CN103305524B - Ear type of crop regulatory gene and application thereof - Google Patents
Ear type of crop regulatory gene and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of ear type of crop regulatory gene and application thereof, disclose a kind of for regulating plant plant height, fringe phenotype or a gene that stalk length is useful, this gene can be applicable to plant hybridization breeding, obtains the plant of plant type change or breed improvement.
Description
Technical field
The invention belongs to biotechnology and phytology field; More specifically, the present invention relates to a kind of ear type of crop regulatory gene and application thereof.
Background technology
Paddy rice is one of most important food crop in the world, and have half population to take rice as staple food in the world, Chinese paddy Year's consumption and output account for 1/3rd of world's total amount.China is populous nation, pays much attention to production and the scientific research of paddy rice always, along with the minimizing of arable area and the increase of population, improves grain yield and becomes great social concern.At the end of the eighties, scientist proposes the Rice Super-yield Breeding theory that Ideal Rice Plant Type combines with use of advantage.The formation of plant type of rice comprises the factors such as plant height, tillering number, tillering angle and fringe type, and fringe type is one of important content of Ideal Rice Plant Type research.Investigation and application affects the gene of fringe type on a molecular scale, not only have theory significance for the developmental mechanism of understanding Rice Panicle in depth, and for applying molecular marker assisted selection in Instructing manufacture and screen the strain of appropriate dense cluster phenotype, increase yield has practical significance.At present, in paddy rice, the existing gene much affecting the growth of fringe type is cloned.FZP gene, by suppressing the hypertrophy of axillary meristem in small ear meristematic tissue, maintains the Feature Conversion from small ear to inflorescence.The homologous gene of FZP in corn is BD1, is grown by the mitogenetic properties influence fringe type of the lateral organ in regulation and control inflorescence structure.LAX gene passes through to regulate the growth that in the Growth adjustment fringe of lateral meristem, branch obstructs in fringe growth course, thus affects fringe type.RCN1 and RCN2 gene is the homologous gene of CEN gene in paddy rice of TFL1-like gene and Common Snapdragon in Arabidopis thaliana respectively.The transfer-gen plant of RCN1 and RCN2 overexpression is mainly through postponing the conversion of vegetative point to the vegetative point of little floral structure of apparatus derivatorius, and increase secondary and secondary above branch stalk number to increase seed number, being embodied in fringe type is exactly dense cluster phenotype.
The function that research affects the gene of fringe type not only has theory significance for the deep growth understanding Rice Panicle, and has practical significance for the strain applying molecular marker assisted selection screening dense cluster phenotype in Instructing manufacture.Therefore, this area is necessary to further investigate and excavate the gene affecting Rice Panicle phenotype.
Summary of the invention
The object of the present invention is to provide a kind of ear type of crop regulatory gene and application thereof.
In a first aspect of the present invention, provide the purposes of a kind of RAMOSA2 polypeptide or its conditioning agent, for regulating plant plant height, fringe phenotype or a stalk (comprising at different levels stalks, as Their First Branch stalk, secondary prop up stalk) length; Described RAMOSA2 polypeptide is selected from:
The polypeptide of aminoacid sequence shown in (a) SEQIDNO:2;
(b) by aminoacid sequence shown in SEQIDNO:2 through one or more (as 1-20; Preferably 1-10; More preferably 1-5; More preferably 1-3) replacement of amino-acid residue, disappearance or interpolation form, and have the polypeptide derivative by (a) of (a) polypeptide function; Or
C aminoacid sequence shown in () Yu SEQIDNO:2 has 80% (preferably 88%; More preferably 90%; More preferably 95%; More preferably 99%) above homogeny, and there is the polypeptide derivative by (a) of (a) polypeptide function.
In a preference, described plant is grass; More preferably, described plant is paddy rice.
In another preference, described RAMOSA2 polypeptide is the RAMOSA2 polypeptide deriving from paddy rice.
In another preference, described RAMOSA2 polypeptide coding genes is polynucleotide sequence or its complementary sequence of encoding such polypeptides.
In another preference, the encoding gene of described RAMOSA2 polypeptide has the nucleotide sequence shown in SEQIDNO:1, or its complementary sequence.
In another preference, described RAMOSA2 polypeptide is used for:
Dwarf plant plant type;
The fringe of plant is made to present dense cluster phenotype; Or
Shorten plant and prop up stalk length.
In another preference, the conditioning agent of described RAMOSA2 polypeptide is its lower adjustment, for:
The fringe of plant is made to present rare fringe phenotype; Or
Extend plant and prop up stalk length.
In another preference, the lower adjustment of described RAMOSA2 polypeptide is the material lowering RAMOSA2 genetic transcription, expression of polypeptides or polypeptide active; Preferably, the lower adjustment of described RAMOSA2 polypeptide is the disturbing molecule of specificity interference RAMOSA2 genetic transcription.
In another preference, described disturbing molecule be with the encoding gene of RAMOSA2 polypeptide or its transcript for suppressing or dsRNA, antisense nucleic acid, siRNA, the Microrna of reticent target, maybe can express or be formed the construction of described dsRNA, antisense nucleic acid, siRNA, Microrna; Preferably, described disturbing molecule is using SEQIDNO:1 or its transcript as the dsRNA of reticent target or construction.
In another preference, described disturbing molecule is construction, the structure containing shown in formula (I):
Seq
forward-X-Seq
oppositelyformula (I),
In formula (I),
Seq
forwardfor the polynucleotide shown in 173-332 position in SEQIDNO:1, Seq
oppositelyfor with Seq
forwardcomplementary polynucleotide;
X is for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelynot complementary.
In another preference, described intervening sequence (X) itself does not form complementary duplex structure, and its length is 200-2000nt; Preferably 500-1500nt; More preferably 1000-1200nt (according to appointment 1163nt).
In another aspect of this invention, a kind of method of regulating plant plant height, fringe phenotype or a stalk length is provided, comprises: the expression of RAMOSA2 polypeptide or its encoding gene or activity in regulating plant; Wherein said RAMOSA2 polypeptide is selected from:
The polypeptide of aminoacid sequence shown in (a) SEQIDNO:2;
(b) by aminoacid sequence shown in SEQIDNO:2 through one or more (as 1-20; Preferably 1-10; More preferably 1-5; More preferably 1-3) replacement of amino-acid residue, disappearance or interpolation form, and have the polypeptide derivative by (a) of (a) polypeptide function; Or
C aminoacid sequence shown in () Yu SEQIDNO:2 has 80% (preferably 88%; More preferably 90%; More preferably 95%; More preferably 99%) above homogeny, and there is the polypeptide derivative by (a) of (a) polypeptide function.
In a preference, described method comprises: the expression of raising RAMOSA2 polypeptide or its encoding gene in plant, thus dwarf plant plant type, makes fringe present dense cluster phenotype or shorten a stalk length.
In another preference, described method comprises: proceeded in plant by the encoding gene of RAMOSA2 polypeptide.
In another preference, described method comprises:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the encoding gene of RAMOSA2 polypeptide;
(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thus make the encoding gene of RAMOSA2 polypeptide proceed to plant.
In another preference, described method comprises: the expression of lowering RAMOSA2 polypeptide or its encoding gene in plant, thus makes fringe present rare fringe phenotype or extend a stalk length.
In another preference, described method comprises: by lowering RAMOSA2 genetic transcription, the lower adjustment of expression of polypeptides or polypeptide active proceeds in plant; Preferably, described lower adjustment is the disturbing molecule of specificity interference RAMOSA2 genetic transcription.
In another preference, described method comprises:
A () provides the Agrobacterium of carrying expression vector, described expression vector contains the disturbing molecule of interference RAMOSA2 genetic transcription;
B vegetable cell or tissue or organ contact with the Agrobacterium in step (a) by (), thus make the disturbing molecule of interference RAMOSA2 genetic transcription proceed to plant.
In another aspect of this invention, provide a kind of plant, it is prepared by the method for arbitrary described regulating plant plant height, fringe phenotype or a stalk length above.
In another aspect of this invention, provide the purposes of a kind of RAMOSA2 polypeptide or its encoding gene, as the marker of the plant height of plant identification, fringe phenotype or a stalk length.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The RT-PCR of Fig. 1, OsRAMOSA2 gene overexpression transfer-gen plant detects.Wherein 1,2,3 is 3 random strain overexpression transfer-gen plants, and that detects is organized as blade.
The plant height of Fig. 2, OsRAMOSA2 gene overexpression plant (OsRAMOSA2OE is called for short OsRA2OE) and fringe type.
The Subcellular Localization of the albumen of Fig. 3, OsRAMOSA2 genes encoding.
The expression of Fig. 4, OsRAMOSA2 gene at paddy rice different tissues position detects.
The expression of Fig. 5, OsRAMOSA2 gene in Rice Panicle growth course (bar=100 μm).A, one-level prop up the former base of stalk; B, one-level prop up the former base of stalk; C, secondary prop up the former base of stalk; D, clever flower primordium.
The expression analysis of Fig. 6, OsRAMOSA2 gene in RNAi transfer-gen plant, wherein 1-5 is transfer-gen plant.
Spend 11 in Fig. 7, wild-type, the fringe type of OsRAMOSA2 gene overexpression transfer-gen plant (OsRA2OE) and RNAi transfer-gen plant (OsRA2RNAi).
Spend 11 in Fig. 8, wild-type, a stalk length of OsRAMOSA2 gene overexpression transfer-gen plant (OsRA2OE) and RNAi transfer-gen plant (OsRA2RNAi).
The structure collection of illustrative plates of Fig. 9, p1301-35SNOS expression vector.
Figure 10, p1301RNAi plasmid map.
Embodiment
The present inventor is through studying widely, and find a kind of for regulating plant plant height, fringe phenotype or a gene that stalk length is useful, it is called ear type of crop regulatory gene (RAMOSA2 gene) by the present inventor.Gene of the present invention can be applicable to plant hybridization breeding, obtains the plant of plant type (particularly fringe type) change or breed improvement.
In the present invention, have no particular limits for being applicable to plant of the present invention (or crop), as long as it is applicable to the conversion operation of carrying out gene, as various farm crop, flower plant or forestry plant etc.Described plant can be such as (being not limited to): dicotyledons, monocotyledons or gymnosperm.More specifically, described plant includes, but is not limited to: wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olea, Sunflower Receptacle, coconut, castor oil plant, cocoa beans, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, natsudaidai, spinach, piemarker lettuce, asparagus, cabbage, Chinese cabbage, Plantula Brassicae chinensis, Radix Dauci Sativae, onion, potato, tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, nut, coffee, eggplant, sugarcane, tealeaves, pepper, grapevine, oyster fiber crops grass, banana, natural rubber tree and ornamental plant etc.
As a kind of optimal way, described " plant " includes but not limited to graminaceous plant.Such as, paddy rice gramineous, wheat, barley, rye, Chinese sorghum, corn.Preferably, described " plant " is paddy rice.
Polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, improvement on synthesis, preferably recombinant polypeptide.Polypeptide of the present invention can be native purified product, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (such as, bacterium, yeast, higher plant, insect and mammalian cell).
The present invention also comprises the fragment of RAMOSA2 polypeptide, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " refer to the polypeptide substantially keeping biological function that RAMOSA2 polypeptide of the present invention is identical or activity.Polypeptide fragment of the present invention, derivative or analogue can be the polypeptide that (i) has one or more conservative or non-conservative amino acid residue (preferred conservative amino acid) and be substituted, and the amino-acid residue of such replacement can may not be and encoded by genetic code, or (ii) has the polypeptide of substituted radical in one or more amino-acid residue, or (iii) mature polypeptide and another compound (such as extend the compound of polypeptide transformation period, such as polyoxyethylene glycol) merge the polypeptide formed, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide formed (as leader sequence or secretion sequence or be used for the sequence of this polypeptide of purifying or proprotein sequence, or fusion rotein).The known scope of those skilled in the art is belonged to according to these fragments of definition herein, derivative and analogue.
In the present invention, term " RAMOSA2 polypeptide (RAMOSA2 albumen) " refers to the polypeptide of the SEQIDNO:2 sequence of the function with regulating plant plant height, fringe phenotype or a stalk length.This term also comprise have regulating plant plant height, fringe phenotype or a stalk length function, the variant form of SEQIDNO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-20, more preferably 1-10, also better for 1-8 or 1-5) amino acid whose disappearance, insertion and/or replacement, and to add or disappearance one or several (is generally within 20 at C-terminal and/or N-terminal, within being preferably 10, within being more preferably 5) amino acid.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, to add or disappearance one or several amino acid also can not change the function of protein usually at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of RAMOSA2 polypeptide.
The variant form of polypeptide comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, under high or low stringency condition can with the polypeptide coded by the DNA of the DNA hybridization of RAMOSA2 polypeptide and the polypeptide utilizing the antiserum(antisera) of anti-RAMOSA2 polypeptide to obtain or albumen.Present invention also offers other polypeptide, as comprised the fusion polypeptide of RAMOSA2 polypeptide or its fragment.Except the polypeptide of almost total length, present invention includes the soluble fragments of RAMOSA2 polypeptide.Usually, this fragment have RAMOSA2 peptide 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.
The present invention also provides the analogue of RAMOSA2 polypeptide.The difference of these analogues and natural RAMOSA2 polypeptide can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that RAMOSA2 polypeptide of the present invention is not limited to the above-mentioned representational polypeptide exemplified.
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.
In the present invention, " RAMOSA2 polypeptide conservative variation polypeptide " refers to compared with the aminoacid sequence of SEQIDNO:2, has 20 at the most, preferably at the most 10, more preferably at the most 5, best at the most 3 amino acid replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out amino acid replacement according to table 1 and produce.
Table 1
| Amino-acid residue | Representational replacement | 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 |
Present invention also offers the polynucleotide sequence of code book invention RAMOSA2 polypeptide or its conservative variation's 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-strand.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can the varient of or degeneracy identical with the coding region sequence shown in SEQIDNO:1.As used herein, " varient of degeneracy " refers to that coding has polypeptide or its derivative polypeptide of SEQIDNO:2 in the present invention, but shown coding region sequence differentiated nucleotide sequence arbitrary with SEQIDNO:1.
The polynucleotide of the mature polypeptide of coding SEQIDNO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional coding sequence; The encoding sequence (with optional additional coding sequence) of mature polypeptide and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide comprising encoding such peptides, also can be the polynucleotide also comprising 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, the sum analogous to general Dedekind sum of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient of non-natural generation.These nucleotide variants comprise and replace varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of one or more Nucleotide, disappearance or insertion, but can not from the function of polypeptide changing in fact its coding.
The invention still further relates to and above-mentioned sequence hybridization and have at least 80% between two sequences, the more preferably polynucleotide of at least 88% homogeny.The present invention be more particularly directed to polynucleotide interfertile with polynucleotide of the present invention under strict conditions.In the present invention, " stringent condition " refers to: (1) compared with the hybridization under low ionic strength and comparatively high temps and wash-out, as 0.2 × SSC, 0.I%SDS, 60 DEG C; Or be added with denaturing agent during (2) hybridization, and as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.; Or (3) homogeny only between two sequences is at least more than 80%, better more than at least 90%, just hybridizes when being more preferably more than 95%.Further, the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in SEQIDNO:2.
The present invention also relates to the carrier comprising polynucleotide of the present invention, and with the host cell that carrier of the present invention or RAMOSA2 polypeptid coding sequence produce through genetically engineered, and the method for polypeptide of the present invention is produced through recombinant technology.
RAMOSA2 polypeptide polynucleotide sequence can be inserted in recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, as long as can copy in host and stablize, any plasmid and carrier can be used.A key character of expression vector is usually containing replication orgin, promotor, marker gene and translation controlling elements.Method well-known to those having ordinary skill in the art can be used for building containing RAMOSA2 peptide coding DNA sequence dna and the suitable expression vector of transcribing/translating control signal.
Comprise the carrier of above-mentioned suitable DNA sequence dna and suitably promotor or control sequence, may be used for transforming suitable host cell, with can marking protein.Host cell can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces, Agrobacterium; Fungal cell is as yeast; Vegetable cell etc.
When the gene of coding RAMOSA2 polypeptide is expressed in higher eucaryotic cells, if will make to transcribe to be enhanced when inserting enhancer sequence in the carrier.Enhanser is the cis-acting factors 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 suitable carrier, promotor, enhanser and host cell.
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.Conversion of plant can use the method such as Agrobacterium-mediated Transformation or via Particle Bombardment Transformation, such as leaf disk method, Rice Young Embryo conversion method etc.Can ordinary method regeneration plant be used for the vegetable cell transformed, tissue or organ, thus the plant that acquired character changes.
The invention provides described RAMOSA2 polypeptide or the purposes of its encoding gene, for regulating plant plant height, fringe phenotype or a stalk length.Under a kind of mode, the expression or the activity that raise RAMOSA2 polypeptide can be used for dwarf plant plant type; The fringe of plant is made to present dense cluster phenotype; Or shortening plant props up stalk length.Under another kind of mode, expression or the activity of lowering RAMOSA2 polypeptide can make the fringe of plant present rare fringe phenotype; Or prolongation plant props up stalk length.
Therefore, plant can be changed based on RAMOSA2 polypeptide for the above-mentioned influence of plant, thus reach the object according to needs of production improvement plant quality.Preferably, described plant is grass; Best, described plant is paddy rice.
The invention still further relates to the upper adjustment of RAMOSA2 polypeptide or its encoding gene or lower adjustment (as the RAMOSA2 gene of antisense, or siRNA, miRNA, shRNA, antisense nucleotide) and uses thereof.Due to the upper adjustment of RAMOSA2 or the expression of the adjustable RAMOSA2 of lower adjustment and/or the activity etc. regulating RAMOSA2, therefore, the upper adjustment of described RAMOSA2 or lower adjustment by carrying out the phenotype (comprising plant height, fringe phenotype and branch stalk length) of regulating plant to the impact of RAMOSA2, thus reach the object of improvement plant.
The activity of any adjustable RAMOSA2 polypeptide, regulate the stability of RAMOSA2 polypeptide, promotion or suppress the expression of RAMOSA2 gene, prolongation or reduce RAMOSA2 polypeptide effective acting time or to promote or the material of transcribing and translating that reduces RAMOSA2 gene all can be used for the present invention, as the active substance of phenotype (comprising plant height, fringe phenotype and branch stalk length) that can be used for regulating plant.
The invention still further relates to the method for a kind of regulating plant plant height, fringe phenotype and branch stalk length, the method comprises the expression regulating RAMOSA2 polypeptide in described plant.
On the one hand, the invention provides a kind of dwarf plant plant type, make fringe present dense cluster phenotype or shorten a method for stalk length, described method comprises: the expression of raising RAMOSA2 polypeptide or its encoding gene in plant.
On the other hand, present invention also offers a kind of fringe that makes and present rare fringe phenotype or extend a method of propping up stalk length, described method comprises: the expression (comprise and RAMOSA2 polypeptide is not expressed or low expression) of lowering RAMOSA2 polypeptide in described plant.The prolongation that firsts and seconds props up the length of stalk can provide space structure more fully for the growth of small ear and seed, thus provides space for increasing crop yield aspect.Plant high to the plant and output that prop up stalk extension is hybridized, is expected to the plant that acquisition output increases further.Described plant is preferably grass, is more preferably paddy rice.
After the purposes obtaining the RAMOSA2 polypeptide described in cicada, multiple method well known in the art can be adopted to regulate the expression of described RAMOSA2 polypeptide.Such as by certain approach, the ceneme (such as expression vector or virus etc.) carrying RAMOSA2 encoding gene is delivered on target spot, and makes it the RAMOSA2 polypeptide of expression activity.In addition, also multiple method well known in the art can be adopted to reduce the expression of RAMOSA2 polypeptide or to make it loss of expression, such as the ceneme (such as expression vector or virus etc.) of carrying antisense RAMOSA2 gene is delivered on target spot, cell or plant tissue is not expressed or reduces to express RAMOSA2 polypeptide.Maybe by lowering RAMOSA2 genetic transcription, the lower adjustment of expression of polypeptides or polypeptide active proceeds in plant; Such as, described lower adjustment is the disturbing molecule of specificity interference RAMOSA2 genetic transcription.
As one embodiment of the present invention, the encoding gene of RAMOSA2 polypeptide is cloned in suitable carrier by conventional method, the described recombinant vectors with foreign gene is imported in the vegetable cell can expressing described RAMOSA2 polypeptide, makes described vegetable cell express RAMOSA2 polypeptide.By described Plant cell regeneration is become plant, obtain the plant of overexpression RAMOSA2 polypeptide.
Preferably, a kind of dwarf plant plant type, make fringe present dense cluster phenotype or shorten stalk length a method comprise:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the encoding gene of the RAMOSA2 be connected with expression vector forward;
(2) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (1), thus make the encoding gene of RAMOSA2 proceed to vegetable cell;
(3) vegetable cell, tissue, the organ of the encoding gene having proceeded to RAMOSA2 is selected; With
(4) by the vegetable cell in step (3), tissue, neomorph select transgenic plant, required transgenic plant are.
Preferably, a kind of method making fringe present rare fringe phenotype or a prolongation stalk length comprises:
(s1) Agrobacterium of carrying expression vector is provided, described expression vector contains the disturbing molecule (include but not limited to dsRNA, antisense nucleic acid, siRNA, Microrna, maybe can express or be formed the construction of described dsRNA, antisense nucleic acid, siRNA, Microrna) of RAMOSA2 gene;
(s2) vegetable cell, tissue or organ are contacted with the Agrobacterium in step (s1), thus make the disturbing molecule of described RAMOSA2 gene proceed to vegetable cell;
(s3) vegetable cell, tissue, the organ of the disturbing molecule having proceeded to RAMOSA2 gene is selected; With
(s4) by the vegetable cell in step (s3), tissue, neomorph select transgenic plant, required transgenic plant are.
As used herein, described forward connects and refers to: the encoding gene of RAMOSA2 and the connection of expression vector are just connections, namely encoding gene according to 5 ' → 3 ' direction be connected on carrier.Usually, the encoding gene of RAMOSA2 is arranged in the downstream of expression vector promotor, and also namely 3 ' end downstream of promotor connects 5 ' end of this encoding gene.Described encoding gene is connected on expression vector operably.Described " operability connection " or " being operationally connected in " refers to so a kind of situation, and namely some part of linear DNA molecule can regulate or control the activity of same linear DNA molecule other parts.Such as, if the transcribing of promotor control sequence, so it is exactly operationally be connected in encoding sequence.
Any suitable conventional means can be adopted, comprise reagent, temperature, pressure condition etc. and implement described method.Other method increasing RAMOSA2 expression is that this area is known.Such as, by driving with strong promoter thus strengthening the expression of RAMOSA2.Or the expression of this RAMOSA2 gene is strengthened by enhanser.The strong promoter being applicable to the inventive method includes but not limited to: the Ubi promotor etc. of 35s promotor, paddy rice, corn.
The Another application of the encoding gene of RAMOSA2 polypeptide is as plant identification plant height, fringe phenotype or the molecule marker propping up stalk length.By analyzing the expression treating RAMOSA2 polypeptide in measuring plants (seed or seedling as plant), judge its plant height, fringe phenotype or a stalk length.Such as, identify the low or nothing of the expression of RAMOSA2 polypeptide in plant, then this plant can present the phenotype of rare fringe phenotype or a stalk extension; And if identify the comparatively high expression level (overexpression) of RAMOSA2 polypeptide, then this plant can present plant type downgrade, dense cluster phenotype or stalk contraction in length phenotype.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, conveniently condition such as J. Pehanorm Brooker etc. is write usually, Molecular Cloning: A Laboratory guide, Science Press, the condition described in 2002, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.
Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
The clone of OsRAMOSA2 gene in embodiment 1, paddy rice
In paddy rice, OsRAMOSA2 gene gene order number in NCBI (http://www.ncbi.nlm.nih.gov/) is NC_008394, and the sequence number in tigr (www.tigr.org) is LOC_Os01g07480.Its cDNA sequence is as shown in SEQIDNO:1, and peptide sequence is as shown in SEQIDNO:2.
The phenotype analytical of OsRAMOSA2 gene overexpression in embodiment 2, paddy rice
By the method for PCR, take rice genome as template, OsRAMOSA2 full-length genome sequence fragment is obtained with primer gggatccaaatggcatcctcgtcgagcacc (SEQIDNO:3) and gggtacccatcaaggccaaagcgcagat (SEQIDNO:4) amplification, be cloned in the BamHI/KpnI site of overexpression vector p1301-35SNOS (Fig. 9), in the method transformed wild type paddy rice mediated with Agrobacterium (EHA105), spend 11 (Zh11).
Random selecting three strain transfer-gen plants (OsRAMOSA2OE), RT-PCR detection is carried out with primer cgcagaagttcgccaacgtc (SEQIDNO:5) and atcaaggccaaagcgcagat (SEQIDNO:6), confirm that the expression of OsRAMOSA2 gene in transfer-gen plant is obviously raised, as Fig. 1.
Observe the character mutation of transfer-gen plant, transfer-gen plant presents the phenotype of dwarfing and dense cluster.Plant the T1 of transfer-gen plant generation in Hainan Datian (winter in 2010), the individual plant choosing dense cluster phenotype carries out economical character statistics, and result is as table 2.Transfer-gen plant is obviously short in contrast (Fig. 2 A); The one-level of transfer-gen plant is propped up the length that stalk and secondary prop up stalk and is significantly shorter than contrast, and these factors cause transfer-gen plant obviously dense cluster phenotype (Fig. 2 B-C).
The Some Agronomic Traits statistics of table 2, OsRAMOSA2 gene overexpression transfer-gen plant
The Subcellular Localization of embodiment 3, OsRAMOSA2 gene coded protein
By the method for PCR, take rice genome as template, the full length sequence fragment of OsRAMOSA2 gene is obtained with primer gggatccaaatggcatcctcgtcgagcacc (SEQIDNO:3) and gggtacccatcaaggccaaagcgcagat (SEQIDNO:4) amplification, then cut by BamHI and KpnI enzyme, be cloned in XhoI and the SpeI site in protein expression vector pA7-GFP, and be transformed in Arabidopis thaliana (Col) protoplastis, by the Subcellular Localization situation of laser scanning co-focusing microscope (Confocol) detection fusion albumen.
The foundation of expression vector pA7-GFP: CaMV35S-MCS-GFP-MCS-Nos3 ' cassette (HindIII/EcoRIfragment) is inserted into plasmid pUC18 (NewEnglandBiolabs, FrankfurtAmMain, Germany) in.CaMV35S-MCS-GFP-MCS-Nos3 ' utilizes HindIII/EcoRI from p35S-GFP-JFH1 (Hongetal., 1999, Identificationofacalmodulin-regulatedCa2+-ATPaseintheend oplasmaticreticulum.PlantPhysiol.119,1165-1175) enzyme cuts acquisition.
Detection display, protein localization in tenuigenin, as Fig. 3.
Embodiment 4, the express spectra of OsRAMOSA2 gene in rice growth process
At the different times of rice growth, get different tissue sites, with primer cgcagaagttcgccaacgtc (SEQIDNO:5) and atcaaggccaaagcgcagat (SEQIDNO:6), carry out the experiment of RT-PCR, have detected OsRAMOSA2 gene at paddy rice different tissues position and the express spectra in period, result is as Fig. 4.OsRAMOSA2 gene all has expression at paddy rice different tissues position, and the expression wherein in stem, fringe, vegetative point and little Hua is strong.
For the effect that deep announcement OsRAMOSA2 gene pairs fringe type is grown, the present inventor has carried out the analysis of in situ hybridization to the expression of OsRAMOSA2 gene in fringe growth course, result display OsRAMOSA2 gene mainly props up the former base of stalk (Fig. 5 A, B) in one-level, secondary props up the former base of stalk (Fig. 5 C) and clever flower primordium position (Fig. 5 D) and expresses, visible one-level prop up the former base of stalk and secondary prop up obstruct former base portion position expression by force, at clever flower primordium position, expression is weak.
The RNAi transgenic analysis of embodiment 5, OsRAMOSA2 gene
The present inventor chooses the more special fragment (in SEQIDNO:1 173-332 position) of of OsRAMOSA2 gene, with pCAMBIA1301 (purchased from Cambia company) for skeleton, construct the RNAi carrier pCAMBIA1301RNAi (Figure 10) of OsRAMOSA2 gene, primer gggagctcggatccGAAATGGCATCCTCGTCGAGCACC (SEQIDNO:7) and gggactagtggtaccGCGGGAAGTAAGGAGCGAACACG (SEQIDNO:8) amplification is utilized to obtain the fragment comprising 173-332 position in SEQIDNO:1, in BamHI and the KpnI site of the pCAMBIA1301RNAi be inserted into, then this fragment is oppositely inserted with SpeI and SacI.The ring structure of hairpin structure is the uncorrelated fragment be present on plasmid.
By the genetic transforming method that Agrobacterium (EHA105) mediates, the recombinant plasmid transformed that leading portion obtains is spent in 11 in wild rice.Obtain transfer-gen plant at present, random selecting 5 strain transfer-gen plants, by the method for RT-PCR, confirm that the expression of wherein OsRAMOSA2 gene is lowered, as Fig. 6.
Observe the phenotype of transfer-gen plant, find that the overall phenotype of OsRAMOSA2RNAi transfer-gen plant is rare fringe phenotype, compared with wild-type, the firsts and seconds of OsRAMOSA2RNAi transfer-gen plant props up stalk and obviously extends, as Fig. 7-8.And the secondary of OsRAMOSA2 gene overexpression transfer-gen plant props up stalk obviously shortening, as Fig. 7-8.
Conclusion
To sum up, overexpression OSRAMOSA2 gene causes obviously dense cluster phenotype, and analyzing reason is that the shortening that firsts and seconds props up stalk causes; Meanwhile, the present inventor has carried out RNAi function to OSRAMOSA2 gene and has lowered research, find that the fringe of transfer-gen plant occurs that firsts and seconds props up the phenotype of stalk prolongation, thus entirety is presented as the phenotype of relatively rare fringe.OSRAMOSA2 protein localization is in tenuigenin; The function of growing with fringe type adapts, and the expression of OSRAMOSA2 gene in stem, fringe, vegetative point and little Hua is strong.Can think: the RNAi of OSRAMOSA2 gene props up the length of stalk owing to extending firsts and seconds, thus provide space structure more fully for the growth of small ear and seed, thus in increase yield, there are potentiality, significant for increase crop yield.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that 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 limited range equally.
Claims (12)
1. a purposes for the conditioning agent of RAMOSA2 polypeptide or its encoding gene or RAMOSA2 polypeptide, for regulating plant plant height, fringe phenotype and a stalk length; Described RAMOSA2 polypeptide is the polypeptide of aminoacid sequence shown in SEQIDNO:2;
Wherein, described RAMOSA2 polypeptide or its encoding gene are used for: dwarf plant plant type, make the fringe of plant present dense cluster phenotype, shorten plant and prop up stalk length;
Wherein, the conditioning agent of described RAMOSA2 polypeptide is the disturbing molecule of specificity interference RAMOSA2 genetic transcription, described disturbing molecule is for suppressing or dsRNA, the antisense nucleic acid of reticent target with the encoding gene of RAMOSA2 polypeptide or its transcript, maybe can express or be formed the construction of described dsRNA, antisense nucleic acid, for: make the fringe of plant present rare fringe phenotype, extend plant and prop up stalk length;
Wherein, described plant is paddy rice.
2. purposes as claimed in claim 1, it is characterized in that, described antisense nucleic acid comprises: siRNA, Microrna.
3. purposes as claimed in claim 1, is characterized in that, described disturbing molecule is dsRNA using SEQIDNO:1 as reticent target or construction.
4. purposes as claimed in claim 3, it is characterized in that, described disturbing molecule is construction, the structure containing shown in formula (I):
Seq
forward-X-Seq
oppositelyformula (I),
In formula (I),
Seq
forwardfor the polynucleotide shown in 173-332 position in SEQIDNO:1, Seq
oppositelyfor with Seq
forwardcomplementary polynucleotide;
X is for being positioned at Seq
forwardand Seq
oppositelybetween intervening sequence, and described intervening sequence and Seq
forwardand Seq
oppositelynot complementary.
5. a method for regulating plant plant height, fringe phenotype and a stalk length, comprising: the expression of RAMOSA2 polypeptide or activity in regulating plant; Wherein said RAMOSA2 polypeptide is the polypeptide of aminoacid sequence shown in SEQIDNO:2;
Wherein, described plant is paddy rice.
6. method as claimed in claim 5, it is characterized in that, described method comprises: the expression or the activity that raise RAMOSA2 polypeptide in plant, thus dwarf plant plant type, makes fringe present dense cluster phenotype or shorten a stalk length.
7. method as claimed in claim 6, it is characterized in that, described method comprises: proceeded in plant by the encoding gene of RAMOSA2 polypeptide.
8. method as claimed in claim 7, it is characterized in that, described method comprises:
(1) provide the Agrobacterium of carrying expression vector, described expression vector contains the encoding gene of RAMOSA2 polypeptide; With
(2) vegetable cell or tissue or organ are contacted with the Agrobacterium in step (1), thus make the encoding gene of RAMOSA2 polypeptide proceed to plant.
9. method as claimed in claim 5, it is characterized in that, described method comprises: expression or the activity of lowering RAMOSA2 polypeptide in plant, thus makes fringe present rare fringe phenotype or extend a stalk length.
10. method as claimed in claim 9, it is characterized in that, described method comprises: specificity disturbed the disturbing molecule of RAMOSA2 genetic transcription to proceed in plant.
11. methods as claimed in claim 10, it is characterized in that, described method comprises:
A () provides the Agrobacterium of carrying expression vector, described expression vector contains the disturbing molecule of interference RAMOSA2 genetic transcription; With
B vegetable cell or tissue or organ contact with the Agrobacterium in step (a) by (), thus make the disturbing molecule of interference RAMOSA2 genetic transcription proceed to plant.
The purposes of 12. 1 kinds of RAMOSA2 polypeptide or its encoding gene, as the marker of the plant height of plant identification, fringe phenotype and a stalk length.
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| Architecture of floral branch systems in maize and related grasses;Erik Vollbrecht et al;《NATURE》;20050825;第436卷(第25期);1119-1126 * |
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| ramosa2 encodes a LATERAL ORGAN BOUNDARY domain protein that determines the fate of stem cells in Branch Meristems of Maize;Esteban Bortiri et al;《The Plant Cell》;20060106;第18卷(第3期);574-585 * |
| ramosa2 encodes a LATERAL ORGAN BOUNDARY domain protein that determines the fate of stem cells in Branch Meristems of Maize;唐如春 等;《中国农业科学》;20111231;第44卷(第3期);439-446 * |
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