodiment

With reference to specific embodiment, the present invention is expanded on further.These embodiments be merely to illustrate the present invention and without In limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, for example Sambrook equimolecular cloning experimentations room handbook (New York：Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.

Term " pollen germination pore is developed and pollen fertility gene " refers to encode have regulation and control pollen germination pore, cell membrane, carefully Kytoplasm and endhymenine normal development, can finally make the nucleotide sequence of the normal GDSL Lipase proteins of pollen fertility, base sequence Arrange the nucleotide sequence and its degenerate sequence as shown in 1-1200 in SEQ ID NO.2.Degenerate sequence refers to, positioned at SEQ In 1-1200 nucleotides of encoder block of ID NO.2 sequences, there are one or more codons to be encoded the letter of same amino acid And codon replace after produce sequence.Due to the degeneracy of codon, thus with SEQ ID NO.2 in 1-1200 The degenerate sequence of nucleotide sequence homology as little as about 70% can also encode out the sequence described in SEQ ID NO.1.The term is also Including can under moderate stringency conditions, more preferably under the high stringent condition with SEQ ID NO.2 in from nucleotides 1-1200 The nucleotide sequence of the nucleotide sequence hybridization of position.The term also include with SEQ ID NO.2 from 1-1200, nucleotides Nucleotide sequence homology at least 70%, preferably at least 80%, more preferably at least 90%, most preferably at least 95% core Nucleotide sequence.The term also includes encoding have and natural regulation and control pollen germination pore, cell membrane, cytoplasm and endhymenine Identical function albumen SEQ ID NO.2 in open reading frame sequence variant form.These variant forms are included (but simultaneously It is not limited to)：Several (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotides lack Lose, insertion and/or replace, and it is several (usually within 60, within preferably 30, more in 5 ' and/or 3 ' end additions Goodly within 10, most preferably within 5) nucleotides.

Term " promoter " refer to for RNA polymerase identification, with reference to and start transcription section of DNA sequence.Under DNA guidances The synthesis of RNA is referred to as transcription, and the starting of transcription is controlled by the promoter of DNA.

In embodiment, various carriers known in the art, such as commercially available carrier, including plasmid, clay etc. are can select.In life When producing the GDSL lipase of adjusting and controlling rice pollen germination pore development of the present embodiment, can be operable by the coded sequence of the albumen Ground is connected in expression regulation sequence, so as to form the development of adjusting and controlling rice pollen germination pore, pollen cytoplasmic, cell membrane and endhymenine The expression vector of GAP-associated protein GAP.

Associated nucleotide full length sequence or its fragment can generally use PCR TRAPs, recombination method or artificial synthesized method Obtain.For PCR TRAPs, relevant nucleotide sequence that can be according to the present embodiment designs primer, and with commercially available CDNA storehouses or the cDNA storehouses as prepared by conventional method well known by persons skilled in the art, or genomic DNA is used as template, amplification And obtain relevant sequence.When sequence is more long, it is often necessary to carry out twice or repeatedly PCR amplifications, then will amplify for each time again Fragment is stitched together by proper order.Once obtain relevant sequence, it is possible to had in large quantity with recombination method Close sequence.This is typically to be cloned into carrier, then is transferred to cell, then by conventional method from the host cell after propagation Isolated relevant sequence.

Embodiment 1, the acquisition of osaom mutant plants and morphologic observation

Osaom mutant derives from bright extensive 63 mutation of long-grained nonglutinous rice, makees male parent with normal bright extensive 63, with osaom mutant Hybridization, mutator is stored in heterozygote, it is necessary to allow heterozygote self propagated during mutant, offspring is that may separate out mutation Body osaom.Osaom mutant and bright extensive 63 hybridization, F₁Educated for all, selfing F₂Occur separating in generation, wherein normal plant It is 293, mutant strain is 120, and ratio meets 3:1Show the malesterile mutants phenotype by a core Gene mutation is caused.To the morphological observation of osaom mutant plants：Bright extensive 63 spike of rice is solid rear sagging (Fig. 1, A), and Osaom mutant small ears are shaky in vertical (Fig. 1, B)；Bright extensive 63 flower pesticide is sturdy full (Fig. 1, C), osaom mutant flower Medicine is relatively thin (Fig. 1, D)；Bright extensive 63 pollen iodine dye is black (Fig. 1, E), and osaom mutant pollen iodine dye after stain is shallow, and pollen is small And it is lopsided (Fig. 1, F)；Bright extensive 63 pollen has an obvious stopper shape port lid (Fig. 1, G) under ESEM at pollen hole, and is mutated The port lid missing of body osaom so that formed at germinal aperature empty (Fig. 1, H).

Embodiment 2, the positioning of OsAOM genes and clone

First, target group

OsAOM mutant and japonica rice Nipponbare are hybridized, selfing obtains F₂Generation, selection male sterile plants are positioning group Body.

2nd, paddy DNA is extracted

Parent is extracted using improved CTAB methods, is comprised the following steps：0.1-0.2 grams of blade (about half) is taken to be put into In small mortar, appropriate liquid nitrogen is added, powdery is ground at once, load 2m1 centrifuge tubes, add 700 μ L 100 DEG C the 1.5 of preheating × CTAB solution is put into 65 DEG C of water-baths in centrifuge tube after careful mixing, and centrifuge tube is taken out after 20 minutes, adds isometric chlorine Imitative/isoamyl alcohol, fierce to mix, 13000rpm is centrifuged 10 minutes, takes supernatant in new pipe, after adding 900 μ L absolute ethyl alcohols to mix- 20 DEG C put more than half an hour.The DNA centrifugations that will be separated out, 14000rpm is centrifuged 10 minutes.Remove supernatant, precipitation is used into 1mL volumes Fraction is that 70% ethanol is cleaned once, and centrifugal drying is dissolved in 200 μ L TE solution, 4 DEG C of Refrigerator stores.The individual plant of target group DNA is extracted using improved alkaline-heating method, is comprised the following steps：Shred leaflet tablet 1-2cm²0.5m1 centrifuge tubes are put into, 100 μ are added L concentration is the NaOH solution of 0.125M, and boiling water bath 30 seconds is subsequently adding the Tris-HCl (pH8.0) that 50 μ L concentration are 1.0M, most Add afterwards 100 μ L concentration be 0.125M HCl, boiling water bath after 2 minutes 4 DEG C of Refrigerator stores it is standby.

3rd, separation of group analysis is just positioned

137 pairs of polymorphism marks of design, including 42 pairs of SSR primers and 95 pairs of InDel molecular labeling primers.Wherein SSR primers according to delivered sequent synthesis (referring specifically tohttp://www.gramene.org.microsat/ ssr.html), the design of other InDel molecular labelings is according to comparing having announced for japonica rice Nipponbare and long-grained nonglutinous rice 9311 liang of strains Primer is designed in nucleotide sequence, the part to difference, many between 2 parent japonica rice Nipponbares of checking and long-grained nonglutinous rice OsAOM mutant State property.Parent and gene pool are expanded simultaneously with 137 pairs of designed InDel primers, PCR amplification programs are:10 μ L bodies In system, l μ L templates, l μ L 10pmol/ μ L sense primers, l μ L10pmol/ μ L anti-sense primers, l μ 10 × Buffer of L (Mg²⁰), L μ L 2mM dNTP, 0.l μ L Taq, 3.9 μ L water；PCR primer quality volume fraction is 10% PAGE glue electrophoresis, silver staining side Method is detected.Result finds that the primer R02004 on the 2nd chromosome shows polymorphism between parent and gene pool simultaneously, and individual plant is tested Card shows the primer and OsAOM gene linkages, in order to by the assignment of genes gene mapping between two primer sites, near primer R02004 Synthesize SSR or InDel primers 12 pairs in the range of 10.0Mb altogether, screened with parent osaom and Nipponbare, as a result show InDel primers R02001, SSR primer RM452, RM300, RM8254 show polymorphism between parent, are verified through individual plant, find R02001 has 18 plants of recon, and genetic distance is 4.66cM, and RM8524 has 49 plants of recon, and genetic distance is 12.69cM, Both exchange strain and do not include mutually, illustrate target gene between both.The work of Primary Location most OsAOM genes at last Be positioned between R02004 and RM300, both recons are respectively 3 plants and 18 plants, genetic distance be respectively 0.78cM and 4.66cM (Fig. 2, A), Primary Location primer sequence is as shown in table 1.

4th, finely positioning

In order to further reduce the screening scope of candidate gene, we have done finely positioning later, construct mutant The F of osaom × JH1₂For colony, 2688 plants of sterile individual plants are obtained altogether.The primer R02004 and RM300 of Primary Location it Between, design has synthesized 34 pairs of primers, using sterile parent, can educate parent and F₁Discrepant primer is screened, 5 pairs are selected altogether has Notable difference and with primer R02004, RM1106, RM13010, RM13013 and R02013 of the gene linkage, recessive individual plant Genotyping result show, total 187 plants of the single-swap strains of R02004, genetic distance is 3.48cM, and RM13013 has single cross 100 plants of strain is changed, genetic distance is 1.86cM, both single-swap strains do not include mutually, total 1 plant of the single-swap strains of RM1106, heredity Distance is 0.02cM, and total 11 plants of the single-swaps plant of RM13010, genetic distance is 0.20cM, and R02013 has 2 plants of single-swap strain, loses It is 0.04cM to pass distance, wherein, the exchange strain of RM1106 is comprised in the exchange strain of R02004, the single-swap strain of RM02013 It is comprised in the exchange strain of RM13010, the exchange strain of RM13010 is comprised in the exchange strain of RM13013, and R02004 Exchange strain not including mutually with the strain that exchanges of RM13013, illustrate OsAOM between two marks of RM1106 and R02013 (Fig. 2, A). http://www.gramene.org/On the chromosome of website paddy rice Nipponbare the 2nd, physical locations of the two marks are respectively 10997625 and 11045788 (version numbers：Oryza_sativa.IRGSP-1.0.21), both are at a distance of about 48Kb, its prediction There was only seven gene locis in the range of this.By BAR (http://bar.utoronto.ca/efprice/cgi-bin/ EfpWeb.cgi) analysis finds that wherein Loc_Os02g18870 is expressed in the inflorescence middle and later periods, is most likely to be in seven genes Our candidate gene OsAOM.Hair is compared by the sequencing to Loc_Os02g18870 genes in bright extensive 63 and mutant osaom It is existing：T → C base mutation is there occurs in the 4th extron of Loc_Os02g18870 genes in osaom mutant, causes to compile The amino acid of code becomes arginine (Arg) by cysteine acid (Cys).Therefore, we primarily determine that Loc_Os02g18870 is Our target gene OsAOM (Fig. 2, B).Gene code GDSL Lipase proteins, encode the amino acid sequence of the protein As shown in SEQ ID NO.1, nucleotide sequence is as shown in SEQ ID NO.2.

Table 1, assignment of genes gene mapping molecular labeling and its primer sequence

Primer	Sense primer (5 ' → 3 ')	Anti-sense primer (5 ' → 3 ')
			R02001	5’-cgataggcaactaaaacatt-3’(SEQ ID NO.3)	5’-cttgtcctcctgctctgta-3’(SEQ ID NO.4)
R02004	5’-gcaatttaacccttattcctg-3’(SEQ ID NO.5)	5’-gggaagaagaaagccattag-3’(SEQ ID NO.6)
			RM1106	5’-cggaaagtgaatcggagaac-3’(SEQ ID NO.7)	5’-gcaccacgctaagctaaacc-3’(SEQ ID NO.8)
R02013	5’-tgcctataatctcaac-3’(SEQ ID NO.9)	5’-ttctacactgcctgtg-3’(SEQ ID NO.10)
			RM13010	5’-cagtatggtcacaggaaacaacc-3’(SEQ ID NO.11)	5’-ctttgtgatcctctaatggtctgc-3’(SEQ ID NO.12)
RM13013	5’-ccgttaggagcctttcact-3’(SEQ ID NO.13)	5’-ctgagatgtttatgcctttcc-3’(SEQ ID NO.14)
			RM300	5’-gcttaaggacttctgcgaacc-3’(SEQ ID NO.15)	5’-caacagcgatccacatcatc-3’(SEQ ID NO.16)
RM8254	5’-aaagggacccacttgtcagc-3’(SEQ ID NO.17)	5’-gtcgaggatggatcgatgg-3’(SEQ ID NO.18)

Embodiment 3, flower pesticide and seed efficient promoter POsAOM are analyzed

Analysis finds that the OsAOM upstream region of gene 1254bp positioned at finely positioning has RNA polymerase recognition site, nucleosides Acid sequence is named as flower pesticide and seed efficient promoter, abbreviation POsAOM as shown in SEQ ID NO.19.

The Space-time speciality of RT-qPCR detection OsAOM expression.The sense primer of RT-qPCR reactions is RT-qPCRF:5'- Ggaatgcatcgactacatc-3 ' (SEQ ID NO.20), anti-sense primer is RT-qPCRR:5'- tggtggtcagaaaaccgtag-3’(SEQ ID NO.21).PCR reaction conditions：95 DEG C are denatured 30 seconds, and 60 DEG C are annealed 30 seconds, 72 DEG C extend 30 seconds, 40 circulations.Result shows that the gene expresses relatively low in root, stem, leaf, and highest is expressed in seed, in Expressed in later stage flower pesticide (Fig. 3) high.Showing the promoter of the gene has regulation and control downstream gene efficient table in flower pesticide and seed The ability for reaching, is flower pesticide and seed high efficient expression gene.

GUS detects the specificity of POsAOM.According to the primers as shown in SEQ ID NO.19, sense primer is POsAOMF:5'-ccaagcttgctctcgcaccaatcttgatc-3 ' (SEQ ID NO.22), underscore is the digestions of Hind III Site；Anti-sense primer is POsAOM R：5'-gctctagaTtccggccggcgagcgagctt-3 ' (SEQ ID NO.23), under The restriction enzyme sites of Xba I are scribed ss, are expanded with PCR, PCR reaction conditions are：95 DEG C of predegenerations 3 minutes；94 DEG C are denatured 45 seconds, 60 DEG C are annealed 45 seconds, and 72 DEG C extend 2 minutes, 28 circulations, and last 72 DEG C extend 10 minutes.PCR primer is through Ago-Gel electricity Swimming identification, reclaims the fragment of the 1254bp sizes containing flower pesticide and seed efficient promoter POsAOM, same with Hind III and Xba I When the fragment that reclaims of digestion and pCAMBIA1300-221 carriers and reclaim flower pesticide and seed efficient promoter POsAOM and PCAMBIA1300221 carrier frameworks, the flower pesticide that will be reclaimed and seed efficient promoter POsAOM and pCAMBIA1300221 carriers Skeleton is connected, and before POsAOM promoters is loaded into the gus gene of pCAMBIA1300221 carriers, obtains high containing flower pesticide and seed The recombinant expression carrier of effect promoter POsAOM, is named as：POsAOM:GUS carriers.By POsAOM:GUS carriers convert Agrobacterium LBA4404 competent cells, acquisition contains POsAOM:The LBA4404 of GUS carriers, is named as LBA4404：POsAOM:GUS.Will LBA4404：POsAOM:GUS infects rice callus, obtains transgenic paddy rice.By the activity analysis of beta-galactosidase (GUS) The expression pattern of POsAOM, takes transgenic paddy rice root, stem, the Ye Hehua of different developmental phases, is dyeed by GUS dyeing liquors, dye Tissue after color is processed with 75% ethanol decolorization, is then carried out GUS dyeing and is compared, and as a result display is in development early stage and mid-term Seed GUS dyeing it is most deep, the flower pesticide GUS in the development middle and later periods dyes deep, and the dyeing of other positions is shallower, indicates separation and obtains There is promoter activity positioned at OsAOM upstream region of gene 1254bp nucleotides, and can regulate and control that downstream gene is high to be expressed in Flower pesticide, therefore be flower pesticide efficient promoter.

In situ hybridization detects the tissue specificity of POsAOM downstream OsAOM gene expressions, and the probe that in situ hybridization is used is closed Into as follows：With rice cDNA as template, sense primer is OsAOMIF:5'-tgatgggatgatacaagg-3’(SEQ ID NO.24), anti-sense primer is OsAOMIR:5'-agatttaggtgacactatagaatcagagaaagtgaggtgcg-3’(SEQ ID NO.25), enter performing PCR amplification, it is marked with digoxin during amplification, prepared in situ hybridization probe.In situ hybridization is used Roche (Roche) company digoxin ribonucleic acid labelling kit (DIG RNA Labeling Kit (SP6/T7)) and digoxin Kit for detecting nucleic acid (DIG Nucleic Acid Detection Kit), specific steps are carried out according to kit specification, Wherein as a result in situ hybridization result as shown in figure 4, show that OsAOM gene expressions, in anther tapetum and sporidiole, are also demonstrated that Positioned at OsAOM upstream region of gene 1234bp nucleotides there is promoter activity, and downstream OsAOM gene specific tables can be regulated and controled Up in anther tapetum, being flower pesticide efficient promoter.

The pCAMBIA1300 carriers using transformation are not limited in the present invention, it is also possible to use other acceptable carriers；Build POsAOM:Genes of interest can be connected to POsAOM downstreams by GUS carriers as the expression vector of other genes, be replaced POsAOM:Gus gene in GUS carriers can in anther tapetum specifically expressing.

Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical Cross above preferred embodiment to be described in detail the present invention, it is to be understood by those skilled in the art that can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

<110>Southwest University；

<120>Rice Anther and seed efficient promoter POsAOM and its recombinant expression carrier and application

<160> 25

<210> 1

<211> 399

<212> PTR

<213>Paddy rice（Oryza sativa）

<220>

<223>Pollen germination pore is developed and pollen fertility gene amino acid sequence

<400> 1

Met Ala Leu Pro Phe Leu Leu Leu Leu Ala Phe Ala Leu Leu Phe

1 5 10 15

Pro Leu Ser Ala Pro Pro Arg Cys Cys Ser Ala Ala Pro Ala Ser

20 25 30

Ser Pro Pro Pro Ser Pro Pro Pro Ser Pro Ala Ala Ala Ala Ala

35 40 45

Ala Pro Arg Arg Thr Pro Leu Val Pro Ala Leu Phe Val Ile Gly

50 55 60

Asp Ser Thr Ala Asp Val Gly Thr Asn Asn Tyr Leu Gly Thr Leu

65 70 75

Ala Arg Ala Asp Arg Glu Pro Tyr Gly Arg Asp Phe Asp Thr Arg

80 85 90

Arg Pro Thr Gly Arg Phe Ser Asn Gly Arg Ile Pro Val Asp Tyr

95 100 105

Ile Ala Glu Lys Leu Gly Leu Pro Phe Val Pro Pro Tyr Leu Glu

110 115 120

Gln Asn Met Arg Met Gly Val Gly Ser Val Asp Leu Ser Asn Ile

125 130 135

Asp Gly Met Ile Gln Gly Val Asn Tyr Ala Ser Ala Ala Ala Gly

140 145 150

Ile Leu Ser Ser Ser Gly Ser Glu Leu Gly Met His Val Ser Leu

155 160 165

Ser Gln Gln Val Gln Gln Val Glu Asp Thr Tyr Glu Gln Leu Ser

170 175 180

Leu Ala Leu Gly Glu Ala Ala Thr Thr Asp Leu Phe Arg Lys Ser

185 190 195

Val Phe Phe Phe Ser Ile Gly Ser Asn Asp Phe Ile His Tyr Tyr

200 205 210

Leu Arg Asn Val Ser Gly Val Gln Met Arg Tyr Leu Pro Trp Glu

215 220 225

Phe Asn Gln Leu Leu Val Asn Ala Met Arg Gln Glu Ile Lys Asn

230 235 240

Leu Tyr Asn Ile Asn Val Arg Lys Val Val Met Met Gly Leu Pro

245 250 255

Pro Val Gly Cys Ala Pro His Phe Leu Trp Glu Tyr Gly Ser Gln

260 265 270

Asp Gly Glu Cys Ile Asp Tyr Ile Asn Asn Val Val Ile Gln Phe

275 280 285

Asn Tyr Ala Leu Arg Tyr Met Ser Ser Glu Phe Ile Arg Gln His

290 295 300

Pro Gly Ser Met Ile Ser Tyr Cys Asp Thr Phe Glu Gly Ser Val

305 310 315

Asp Ile Leu Lys Asn Arg Asp Arg Tyr Gly Phe Leu Thr Thr Thr

320 325 330

Asp Ala Cys Cys Gly Leu Gly Lys Tyr Gly Gly Leu Phe Met Cys

335 340 345

Val Leu Pro Gln Met Ala Cys Ser Asp Ala Ser Ser His Val Trp

350 355 360

Trp Asp Glu Phe His Pro Thr Asp Ala Val Asn Arg Ile Leu Ala

365 370 375

Asp Asn Val Trp Ser Gly Glu His Thr Lys Met Cys Tyr Pro Val

380 385 390

Asp Leu Gln Gln Met Val Lys Leu Lys

395 399

<210> 2

<211> 1200

<212> DNA

<213>Paddy rice（Oryza sativa）

<220>

<223>Pollen germination pore is developed and pollen fertility gene sequence

<400> 2

atggcgctcc ccttcctcct cctcctcgcc ttcgccctgc tcttcccgct ctccgctccc 60

ccgcgctgct gctccgcggc ccccgcctcc tcgccgcccc cgtccccgcc cccttcccct 120

gcggcggcgg cggcggcccc gcgccgcacg ccgctcgtcc cggcgctctt cgtgatcggc 180

gactccacgg cggacgtcgg caccaacaac tacctcggca cgctcgcccg cgccgaccgc 240

gagccgtacg gccgcgactt cgacacccgc cgccccacgg ggcgcttctc caacggccgc 300

atccccgtcg actacatcgc agagaagctg gggcttcctt ttgtgcctcc ataccttgaa 360

cagaacatgc gcatgggtgt cggcagtgtc gacctcagca acattgatgg gatgatacaa 420

ggtgtcaact atgcatccgc ggcagctggc attctctcca gcagtggttc tgagctggga 480

atgcatgtgt cgctgagcca gcaggtgcag caggttgagg acacatatga gcagctctct 540

ctggctctcg gggaggcagc aacaactgac cttttcagaa agtccgtgtt ctttttctca 600

atcgggagca acgacttcat ccactattac ctgcgcaatg tgtctggcgt ccagatgcgt 660

tacctcccat gggagttcaa ccagcttctt gtcaatgcaa tgaggcagga aatcaagaat 720

ttgtacaata tcaatgttcg gaaggtcgtc atgatgggcc tccctcctgt tggctgcgca 780

cctcactttc tctgggagta cggcagtcaa gacggggaat gcatcgacta catcaataac 840

gtcgtgattc agttcaacta tgccctgaga tacatgtcta gtgaattcat ccgccagcac 900

ccaggctcta tgatcagtta ctgtgatact tttgaggggt ctgtggacat actgaagaat 960

cgtgaccgct acggttttct gaccaccact gatgcctgct gtgggctggg gaagtatggg 1021

ggcctgttca tgtgtgttct tccacagatg gcgtgcagcg acgcgtcgag ccatgtctgg 1081

tgggacgagt tccaccccac ggatgctgtg aaccgaatcc tggctgataa tgtgtggtct 1141

ggtgagcata ccaagatgtg ctatcctgtg gatttgcagc agatggtaaa actcaagtag 1200

<210> 3

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>R02001 sense primers

<400> 3

cgataggcaa ctaaaacatt 20

<210> 4

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>R02001 anti-sense primers

<400> 4

cttgtcctcc tgctctgta 19

<210> 5

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>R02004 sense primers

<400> 5

gcaatttaac ccttattcct g 21

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>R02004 anti-sense primers

<400> 6

gggaagaaga aagccattag 20

<210> 7

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RM1106 sense primers

<400> 7

cggaaagtga atcggagaac 20

<210> 8

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RM1106 anti-sense primers

<400> 8

gcaccacgct aagctaaacc 20

<210> 9

<211> 16

<212> DNA

<213>Artificial sequence

<220>

<223>R02013 sense primers

<400> 9

tgcctataat ctcaac 16

<210> 10

<211> 16

<212> DNA

<213>Artificial sequence

<220>

<223>R02013 anti-sense primers

<400> 10

ttctacactg cctgtg 16

<210> 11

<211> 23

<212> DNA

<213>Artificial sequence

<220>

<223>RM13010 sense primers

<400> 11

cagtatggtc acaggaaaca acc 23

<210> 12

<211> 24

<212> DNA

<213>Artificial sequence

<220>

<223>RM13010 anti-sense primers

<400> 12

ctttgtgatc ctctaatggt ctgc 24

<210> 13

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>RM13013 sense primers

<400> 13

ccgttaggag cctttcact 19

<210> 14

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>RM13013 anti-sense primers

<400> 14

ctgagatgtt tatgcctttc c 21

<210> 15

<211> 21

<212> DNA

<213>Artificial sequence

<220>

<223>RM300 sense primers

<400> 15

gcttaaggac ttctgcgaac c 21

<210> 16

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RM300 anti-sense primers

<400> 16

caacagcgat ccacatcatc 20

<210> 17

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RM8254 sense primers

<400> 17

aaagggaccc acttgtcagc 20

<210> 18

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>RM8254 anti-sense primers

<400> 18

gtcgaggatg gatcgatgg 19

<210> 19

<211> 1254

<212> DNA

<213>Paddy rice（Oryza sativa）

<220>

<223>Promoter POsAOM

<400> 19

gctctcgcac caatcttgat caagacaaca tcatgaccgc atattatgga gagttgttga 60

aaactgatcg ccaggtgttt gagactcaac ttgaagaatt gcttcaaaag atggcttcgt 120

actaccgtaa gacgaggcaa ggcatcatca agcaacaaaa attcatgtta cccatccatg 180

caaaatcaaa ggtaacaacg attctagcag tgcctattct aacatatgaa gttgtgatgg 240

tgatgttatt tctagtttaa ataatcggtt tacttctttg aatactacct ttgagaaaac 300

ctttagtact accttggaaa gccctagatg atcacatgag tgagttagag aatcgactaa 360

atagccggtt ccttggtggt gcatgtattt catgcaacaa cgagcatatg catggtattc 420

cttcatattt tcatatacca aaattttctg tacaacaaga ttggccgatg aaaaataccc 480

acaattccct cgatacaata gatcctagat tcactcctac gatactgtat gctggtccga 540

ccgatgtgtt attgctggtc agaccggacg catcgatcgg accgacgggt gtaataccac 600

cagtccgact agaggcagca gccgatccga ttggagggac atcaccggtc agactacgct 660

cggcatgttt cgtagaatca gctcatgtct ggtgctcctc taaaagacat tgacatgtgg 720

ggtccacggg ctgactcaac aagattggat aaactgcctc ctaaaccgtt caaggagtca 780

atttgtaatg gttttgtgag ttggaggata ggttataggg atgcgattca accatgggca 840

tgagttgagg gaagcaaagt agacttaggg cctgttcact ttgatgaaaa aaaaaacctt 900

accaaatttt ggtaggcaac ttgccaaaat tttggcagga tttcttatat agttatcaaa 960

atttggcagc aaattaaata tagtcacttt tttggcaaat ttactaaaat ttggtaaggt 1020

tgaaaatgac atcaaagtaa acaggccctt attacccacg ggaatggtgt ggggtgggct 1080

ggtaatataa gcccagaaac caatccatcc agcccagcaa ctgcgaggtc ggctgctagt 1140

ctaacgtgca cccaagccat caccccacac gtgaaaaatc cccgctccac ccgcgccgcg 1200

ccgcgcccag gtagttcagc cgcgcgccaa cccaagctcg ctcgccggcc ggaa 1254

<210> 20

<211> 19

<212> DNA

<213>Artificial sequence

<220>

<223>RT-qPCRF sense primers

<400> 20

ggaatgcatc gactacatc 19

<210> 21

<211> 20

<212> DNA

<213>Artificial sequence

<220>

<223>RT-qPCRR anti-sense primers

<400> 21

tggtggtcag aaaaccgtag 20

<210> 22

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>POsAOMF sense primers

<400> 22

ccaagcttgc tctcgcacca atcttgatc 29

<210> 23

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>POsAOM R anti-sense primers

<400> 23

gctctagatt ccggccggcg agcgagctt 29

<210> 24

<211> 18

<212> DNA

<213>Artificial sequence

<220>

<223>OsAOMIF sense primers

<400> 24

tgatgggatg atacaagg 18

<210> 25

<211> 41

<212> DNA

<213>Artificial sequence

<220>

<223>OsAOMIF sense primers

<400> 25

agatttaggt gacactatag aatcagagaa agtgaggtgc g 41

Claims

1. flower pesticide and seed efficient promoter POsAOM, it is characterised in that：The flower pesticide and seed efficient promoter POsAOM's Nucleotide sequence is as shown in SEQ ID NO.23；Or sequence shown in SEQ ID NO.23 it is added, substitution, insertion or missing one Or more than one nucleotides generation with POsAOM promoter function identical nucleotide sequences；Or with SEQ ID NO.23 Shown sequence has the nucleotide sequence of 80% or more than 90% homology.

2. the recombinant expression carrier containing flower pesticide described in claim 1 and seed efficient promoter POsAOM.

3. the recombinant expression carrier of flower pesticide and seed efficient promoter POsAOM according to claim 2, it is characterised in that：Institute It is loading flower pesticide and seed efficient promoter POsAOM before genes of interest to state recombinant expression carrier.

4. the recombinant expression carrier of flower pesticide and seed efficient promoter POsAOM according to claim 3, it is characterised in that：Institute Genes of interest is stated for gus gene or with the gene or genetic fragment for regulating and controlling pollen fertility and seed properties.

5. flower pesticide described in claim 1 and seed efficient promoter POsAOM are in regulation and control plant pollen fertility and improved seed proterties In application.

6. application according to claim 5, it is characterised in that：The plant is cereal plantses.

7. application according to claim 6, it is characterised in that：The cereal plantses be paddy rice, wheat, sorghum, barley, One or more in oat and rye.