CN101863969B - Separated rice female fertility relevant protein as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses a separated rice female fertility relevant protein. The primary structure of the separated rice female fertility relevant protein is an animo acid sequence as shown in SEQ ID NO.2, or the animo acid sequence shown in SEQ ID NO.2 forms a derived protein with the same function by substitution, missing or adding of one or a plurality of animo acid residues, or the animo acid sequence shown in SEQ ID NO.2 has at least 70% of homology and has the derived protein with the same function. The invention also provides a gene for encoding the protein and derivatives thereof. The protein and the encoding gene thereof can be used for controlling the fertility of rice female organs, or controlling the growth of pollen tubes of the rice, or identifying the rich female fertility.

Description

Isolating rice female fertility relevant protein, its encoding sox and application thereof

Technical field

The present invention relates to plant genetic engineering, particularly relate to a kind of isolating rice female fertility relevant albumen, its encoding sox and application thereof.

Background technology

Plant female sterile phenomenon extensively exists at nature, on plants such as Chinese pine, soybean, corn and paddy rice, is seen in report.The female sterile of plant is normally caused by the heteroplasia of female organ, comprises the heteroplasia of ovule, blastular, ovum.According to angiosperm reproduction fetology, female sterile can be divided three classes, and the first kind is no female organ or the differentiation of incomplete female organ; Second type for female organ lacks normal blastular, and reason is ovule or megaspore impaired development; Grow normally for blastular for the 3rd type, but the fetal development of after fertilization is unusual.With regard to paddy rice, what study at most is the female sterile phenomenon that the indica rice filial generation produces, and the overwhelming majority belongs to blastular to grow abnormal structural abortion.

At present, people mainly concentrate on generation, cytology, the fetology aspect of plant female sterile to plant female sterile phenomenon Study.Although the gene that more existing plant female fertilities are relevant is cloned successively, the molecular mechanism of plant female fertility control is also clear generally.Therefore, the clone and the functional study of plant female fertility controlling gene, molecule mechanism and the application of sterile gene in hybridisation rice production controlled for clear and definite plant female fertility have great importance.

Summary of the invention

The purpose of this invention is to provide a kind of rice female fertility relevant albumen, its encoding sox, the carrier that contains this encoding sox and cell, and use.

For achieving the above object, technical scheme of the present invention is:

The contriver is through extensive studies, and application drawing position clone technology obtains the gene that a kind of female fertility is correlated with from paddy rice, and the disappearance of this gene causes the misgrowth in style of paddy pollen pipe, inventor general's the called after pollen tube growth gene (PTB1) that is obstructed.This expression of gene is rice female fertility basis normally, disturbs this expression of gene or reduce the PTB1 protein-active to cause the sterile or setting percentage reduction of paddy rice.Therefore this gene can be used as the molecule marker of rice female fertility index, in rice heterosis utilization, is with a wide range of applications.

On this basis, the present invention provides a kind of isolating rice female fertility relevant protein, and its primary structure is the aminoacid sequence shown in SEQ ID NO.2; Or has the deutero-albumen identical through what replacement, disappearance or the interpolation of one or more amino-acid residues formed for the aminoacid sequence shown in SEQ ID NO.2 with its function; Or for to have 70% homology at least and to have the deutero-albumen identical with its function with the aminoacid sequence shown in the SEQ ID NO.2.

Simultaneously, the present invention provides a kind of isolating rice female fertility genes involved, and the nucleotides sequence of said gene is classified as: the above-mentioned proteic polynucleotide of encoding; Or with the coding above-mentioned proteic polymerized nucleoside acid sequence complementary polynucleotide; Or with continuous 100 the Nucleotide identical deutero-polynucleotide of coding in the above-mentioned proteic polynucleotide.

Other aspects of the present invention are because the disclosed content of this paper is conspicuous to those skilled in the art.

Among this paper, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification like polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with other materials that exist separately, then for separation and purification.

Among this paper, " isolating female fertility albumen ", " isolating PTB1 albumen " or " isolating PTB1 polypeptide " are meant that PTB1 albumen does not contain natural relative other albumen, lipid, carbohydrate or other materials basically.Those of ordinary skill in the art can use the purified technology of protein purifying PTB1 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.

Among this paper, term " contains ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute ".

Among this paper, the plant of described " sterile " is meant a kind of plant, when it grows to certain stage (like the stage of maturity) under suitable growth conditions, significantly reduces (as reducing by 40% than the setting percentage of similar plant of growth under the same conditions; Preferably reduce by 60%; More preferably reduce by 80%; Reduce by 95% or more best).

Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferably recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.

The present invention also comprises the proteic fragment of PTB1, verivate and analogue.Among this paper, term " fragment ", " verivate " are meant with " analogue " and keep identical biological function of PTB1 albumen of the present invention or active polypeptide basically.Polypeptide fragment of the present invention, verivate or analogue can be that (1) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferably conservative type amino-acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (2) have the polypeptide of substituted radical in one or more amino-acid residues; Or (3) mature polypeptide and another compound are (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (4) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the protein sequence of this polypeptide of purifying or fusion rotein).According to the definition of this paper, these fragments, verivate and analogue belong to the scope of well known to a person skilled in the art.

In the present invention, term " PTB1 albumen " refers to have SEQ ID NO.2 polypeptide of sequence.This term also comprises the variant form albumen identical function, SEQ ID NO.2 sequence with SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 or still less 1-8 or 1-5 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in this area, when replacing, can not change proteinic function usually with the similar amino acid of performance.Again such as, add one or several general designations at C-terminal and/or N-terminal and also can not change proteinic function.This term also comprises proteic active fragments of PTB1 and reactive derivative.

The variant form of polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low rigorous degree condition can with coded albumen of the DNA of PTB1 encoding histone DNA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of PTB1 to obtain.Described homologous sequence is meant to have with SEQ ID NO.2 sequence has at least 50%, and preferably at least 60%, 70%, 80%, the polypeptide of at least 85%, 90%, 95% homogeny more preferably.The present invention also provides other polypeptide, as comprises PTB1 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of PTB1.Usually, it is proteic at least about 20 successive amino acid that this fragment has PTB1, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 100 successive amino acid, best at least about 150 continuous amino acids.

The present invention also provides the analogue of PTB1 albumen or polypeptide.These analogues and the natural proteic difference of PTB1 can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain through various technology, as through radiation or be exposed to the random mutagenesis that mutagenic compound produce, can also pass through site-directed mutagenesis method or other known molecular biological technology.Analogue also comprises having the analogue that is different from natural L-amino acid residue (like D-amino acid), and has non-natural analogue that exist or synthetic amino acid (like beta-amino acids).Should be understood that polypeptide of the present invention is not limited to above-mentioned representative polypeptide of giving an example.

(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modify and also comprise glycosylation.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the polypeptide that has improved its anti-proteolyze performance or optimized solubility property.

In the present invention; " PTB1 albumen conservative property variant protein (polypeptide) " refers to compare with the aminoacid sequence of SEQ IDNO.2; There are 20 at the most, preferably at the most 10, more preferably at the most 5; 3 amino acid is substituted by similar performance or close amino acid and the polypeptide that forms at the most best, and the albumen identical functions of reservation and SEQ ID NO.2 sequence.

The present invention also provides the polynucleotide sequence of code book invention PTB1 albumen or its conservative property variation polypeptide.

Polynucleotide of the present invention (gene) can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or two strands.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO.2 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO.2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO.2.

The polynucleotide of the mature polypeptide of coding SEQ ID NO.2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.

Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.

The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the verivate of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and he possibly be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.

The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, further preferably at least 80%, the polynucleotide of at least 90% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) at lower ionic strength and hybridization under the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (V/V) methane amide, 0.1% calf serum/0.1Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 70%, preferably more than at least 80%, more preferably more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is at least 50 Nucleotide better, preferably more than at least 100 Nucleotide.The amplification technique (like PCR) that the accounting fragment can be used for adjusting is to confirm and/or the proteic polynucleotide of separation coding PTB1.

PTB1 pyrenoids thuja acid full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For the pcr amplification method; Can be according to relevant nucleotide sequence disclosed by the invention; Especially ORFs designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and obtain relevant sequence.When sequence is longer, usually need carries out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by correct order.

In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, then through ordinary method from increment with host cell separate and obtain relevant sequence.

In addition, can also utilize the method for synthetic to synthesize relevant sequence, especially fragment length more in short-term.Usually, earlier synthetic a plurality of small segments can obtain the very long fragment of sequence connecting then.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change in the introducing protein sequence of the present invention with crossing chemosynthesis.

The present invention also relates to comprise polynucleotide carrier of the present invention, and produce host cell through genetically engineered, and produce the method for polypeptide according to the invention through recombinant technology with carrier of the present invention or PTB1 albumen coded sequence.

Through the DNA recombinant technology of routine, the PTB1 albumen of reorganization is expressed or produced to polymerized nucleoside acid sequence of the present invention capable of using.Generally speaking following steps are arranged:

The proteic polynucleotide of coding PTB1 or the varient that get with the present invention, or transform or the transduction proper host cell with the recombinant expression vector of these polynucleotide;

In suitable medium, cultivate host cell;

Separation, protein purification from substratum or cell.

In the present invention, PTB1 albumen polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell is viral or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can use.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.

Method well-known to those having ordinary skill in the art can be used to make up and contains PTB1 encoding histone dna sequence dna and the suitable expression vector of transcribing, translate wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the promotor suitable in the expression vector, and is synthetic to instruct mRNA's.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.

In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate with Tetrahydrofolate dehydrogenase, neomycin resistance and green fluorescent protein (GFP) like eukaryotic cell, or be used for colibacillary kantlex or amicillin resistance.

Comprise above-mentioned suitable dna sequence dna and suitable promotor or control sequence carrier, can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like vegetable cell.Representational example has: intestinal bacteria, streptomyces, Agrobacterium; Eukaryotic cell such as yeast; Vegetable cell etc.

The purposes of the albumen that provides of the present invention or its encoding sox is used for:

Control paddy female organ fertility;

The growth of control paddy pollen pipe;

Identify the molecule marker of female fertility.

The invention still further relates to a kind of fertility through the adjusting plant and improve the method for plant, this method comprises: regulate PTB1 expression of gene or activity in the said plant.The expression activity that promotes still to suppress PTB1 depends primarily on the proterties of the required improvement of plant and decides.When needs increase the setting percentage of plant, can realize through improving in the said plant PTB1 expression of gene or activity; When needs reduce the setting percentage of plant, even it is thorough when sterile that plant is showed, and can realize through reducing in the said plant PTB1 expression of gene or activity.

The method that increases the PTB1 expression of gene is that this area is known.For example, can make plant overexpression PTB1 through changing the expression vector that carries the PTB1 encoding sox over to; Thereby maybe can drive and strengthen PTB1 gene or its homogenic expression through strong promoter; Perhaps strengthen this PTB1 expression of gene through enhanser (like paddy rice waxy gene first intron, Actin gene first intron).The strong promoter that is applicable to the inventive method includes but not limited to: the Ubi promotor of the Actin1 of 35S promoter, paddy rice, Actin2 promotor, corn etc.

The method that suppresses PTB1 genetic expression also is that this area is known, for example, can disturb (RNAi) or clpp gene to come out to realize through antisense or RNA.

As a kind of optimal way of the present invention, the method for the plant of acquisition PTB1 gene high expression is following:

The Agrobacterium of carrying expression vector is provided, and described expression vector contains the proteic encoding sequence of described PTB1;

Vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make said PTB1 albumen coded sequence change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;

Select the vegetable cell, tissue or the organ that change said PTB1 albumen coded sequence over to;

Vegetable cell, tissue or neomorph in the step (3) are become plant.

Wherein, can adopt any suitable conventional means, comprise that reagent, temperature, pressure condition implement this method.

The present invention also comprises the antagonist or the agonist of PTB1 albumen or its encoding sox.Because the antagonist of PTB1 or agonist can be regulated the active of PTB1 or express, therefore, the antagonist of described PTB1 or agonist also can be through regulating the fertility of plant to the influence of PTB1, thereby reach the purpose of plant trait improvement.

The antagonist of described PTB1 is meant the proteic activity of any PTB1 of reduction, reduction PTB1 gene or protein stability, the proteic expression of downward modulation PTB1; Reduce the material of PTB1 albumen effective acting time or inhibition PTB1 gene transcription and translation; These materials all can be used for the present invention, as the useful material of coordinate plant growth.The agonist of described PTB1 is meant the proteic activity of any PTB1 of raising, keep PTB1 gene or protein stability, the proteic expression of rise PTB1; Improve the material of PTB1 albumen effective acting time or promotion PTB1 gene transcription and translation; These materials all can be used for the present invention, as the useful material of coordinate plant growth.

According to an embodiment of the present invention, a kind of PTB1 albumen is provided, the ORFs of its total length (ORF) sequence is encoded one and is contained 384 amino acid whose protein (SEQ ID NO.2) shown in SEQ ID NO.1.Sequential analysis at wild-type paddy rice and female-sterile mutant shows that 252 nucleotide deletions are arranged in two mutants ORF, caused a terminator codon that shifts to an earlier date, and makes to translate the brachymemma of premature termination expressing protein, thereby causes the phenotype that paddy rice is sterile.Through transgenic method, normal wild type gene PTB1 imported to recover in the two mutants normally can educate phenotype.

Technique scheme has following advantage:

Find rice female fertility controlling gene (PTB1) from the paddy rice kind first, the discovery of this gene can be produced evidence for disclosing rice female fertility control molecule mechanism, also can new technological approaches be provided for the female fertility control improvement of plants such as paddy rice.In addition, consider that fertility is controlled at the importance in the heterosis utilization, the PTB1 gene particularly is with a wide range of applications aspect the sterile line propagation at heterosis utilization.

Description of drawings

Fig. 1 is the phenotype after the PTB1 transgenation;

Fig. 2 mainly is positioned in the tenuigenin for wild-type PTB1 albumen;

Fig. 3 mainly expresses in gynoecium and vascular bundle for wild-type PTB1 gene;

Fig. 4 is a PTB1 gene major control rice fertility.

Embodiment

Below in conjunction with accompanying drawing and embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.The TP of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Unless stated otherwise, otherwise per-cent and mark calculate by weight.

The clone of embodiment 1 PTB1 gene

1, the map based cloning of PTB1 gene

In rice restorer 202R, find a sterile two mutants, find that through cytology and pollen tube observation two mutants shows as specificity female sterile (Figure 1A), its sterile basic reason is two mutants pollen tube growth retardation (Figure 1B) in style.Utilize G630 etc. to do maternal and two mutants hybridization, make up segregating population,, and cloned this gene (PTB1) through Fine Mapping and map based cloning technology with the assignment of genes gene mapping.

Sequence comparing analysis shows that 252 nucleotide deletions are arranged in two mutants ORF, caused a terminator codon that shifts to an earlier date, and makes to translate the brachymemma of premature termination expressing protein, thereby causes the phenotype that paddy rice is sterile.Through transgenic method, normal wild type gene PTB1 imported to recover in the two mutants normally can educate phenotype.

The sequence of wild-type PTB1 gene shown in SEQ ID NO.1, the brand-new new albumen of encoding, its sequence is shown in SEQ ID NO.2.Further discover in rice genome, there is not the homology copy of PTB1.The total length ORF length of PTB1 gene is 1155bp, 384 amino acid of encoding.

2, PTB1 full length gene cDNA and promotor clone

RNA with the fine kind of normal wild type paddy rice Japan is a template; The synthetic first chain cDNA; The oligonucleotide of 5 ' and 3 ' end of this PTB1 gene of usefulness ORF sequence is as PCR primer (SEQ ID NO:3 and SEQ ID NO:4); Primerstar increases with high-fidelity Taq enzyme, obtains the full-length cDNA amplified production of the PTB1 gene of about 1.5K.This amplified production through Bamh I and the Spe I restriction enzyme digestion into respective limits property restriction endonuclease MCS of carrier pBluescript sk+ of recombinating, and is carried out sequence verification to recon.The transition plasmid vector of this reorganization is called pBS-PTB1.

The oligonucleotide sequence of 5 ' end is (SEQ ID NO.3):

5’-GCCGGATCCATGGCGATGCGGGGGGTCGA-3’

The oligonucleotide sequence of 3 ' end is (SEQ ID NO.4):

5’-GGACTAGTGCGATCCGGCAGCTGATGC-3’

3, PTB1 gene promoter clone

In the present embodiment; DNA with the fine kind of normal wild type paddy rice Japan is a template; With the about 1.5K segmental 5 ' in said PTB1 gene start codon front and 3 ' end oligonucleotide as PCR primer (SEQ ID NO:5 and SEQ ID NO:6); Increase with high-fidelity Taq enzyme KOD Plus, obtain the promotor amplified production of the PTB1 gene of about 1.5K.This amplified production through Pst I and the Bamh I restriction enzyme digestion into respective limits property restriction endonuclease MCS of carrier pBluescriptsk+ (being purchased) of recombinating, and is carried out sequence verification to recon.The transition plasmid vector of this reorganization is called pBS-PPTB1.

The oligonucleotide sequence of 5 ' end is (SEQ ID NO.5):

5’-AAACTGCAGAACATCTAATCTCATTAAATTTAAC-3’

The oligonucleotide sequence of 3 ' end is (SEQ ID NO.6):

5’-GCCGGATCCGGCCTGCTAGCTAGATCTGGC-3’

Embodiment 2PTB 1 expression of gene pattern

In the present embodiment; With Bamh I and Spe I restriction enzyme digestion pBS-PTB1 and PA7-YFP; Reclaim the PTB1 full length gene cDNA fragment that the pBS-PTB1 enzyme is cut postdigestive about 1.5K; Be connected into the identical restriction enzyme site of cutting postdigestive PA7-YFP through enzyme, recon is carried out the enzyme evaluation of cutting and check order, make up cellular localization transient expression carrier PA7-PT-YFP.Utilize particle gun bombardment onion epidermis cell, the Arabidopis thaliana of instantaneous conversion simultaneously protoplastis is observed luciferase expression with Laser Scanning Confocal Microscope.The result shows that like figure the PTB1 gene is mainly expressed (Fig. 2) in cell on tenuigenin.

The inventor has also carried out the tissue expression The Location to this gene.With Pst I and Bamh I restriction enzyme digestion pBS-P _PTB1And P1300-GUS, reclaim pBS-P _PTB1Enzyme is cut the PTB1 gene promoter fragment of postdigestive about 1.5K, is connected into the identical restriction enzyme site of cutting postdigestive P1300-GUS through enzyme, and recon is carried out the enzyme evaluation of cutting and check order, and makes up tissue positioned expression vector P1300-P _PTB1-GUS.The expression of gus gene has the PTB1 gene promoter to drive in this expression vector.With this carrier arabidopsis thaliana transformation, find that GUS mainly efficiently expresses (Fig. 3 A) in pistil stigma, in stamen and vascular bundle, also have than high expression level (Fig. 3 B) simultaneously, confirm that said PTB1 gene is relevant with the plant female fertility.

The test of embodiment 3PTB1 trans-genetic hybrid rice transgenic

Present embodiment employing expression vector PHB (Mao etc., 2005, PNAS102:12270-12275, commercially available) as the genetically modified carrier of paddy rice.A bacterium replication orgin of this vector encoded (ori), kalamycin resistance gene (Kan ^r), hygromycin gene (Hyg ^r), herbicide resistance gene (Bar ^r), 2 * 35S promoter, NOS gene termination signal sequence and be used to connect the segmental MCS of purpose (MCS).Can insert the PTB1 gene or its fragment is built into the transgenic plasmid at restriction endonuclease sites.

1, PTB1 gene complementation expression vector establishment

In the present embodiment, at first use EcoR I and Bamh I restriction enzyme digestion pBS-P _PTB1And PHB, reclaim pBS-P _PTB1Enzyme is cut the promoter fragment of the PTB1 gene of postdigestive about 1.5K, and the carrier framework (getting rid of 2 * 35S promoter) of cutting postdigestive about 12K through enzyme with the PHB that reclaims is connected, and recon is carried out the enzyme evaluation of cutting and check order, structure intermediate carrier PHB-P _PTB1

With Bamh I and Sac I restriction enzyme digestion pBS-PTB1 and PHB-P _PTB1, the full-length cDNA fragment of the postdigestive about 1.5K of pBS-PTB1 is connected into PHB-P _PTB1Bamh I and Sac I site in, recon is carried out the enzyme evaluation of cutting and check order.So successfully make up PHB-P _PTB1-PTB1, wherein the PTB1 expression of gene is by self promoters driven.

2, PTB1 gene overexpression vector construction

With Hind III and Sac I restriction enzyme digestion pBS-PTB1 and PHB, the full-length cDNA fragment of the postdigestive about 1.5K of pBS-PTB1 is connected in the Hind III and Sac I site of PHB, recon is carried out the enzyme evaluation of cutting and check order.So successfully make up PHB-PTB1, wherein the PTB1 expression of gene is driven by 2 * 35S strong promoter.

3, PTB1 gene interference carrier makes up

In the present embodiment; RNA with the fine kind of normal wild type paddy rice Japan is a template; The synthetic first chain cDNA; The terminal oligonucleotide of fragment of the about 500bp of 5 ' end of this PTB1 gene of usefulness ORF sequence increases with high-fidelity Taq enzyme KOD Plus as PCR primer (SEQ ID NO:7 and SEQ ID NO:8), obtains the forward interference fragment amplified production of the PTB1 gene of about 500bp.Use PCR primer (SEQ ID NO:9 and SEQ IDNO:10) amplification to obtain the reverse interference fragment simultaneously.This forward amplified production through Hind III and the EcoR I restriction enzyme digestion into respective limits property restriction endonuclease MCS of carrier pBs-G (the Sma I of pBs has been inserted GUS intron fragment) of recombinating, and is carried out sequence verification to recon.The transition plasmid vector of this reorganization is called pBS-R1-G.This reverse amplified production through Spe I and the Sac I restriction enzyme digestion into respective limits property restriction endonuclease MCS of carrier pBS-R1-G of recombinating, and is carried out sequence verification to recon.The transition plasmid vector interference structure intermediate carrier pBS-R1-G-R2 of this reorganization.Use Hind III and Sac I restriction enzyme digestion pBS-R1-G-R2 and PHB at last, the interference structure fragment of the postdigestive about 2K of pBS-R1-G-R2 is connected in the Hind III and Sac I site of PHB, recon is carried out the enzyme evaluation of cutting and check order.So successfully make up PHB-R1-G-R2, wherein the expression of this interference structure is driven by 2 * 35S strong promoter.

The oligonucleotide sequence of 5 ' end is (SEQ ID NO.7):

5’-CCCAAAGCTTCTTCATCTTCCGCCTTCTCA-3’

The oligonucleotide sequence of 3 ' end is (SEQ ID NO.8):

5’-CGGAATTCTGCCTCAACTGCCTCTCTTT-3’

The oligonucleotide sequence of 5 ' end is (SEQ ID NO.9):

5’-CGGAGCTCCTTCATCTTCCGCCTTCTCA-3’

The oligonucleotide sequence of 3 ' end is (SEQ ID NO.10):

5’-GGACTAGTTGCCTCAACTGCCTCTCTTT-3’

4PTB1 gene transformation rice test

With above-mentioned PHB-P _PTB1-PTB1, PHB-PTB1 and PHB-R1-G-R2 import Agrobacterium EHA105 through freeze-thaw method.From dull and stereotyped (4 ℃ of preservations) the picking list bacterium colony of Agrobacterium tumefaciens in YEP liquid nutrient medium (each 50mg/L of Km and Rif); 28 ℃ of shaking culture 12～18h; Get 1～5mL bacterium liquid then and receive in the 100mL YEP liquid nutrient medium (containing Syringylethanone 100 μ mol/L), it is rare to respective concentration (OD=0.5) to survey the OD value behind the shaking culture 4h.Fresh bacterium liquid is collected thalline in 8000rpm, 4 ℃, 5min, and be resuspended in 1/3rd AAM substratum mentioned.Add in the sterilization triangular flask that is placed with eugonic embryo callus and soak 25min, dry up surperficial bacterium liquid again, callus is transferred in being total on the substratum 28 ℃ of dark 2～3d that cultivate.Cultured calli is being blown on the worktable about 4h through sterilized water with after containing the rinsed with sterile water of Cef 500mg/L altogether, and 28 ℃ of dark 5～7d of cultivation in pre-culture medium transfer.Pre-incubated callus is transferred continuation 3～4 weeks of cultivation on the screening culture medium that contains Hyg and Cef, again resistant calli is transferred on the screening culture medium that only contains Hyg, screen 1～2 time.Get resistant calli and transfer on division culture medium, 28 ℃ of illumination differentiation.The differentiation seedling is transferred in root media, in 28 ℃ of 3～4 weeks of illumination cultivation, about open culture refining seedling 7d, is transplanted to the land for growing field crops at last.

Wherein, PHB-P _PTB1-PTB1 transforms female-sterile mutant FS, and PHB-PTB1 and PHB-R1-G-R2 transform normal wild type material 202R, and PHB-PTB1 also part transforms female-sterile mutant FS.The female-sterile mutant callus obtains to adopt young fringe to cultivate, and all the other orthodox materials are mature embryo source tissue culture and induce.

Use the herbicide screening transformed plant behind the regeneration plant transplant survival that obtains; Positive plant extracts the total DNA of blade, further identifies transformed plant through PCR.For investigation fertility phenotype, verify the PTB1 gene function with transgenic T1.

Embodiment 4PTB1 gene function analysis

Obtain the complementary FS two mutants transfer-gen plant of expressing of paddy rice PTB1 like embodiment 2 described methods, T1 observes rice fertility for plant with transgenic.Result such as Fig. 4 A, the fertility of changeing the plant of the complementary expressing gene of PTB1 has obtained recovery, becomes normally and can educate, and it is necessary to show that the PTB1 expression of gene is that paddy rice is kept normal fertility.

Like the transfer-gen plant of embodiment 2 described methods acquisition paddy rice PTB1 overexpressions, T1 observes rice fertility for plant with transgenic.Result such as Fig. 4 C, the setting percentage of the FS plant of commentaries on classics PTB1 overexpression gene improves than the FS plant of the complementary expression of PTB1.The high expression level that shows the PTB1 gene helps improving the paddy rice setting percentage.

Obtain paddy rice PTB1 expression like embodiment 2 described methods and receive the interferential transfer-gen plant, T1 observes rice fertility for plant with transgenic.Result such as Fig. 4 B, the setting percentage of the plant of PTB1 down regulation of gene expression reduces.Simultaneously, like Fig. 4 D, in the strain system of almost detecting less than PTB1 genetic expression, the plant setting percentage shows sterile near two mutants FS level.The downward modulation expression that shows the PTB1 gene can reduce the paddy rice setting percentage.

Can find out that from above-mentioned analysis PTB1 is that paddy rice is kept the necessary gene of normal fertility, lacks this gene and can cause plant sterile, raise this genetic expression and help the raising of plant setting percentage, reduce the setting percentage that this genetic expression then reduces plant.

The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.

Sequence table

< 110>Sichuan Agricultural University

< 120>isolating rice female fertility relevant protein, its encoding sox and application thereof

<130>KHP10112596.4

<160>10

<170>PatentIn version 3.5

<210>1

<211>1155

<212>DNA

<213>PTB1

<400>1

atggcgatgc ggggggtcga tttcaagtgg tacgatggat tcttcctctc catgctcgcc 60

accagcctaa tcattgtctc catcaactgg aagaggtatc gtctctgcgc ccacccgttg 120

cacatatgga tcgtggtcga ctacaccacc gtcttcatct tccgccttct catgttcgtc 180

gataatggcc ttgccgccgg catgggattg gatcttggat ggcaacagag atatgctcgt 240

ttttgtggga gaattattgt cttgtcggtt cttgtgcttc ttctctatcc ctttctttgg 300

gtttggactg tgataggaac attgtggttt agcactgcaa gaggctgttt acccgaggaa 360

ggacaaaaat ggggcttcct tatatggctg cttttcagct actgtggtct cgcctgtatt 420

gcatgtgtgg ctgttggaaa gtggctaagc cgaaggcatg ctctccagca gagggcacaa 480

cagggaatac cagtctctga atatggggtt ttggttgaca tgatccgtgt gcctgattgg 540

gcatttgagg ccgttggctt ggaaatgaga ggaatgggcc aagatactgc atatcatcct 600

ggtctttatt taacagctgc tcaaagagag gcagttgagg cactgattca agaactcccg 660

aagttcagac tgaaagctgt tcctacagac tgcagtgagt gtccaatctg ccttgaagaa 720

ttccatgttg gcaacgaggt ccgtgggctc ccgtgcgcgc acaatttcca tgtggagtgc 780

atcgaccagt ggctccggct gaacgtcaag tgcccccgtt gccggtgctc cgtcttcccc 840

aacctcgacc tgagcgcgct caacaacctc cggccatcct ccgagccgga ccggccgtcg 900

gccagcgagg tgacagcggc gacgatggcg cggtacgtga ggtcgtcgca gccggctggg 960

cagagctacc tgctgcggct gcaggggctt ctgctgcggc aggtggtggt tcggcatggc 1020

ggcagcgacg acatggcgag cgccggcgct cttcatgtgg ccgccgcggt gactgcgccg 1080

gcgaccaccg gcggcgtgga gagcgagctg cctagcatag tggttgatgg tgggcatcag 1140

ctgccggatc gctga 1155

<210>2

<211>384

<212>PRT

<213>PTB1

<400>2

Met Ala Met Arg Gly Val Asp Phe Lys Trp Tyr Asp Gly Phe Phe Leu

1 5 10 15

Ser Met Leu Ala Thr Ser Leu Ile Ile Val Ser Ile Asn Trp Lys Arg

20 25 30

Tyr Arg Leu Cys Ala His Pro Leu His Ile Trp Ile Val Val Asp Tyr

35 40 45

Thr Thr Val Phe Ile Phe Arg Leu Leu Met Phe Val Asp Asn Gly Leu

50 55 60

Ala Ala Gly Met Gly Leu Asp Leu Gly Trp Gln Gln Arg Tyr Ala Arg

65 70 75 80

Phe Cys Gly Arg Ile Ile Val Leu Ser Val Leu Val Leu Leu Leu Tyr

85 90 95

Pro Phe Leu Trp Val Trp Thr Val Ile Gly Thr Leu Trp Phe Ser Thr

100 105 110

Ala Arg Gly Cys Leu Pro Glu Glu Gly Gln Lys Trp Gly Phe Leu Ile

115 120 125

Trp Leu Leu Phe Ser Tyr Cys Gly Leu Ala Cys Ile Ala Cys Val Ala

130 135 140

Val Gly Lys Trp Leu Ser Arg Arg His Ala Leu Gln Gln Arg Ala Gln

145 150 155 160

Gln Gly Ile Pro Val Ser Glu Tyr Gly Val Leu Val Asp Met Ile Arg

165 170 175

Val Pro Asp Trp Ala Phe Glu Ala Val Gly Leu Glu Met Arg Gly Met

180 185 190

Gly Gln Asp Thr Ala Tyr His Pro Gly Leu Tyr Leu Thr Ala Ala Gln

195 200 205

Arg Glu Ala Val Glu Ala Leu Ile Gln Glu Leu Pro Lys Phe Arg Leu

210 215 220

Lys Ala Val Pro Thr Asp Cys Ser Glu Cys Pro Ile Cys Leu Glu Glu

225 230 235 240

Phe His Val Gly Asn Glu Val Arg Gly Leu Pro Cys Ala His Asn Phe

245 250 255

His Val Glu Cys Ile Asp Gln Trp Leu Arg Leu Asn Val Lys Cys Pro

260 265 270

Arg Cys Arg Cys Ser Val Phe Pro Asn Leu Asp Leu Ser Ala Leu Asn

275 280 285

Asn Leu Arg Pro Ser Ser Glu Pro Asp Arg Pro Ser Ala Ser Glu Val

290 295 300

Thr Ala Ala Thr Met Ala Arg Tyr Val Arg Ser Ser Gln Pro Ala Gly

305 310 315 320

Gln Ser Tyr Leu Leu Arg Leu Gln Gly Leu Leu Leu Arg Gln Val Val

325 330 335

Val Arg His Gly Gly Ser Asp Asp Met Ala Ser Ala Gly Ala Leu His

340 345 350

Val Ala Ala Ala Val Thr Ala Pro Ala Thr Thr Gly Gly Val Glu Ser

355 360 365

Glu Leu Pro Ser Ile Val Val Asp Gly Gly His Gln Leu Pro Asp Arg

370 375 380

<210>3

<211>29

<212>DNA

< 213>artificial sequence

<400>3

gccggatcca tggcgatgcg gggggtcga 29

<210>4

<211>27

<212>DNA

< 213>artificial sequence

<400>4

ggactagtgc gatccggcag ctgatgc 27

<210>5

<211>34

<212>DNA

< 213>artificial sequence

<400>5

aaactgcaga acatctaatc tcattaaatt taac 34

<210>6

<211>30

<212>DNA

< 213>artificial sequence

<400>6

gccggatccg gcctgctagc tagatctggc 30

<210>7

<211>30

<212>DNA

< 213>artificial sequence

<400>7

cccaaagctt cttcatcttc cgccttctca 30

<210>8

<211>28

<212>DNA

< 213>artificial sequence

<400>8

cggaattctg cctcaactgc ctctcttt 28

<210>9

<211>28

<212>DNA

< 213>artificial sequence

<400>9

cggagctcct tcatcttccg ccttctca 28

<210>10

<211>28

<212>DNA

< 213>artificial sequence

<400>10

ggactagttg cctcaactgc ctctcttt 28

Claims (7)

1. an isolating rice female fertility relevant protein is characterized in that, its primary structure is the aminoacid sequence shown in SEQ ID NO.2.

2. an isolating rice female fertility genes involved is characterized in that the nucleotides sequence of said gene is classified as: the said proteic polynucleotide of coding claim 1; Or with coding claim 1 said proteic polymerized nucleoside acid sequence complementary polynucleotide.

3. rice female fertility genes involved as claimed in claim 2 is characterized in that, its nucleotide sequence is shown in SEQ ID NO.1.

4. contain claim 2 or 3 described expression carrier or recombinant vectorss.

5. contain the host cell that is integrated with claim 2 or 3 described genes in the described carrier of claim 4 or its genome.

6. claim 2 or the 3 described genes application in control paddy female organ fertility or pipe growth of control paddy pollen or evaluation rice female fertility.

7. a method of regulating rice female fertility is characterized in that, said proteic activity of claim 1 or the said expression of gene of claim 2 improve the paddy rice rice fertility in the raising paddy rice; Said proteic activity of claim 1 or the said expression of gene of claim 2 descend the paddy rice rice fertility in the reduction paddy rice.

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