CN101161675A - Rice big grain gene and uses thereof - Google Patents
Rice big grain gene and uses thereof Download PDFInfo
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- CN101161675A CN101161675A CNA2006101171135A CN200610117113A CN101161675A CN 101161675 A CN101161675 A CN 101161675A CN A2006101171135 A CNA2006101171135 A CN A2006101171135A CN 200610117113 A CN200610117113 A CN 200610117113A CN 101161675 A CN101161675 A CN 101161675A
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Abstract
The present invention discloses a rice large-grain gene GW2 and the application thereof, the GW2 gene can be used to control the grain size of the crop, improve the yield or quality of the crop, regulate the cycle duration of cellular mitosis, and used as a molecular marker to identify the species of the crop to be a large-grain one or a small-grain one. The present invention also discloses a method to ameliorate the crop. The gene GW2 has a wide prospect on high-yield breeding of crops such as rice.
Description
Technical field
The invention belongs to gene engineering and phytology field.More particularly, the present invention relates to a kind of new rice big grain gene and application thereof.
Background technology
Rice yield is heavy by grain, spike number, grain several three big proterties determine.These proterties are the quantitative characters by a plurality of quantitative character genes (QTL) control.Use the QTL that molecular marking technique has been located many output correlated character over nearly 10 years, but by successful cloned genes also seldom.
Japanese scientists in 2005 have successfully cloned the gene (Gnla) of 1 control paddy rice grain number, and have illustrated the molecule mechanism of this gene, and relevant achievement is published on the Science, causes international extensive concern.In addition, be positioned a trisomic report that control grain length gene GS3 is cloned, it does not carried out the correlation function analysis though have recently.
Yet the clone and the functional study of the genes involved that the control grain is heavy also do not obtain real success so far.Grain heavily is one of most important proterties of decision output, carries out the clone and the functional study of a heavy gene, so that be applied to the high crop yield molecular breeding, has great theory significance and practical value.
Summary of the invention
The object of the present invention is to provide a kind of new rice big grain gene and the application thereof that can be used for controlling the crop kernel size.
In a first aspect of the present invention, a kind of isolating rice big grain albumen is provided, this albumen is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the wide or heavy function of grain of control crop grain by (a) polypeptides derived.
In another preference of the present invention, described dietary protein origin is in paddy rice.
In another preference of the present invention, described albumen has E3 ligase enzyme activity.
In a second aspect of the present invention, a kind of isolating polynucleotide are provided, these polynucleotide are selected from down group:
(i) the proteic polynucleotide of the described rice big grain of coding; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
In another preference of the present invention, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.
In another preference of the present invention, these polynucleotide are selected from down group:
(1) nucleotide sequence shown in the SEQ ID NO:1; Or
(2) nucleotide sequence shown in the 255-1532 position among the SEQ ID NO:1.
In a third aspect of the present invention, a kind of carrier is provided, it contains described polynucleotide.
In a fourth aspect of the present invention, a kind of genetically engineered host cell is provided, it contains described carrier; Or be integrated with described polynucleotide in its genome.
In a fifth aspect of the present invention, the purposes of described rice big grain albumen or its encoding gene is provided, be used for: the grain of control crop kernel is wide or grain is heavy (preferred, wide or grain is heavy for the grain that increases crop kernel, thereby increase crop yield); Regulate cell processes; Or as the molecule marker of identifying crop big grain kind and granule kind.
In another preference of the present invention, described cell processes includes but not limited to: cell fission, signal conduction, cell elongation.
In a sixth aspect of the present invention, the method for a kind of improvement crop (preferred, wide or grain is heavy for the grain that increases crop kernel) is provided, this method comprises: (A) reduce the expression of rice big grain gene in the described crop; Or (B) function is reduced or the rice big grain gene or the albumen of afunction import in the crop.
In another preference of the present invention, function is reduced or the rice big grain gene or the albumen of afunction import in the crop of granule kind, thereby can promote crop kernel to become big.Preferred, in the rice big grain gene or albumen importing crop with afunction.
In another preference of the present invention, the GW2 gene fragment of selecting technology to obtain from the crop of big grain kind with molecule marker imports in the crop of granule kind.This method is a non-transgenic method, does not have potential safety hazard.
In a seventh aspect of the present invention, provide the agonist or the antagonist of a kind of described rice big grain albumen or its encoding gene.
In a eighth aspect of the present invention, provide a kind of can with described rice big grain gene or its coded protein specific bonded antibody.
In a ninth aspect of the present invention, a kind of method for preparing transgenic plant is provided, described method comprises step: described polynucleotide are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
In another preference of the present invention, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains described proteic encoding gene;
(s2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (s1), thereby make described proteic encoding gene change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell or tissue or the organ that changes described protein coding gene over to; And
(s4) vegetable cell in the step (S3) or tissue or neomorph are become plant.
In a tenth aspect of the present invention, provide a kind of preparation described proteic method, described method comprises: cultivate described host cell, collect and obtain described albumen.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown paddy rice GW2 antisense transgene plant (AS3, AS24, the comparison of the grain of grain AS25) and acceptor kind " in spend 11 (ZH11) ".
Fig. 2 has shown paddy rice GW2 justice transfer-gen plant (S21, S22, the comparison of the grain of grain S24) and acceptor kind " in spend 11 (ZH11) ".
Fig. 3 has shown will the influence heavy to granule kind grain of big grain GW2 gene fragment importing granule kind with method for selecting molecular marker (non-transgenic method).
Embodiment
The inventor has at first found a kind of new gene that the crop kernel grain is wide and/or grain is heavy of controlling through extensive and deep research, and the disappearance of the function of this gene can produce the phenotype of big grain, inventor general's called after rice big grain gene (GW2).Test confirms, the grain type of the transfer-gen plant of GW2 gene overexpression diminishes, grain heavily reduces, the grain type that the GW2 gene reduces the transfer-gen plant of expressing becomes greatly, grain is brought up again height, and visible GW2 gene will play an important role in the higher yield of crops breeding, be with a wide range of applications.Finished the present invention on this basis.
As used herein, described " crop " includes but not limited to: grass.Preferred, described grass includes but not limited to: wheat, barley, corn, Chinese sorghum etc.
As used herein, " 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 as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating GW2 albumen or polypeptide " is meant that described GW2 albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use the purified technology of protein purifying GW2 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).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 GW2, derivative and analogue.As used herein, term " fragment ", " derivative " are meant biological function or the active polypeptide that keeps natural GW2 albumen of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying or fusion rotein).These fragments of definition, derivative and analogue according to this paper belong to the known scope of those skilled in the art.
In the present invention, term " GW2 albumen " refers to have the SEQ ID NO:2 polypeptide of sequence of GW2 protein-active.This term also comprises having and variant form GW2 albumen identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best, also better for 1-8,1-5) amino acid whose disappearance, insertion and/or replacement, and at C-terminal and/or N-terminal interpolation one or several (being generally in 20, preferably is in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of GW2 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 tight degree condition can with coded albumen of the DNA of GW2 protein D NA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-GW2 to obtain.The present invention also provides other polypeptide, as comprises GW2 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of GW2.Usually, this fragment have the GW2 protein sequence at least about 20 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of GW2 albumen or polypeptide.These analogues and the proteic difference of natural GW2 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 by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(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 (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " GW2 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.For example, these conservative propertys variation polypeptide can carry out amino acid according to table 1 and replace and produce.
Table 1
| Amino-acid residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also provides the polynucleotide sequence of code book invention GW2 albumen or its conservative property variation polypeptide.
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.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:1 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:1.
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 derivative 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 it may 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%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least 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 more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or the proteic polynucleotide of separation coding GW2.
Should understand, though GW2 gene of the present invention preferably derives from paddy rice, but that derive from other plant and other gene paddy rice GW2 gene height homology (as have more than 80%, as 85%, 90%, 95% even 98% sequence homogeny) are also within the scope that the present invention considers.The Method and kit for of aligned sequences homogeny also is that this area is known, for example BLAST.
GW2 pyrenoids thuja acid full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence 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 must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and the host cell that produces through genetically engineered with carrier of the present invention or GW2 albumen coded sequence, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the GW2 albumen of reorganization.In general following steps are arranged:
(1). with the proteic polynucleotide of coding GW2 of the present invention (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, GW2 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 virus or other carriers.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.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 GW2 encoding histone dna sequence dna and suitable transcribing/the translate expression vector of control signal.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 suitable promotor in the expression vector, and is synthetic to instruct mRNA.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 Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary kantlex or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell such as yeast; Vegetable cell etc.
When polynucleotide of the present invention are expressed in higher eucaryotic cells, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.
Persons skilled in the art all know how to select appropriate carriers, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method, paddy rice rataria conversion method etc.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain genetically modified plant.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The GW2 albumen or the polypeptide of reorganization are of use in many ways.For example be used to screen antibody, polypeptide or other part that promotes or resist the GW2 protein function.Can be used for seeking the valuable peptide molecule that can suppress or stimulate the GW2 protein function with the reorganization GW2 protein screening peptide library of expressing, proteic antagonist of the GW2 that is obtained or agonist are also included within the scope of the present invention.
On the other hand, the present invention also comprises GW2 DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into GW2 gene product or fragment.Preferably, refer to that those can combine with GW2 gene product or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Preferable, among the present invention antibody be those can in conjunction with and suppress the molecule of GW2 function.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the GW2 gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing GW2 albumen or its has antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology prepare (see people such as Kohler,
Nature256; 495,1975; People such as Kohler,
Eur.J.Immunol.6:511,1976; People such as Kohler,
Eur.J.Immunol.6:292,1976; People such as Hammerling,
In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).Each antibody-like of the present invention can utilize the fragment or the functional zone of GW2 gene product, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of GW2 gene product; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
Antibody among the present invention can be used for suppressing the function of GW2 in the crop.
Utilize albumen of the present invention,, can filter out with GW2 albumen or gene interactional material takes place, as acceptor, inhibitor, agonist or antagonist etc. by various conventional screening methods.
Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (Microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) amplification in vitro with the special primer of GW2 albumen and also can detect the proteic transcription product of GW2.
The invention still further relates to a kind of crop kernel that makes and become big method, this method comprises GW2 gene or its homogenic expression in the described plant of reduction.The method that reduces the expression of GW2 gene or its homologous gene is that this area is known, such as waiting the expression that neutralizes or stop GW2 by adopting the proteic antagonist of GW2, antibody.Perhaps, can realize that the seed of crop becomes big by in plant, importing the GW2 gene fragment of forfeiture GW2 gene function.
GW2 gene of the present invention also can be used as the cue mark of true hybrid in gene transformation plant offspring's tracking mark and the hybrid seeding process.
In addition, the invention still further relates to the application of molecule marker selection technology in the big grain of farm crop SOYBEAN IN HIGH-YIELD BREEDING of GW2 gene.
As an example of the present invention, the inventor is with WY3 (especially big grain kind) and rich short No. 1 (granule kind) hybridization structure genetic group that accounts for, the application molecular marking technique has been located one and has been positioned at the second new gene GW2 that chromosomal control paddy rice grain is wide and grain is heavy, and has cloned this gene by the map based cloning technology.The genome length of GW2 gene is 5.877kb, includes 8 exons, 7 introns, its cDNA length is 1.634Kb, total length ORF (Open Reading-Frame) length is 1.278Kb, 1 albumen that contains 425 bases of encoding, and the protein product molecular weight is estimated as 47KDa.
In an example of the present invention, find to take place on a kind of the 4th exon of GW2 gene the disappearance of 1 base, produce the premature termination codon, cause to lack 310 amino acid, make GW2 gene function disappearance, thereby produce the phenotype of big grain.
As another example of the present invention, the inventor obtains the mutant gene of a GW2 from rice varieties " spend 11 ", (be ORF the 787th) C → T variation causes occurring in advance terminator codon because the 1041st of the base of this gene, causes its encoded protein disappearance partial amino-acid series.Yet this plant still shows the granule phenotype, although prove deletion mutation has taken place, yet has still kept the proteic function of GW2.
As another example of the present invention, the inventor obtains the mutant gene of a GW2 from rice varieties " Huang Huazhan ", C → T variation has also taken place in the 1041st of the base of this gene (be ORF the 787th), occur terminator codon in advance, cause its encoded protein disappearance partial amino-acid series.Yet this plant still shows the granule phenotype, has also promptly kept the proteic function of GW2.
GW2 albumen contains a RING functional domain, and in an example of the present invention, the inventor shows that through protease activity functional analysis result GW2 has the E3 ligase enzyme activity in the proteolytic degradation approach.The proteolytic degradation approach participates in cell processes such as cell fission, so the size of seed may to have E3 ligase enzyme activity relevant with GW2.
In an example of the present invention, the inventor places the full-length cDNA of the GW2 gene of granule rice varieties under the control of CaMV 35S promoter by justice and antisense orientation respectively, is building up to respectively on the paddy rice expression vector.To spend in 11 in 2 kinds of expression vectors importing rice varieties by agrobacterium-mediated transformation.Positive transformed plant is carried out the phenotype observation to be shown, antisense GW2 obviously increases owing to this expression of gene of reduction causes the grain type of transfer-gen plant, grain is heavy obviously increases weight (Fig. 1), and just overexpression GW2 then causes that owing to increasing this expression of gene the grain type of transfer-gen plant diminishes, grain heavily reduces (Fig. 2).Show that the GW2 gene plays an important role, and can significantly improve rice yield by this gene of molecular designing in control paddy rice grain is heavy.
In an example of the present invention, the inventor by molecule marker selection technology will big grain kind the GW2 gene fragment import that to improve grain in the granule kind significantly heavy, thousand seed weight is increased to 32 grams by 22 original grams, amplification is up to 46% (Fig. 3).This technology can be exempted the transgenosis safety evaluation owing to be non-transgenic method, does not have potential safety hazard.Show that the GW2 gene is with a wide range of applications in high-yield breeding of crops such as paddy rice.
Major advantage of the present invention is:
(1) separation obtains a kind of new rice big grain gene first, reduces this expression of gene and can make the seed of crop (as paddy rice) become big, thereby can increase the output of crop.
(2) rice big grain gene GW2 of the present invention can be used as control crop kernel size, and a gene that improves the yield and quality is applied to the improvement of crop varieties.And, the molecule marker selection technology of GW2 gene can be used for the big grain of farm crop SOYBEAN IN HIGH-YIELD BREEDING.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the method for announcing in the following document: Carl W.Dieffenbach and Gabriela S.Devksler eds.PCRPrimer:A Laboratory Manual.Cold Spring Harbor Laboratory Press, 1995.Or the condition of advising according to manufacturer.
The acquisition of embodiment 1 rice big grain gene
WY3 is huge grain rice varieties (thousand seed weight reaches 48 grams), and richly shortly accounts for No. 1 (FAZ1) and promote fine quality (thousand seed weight only 22 restrains) for the granule paddy rice.The inventor utilizes molecule marker to locate new gene (or QTL) GW2 that control paddy rice grain is wide and grain is heavy with WY3 and the rich short hybridization structure genetic group that accounts for No. 1, and this gene is positioned on second karyomit(e).Further utilize the map based cloning technology to clone the GW2 gene, its gene order is shown in SEQ ID NO:1, and the albumen that contains 425 bases of encoding is shown in SEQ ID NO:2.
Sequence comparing analysis shows that there are 3 nucleotide variations in the GW2 gene of big grain kind, and 2 are the base replacement, but do not cause amino acid change; And in addition 1 variation is the disappearance that 1 base (A316, ORF the 316th) takes place on the 4th exon, causes the reading frame displacement, produce the premature termination codon in 30 base back thereafter, cause to lack 310 amino acid, make GW2 gene function disappearance, thereby produce the phenotype of big grain.
The cDNA sequence of paddy rice GW2 gene (from rich short the accounting for No. 1 of granule kind, being normal gene) following (SEQ ID NO:1), wherein, underscore is depicted as open reading frame.
ccaccccgagaaagccaaaaaaaagaaaaattgtttttcaaaaaaaaagctcgcctagccctcgcgtcgtcgtccccatcaccccccctcct
ccgctccgagtacgcgtgcgtataccaccacctccatctccaccaccgtatgtatctacggtgaggcggcggcggcggaggaggaggaggag
ggggagtggtgagggtttcatctgcggaggaggagggaggaggagggaggagggtagatctgggaggggg
ATGGGGAACAGGATAGGGGGGA
GGAGGAAGGCGGGGGTGGAGGAGAGGTACACGAGGCCGCAGGGGCTGTACGAGCACAGGGACATCGACCAGAAGAAGCTCCGGAAGCTGATT
CTCGAGGCCAAGCTCGCGCCGTGCTACATGGGCGCCGACGACGCCGCCGCCGCCGCCGACCTCGAGGAGTGCCCCATCTGCTTCCTGTACTA
CCCAAGTCTTAACCGATCAAAGTGTTGCTCAAAAGGGATATGCACCGAGTGCTTTCTCCAAATGAAACCAACTCACACTGCTCAGCCTACAC
AATGTCCATTCTGCAAAACTCCCAGTTATGCTGTGGAGTATCGTGGTGTAAAGACAAAGGAGGAAAGGAGCATAGAACAATTTGAAGAGCAG
AAAGTCATAGAAGCACAAATGAGGATGCGCCAGCAAGCACTTCAAGATGAAGAAGATAAGATGAAAAGAAAACAGAACAGGTGCTCTTCTAG
CAGAACAATCACACCGACCAAAGAAGTGGAGTATAGAGATATTTGCAGCACATCCTTTTCAGTGCCGTCATACCGATGTGCTGAGCAAGAAA
CTGAATGCTGTTCATCGGAACCTTCATGCTCTGCCCAGACTAGCATGCGCCCTTTCCATTCTAGGCATAACCGTGATGATAACATTGACATG
AATATAGAGGATATGATGGTTATGGAAGCGATTTGGCGTTCCATTCAGGAGCAGGGAAGTATAGGGAATCCTGTCTGTGGCAACTTTATGCC
TGTAACTGAGCCATCTCCGCGTGAACGCCAGCCATTCGTTCCAGCTGCTTCTCTAGAAATACCTCATGGTGGTGGATTTTCCTGTGCGGTTG
CGGCAATGGCTGAGCACCAGCCACCCAGTATGGACTTCTCTTACATGGCTGGCAGCAGCGCATTCCCAGTTTTCGACATGTTCCGGCGACCA
TGCAACATTGCTGGTGGAAGCATGTGTAATCTGGAGAGCTCACCGGAGAGCTGGAGCGGGATAGCACCAAGCTGCAGCAGGGAAGTGGTAAG
AGAAGAAGGAGAGTGCTCGGCTGACCACTGGTCGGAGGGTGCAGAGGCCGGAACAAGCTACGCGGGCTCAGACATCGTGGCGGATGCCGGGA
CCATGCCGCAGCTGCCTTTCGCCGAGAACTTCGCCATGGCGCCAAGCCACTTCCGCCCGGAGAGCATCGAAGAACAGATGATGTTTTCCATG
GCTCTTTCTTTAGCAGATGGTCATGGAAGAACACACTCGCAAGGGTTGGCATGGTTGTAGgtagagcactctaattttgacgcctTgctgcc
ctctcccttgcgctgctgttgctgcccttctctcccctgcctcctgcttctgcctcctttttgccaccag
The aminoacid sequence of paddy rice GW2 coded by said gene following (SEQ ID NO:2) wherein, after the base deletion variation takes place, produces the premature termination codon, causes to lack 310 amino acid (shown in the underscore), thereby makes the afunction of GW2 gene.
MetGlyAsnArgIleGlyGlyArgArgLysAlaGlyValGluGluArgTyrThrArgProGlnGlyLeuTyrGluHis
ArgAspIleAspGlnLysLysLeuArgLysLeuIleLeuGluAlaLysLeuAlaProCysTyrMetGlyAlaAspAsp
AlaAlaAlaAlaAlaAspLeuGluGluCysProIleCysPheLeuTyrTyrProSerLeuAsnArgSerLysCysCys
SerLysGlyIleCysThrGluCysPheLeuGlnMetLysProThrHisThrAlaGlnProThrGlnCysProPheCys
LysThrProSerTyrAlaValGluTyrArgGly
ValLysThrLysGluGluArgSerIleGluGlnPheGluGluGln
LysValIleGluAlaGlnMetArgMetArgGlnGlnAlaLeuGlnAspGluGluAspLysMetLysArgLysGlnAsn
ArgCysSerSerSerArgThrIleThrProThrLysGluValGluTyrArgAspIleCysSerThrSerPheSerVal
ProSerTyrArgCysAlaGluGlnGluThrGluCysCysSerSerGluProSerCysSerAlaGlnThrSerMetArg
ProPheHisSerArgHisAsnArgAspAspAsnIleAspMetAsnIleGluAspMetMetValMetGluAlaIleTrp
ArgSerIleGlnGluGlnGlySerIleGlyAsnProValCysGlyAsnPheMetProValThrGluProSerProArg
GluArgGlnProPheValProAlaAlaSerLeuGluIleProHisGlyGlyGlyPheSerCysAlaValAlaAlaMet
AlaGluHisGlnProProSerMetAspPheSerTyrMetAlaGlySerSerAlaPheProValPheAspMetPheArg
ArgProCysAsnIleAlaGlyGlySerMetCysAsnLeuGluSerSerProGluSerTrpSerGlyIleAlaProSer
CysSerArgGluValValArgGluGluGlyGluCysSerAlaAspHisTrpSerGluGlyAlaGluAlaGlyThrSer
TyrAlaGlySerAspIleValAlaAspAlaGlyThrMetProGlnLeuProPheAlaGluAsnPheAlaMetAlaPro
SerHisPheArgProGluSerIleGluGluGlnMetMetPheSerMetAlaLeuSerLeuAlaAspGlyHisGlyArg
ThrHisSerGlnGlyLeuAlaTrpLeu
The variant form of embodiment 2 rice big grain genes
The inventor is a research object with rice varieties " in spend 11 ", from " spend 11 " of granule phenotype, obtain the mutant gene of a GW2, (ORF the 787th) C → T variation causes occurring in advance terminator codon because the 1041st of the base of this gene, causes its encoded protein disappearance partial amino-acid series.This plant shows the granule phenotype, although prove deletion mutation has taken place, yet has still kept the proteic function of GW2.
The inventor is a research object with rice varieties " Huang Huazhan ", obtain the mutant gene of a GW2, C → T variation has also taken place in the 1041st of the base of this gene (ORF the 787th), occurs terminator codon in advance, causes its encoded protein disappearance partial amino-acid series.This plant still shows the granule phenotype, has also promptly kept the proteic function of GW2.
In addition, the inventor has also obtained the 368th of GW2 gene (ORF the 114th) T → C variation from the plant of paddy rice granule phenotype, and this variation does not cause the variation of amino acid (Ile).This plant still shows the granule phenotype, also promptly should make a variation for the not influence of function of GW2 gene.
In addition, the inventor has also obtained the 1370th of GW2 gene (ORF the 1116th) G → A variation from the plant of paddy rice granule phenotype, and this variation does not cause the variation of amino acid (Ala).This plant still shows the granule phenotype, also promptly should make a variation for the not influence of function of GW2 gene.
The molecular marker assisted selection breeding experiment of embodiment 3GW2
In the GW2 gene, design PCR Oligonucleolide primers (SEQ ID NO:3 and SEQ IDNO:4) in the present embodiment, carry out the DNA of pcr amplification big grain kind and granule kind with the Taq enzyme, cut amplified production with restriction enzyme EcoR I enzyme, detect between big grain kind and the granule kind through 1% agarose gel electrophoresis and to have dna polymorphism (difference), the molecular weight of big grain kind is 1.159kb, the molecular weight of granule kind is 0.88kb, so this primer develops into the molecule marker that specificity is differentiated big grain GW2 gene and granule GW2 gene.In the filial generation colony of big grain kind and granule kind, can apace the individuality that carries big grain gene be picked out, reach the purpose of the big grain of further cultivation high-yield variety with this molecule marker.
5 ' end Oligonucleolide primers sequence is:
5’-TGTTGTTGCGAGAGTAGGG-3’(SEQ ID NO：3)；
3 ' end primer sequence is:
5’-TCGGTTAAGACTTGGGTAG-3’(SEQ ID NO：4)。
Embodiment 4GW2 paddy rice transgenic experiments
Present embodiment adopts and derives from the binary vector pHB (Mao etc., 2005, PNAS 102:12270-12275) of plant expression vector pCAMBIA3301 as the paddy rice transgene carrier.A bacterium replication orgin of this vector encoded (ori), kalamycin resistance gene (Kan
r), hygromycin gene (Hyg
r), the termination signal sequence of herbicide resistance gene (Bar), CaMV35S promotor, NOS gene and after between the two restriction enzyme cloning site (MCS).The cDNA that can insert GW2 at the restriction enzyme cloning site forward or backwards is built into the transgenosis plasmid.
1.GW2 the transgenosis plasmid construction of just overexpression
In the present embodiment, with the RNA that derives from the granule kind is template, synthetic article one chain cDNA, 5 ' and 3 ' the PCR oligonucleotide of holding with this dna sequence dna is primer (SEQ ID NO:5 and SEQ ID NO:6), increase with high-fidelity Taq enzyme pfu Taq, obtain the full-length cDNA amplified production of 1.634kb.This amplified production by Hind III and the Bam HI restriction enzyme digestion corresponding restriction enzyme cloning site of PMD18-T (available from TAKARA) of recombinating into, and is checked order to a plurality of recons, with the exactness of checking sequence.The transition plasmid vector of this reorganization is called GW2-PMD.
5 ' end Oligonucleolide primers sequence is:
5’-CCACCCCGAGAAAGCCAAAA-3’(SEQ ID NO：5)；
3 ' end primer sequence is:
5’-CTGGTGGCAAAAAGGAGG-3’(SEQ ID NO：6)。
With Hind III and Bam HI digestion GW2-PMD and carrier pHB, the postdigestive 1.634kb purpose of GW2-PMD fragment is connected among the Hind III and Bam HI restriction enzyme site of carrier pHB.Connector transformed into escherichia coli bacterial strain DH5 α, on the LB substratum that contains Kan (50 μ g/ml), screen transformant, select single bacterium colony and extract plasmid, pick out the clone that the 1.6Kb fragment of having an appointment is inserted with Hind III and Bam HI enzymolysis, and whether correct with M13 universal primer order-checking check nucleotide sequence.So successfully make up the pHB-35S-GW2 plasmid.
2.GW2 the transgenosis plasmid construction of antisense overexpression
With the RNA that derives from the granule kind is template, synthetic article one chain cDNA, 5 ' and 3 ' the PCR oligonucleotide of holding with this dna sequence dna is primer (SEQ ID NO:7 and SEQ ID NO:8), increase with high-fidelity Taq enzyme pfu Taq, obtain the total length ORF amplified production of 1.278kb, be connected to then among the Hind III and Bam HI restriction enzyme site of carrier pHB.
The evaluation of recon is identical with preceding method, so successfully makes up pHB-35S-GW2 (antisense) plasmid.
5 ' end Oligonucleolide primers sequence is:
5’-ATGGGGAACAGGATAGGGGGGAG-3’(SEQ ID NO：7)；
3 ' end primer sequence is:
5’-CTACAACCATGCCAACCCTTGCGAG-3’(SEQ ID NO：8)。
3.GW2 rice transformation
Above-mentioned two kinds of recombinant plasmids import agrobacterium strains EHA105 by freeze-thaw method.Per 200 μ l EHA105 competent cells and 0.5-1 μ g (about 10 μ l) plasmid DNA mixing, successively on ice, in the liquid nitrogen and respectively placed 5 minutes in 37 ℃ of water-baths; Be diluted to 1ml with fresh YEB liquid nutrient medium, cultivated 2-4 hour in 28 ℃ of joltings; Get 200 μ l and coat on the YEB flat board that contains microbiotic Kan (50 μ g/ml), cultivated 2-3 days for 28 ℃.The bacterium colony that grows is drawn 3 times continuously containing stroke single bacterium on the antibiotic YEB flat board.The single colony inoculation of picking Agrobacterium contains the antibiotic YEB liquid nutrient medium in 28 ℃ of jolting overnight incubation to 3ml from the YEB flat board, contains in the antibiotic AB liquid nutrient medium by the 1% inoculum size 50ml that transfers in the 2nd day, and 200rpm continues jolting and is cultured to OD
600When being 0.6 to 0.8 left and right sides, fresh Agrobacterium bacterium liquid in centrifugal 5 minutes of 5000rpm, 4 ℃, is collected and is resuspended in the AAM liquid nutrient medium of 1/3 volume, promptly can be used for the various acceptor materials of rice transformation this moment.
Present embodiment adopts in the conventional conversion method for agrobacterium rice transformation and spends 11 rataria callus.Get pollination back 12-15 days in spend 11 immature seeds through 70% alcohol immersion after 1 minute, in NaClO solution, (mix at 1: 3 with water, add 2-3 and drip polysorbas20) sterilization is more than 90 minutes, with aseptic water washing 4-5 time, then with scalper with take the photograph son and choose rataria and be inoculated in N6D
2Evoked callus on the substratum is cultivated under 26 ± 1 ℃, lucifuge condition, can be used for after 4 days transforming.The rataria callus is soaked in the fresh AAM Agrobacterium bacterium liquid and shakes frequently, after 20 minutes rice material is shifted out, on aseptic filter paper, inhale and remove too much bacterium liquid, transfer to N6D immediately
2On the C substratum, cultivated altogether 3 days in 26 ℃.When cultivating altogether, adding Syringylethanone as Agrobacterium Vir gene activation thing in the culture medium altogether, working concentration is 100 μ mol/L.After 3 days, take out callus, cut plumule and change over to and select substratum N6D from being total to culture medium
2S1 (Hyg 25mg/l) selects to cultivate.Forward resistant calli to N6D after 7-12 days
2S2 (Hyg 50mg/l) selects to continue on the substratum screening.Eugonic resistant calli is transferred on the pre-differentiation substratum and is cultivated about a week after 10-12 days, moves to differentiation (12 hours illumination/skies) on the division culture medium again.The regenerated seedling is at 1/2MS
0Strong plantlets and rootage on the H substratum moves into the cultivation of phytotron basin soil subsequently.
Screen transformed plant once more with weedicide behind the regeneration plant transplant survival that obtains; Positive plant extracts the total DNA of blade, further identifies transformed plant through PCR.For observing paddy rice grain type phenotype, verify the GW2 gene function with transgenosis T1.
Embodiment 5
The grain size of paddy rice GW antisense transgene plant and paddy rice GW justice transfer-gen plant and the big grain of molecule marker (non-transgenic) importing GW2 fragment plant relatively
1.GW the comparison of antisense transgene plant and wild-type
Obtain paddy rice GW antisense transgene plant as embodiment 4 described methods, for observing paddy rice grain type phenotype, observe of the influence of GW gene for the grain size with transgenosis T1.
The results are shown in Figure 1.As seen from Figure 1, the grain of paddy rice GW antisense transgene plant (AS3,24,25) is obviously big than spending 11 (ZH11) in the acceptor kind.
2.GW the comparison of just transfer-gen plant and wild-type
Obtain paddy rice GW justice transfer-gen plant as embodiment 2 described methods, for observing paddy rice grain type phenotype, observe of the influence of GW gene for the grain size with transgenosis T1.
The results are shown in Figure 2.As seen from Figure 2, the grain of paddy rice GW justice transfer-gen plant (S21,22,24) is obviously little than spending 11 (ZH11) in the acceptor kind.
3. big grain GW2 gene fragment imports the influence heavy to granule kind grain of granule kind
Big grain kind WY3 and rich short the accounting for of granule kind are hybridized for No. 1 acquisition F1 generation, shortly account for No. 1 and repeatedly backcross so that the granule kind is rich as recurrent parent, select from backcross progeny with the molecule marker described in the embodiment 3 then and carry big grain GW2 gene fragment and genetic background is the plant of granule kind, like this will big grain GW2 gene fragment importing granule kind (FAZ1) in.Method for selecting molecular marker is a non-transgenic method, has the advantage that does not have potential safety hazard.
The result as shown in Figure 3.The result shows, big grain GW2 gene fragment is imported in the granule kind, and the paddy rice grain that can obviously improve the granule kind is heavy.The thousand seed weight of paddy rice is increased to 32 grams by 22 original grams, and amplification is up to 46%.
The analysis of protein of embodiment 6GW2
1. Antibody Preparation
With the 1.278kb total length ORF that the foregoing description was obtained, with Hind III and the Bam HI pET32a (+) (available from Novagen company) that recombinates.Have the prokaryotic expression carrier pET32a (+) of GW2 to be transformed into e. coli bl21 (DE3) reorganization, IPDG induces prokaryotic expression, uses His-tag pearl purifying protein then.With purified protein immunization rabbit, obtain the antibody of anti-GW2 by ordinary method.
2. detection antibody titer
The expression amount of Western Blotting detection GW2 overexpression, and do negative contrast with unconverted parent, empty plasmid and antisense transgene plant simultaneously, the conduct of GW2 prokaryotic expression protein is over against photograph.
3. immunohistochemical analysis
Detect the variation of GW2 protein content, and carry out tissue and the Subcellular Localization of GW2.
4.E3 the active research of ligase enzyme
There is the prokaryotic expression carrier pET32a (+) of GW2 to be transformed into e. coli bl21 (DE3) reorganization, 0.5% lactose, low temperature (12 ℃) are induced prokaryotic expression, whether extract from the prokaryotic expression bacterial strain and the active GW2 albumen of purifying with the not damaged method, being used to study GW2 has E3 ligase enzyme activity.
Ubiquitin ligase active reaction employing Xiuren Zhang in the present embodiment etc. similarly method (see Xiuren Zhang etc., Genes﹠amp; Dev.200519:1532-1543).Be summarized as follows: the 30ul system includes 5mM MgCl
2, 0.33mM DTT, 50mM Tris-HCl (PH7.4), 2mM ATP, 0.125ug E1,0.2ug E2 (UbcH5b), 1ug E3 adds 2ul intestinal bacteria crude extract in addition in reaction system.Be reflected at warm the bath 3 hours in 30 degrees centigrade.Add 100 degrees centigrade of temperature of 1 * SDS-PAGE sample loading buffer afterwards and bathe 5 minutes termination reactions.Reaction effect is identified by the 12%SDS-PAGE gel electrophoresis.Ubiquitin protein utilizes Western blotting to detect by anti-Ubiquitin.
Above-mentioned experimental result shows that GW2 has the E3 ligase enzyme activity in the proteolytic degradation approach, thereby points out it relevant with cell incident such as cell fission.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉rice big grain gene and application thereof
<130>065312
<160>9
<170>PatentIn version 3.3
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＜213〉Oryza (Oryza sativa L.)
<220>
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ccaccccgag aaagccaaaa aaaagaaaaa ttgtttttca aaaaaaaagc tcgcctagcc 60
ctcgcgtcgt cgtccccatc accccccctc ctccgctccg agtacgcgtg cgtataccac 120
cacctccatc tccaccaccg tatgtatcta cggtgaggcg gcggcggcgg aggaggagga 180
ggagggggag tggtgagggt ttcatctgcg gaggaggagg gaggaggagg gaggagggta 240
gatctgggag gggg atg ggg aac agg ata ggg ggg agg agg aag gcg ggg 290
Met Gly Asn Arg Ile Gly Gly Arg Arg Lys Ala Gly
1 5 10
gtg gag gag agg tac acg agg ccg cag ggg ctg tac gag cac agg gac 338
Val Glu Glu Arg Tyr Thr Arg Pro Gln Gly Leu Tyr Glu His Arg Asp
15 20 25
atc gac cag aag aag ctc cgg aag ctg att ctc gag gcc aag ctc gcg 386
Ile Asp Gln Lys Lys Leu Arg Lys Leu Ile Leu Glu Ala Lys Leu Ala
30 35 40
ccg tgc tac atg ggc gcc gac gac gcc gcc gcc gcc gcc gac ctc gag 434
Pro Cys Tyr Met Gly Ala Asp Asp Ala Ala Ala Ala Ala Asp Leu Glu
45 50 55 60
gag tgc ccc atc tgc ttc ctg tac tac cca agt ctt aac cga tca aag 482
Glu Cys Pro Ile Cys Phe Leu Tyr Tyr Pro Ser Leu Asn Arg Ser Lys
65 70 75
tgt tgc tca aaa ggg ata tgc acc gag tgc ttt ctc caa atg aaa cca 530
Cys Cys Ser Lys Gly Ile Cys Thr Glu Cys Phe Leu Gln Met Lys Pro
80 85 90
act cac act gct cag cct aca caa tgt cca ttc tgc aaa act ccc agt 578
Thr His Thr Ala Gln Pro Thr Gln Cys Pro Phe Cys Lys Thr Pro Ser
95 100 105
tat gct gtg gag tat cgt ggt gta aag aca aag gag gaa agg agc ata 626
Tyr Ala Val Glu Tyr Arg Gly Val Lys Thr Lys Glu Glu Arg Ser Ile
110 115 120
gaa caa ttt gaa gag cag aaa gtc ata gaa gca caa atg agg atg cgc 674
Glu Gln Phe Glu Glu Gln Lys Val Ile Glu Ala Gln Met Arg Met Arg
125 130 135 140
cag caa gca ctt caa gat gaa gaa gat aag atg aaa aga aaa cag aac 722
Gln Gln Ala Leu Gln Asp Glu Glu Asp Lys Met Lys Arg Lys Gln Asn
145 150 155
agg tgc tct tct agc aga aca atc aca ccg acc aaa gaa gtg gag tat 770
Arg Cys Ser Ser Ser Arg Thr Ile Thr Pro Thr Lys Glu Val Glu Tyr
160 165 170
aga gat att tgc agc aca tcc ttt tca gtg ccg tca tac cga tgt gct 818
Arg Asp Ile Cys Ser Thr Ser Phe Ser Val Pro Ser Tyr Arg Cys Ala
175 180 185
gag caa gaa act gaa tgc tgt tca tcg gaa cct tca tgc tct gcc cag 866
Glu Gln Glu Thr Glu Cys Cys Ser Ser Glu Pro Ser Cys Ser Ala Gln
190 195 200
act agc atg cgc cct ttc cat tct agg cat aac cgt gat gat aac att 914
Thr Ser Met Arg Pro Phe His Ser Arg His Asn Arg Asp Asp Asn Ile
205 210 215 220
gac atg aat ata gag gat atg atg gtt atg gaa gcg att tgg cgt tcc 962
Asp Met Asn Ile Glu Asp Met Met Val Met Glu Ala Ile Trp Arg Ser
225 230 235
att cag gag cag gga agt ata ggg aat cct gtc tgt ggc aac ttt atg 1010
Ile Gln Glu Gln Gly Ser Ile Gly Asn Pro Val Cys Gly Asn Phe Met
240 245 250
cct gta act gag cca tct ccg cgt gaa cgc cag cca ttc gtt cca gct 1058
Pro Val Thr Glu Pro Ser Pro Arg Glu Arg Gln Pro Phe Val Pro Ala
255 260 265
gct tct cta gaa ata cct cat ggt ggt gga ttt tcc tgt gcg gtt gcg 1106
Ala Ser Leu Glu Ile Pro His Gly Gly Gly Phe Ser Cys Ala Val Ala
270 275 280
gca atg gct gag cac cag cca ccc agt atg gac ttc tct tac atg gct 1154
Ala Met Ala Glu His Gln Pro Pro Ser Met Asp Phe Ser Tyr Met Ala
285 290 295 300
ggc agc agc gca ttc cca gtt ttc gac atg ttc cgg cga cca tgc aac 1202
Gly Ser Ser Ala Phe Pro Val Phe Asp Met Phe Arg Arg Pro Cys Asn
305 310 315
att gct ggt gga agc atg tgt aat ctg gag agc tca ccg gag agc tgg 1250
Ile Ala Gly Gly Ser Met Cys Asn Leu Glu Ser Ser Pro Glu Ser Trp
320 325 330
agc ggg ata gca cca agc tgc agc agg gaa gtg gta aga gaa gaa gga 1298
Ser Gly Ile Ala Pro Ser Cys Ser Arg Glu Val Val Arg Glu Glu Gly
335 340 345
gag tgc tcg gct gac cac tgg tcg gag ggt gca gag gcc gga aca agc 1346
Glu Cys Ser Ala Asp His Trp Ser Glu Gly Ala Glu Ala Gly Thr Ser
350 355 360
tac gcg ggc tca gac atc gtg gcg gat gcc ggg acc atg ccg cag ctg 1394
Tyr Ala Gly Ser Asp Ile Val Ala Asp Ala Gly Thr Met Pro Gln Leu
365 370 375 380
cct ttc gcc gag aac ttc gcc atg gcg cca agc cac ttc cgc ccg gag 1442
Pro Phe Ala Glu Asn Phe Ala Met Ala Pro Ser His Phe Arg Pro Glu
385 390 395
agc atc gaa gaa cag atg atg ttt tcc atg gct ctt tct tta gca gat 1490
Ser Ile Glu Glu Gln Met Met Phe Ser Met Ala Leu Ser Leu Ala Asp
400 405 410
ggt cat gga aga aca cac tcg caa ggg ttg gca tgg ttg tag 1532
Gly His Gly Arg Thr His Ser Gln Gly Leu Ala Trp Leu
415 420 425
gtagagcact ctaattttga cgccttgctg ccctctccct tgcgctgctg ttgctgccct 1592
tctctcccct gcctcctgct tctgcctcct ttttgccacc ag 1634
<210>2
<211>425
<212>PRT
＜213〉Oryza (Oryza sativa L.)
<400>2
Met Gly Asn Arg Ile Gly Gly Arg Arg Lys Ala Gly Val Glu Glu Arg
1 5 10 15
Tyr Thr Arg Pro Gln Gly Leu Tyr Glu His Arg Asp Ile Asp Gln Lys
20 25 30
Lys Leu Arg Lys Leu Ile Leu Glu Ala Lys Leu Ala Pro Cys Tyr Met
35 40 45
Gly Ala Asp Asp Ala Ala Ala Ala Ala Asp Leu Glu Glu Cys Pro Ile
50 55 60
Cys Phe Leu Tyr Tyr Pro Ser Leu Asn Arg Ser Lys Cys Cys Ser Lys
65 70 75 80
Gly Ile Cys Thr Glu Cys Phe Leu Gln Met Lys Pro Thr His Thr Ala
85 90 95
Gln Pro Thr Gln Cys Pro Phe Cys Lys Thr Pro Ser Tyr Ala Val Glu
100 105 110
Tyr Arg Gly Val Lys Thr Lys Glu Glu Arg Ser Ile Glu Gln Phe Glu
115 120 125
Glu Gln Lys Val Ile Glu Ala Gln Met Arg Met Arg Gln Gln Ala Leu
130 135 140
Gln Asp Glu Glu Asp Lys Met Lys Arg Lys Gln Asn Arg Cys Ser Ser
145 150 155 160
Ser Arg Thr Ile Thr Pro Thr Lys Glu Val Glu Tyr Arg Asp Ile Cys
165 170 175
Ser Thr Ser Phe Ser Val Pro Ser Tyr Arg Cys Ala Glu Gln Glu Thr
180 185 190
Glu Cys Cys Ser Ser Glu Pro Ser Cys Ser Ala Gln Thr Ser Met Arg
195 200 205
Pro Phe His Ser Arg His Asn Arg Asp Asp Asn Ile Asp Met Asn Ile
210 215 220
Glu Asp Met Met Val Met Glu Ala Ile Trp Arg Ser Ile Gln Glu Gln
225 230 235 240
Gly Ser Ile Gly Asn Pro Val Cys Gly Asn Phe Met Pro Val Thr Glu
245 250 255
Pro Ser Pro Arg Glu Arg Gln Pro Phe Val Pro Ala Ala Ser Leu Glu
260 265 270
Ile Pro His Gly Gly Gly Phe Ser Cys Ala Val Ala Ala Met Ala Glu
275 280 285
His Gln Pro Pro Ser Met Asp Phe Ser Tyr Met Ala Gly Ser Ser Ala
290 295 300
Phe Pro Val Phe Asp Met Phe Arg Arg Pro Cys Asn Ile Ala Gly Gly
305 310 315 320
Ser Met Cys Asn Leu Glu Ser Ser Pro Glu Ser Trp Ser Gly Ile Ala
325 330 335
Pro Ser Cys Ser Arg Glu Val Val Arg Glu Glu Gly Glu Cys Ser Ala
340 345 350
Asp His Trp Ser Glu Gly Ala Glu Ala Gly Thr Ser Tyr Ala Gly Ser
355 360 365
Asp Ile Val Ala Asp Ala Gly Thr Met Pro Gln Leu Pro Phe Ala Glu
370 375 380
Asn Phe Ala Met Ala Pro Ser His Phe Arg Pro Glu Ser Ile Glu Glu
385 390 395 400
Gln Met Met Phe Ser Met Ala Leu Ser Leu Ala Asp Gly His Gly Arg
405 410 415
Thr His Ser Gln Gly Leu Ala Trp Leu
420 425
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＜213〉artificial sequence
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＜223〉primer
<400>5
ccaccccgag aaagccaaaa 20
<210>6
<211>18
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
ctggtggcaa aaaggagg 18
<210>7
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>7
atggggaaca ggataggggg gag 23
<210>8
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>8
ctacaaccat gccaaccctt gcgag 25
Claims (16)
1. an isolating albumen is characterised in that altogether, and this albumen is selected from down group:
(a) has the polypeptide of SEQ ID NO:2 aminoacid sequence; Or
(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the wide or heavy function of grain of control crop grain by (a) polypeptides derived.
2. albumen as claimed in claim 1 is characterized in that described dietary protein origin is in paddy rice.
3. albumen as claimed in claim 1 is characterized in that, described albumen has E3 ligase enzyme activity.
4. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(i) the described proteic polynucleotide of coding claim 1; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
5. polynucleotide as claimed in claim 4 is characterized in that this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQID NO:2.
6. polynucleotide as claimed in claim 4 is characterized in that, these polynucleotide are selected from down group:
(1) nucleotide sequence shown in the SEQ ID NO:1; Or
(2) nucleotide sequence shown in the 255-1532 position among the SEQ ID NO:1.
7. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
8. a genetically engineered host cell is characterized in that,
It contains the described carrier of claim 7; Or
Be integrated with the described polynucleotide of claim 4 in its genome.
9. the purposes of the described albumen of claim 1 or its encoding gene is characterized in that, is used for:
The grain of control crop kernel is wide or grain is heavy;
Regulate cell processes; Or
As the molecule marker of identifying crop big grain kind and granule kind.
10. purposes as claimed in claim 9 is characterized in that, described cell processes comprises: cell fission, signal conduction or cell elongation.
11. a method that improves crop is characterized in that, this method comprises:
(A) reduce the described proteic expression of claim 1 in the described crop; Or
(B) the described albumen of claim 1 or its encoding gene with function reduction or afunction imports in the crop.
12. the agonist or the antagonist of the described albumen of claim 1 or its encoding gene.
13. an energy and the described albumen of claim 1 or its encoding gene specificity bonded antibody.
14. a method for preparing transgenic plant is characterized in that, described method comprises step: the described polynucleotide of claim 4 are imported in the vegetable cell, cultivate described vegetable cell, the regeneration plant.
15. method as claimed in claim 14 is characterized in that, described method comprises step:
(s1) provide the Agrobacterium of carrying expression vector, described expression vector contains the described proteic encoding gene of claim 1;
(s2) vegetable cell or tissue or organ are contacted with Agrobacterium in the step (s1), thereby make the described proteic encoding gene of claim 1 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(s3) select vegetable cell or tissue or the organ that changes the described protein coding gene of claim 1 over to; And
(s4) vegetable cell in the step (s3) or tissue or neomorph are become plant.
16. one kind prepares the described proteic method of claim 1, it is characterized in that, cultivates the described host cell of claim 8, collects to obtain the described albumen of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2006101171135A CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2006101171135A CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
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| CN101161675A true CN101161675A (en) | 2008-04-16 |
| CN101161675B CN101161675B (en) | 2012-07-04 |
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|---|---|---|---|
| CN2006101171135A Active CN101161675B (en) | 2006-10-13 | 2006-10-13 | Rice big grain gene and uses thereof |
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