CN101575366A - Rice plant type gene and application thereof - Google Patents
Rice plant type gene and application thereof Download PDFInfo
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- CN101575366A CN101575366A CNA2008100370427A CN200810037042A CN101575366A CN 101575366 A CN101575366 A CN 101575366A CN A2008100370427 A CNA2008100370427 A CN A2008100370427A CN 200810037042 A CN200810037042 A CN 200810037042A CN 101575366 A CN101575366 A CN 101575366A
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Abstract
The invention discloses a rice plant type protein and the application thereof. The rice plant type protein is a protein of a sequence showed by SEQ ID NO: 2 or a derived protein having the same functions with the protein of the sequence showed by the SEQ ID NO: 2. The invention also discloses a coding gene of the rice plant type protein, a carrier containing the coding gene and a host cell. The rice plant type protein can be used for regulating the erecting characteristic and/or the tillering characteristic of a plant, and can also be used for identifying molecular markers of the erecting characteristic and/or the tillering characteristic of the plant. The rice plant type protein has wide application prospect on plant types and high yield breeding.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to a kind of new rice plant type gene and application thereof.
Background technology
Asia cultivated rice (Oryza sativa)-paddy rice is to be come through secular artificial selection domestication by her ancestors' common wild-rice (Oryza rufipogon).American scholar has been cloned the shattering gene Sh4 of control wild-rice recently, and this gene and paddy rice are evolved, and are controlling the cultivated rice that is domesticated for more difficult shattering by the easy seed holding of wild-rice.Wild-rice has the habit of growing, tillering more of crawling, and such plant type is unfavorable for dense planting and high-yield culturing.This unfavorable plant type of wild-rice by ancient times people as a kind of improvement target, carry out artificial selection and domestication, the most unfavorable plant type of wild-rice changes upright, less cultivated rice of tillering into, such plant type helps dense planting, high-yield culturing.
The plant type of taming into cultivated rice from the plant type of wild-rice is the most important events on the paddy rice evolutionary history, but the genes involved of control wild-rice plant type yet there are no report so far.Because the plant type characteristic relation can impact output to dense planting, high yield or the outward appearance of plant, so this area is necessary to study the gene of regulating the plant plant type, thereby provides new approach for the plant orderly improvement.
Summary of the invention
The object of the present invention is to provide the relevant PA7 albumen of a kind of plant type of rice, its encoding gene contains the carrier and the cell of this encoding gene, and uses.
In a first aspect of the present invention, a kind of isolating PA7 albumen is provided, this albumen is:
(a) 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 with the aminoacid sequence identical function shown in the SEQ ID NO:2 by (a) deutero-albumen;
(c) (preferably 80%, more preferably 90%, the best 95%) homogeny that has at least 70% with SEQ ID NO:2 aminoacid sequence, and have with the aminoacid sequence identical function shown in the SEQ ID NO:2 by (a) deutero-albumen.
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 PA7 of coding; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
In another preference, this polynucleotide encoding contains the albumen of aminoacid sequence shown in the SEQ ID NO:2.
In another preference, these polynucleotide have the nucleotide sequence shown in 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 and is integrated with described polynucleotide in described carrier or the genome.
In a fifth aspect of the present invention, the purposes of described PA7 albumen or its encoding gene is provided,
Be used to regulate upright characteristic and/or the shooting property of plant; Or
As the upright characteristic of plant identification and/or the molecular marked compound of shooting property; Or
Be used to prepare the upright characteristic of plant identification and/or the molecular marked compound of shooting property; Or
Be used for as activating transcription factor the expression of regulation and control downstream gene.
In another preference, described PA7 albumen or its encoding gene are used to increase the tiller number of plant, or are used to make plant to crawl to grow.
In another preference, described plant is a paddy rice.
In a sixth aspect of the present invention, a kind of upright characteristic of plant and/or method of shooting property of regulating is provided, this method comprises regulates proteic expression of PA7 or the activity described in the described plant.
In another preference, a kind of method that increases the tiller number of plant or make plant crawl to grow is provided, described method comprises proteic expression of PA7 or activity in the described plant of raising.
In another preference, a kind of method that reduces the tiller number of plant or make the plant vertical growth is provided, described method comprises proteic expression of PA7 or activity in the described plant of reduction.
In a seventh aspect of the present invention, a kind of upright characteristic of plant identification and/or the molecular marked compound of shooting property are provided, described molecular marked compound is that primer is right, has the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
In a eighth aspect of the present invention, provide the agonist or the antagonist of a kind of described PA7 or its encoding gene.
In a ninth aspect of the present invention, provide tiller or the method for the growing plants that crawls a kind of the preparation more, it comprises step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the proteic encoding sequence of described PA7;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make the proteic encoding sequence of described PA7 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell, tissue or the organ that changes the proteic encoding sequence of described PA7 over to;
(4) vegetable cell, tissue or neomorph in the step (3) are become plant.
In a tenth aspect of the present invention, the upright characteristic of a kind of plant identification (as plant seed) and/or the method for shooting property are provided, described method comprises step:
(s1) genomic dna with described plant is a template, carries out pcr amplification with the primer with the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, obtains amplified production;
(s2) restriction enzyme EheI is joined in the described amplified production, enzyme analysis is cut situation;
Wherein, if amplified production is not digested, then described plant has the characteristic of tillering more or crawling to grow;
If amplified production is digested, then described plant has and tillers less or the characteristic of vertical growth.
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 wild-rice PA7 gene order (A) and amino acid sequence coded (B) thereof.
Fig. 2 has shown by transgenic method and wild-rice PA7 gene fragment has been imported in the rice-cultivating kind plant type of transgenic line performance wild-rice.Wherein, contrast and be cultivated rice kind " in spend 11 ", T781 and T790 are two transgenic lines that change PA7 over to.
Fig. 3 is positioned in the nucleus with having shown the PA7 protein-specific, and prompting PA7 is a transcription factor gene.
Fig. 4 shown by molecule marker select PA7 gene fragment with wild-rice import the cultivated rice kind (special blue or green, TQ) in, the plant type of near isogenic line NIL (PA7) the performance wild-rice of cultivation.
Embodiment
The inventor is through extensive studies, use molecular marking technique and from wild-rice, obtain the relevant gene of a kind of plant type first, it is the new gene of a kind of transcription factor, has the effect of regulation and control downstream gene expression, inventor general's called after rice plant type gene (PA7).This expression of gene can make that paddy rice crawls to grow, and it is many to tiller; After the functional site of this gene is undergone mutation and is made albumen lose function, but the paddy rice vertical growth, and it is few to tiller.Therefore this gene can be used as the molecule marker of plant identification plant type, and is with a wide range of applications in control of plant plant type and SOYBEAN IN HIGH-YIELD BREEDING.
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 rice plant type protein ", " isolating PA7 albumen " or " isolating PA7 polypeptide " are meant that PA7 albumen is substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those of ordinary skill in the art can use the purified technology of protein purifying PA7 albumen of standard.Basically pure polypeptide can produce single master tape on non-reduced polyacrylamide gel.
As used herein, term " contains ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute (making) ", " basically by ... constitute " and " by ... constitute ".
As used herein, the plant of described " tillering " is meant a kind of plant more, and it grows to certain phase when (as the stage of maturity) under suitable growth conditions, and is significantly more (as many 20% than the tiller number of the similar plant that grows under the same conditions; Preferably many 40%, more preferably many 60% or more).The plant of described " tillering less " is meant a kind of plant, when it grows to certain phase under suitable growth conditions, significantly lack (as less 20% than the tiller number of similar plant of growth under the same conditions; Preferably lack 40%, more preferably lack 60% or still less).
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 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 PA7, derivative and analogue.As used herein, term " fragment ", " derivative " are meant with " analogue " and keep identical biological function of PA7 albumen of the present invention or active polypeptide basically.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).According to the definition of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, term " PA7 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, more preferably 1-20,1-10 best, also better for 1-8 or 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 PA7 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 PA7 protein D NA hybridization and polypeptide or the albumen that utilizes the proteic antiserum(antisera) of anti-PA7 to obtain.Described homologous sequence is meant to have with SEQ ID NO:2 sequence 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 PA7 albumen or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the proteic soluble fragments of PA7.Usually, this fragment have the PA7 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 100 continuous amino acids, best at least about 150 continuous amino acids.
The present invention also provides the analogue of PA7 albumen or polypeptide.These analogues and the proteic difference of natural PA7 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, " PA7 albumen conservative property variant protein (polypeptide) " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 20 at the most, preferably at the most 10, 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.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce, and the albumen identical functions of reservation and SEQ ID NO:2 sequence.Yet, guard in the key function site upper amino acid sequence of SEQ ID NO:2 sequence, for example the 152nd amino acid is Thr in the SEQ ID NO:2 sequence, it can not be replaced by Ser.
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 PA7 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: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 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%, 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 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 70%, preferably more than at least 80%, more preferably more than at least 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 PA7.
As optimal way of the present invention, a kind of upright characteristic of plant identification and/or the molecular marked compound of shooting property are provided, described molecular marked compound is a primer, the described primer encoding sequence of the proteic key function site areas of PA7 that can increase, thus can be tested and appraised upright characteristic and/or the shooting property that this encoding sequence is learnt plant.Preferably, described primer has the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.Therefore, a kind of upright characteristic of plant identification and/or the method for shooting property comprise:
(s1) genomic dna with described plant is a template, carries out pcr amplification with the primer with the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, obtains amplified production;
(s2) restriction enzyme EheI is joined in the described amplified production, enzyme analysis is cut situation; Wherein, if amplified production is not digested, then described plant has the characteristic of tillering more or crawling to grow; If amplified production is digested, then described plant has and tillers less or the characteristic of vertical growth.
Observe whether digested can being undertaken of amplified production by the method for routine, a kind of simple method is to utilize gel electrophoresis technology, cut the band number that produces behind the product electrophoresis by observing enzyme, thereby learn that whether effective enzyme takes place is cut, this technology is well known in the art.
PA7 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 PA7 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 express or produce the PA7 albumen of reorganization.In general following steps are arranged:
(1). with the proteic polynucleotide of coding PA7 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, PA7 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.A kind of suitable expression vector is pCAMIA1301 for example.
Method well-known to those having ordinary skill in the art can be used to make up and contains PA7 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 (especially being non-reproduction 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, thus the plant that acquired character changes.
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 PA7 albumen of reorganization is of use in many ways.For example be used to screen antibody, polypeptide or other part that promotes or resist the PA7 protein function.Can be used for seeking the valuable peptide molecule that can suppress or stimulate the PA7 protein function with the reorganization PA7 protein screening peptide library of expressing.
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 PA7 albumen and also can detect the proteic transcription product of PA7.
The invention still further relates to a kind of upright characteristic and/or shooting property by the adjusting plant and improve the method for plant, this method comprises: regulate PA7 expression of gene or activity in the described plant.Promote still to suppress the expression of PA7 or actively depend primarily on the proterties of the required improvement of plant and decide.When needs increase the tiller number of plant or make the plant growth of crawling, can realize by improving in the described plant PA7 expression of gene or activity; When needs reduce the tiller number of plant or make the method for plant vertical growth, then can realize by reducing in the described plant PA7 expression of gene or activity.
The method that increases PA7 genetic expression is that this area is known.For example, can make plant cross expression PA7 by changing the expression constructs of carrying the PA7 encoding gene over to; Thereby maybe can strengthen PA7 gene or its homogenic expression by driving with strong promoter; Perhaps strengthen this PA7 expression of gene by enhanser (as paddy rice waxy gene first intron, Actin gene first intron etc.).The strong promoter that is applicable to the inventive method includes but not limited to: the Ubi promotor of 35s promotor, paddy rice, corn etc.
The method that suppresses PA7 genetic expression also is that this area is known.For example, can disturb the technology of (RNAi) or gene knockout to realize by antisense or RNA.
As a kind of optimal way of the present invention, the method for the plant of acquisition PA7 high expression level is as follows:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the proteic encoding sequence of described PA7;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make the proteic encoding sequence of described PA7 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell, tissue or the organ that changes the proteic encoding sequence of described PA7 over to;
(4) 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 wait to implement this method.
The present invention also comprises the antagonist or the agonist of PA7 albumen or its encoding gene.Because the antagonist of PA7 or agonist can be regulated the active of PA7 or express, therefore, the antagonist of described PA7 or agonist also can be by regulating upright characteristic and/or the shooting property of plant to the influence of PA7, thereby reach the purpose that makes the plant trait improvement.
The antagonist of described PA7 is meant the proteic activity of any PA7 of reduction, reduces the material of PA7 gene or proteic stability, the proteic expression of downward modulation PA7, minimizing PA7 albumen effective acting time or inhibition PA7 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 PA7 is meant the proteic activity of any PA7 of raising, keeps PA7 gene or proteic stability, promotes the proteic expression of PA7, prolongs the material of proteic effective acting time of PA7 or promotion PA7 gene transcription and translation, these materials all can be used for the present invention, as upright characteristic and/or the useful material of shooting property of regulating plant.
In an example of the present invention, a kind of PA7 gene is provided, the open reading frame of its total length (ORF) sequence is encoded one and is contained 167 amino acid whose protein (SEQ ID NO:2) shown in SEQ ID NO:1.Sequential analysis in wild-rice and the cultivated rice shows, the coding region of PA7 gene has two bases to replace in cultivated rice, one of them cause on the protein sequence an amino acid whose replacement (the 152nd T among the SEQ ID NO:2 → S), thus cause that being evolved by the plant type of wild-rice is the plant type of cultivated rice; By recovering the plant type of wild-rice in the PA7 fragment importing cultivated rice of transgenic method with wild-rice.
Major advantage of the present invention is:
Find rice plant type gene (PA7) first from wild-rice, the announcement of this gene can provide new approach for the improvement of upright characteristic of paddy rice and/or shooting property.The inventor's research shows that also this gene is a kind of activating transcription factor, thereby it has the relevant characteristic of activating transcription factor, can be used for regulating and control the expression of downstream gene that is:.In addition, the PA7 gene also will play an important role in other plant comprises the directive breedings of wheat, corn, jowar etc., be with a wide range of applications.
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 the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
The clone of embodiment 1PA7 gene
Ancestors' common wild-rice of cultivated rice has the plant type of growing, tillering more of crawling, and the plant type of Asia cultivated rice kind " special blue or green " is upright, less tillering.The inventor as donor parents, with special blue or green as recurrent parent, makes up cross combination with Hainan wild-rice, and carries out many generations and backcross.A gene of controlling the wild-rice plant type, called after PA7 have been located with backcross population.Further utilize the map based cloning technology to clone this gene.
Sequence comparing analysis shows, the special blue or green variation that has 2 bases in the coding region of PA7 gene of wild-rice and cultivated rice, 1 (the 99th, in wild-rice A, in cultivated rice for G) do not cause amino acid change, and 1 (the 454th in addition, in wild-rice A, in cultivated rice, be T) make a variation and cause an amino acid whose replacement, become the Serine (S) of cultivated rice by the Threonine (T152) of wild-rice, cause the function variation of this gene, thereby cause that being evolved by the plant type of wild-rice is the plant type of cultivated rice.
The sequence of the PA7 gene of wild-rice plant type shown in SEQ ID NO:1 (Figure 1A), the coding a brand-new Unknown Function new albumen, its sequence is shown in SEQ ID NO:2 (Figure 1B).Further discover in rice genome, there is not the homology copy of PA7.The total length ORF of PA7 gene (open reading-frame) length is 504bp, 167 the amino acid whose albumen of encoding.
Sequential analysis of protein shows that PA7 albumen is zinc finger protein, be speculated as a transcription factor, in order to prove this supposition, the inventor has carried out Subcellular Localization, green fluorescent protein GFP and PA7 ordinary method are constructed the 35S::GFP-PA7 carrier, bombard onion epidermis cell with particle gun then, observe luciferase expression with Laser Scanning Confocal Microscope.The result is positioned in the nucleus as shown in Figure 3 visible PA7 protein-specific, and prompting PA7 is a transcription factor.5 transcriptional activation activity analysis experiment has confirmed that further PA7 is a transcription factor gene in conjunction with the embodiments.
The molecular marker assisted selection breeding experiment of embodiment 2PA7
In the present embodiment, sequence according to the PA7 gene, design PCR Oligonucleolide primers (SEQ ID NO:3 and SEQ ID NO:4), carry out the DNA of pcr amplification common wild-rice (Hainan wild-rice) and Asia cultivated rice kind (special blue or green or other cultivated rice kind) with the Taq enzyme, cut amplified production with restriction enzyme EheI enzyme, detect the dna polymorphism (difference) that exists between common wild-rice and the Asia cultivated rice kind through 1% agarose gel electrophoresis.As 1 of amplified production electrophoretic band, then be the wild-rice kind; As 2 of amplified production electrophoretic bands, then be special blue or green or other cultivated rice kind (restriction enzyme site is in SEQ ID NO:1 99-105 position).Therefore this primer can be used as the molecule marker that specificity is differentiated wild-rice PA7 gene and cultivated rice PA7 gene.In the filial generation colony of wild-rice and cultivated rice kind, can apace the individuality that carries the PA7 gene be picked out with this molecule marker, reach the purpose of improvement plant type.
5 ' end Oligonucleolide primers sequence is (SEQ ID NO:3):
5’-TCTCCGCCGGTGGAGCTGTC-3’；
3 ' end Oligonucleolide primers sequence is (SEQ ID NO:4):
5’-GGAGGCGATGGGCATGGACG-3’。
Embodiment 3PA7 paddy rice transgenic experiments
Present embodiment adopts expression vector pCAMBIA1301 (available from CAMBIA) 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 and the restriction enzyme cloning site (MCS) of NOS gene.Can insert the PA7 genomic fragment at the restriction enzyme cloning site and be built into the transgenosis plasmid.
1.PA7 the structure of the transgenosis plasmid of genomic fragment
In this embodiment, with the wild-rice genomic dna is template, PCR oligonucleotide with 5 ' and 3 ' end of the dna sequence dna of PA7 is primer (SEQ ID NO:5 and SEQ ID NO:6), and pfuTaq increases with high-fidelity Taq enzyme, the PA7 genomic fragment amplified production of acquisition 2.677kb.This amplified production is recombinated on the PMD18-T carrier, and (TaKaRa Japan), and checks order to a plurality of recons, with the exactness of checking sequence.The transition plasmid vector of this reorganization is called PA7-PMD.
5 ' end Oligonucleolide primers sequence is (SEQ ID NO:5):
5’-GCCCGTTCGGATTTAAGTTCTA-3’；
3 ' end primer sequence is (SEQ ID NO:6):
5’-GCCAGTCCCTATGTGGTGTTCT-3’。
With PstI and KpnI digestion PA7-PMD and carrier pCAMIA1301, the postdigestive 2.677kb purpose of PA7-PMD fragment is connected to carrier pCAMIA1301 respectively.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 2.677kb fragment is inserted with PstI and KpnI enzymolysis, and whether correct with M13 universal primer order-checking check nucleotide sequence.So successfully make up the pCAMIA1301-PA7 plasmid.
2.PA7 rice transformation
The pCAMIA1301-PA7 plasmid by freeze-thaw method import agrobacterium strains EHA105 (referring to Hood, E.E. etc., Transgenic Res., 1993,2,208-218).Per 200 μ l EHA105 competent cells add 0.5-1 μ g (about 10 μ l) plasmid DNA mixing, successively on ice, respectively placed 5 minutes in liquid nitrogen and 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 YEB flat board of microbiotic Kan.The single colony inoculation of picking Agrobacterium contains to 3ml the YEB liquid nutrient medium of microbiotic Kan in 28 ℃ of jolting overnight incubation from the YEB flat board, contained in the condition described in the AB liquid nutrient medium of microbiotic Kan by the 1% inoculum size 50ml that transfers in the 2nd day, when 200rpm continues jolting and is cultured to OD600 and is 0.6 to 0.8 left and right sides, fresh Agrobacterium bacterium liquid is centrifugal 5 minutes in 5000rpm, 4 ℃, collect and be 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 conventional conversion method for agrobacterium to transform the rataria callus of rice-cultivating kind " in spend 11 ".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 (containing Hyg25mg/l) selects to cultivate.Forward resistant calli to N6D after 7-12 days
2S2 (containing 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 the plant type of rice phenotype, verify the PA7 gene function with transgenosis T2.The substratum that more than relates to is referring to " molecular cloning: lab guide ".
As a result, import in the cultivated rice kind, obtained the plant type of wild-rice, see Fig. 2, show that PA7 is the relevant gene of plant type of rice by the PA7 genomic fragment of transgenic method with wild-rice.
The wild-rice chromosome segment that embodiment 4 will comprise PA7 imports the influence in the special blue or green genetic background
The inventor's seed selection near isogenic line NIL (PA7) of PA7, the very short wild-rice chromosome segment that is about to comprise PA7 imports in the genetic background of special blue or green (TQ).Specifically: with Hainan wild-rice and the special blue or green hybridization of cultivated rice kind, carry out continuously backcrossing for 4 times as recurrent parent with special green grass or young crops again, with molecule marker from backcross population, select comprise the short segmental and genetic background (genotype) of wild-rice PA7 for special blue or green near isogenic line NIL (PA7) (Nearly isogenic line, NIL).
The result as shown in Figure 4, the similar wild-rice of plant type of NIL (PA7) shows that PA7 is the key gene of control wild-rice plant type.
Therefore, can improve the plant type of paddy rice by the molecular designing of PA7, reach the target of SOYBEAN IN HIGH-YIELD BREEDING, visible PA7 gene is with a wide range of applications in plant type of rice and SOYBEAN IN HIGH-YIELD BREEDING.
The transcriptional activation activity analysis experiment of embodiment 5PA7
With Matchmaker GAL4 yeast two-hybrid system 3 (available from Clontech) transcriptional activation of PA7 is analyzed.Make up positive control carrier pAD, link after NLS that pGADT7 (Clontech) is carried and AD cut with BamHI and SalI enzyme on the BamHI of pGBKT7 (Clontech) and the SalI site and obtain.Use the total length ORF of PCR method (is primer with SEQ ID NO:7 and SEQ ID NO:8) amplification PA7 then, be building up on the EcoRI and PstI on the pGBKT7 carrier, form the pGBKT7-PA7 carrier.
5 ' end Oligonucleolide primers sequence is (SEQ ID NO:7):
5’-AAGAATTCATGGATCCCTCATCGGCTTC-3’；
3 ' end primer sequence is (SEQ ID NO:8):
5’-AACTGCAGCTAGAGGCCGAGCTCGAGGA-3’。
Carrier with N end that makes up PA7 with quadrat method respectively and C end.At the amino acid whose nucleotide sequence of coding N end 1-69 two ends design primer (SEQ ID NO:9 and SEQ ID NO:10), carry out pcr amplification, the PCR product is inserted on the restriction endonuclease EcoRI and PstI site of pGBKT7 carrier, makes up the pGBKT7-PA7-Nterminal carrier.At the amino acid whose nucleotide sequence of coding C end 70-167 two ends design primer (SEQ ID NO:11 and SEQ ID NO:12), carry out pcr amplification, the PCR product is inserted on the restriction endonuclease EcoRI and PstI site of pGBKT7 carrier, makes up pGBKT7-PA7-C terminal carrier.
5 ' end Oligonucleolide primers sequence is (SEQ ID NO:9):
5’-AAGAATTCATGGATCCCTCATCGGCTTC-3’；
3 ' end primer sequence is (SEQ ID NO:10):
5’-AACTGCAGGTGCGCGTTCTGGTGGCCGC-3’。
5 ' end Oligonucleolide primers sequence is (SEQ ID NO:11):
5’-AAGAATTCCGGAAGGAGCGCGTCGCCGG-3’；
3 ' end primer sequence is (SEQ ID NO:12):
5’-AACTGCAGCTAGAGGCCGAGCTCGAGGA-3’。
Various carriers are transformed among the barms AH109 (available from Clontech), bacterial classification dilution with grow overnight, be coated in and lack Trp amino acid or lack three seed amino acids (SD cultivation base (molecular cloning: lab guide) Trp/-His/-Ade), observe the growth situation then, the transcriptional activation activity of decision PA7.
The result shows that pGBKT7-PA7 has stronger transcriptional activation activity, shows that PA7 is the transcription factor with transcriptional activation activity.In addition, the C end that has only PA7 also has transcriptional activation activity and the N end of PA7 does not have, and shows that the transcription activating domain of PA7 is positioned at the C end.
The function of embodiment 6PA7 protein variant
Adopt conventional site-directed mutagenesis technique, the inventor replaces with Ile with the 18th Leu of the wild-rice PA7 aminoacid sequence shown in the SEQ ID NO:2, constitutes PA7 protein variants (PA7-M).Similar as previously mentioned method utilizes Agrobacterium that the PA7-M encoding gene is imported in the cultivated rice kind, measures the plant type of this transgenic paddy rice.Found that the plant type of described transgenic paddy rice performance wild-rice.
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.
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉rice plant type gene and application thereof
<130>082683
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＜213〉common wild-rice (Oryza rufipogon)
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atggatccct catcggcttc ttggccggct ccggcttctc cgccggtgga gctgtccctg 60
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gcgctcggcg gccaccagaa cgcgcaccgg aaggagcgcg tcgccggcgg cagctggaac 240
cccaacgtct acggcgacgg cggcggatca gcgtccatgc ccatcgcctc ccatggcgtc 300
acggcggcgg ggagtagtac ggcagccgac ggccggtggt gcggcggcgc tgccagcgac 360
gacgacacaa cggcggcgcc catgccttcc ctcggctcag gctcggcggc gctcggcgcc 420
ggcgccggtt tcgcttcgac cgaaaggggc tctaccggcg gcggcgtcgc cggcgaggag 480
cttgtcctcg agctcggcct ctag 504
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35 40 45
Cys Leu Phe Cys Ala Lys Thr Phe Arg Lys Ser Gln Ala Leu Gly Gly
50 55 60
His Gln Asn Ala His Arg Lys Glu Arg Val Ala Gly Gly Ser Trp Asn
65 70 75 80
Pro Asn Val Tyr Gly Asp Gly Gly Gly Ser Ala Ser Met Pro Ile Ala
85 90 95
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100 105 110
Trp Cys Gly Gly Ala Ala Ser Asp Asp Asp Thr Thr Ala Ala Pro Met
115 120 125
Pro Ser Leu Gly Ser Gly Ser Ala Ala Leu Gly Ala Gly Ala Gly Phe
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145 150 155 160
Leu Val Leu Glu Leu Gly Leu
165
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Claims (10)
1. isolating PA7 albumen is characterized in that this albumen is:
(a) 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 with the aminoacid sequence identical function shown in the SEQ ID NO:2 by (a) deutero-albumen; Or
(c) with SEQ ID NO:2 aminoacid sequence at least 70% homogeny is arranged, and have with the aminoacid sequence identical function shown in the SEQ ID NO:2 by (a) deutero-albumen.
2. isolating polynucleotide is characterized in that, these polynucleotide are selected from down group:
(i) the proteic polynucleotide of the coding described PA7 of claim 1; Or
(ii) with (i) in polynucleotide complementary polynucleotide.
3. a carrier is characterized in that, it contains the described polynucleotide of claim 2.
4. a genetically engineered host cell is characterized in that, it contains and is integrated with the described polynucleotide of claim 2 in described carrier of claim 3 or the genome.
5. the purposes of the described PA7 albumen of claim 1 or its encoding gene is characterized in that,
Be used to regulate upright characteristic and/or the shooting property of plant; Or
As the upright characteristic of plant identification and/or the molecular marked compound of shooting property; Or
Be used to prepare the upright characteristic of plant identification and/or the molecular marked compound of shooting property; Or
Be used for as activating transcription factor the expression of regulation and control downstream gene.
6. regulate the upright characteristic of plant and/or the method for shooting property for one kind, it is characterized in that, this method comprises regulates proteic expression of PA7 as claimed in claim 1 or activity in the described plant.
7. the upright characteristic of a plant identification and/or the molecular marked compound of shooting property is characterized in that, described molecular marked compound is that primer is right, have the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4.
8. the agonist or the antagonist of the described PA7 of claim 1 or its encoding gene.
9. one kind prepares and tillers or the method for the growing plants that crawls more, it is characterized in that it comprises step:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the proteic encoding sequence of the described PA7 of claim 1;
(2) vegetable cell, tissue or organ are contacted with Agrobacterium in the step (1), thereby make the proteic encoding sequence of described PA7 change vegetable cell over to, and be incorporated on the karyomit(e) of vegetable cell;
(3) select vegetable cell, tissue or the organ that changes the proteic encoding sequence of described PA7 over to;
(4) vegetable cell, tissue or neomorph in the step (3) are become plant.
10. the upright characteristic of a plant identification and/or the method for shooting property is characterized in that, described method comprises step:
(s1) genomic dna with described plant is a template, carries out pcr amplification with the primer with the nucleotide sequence shown in SEQ ID NO:3 and the SEQ ID NO:4, obtains amplified production;
(s2) restriction enzyme EheI is joined in the described amplified production, enzyme analysis is cut situation;
Wherein, if amplified production is not digested, then described plant has the characteristic of tillering more or crawling to grow;
If amplified production is digested, then described plant has and tillers less or the characteristic of vertical growth.
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| CN102199600A (en) * | 2010-03-26 | 2011-09-28 | 中国科学院上海生命科学研究院 | Gene for adjusting vein color and application thereof |
| CN114350700A (en) * | 2021-10-19 | 2022-04-15 | 深圳大学 | Saccharomyces cerevisiae vector and construction method and application thereof |
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| CN1185256C (en) * | 2002-08-20 | 2005-01-19 | 中国科学院遗传与发育生物学研究所 | Rice tiller control gene MOC1 and its application |
| CN100429310C (en) * | 2006-03-15 | 2008-10-29 | 华中农业大学 | Gene for controlling paddy tillering and usage |
| CN1900280A (en) * | 2006-07-19 | 2007-01-24 | 浙江大学 | Rice tiller regulating gene OsTIL1 and its use |
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| CN102199600A (en) * | 2010-03-26 | 2011-09-28 | 中国科学院上海生命科学研究院 | Gene for adjusting vein color and application thereof |
| CN114350700A (en) * | 2021-10-19 | 2022-04-15 | 深圳大学 | Saccharomyces cerevisiae vector and construction method and application thereof |
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Effective date of registration: 20200611 Address after: 200032 building 4, No. 300 Fenglin Road, Xuhui District, Shanghai Patentee after: Center for excellence and innovation in molecular plant science, Chinese Academy of Sciences Address before: 200031 No. 320, Yueyang Road, Shanghai Patentee before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |