CN103993018B - Control Plant Height of Rice, improve lodging tolerance, increase available tillering and the gene of yield and application thereof - Google Patents

Abstract

The present invention is open reduces Plant Height of Rice, raising lodging tolerance, increase rice tillering number and the SDT gene of yield and application thereof.SDT gene code OsmiR156h.In the sdt rice mutant partly downgrading many tillers, 3 ' the UTR length of the mRNA of sdt genetic transcription are shorter than 3 ' UTR of wild type SDT genetic transcription, cause sdt gene transcription level higher than wild type SDT gene expression dose.Genetic analysis shows that sdt is the sudden change of semidominance, and it can suppress the longitudinal splitting of stem stalk cell to control each panel length and plant height, and then improves the lodging tolerance of Oryza sativa L.；Meanwhile, it can increase Oryza sativa L. available tillering, and then improves Oryza sativa L. every strain grain number per spike and yield.First demonstration that 3 ' the UTR length of regulation and control OsmiR156h genetic transcription mRNA can change the expression of this gene, and then realize cultivating the application prospect of the Oryza sativa L. with merit.

Description

Control Plant Height of Rice, improve lodging tolerance, increase available tillering and the base of yield Cause and application thereof

Technical field

The invention belongs to plant biotechnology field.In particular it relates to a kind of control Plant Height of Rice, improve anti-fall Volt ability, increase available tillering and the gene of yield and application thereof.The invention still further relates to the non-coding area sequence of this gene.

Background technology

Oryza sativa L. (Oryza sativa L.) is one of the most highly important cereal crops, and rice yield improves and protects Barrier grain security is relevant with agricultural sustainable development breath.In current agricultural production, the raising of rice yield depends in a large number Additional fertilising, but so can strengthen the danger producing the lodging underproduction at rice maturity, thus again limit fertilizer Amount of application.The method tackling this contradiction is the extensive application of Oryza sativa L. Semi dwarfism gene sd1^1,2This gene is otherwise known as " green Color revolution " gene, its application result in and starts " the most green leather that the Oryza sativa L. affected so far is significantly increased production the sixties in last century Life "^3,4.On the basis of the first time Green revolution, how to continue to improve rice yield to adapt to ever-increasing population and grain Demand, is pursuing a goal of rice breeding man.Although sd1 gene is widely used in current agricultural production, but lodging Problem remains the bottleneck that a SOYBEAN IN HIGH-YIELD BREEDING faces.During high-yield rice rearing new variety, new in the urgent need to excavating Half Dwarfing Gene of type, it is possible to ensure to continue to improve capacity for the resistance to lodging and yield on the application foundation of sd1 gene.The present invention just retouches State and a kind of new can improve Oryza sativa L. capacity for the resistance to lodging, improved rice tillering number and the paddy gene SDT (SEMID of yield simultaneously WARFAND HIGH-TILLERING)。

Summary of the invention

The present invention relates to a kind of control Plant Height of Rice, improve lodging tolerance, increase available tillering and the gene of yield And application.More specifically, the present inventor utilizes Oryza sativa L. half to downgrade the something lost that More-tiller mutant sdt builds with Oryza sativa L. hybridization in evening 3 Pass colony, use the method for qtl analysis and map based cloning to clone control Plant Height of Rice first, improved lodging tolerance, increase The gene (the mutant sdt that SDT and its 3 '-UTR shortens) of available tillering and yield, and open containing SDT gene by building The positive transgenic plant of vector Anhui rice 44 (wild type) material of SDT and the sdt cDNA under mover driving is verified The function of SDT Yu sdt gene.

It is an object of the invention to provide a kind of from Oryza sativa L. one of separating clone can improve simultaneously lodging tolerance and The critical function gene of yield and application thereof.

SDT gene, by hybridization, the method utilizing map based cloning, is finely navigated to No. six chromosome of Oryza sativa L. by the present invention In the physical extent of long-armed 26.3Kb, this section has three candidate genes predicted, order-checking comparative analysis shows wherein one Detect on second exon of individual gene LOC_Os06g44034 that fragment is inserted and deletion mutation.This gene is aobvious outside having 5 Son, the pre-mRNA of coding OsmiR156h gene.Coding OsmiR156h loop-stem structure (SEQ ID NO:5 sequence) is positioned at first Individual exon, First Exon and the second to five exons afterwards produce 3 ' long-UTR (3 '-noncoding region) tail.

The first aspect of the invention, refers to, by backcrossing and molecular marker assisting sifting, construct containing sudden change half short Change the rice material of many Tillering genes sdt, select the chromosome segment under 6-sdt, 9311 backgrounds to replace including Anhui rice 44-sdt, peace Based material CSSL-sdt and NIL 9311-sdt/sd1,9311-sdt/SD1 and excellent allelic variation OsSPL14^WFPMiscellaneous Hand over the NIL NIL-OsSPL14 obtained^WFP/ sdt etc..

A second aspect of the present invention relates to controlling Plant Height of Rice, improving lodging tolerance, increase available tillering and yield Gene, it is the polynucleotide of a kind of separation, and it comprises the nucleotide sequence selected from following group nucleotide sequence:

(1) any one shown nucleotide sequence in SEQ ID NOs:1-4；

(2) complementary series with the nucleotide sequence of (1) hybridizes under medium stringency condition, preferred high stringent hybridization condition Nucleotide sequence；

(3) have at least 70% with the nucleotide sequence of (1), preferably at least 80%, more preferably at least 90%, the most extremely The nucleotide sequence of few 95% or 98% or 99% sequence iden；

(4) after montage, have at least 70% with SEQ ID NOs:1-4, preferably at least 80%, more preferably at least 90%, the nucleotide sequence of the sequence iden of especially at least 95% or 98% or 99%；；

(5) with the nucleotides sequence of (1) be classified as effect core and derivative nucleotide sequence

(6) active fragment of any one nucleotide sequence in (1)-(5)；Or

(7) with the nucleotide sequence of any one nucleotide sequence complementary in (1)-(5).

Wherein, the wild type shape of the gene of Plant Height of Rice, raising lodging tolerance, increase available tillering and yield is controlled Formula SDT represents, the mutant forms sdt that its 3 '-UTR shortens represents.SDT cDNA sequence and mutant, relevant startup The nucleotide sequence of son and 3 '-UTR is by shown in SEQ ID NOs:1-10, referring specifically to table 1 below.

A third aspect of the present invention includes the gene of control Plant Height of Rice, lodging tolerance, available tillering and yield Non-coding area sequence (includes promoter and 3 '-non-coding area sequence etc.), and it comprises the core selected in following group nucleotide sequence Nucleotide sequence:

(1) nucleotide sequence shown in SEQ ID NOs:6-10；

(2) complementary series with the nucleotide sequence of (1) hybridizes under medium stringency condition, preferred high stringent hybridization condition Nucleotide sequence；

(3) have at least 70% with the nucleotide sequence of (1), preferably at least 80%, more preferably at least 90%, the most extremely The nucleotide sequence of few 95% or 98% homogeneity；

(4) active fragment of any one nucleotide sequence in (1)-(3)；Or

(5) with the nucleotide sequence of any one nucleotide sequence complementary in (1)-(4).

The sequence names of table 1:SEQ ID NOs:1-10 and source

A fourth aspect of the present invention comprises control Plant Height of Rice, improves lodging tolerance, increase available tillering and yield The construct of non-coding area sequence of gene SDT, wherein said non-coding area sequence include SDT gene promoter sequence and 3 '-non-coding area sequence.Carrier used by wherein said construct is cloning vehicle or for expressing described polynucleotide Expression vector.Further, the promoter sequence of wherein said SDT gene as shown in SEQ ID NO:7 or 8,3 '-UTR (3 '-non- Coding region) sequence is as shown in SEQ ID NO:9 or 10.

A fifth aspect of the present invention relates to a kind of recombinant host cell, and it contains recombinant precursor of the present invention, or Its genome is integrated and has polynucleotide of the present invention.Described host cell can be selected from zooblast, plant cell Or microbial cell, such as Bacillus coli cells, agrobatcerium cell, preferred plant cell, most preferably rice cell.Described carefully Born of the same parents can be separation, in vitro, cultivation an or part for plant.

A sixth aspect of the present invention relates to the polynucleotide of the present invention or the recombinant precursor of the present invention or the weight of the present invention Group host cell purposes in improvement Rice Characters.The method that the invention still further relates to improve Rice Characters, the method includes system For the polynucleotide containing the present invention or the Oryza sativa L. of the construct of the present invention, such as, described method can include from the present invention's Rice cell regeneration of transgenic Oryza sativa L., or utilize conventional rotaring dyeing technology to be transfected in rice cell by SDT or sdt gene to obtain Obtain transgenic rice plant, or will be containing control Plant Height of Rice of the present invention, raising lodging tolerance, increase tiller number Plant and another paddy rice cross breeding with the gene of yield.Described character includes but not limited to: plant height, lodging index, tiller number and Yield etc..

The innovation that a seventh aspect of the present invention relates to the SDT described in the present invention and affects rice yield as microRNA is used On the way.

A eighth aspect of the present invention relates to the insertion sequence of the sdt mutant gene of separation in the present invention and makes sdt gene 3 '-UTR length shorten, and cause gene expression dose to increase method and the correlated series of this new regulation and control mrna expression level. The insertion sequence of wherein said sdt mutant gene is as shown in SEQ ID NO:6.

The present inventor finds that the insertion sequence shown in SEQ ID NO:6 is inserted into arbitrary target gene under study for action The 3 '-UTR of (being not limited to SDT gene), make 3 '-UTR of described target gene shorten, thus change the mRNA of described target gene Transcriptional level.It is achieved in the regulation and control that described target gene is expressed." regulation and control " described herein includes that target gene mRNA turns The rise of record level or downward.Regulate the mRNA transcriptional level of described target gene, some character of plant will be affected.

In particular it relates to a kind of method of goal of regulation and control gene expression dose, described method includes: will comprise The construct of SEQ ID NO:6 proceeds in plant cell, makes SEQ ID NO:6 be inserted into 3 '-UTR of described target gene, makes The 3 '-UTR length obtaining described target gene shorten, and cause the mrna expression level of described target gene to change, thus realize institute State the regulation and control that target gene is expressed." regulation and control " described herein includes rise or the downward of target gene mRNA transcriptional level.

It should be appreciated by those skilled in the art that the 3 '-UTR that SEQ ID NO:6 is inserted into described target gene can lead to The molecular cloning method crossing routine realizes, such as, by gene recombination method, utilize SEQ ID NO:6 both end sides connects and mesh , there is homologous recombination with the specific region of target gene in the homologous sequence that mark gene specific region is complementary, so that SEQ ID NO:6 is inserted into suitable position.

Preferably, the invention still further relates to the insertion sequence shown in SEQ ID NO:6 in the crop cultivating output increased Application.A kind of method that the present invention relates to crop cultivating output increased, described method includes: will comprise SEQ ID NO:6's Construct proceeds in crop, makes SEQ ID NO:6 be inserted into 3 '-UTR of OsmiR156h gene so that in described crop The mrna expression level of OsmiR156h gene increases, thus obtains plant height reduction, lodging tolerance enhancing, tiller number and yield The crop increased, wherein said crop is selected from Oryza sativa L. or Semen Tritici aestivi.

A ninth aspect of the present invention relates to a kind of method of Oryza sativa L. cultivating output increased.The method includes: from the present invention The Oryza sativa L. host cell regenerating plants that comprises SDT or sdt gene, or utilize the conventional transgenic method transfection present invention Nucleotide sequence and obtain transgenic paddy rice, or the appropriate expression regulating SDT or sdt gene, or change in right amount The expression of OsmiR156h maturation body, or the plant of the sdt gene containing the present invention is hybridized with another plant；Wherein Described plant is preferably plant height reduction, lodging tolerance strengthens, tiller number increases and yield promotes.

In sum, the present invention provides following embodiment:

1. controlling Plant Height of Rice, improve lodging tolerance, increase Oryza sativa L. available tillering and the gene of yield, it is a kind of The polynucleotide separated, it comprises from the following nucleotide sequence organized and select nucleotide sequence:

(1) any one shown nucleotide sequence in SEQ ID NOs:1-4；

(2) with the complementary series of the nucleotide sequence of (1) under medium stringency condition, preferred high stringent hybridization condition miscellaneous The nucleotide sequence handed over；

(3) have at least 70% with the nucleotide sequence of (1), preferably at least 80%, more preferably at least 90%, the most extremely The nucleotide sequence of few 95% or 98% sequence iden；

(4) after montage, have at least 70% with SEQ ID NOs:1-4, preferably at least 80%, more preferably at least 90%, the nucleotide sequence of the sequence iden of especially at least 95% or 98%；

(5) with the nucleotides sequence of (1) be classified as effect core and derivative nucleotide sequence；

(6) active fragment of any one nucleotide sequence in (1)-(5)；Or

(7) with the nucleotide sequence of any one nucleotide sequence complementary in (1)-(5).

2. a recombinant precursor, it is characterised in that comprise the polynucleotide sequence of the gene of the 1st, described construct institute Carrier be cloning vehicle or for expressing the expression vector of described polynucleotide.

3. a recombinant host cell, it is characterised in that comprise the polynucleotide sequence of the gene of the 1st or the weight of the 2nd Integrating the polynucleotide sequence of the gene having the 1st in group construct, or its genome, wherein said cell is that microorganism is thin Born of the same parents.

4. the 3rd described recombinant host cell, wherein said cell is Bacillus coli cells.

5. the 3rd described recombinant host cell, wherein said cell is agrobatcerium cell.

6. the method cultivating the crop of output increased, described method includes: utilize the recombinant host cell of the 3rd to obtain Genetically modified crops, or the 1st described gene is transfected in crop cell acquisition genetically modified crops plant, maybe will be containing the The crop plant of 1 described gene controlling Plant Height of Rice, improving lodging tolerance, increase tiller number and yield is made with another Thing plant hybridization obtains hybrid crop plant so that the 1st described control Oryza sativa L. in described genetically modified crops or hybrid crop The expression of the gene of plant height, lodging tolerance, tiller number and yield increases, thus obtains plant height reduction, lodging tolerance increasing By force, the crop that tiller number and yield increase.

7. the 6th described method, wherein said crop is selected from Oryza sativa L. or Semen Tritici aestivi.

8. a method for goal of regulation and control gene expression dose, described method includes: will comprise the structure of SEQ ID NO:6 Body proceeds in plant cell, makes SEQ ID NO:6 be inserted into 3 '-UTR of described target gene so that described target gene 3 '-UTR length shorten, and affect the mrna expression level of described target gene, thus realize the regulation and control that described target gene is expressed.

9. the method cultivating the crop of output increased, described method includes: will comprise the construct of SEQ ID NO:6 Proceed in crop, make SEQ ID NO:6 be inserted into 3 '-UTR of OsmiR156h gene, so that in described crop The mrna expression level of OsmiR156h gene increases, and is derived from plant height reduction, lodging tolerance enhancing, tiller number and yield The crop increased, wherein said crop is selected from Oryza sativa L. or Semen Tritici aestivi.

10. the method cultivating the Oryza sativa L. of output increased, described method includes: comprising OsSPL14^WFPThe back of the body of gene Under scape, import control Plant Height of Rice, lodging tolerance, tiller number and the gene of yield shown in SEQ ID NO:2 or 4.

The method of 11. 1 kinds of Oryza sativa L. cultivating output increased, described method includes: under the background comprising sd1 gene, lead Enter control Plant Height of Rice, lodging tolerance, tiller number and the gene of yield shown in SEQ ID NO:2 or 4.

12. the 1st described controls Plant Height of Rice, improve lodging tolerance, increase Oryza sativa L. available tillering and yield The promoter sequence of gene, it is SEQ ID NO:7 or 8.

13. the 1st described controls Plant Height of Rice, improve lodging tolerance, increase Oryza sativa L. available tillering and yield The 3 ' of gene-UTR sequence, it is SEQ ID NO:9 or 10.

The following is the definition to some terms used in the present invention.Except as otherwise noted, term used herein tool There is the known meaning of this area those of ordinary skill.

" be correlated with "/" being operably connected " refer to the nucleotide sequence that two physics or function are relevant.Such as, if promoter, 3 '-noncoding region or regulation DNA sequence and coding RNA or protein DNA sequence are operably connected or position to such an extent as to adjust Joint DNA sequence is by impact coding or the expression of structural DNA sequence, then claim promoter, 3 '-noncoding region or regulation DNA Sequence and coding RNA or protein DNA sequence " relevant ".

" mosaic gene " is recombinant nucleic acid sequence, and wherein promoter, 3 '-noncoding region or regulation nucleotide sequence are operationally Connect coding mRNA or the nucleotide sequence as protein expression, or with coding mRNA or the nucleotide sequence as protein expression Relevant so that regulation nucleotide sequence can regulate transcribing or expressing of associated nucleic acid sequences.The regulation nucleotide sequence of mosaic gene is not It it is the associated nucleic acid sequences that is normally operably connected as found in nature.

" coded sequence " is to be transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, has the nucleic acid sequence of adopted RNA or antisense RNA Row.Preferably, subsequently in organism antisense RNA to produce protein.

In the context of the present invention, " correspond to " mean when nucleic acid coding and the non-coding sequence of different SDT genes are mutual During comparison, the nucleic acid of " corresponding to " some counting position is and these position comparisons, but needs not to be relative to specific SDT each Nucleic acid in these exact numerical positions of nucleic acid coding and non-coding sequence.Equally, when coding and the non-coding sequence of specific SDT When row and the coding of reference SDT and non-coding sequence comparison, this of " corresponding to " reference SDT some counting position of sequence is specific SDT sequence amplifying nucleic acid is and with reference to these position comparisons of SDT sequence, but need not to be at this specific SDT each nucleic acid coding and Nucleic acid in these exact numerical positions of non-coding sequence.

Mean to instruct used herein of " expression cassette " and be suitable for the nucleic acid sequence that in host cell, specific nucleotide sequence is expressed Row, comprise the promoter being operably connected with purpose nucleotide sequence, and described purpose nucleotide sequence is operably connected end Stop signal.Generally, it also comprises nucleotide sequence and correctly translates required sequence.The expression cassette comprising purpose nucleotide sequence can Being chimeric, it is intended that at least one of its composition is allos relative at least its one of other composition.Expression cassette can also It is naturally-occurring, but obtains the expression cassette for heterogenous expression in recombinant form.But, generally, expression cassette is relative to host It is allos, i.e. the specific nucleic acid sequence non-natural of expression cassette occurs in host cell, it is necessary to drawn by transformation event Enter the precursor of host cell or host cell.The expression of expression cassette nucleotide sequence can be by constitutive promoter or induction type Promoter controls, and the most only when host cell is exposed to some specific outside stimuluss, described inducible promoter is just initial to be turned Record.If the situation of multicellular organisms, such as plant, promoter can also be to particular organization, or organ or stage of development Special.

The restriction region that " gene " is in genome, in addition to aforementioned coding nucleic acid sequence, comprises other mainly The nucleotide sequence of modulability, described modulability nucleotide sequence is responsible for the expression of coded portion, i.e. transcription and translation and is controlled.Gene is also Other 5 ' and 3 ' non-translated sequence and terminator sequence can be comprised.The element that can exist further is, such as intron.

" allos " nucleotide sequence is the nucleotide sequence that the host cell non-natural being introduced into it is relevant, comprises non-natural and deposits The multicopy naturally occurring nucleotide sequence.

" homology " nucleotide sequence is the natural relevant nucleotide sequence of the host cell being introduced into it.

" homologous recombination " is being exchanged with each other of nucleic acid fragment between homologous nucleic acid molecules.

When nucleic acid sequence encoding has the polypeptide of same acid sequence with the polypeptide with reference to nucleic acid sequence encoding, this core Acid sequence be " isocoding " with reference to nucleotide sequence.

The protein of " separation " nucleic acid molecules or separation is artificially to separate with its natural surroundings and exist, and is not The nucleic acid molecules of natural product or protein.The nucleic acid molecules or the protein that separate can exist with purified form, or permissible It is present in non-natural environment such as, such as in recombinant host cell or transgenic plant.

" natural gene " refers to gene present in the genome of no transformed cells.

Term " naturally occurs " for describing the object that can find in nature, and it is with artificially generated object not With.For example, it is possible to separate from natural source, not it is not intended for manually modified, organism (including virus) at laboratory Present in protein or nucleotide sequence be " naturally occurring ".

" nucleic acid molecules " or " nucleotide sequence " is the single or double chain DNA or the linear sheet of RNA that can separate from any source Section.In the context of the present invention, it is preferable that nucleic acid molecules is DNA fragmentation." nucleic acid molecules " is also referred to as polynucleotide molecule.

" plant " is any plant in any stage of development, particularly seed plant.

" plant cell " is structure and the physiological unit of plant, comprises protoplast and cell wall.Plant cell is permissible It is the individual cells separated or cultivation cellular forms, or as high organized unit such as, such as plant tissue, plant organ Or a part for whole plant.

" plant cell cultures " means the plant units of various stage of development such as, such as protoplast, and cell is cultivated thin Born of the same parents, the cell in plant tissue, pollen, pollen tube, ovule, blastular, zygote and the culture of embryo.

" vegetable material " refers to leaf, stem, root, spends or the part of flower, fruit, pollen, ovum, zygote, seed, cutting, carefully Born of the same parents or tissue culture, or any other parts of plant or product.

" plant organ " be plant clearly with obvious structuring and the part of differentiation, such as root, stem, leaf, alabastrum or embryo.

Mean to be organized into one group of plant cell of 26S Proteasome Structure and Function unit used herein of " plant tissue ".Including in plant Or any tissue of plant in culture.This term includes but not limited to whole plant, plant organ, plant seed, tissue training Support thing and be organized into any plant cell group of structure and/or functional unit.This term comprises with maybe this definition listed above The use in conjunction of any particular type plant tissue or be used alone it is not intended that get rid of other type of plant tissue any.

" promoter " is the DNA sequence of coding region upstream untranslated, and it comprises the binding site of RNA polymerase 11, and Initial DNA transcribes.Promoter region can also comprise other element as Gene expression and regulation thing.

" protoplast " is not have cell wall or the plant cell of the only separation of part cell wall.

" regulating element " refers to participate in controlling the sequence that nucleotide sequence is expressed.Regulating element comprises the purpose that is operably connected The promoter of nucleotide sequence and termination signal.Generally they also comprise nucleotide sequence and correctly translate required non-coding sequence Row.

" reorganization " nucleic acid is by Shuffling Method, the nucleic acid that any Shuffling Method as described herein produces.Pass through people Work and the mode (physically or actually) that circulates alternatively two or more nucleic acid (or character string) of recombinating produce reorganization core Acid.Usually, utilize one or multi-step screening step to identify purpose nucleic acid in Shuffling Method；Can be in any reconstitution steps Before or after carry out this screening step.In some (but not all) shuffling embodiments, it is desirable to carry out many wheel loads before screening Group is to increase the multiformity in storehouse to be screened.It is alternatively possible to circulation repeats restructuring and all processes of screening.Based on context, Reorganization can refer to restructuring and all processes of screening, or alternately, can only refer to the restructuring part of all processes.

Phrase " essentially identical " in two nucleic acid or sequence alignment of protein refers to when comparing with comparison to obtain maximum To during correspondence as utilize one of sequence below comparison algorithm or range estimation measure, have at least 60%, preferably 80%, more preferably 90%, even more preferably 95% and most preferably at least 99% nucleotide or two or more sequences of amino acid residue identity or Subsequence.Preferably, basic identity is present in the sequence area of at least about 50 residues in length, more preferably at least about 100 On the region of residue, most preferably, the sequence at least about 150 residues is essentially identical.In particularly preferred embodiments, In the whole length in coding region, sequence is essentially identical.And, essentially identical nucleic acid or protein sequence have essentially identical Function.

In order to carry out gene comparision, generally, sequence as canonical sequence with detection gene comparision.When utilizing sequence During comparison algorithm, detection and canonical sequence are input in computer, if necessary specify the coordinate of subsequence, and specify The parameter of sequence algorithm program.Then, according to selected program parameter, sequence comparison algorithm will calculate detection sequence relative to The percent sequence identity of canonical sequence.

Such as, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482 (1981), By the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), by Pearson & Lipman, Proc.Nat ' the similarity retrieval method of 1.Acad.Sci.USA85:2444 (1988), by the meter of these algorithms Calculation machineization implements (GAP, BESTFIT, FASTA and TFASTA, Genetics in Wisconsin Genetics software kit Computer Group, 575Science Dr., Madison, WI) or (generally seen, Ausubel etc., hereafter) by range estimation The optimal comparison of sequence for comparing can be carried out.

The examples of algorithms being suitable to measure percent sequence identity and sequence similarity is BLAST algorithm, Altschul etc., J.Mol.Biol.215:403-410 describe this algorithm in (1990).By in country's Biotechnology Information The heart (http://www.Ncbi.nlm.nih.gov/) public can obtain carrying out the software of BLAST analysis.This algorithm includes: pass through Identify and search the short word of a length of W in sequence and first identify high scoring sequence to (HSPs), described short word with data Coupling or meet some on the occasion of threshold score T during the word comparison of equal length in the sequence of storehouse.T is referred to as neighborhood word score threshold (Altschul etc., 1990).These initial neighborhood word hits go to find to comprise the longer of them as the clue starting to search HSPs.Then, the hit of these words is by the extension as far as possible of the both direction along each sequence, until accumulation comparison score value is not It is further added by.For nucleotide sequence, (mate the award score value of residue in pairs by parameter M；Always greater than zero) and N (mismatched residue Penalty value；Always less than zero) calculate accumulation score value.For aminoacid sequence, calculate accumulation score value with Scoring matrix.Work as accumulation Comparison score value falls quantity X after rise from the maximum obtained, and owing to one or more negative scoring residue comparisons accumulate, accumulation score value reaches Or less than zero, or when any one of two sequences is reached home, the word hit in each direction extends and stops.The ginseng of BLAST algorithm Number W, T and X determine sensitivity and the speed of comparison.BLASTN program (for nucleotide sequence) uses word length value (W) 11, phase Prestige value (E) 10, cutoff value 100, M=5, N=-4 and two chains are relatively default value.For aminoacid sequence, BLASTP journey Sequence uses word length value (W) 3, it is desirable to value (E) 10 and BLOSUM62 Scoring matrix is that default value (sees, Henikoff & Henikoff, Proc.Natl.Acad.Sci.USA89:10915 (1989)).

In addition to calculating percent sequence identity, BLAST algorithm is also carried out the statistical analysis of similarity between two sequences (see, e.g. Karlin&Altschul, Proc.Nat ' l.Acad.Sci.USA90:5873-5787 (1993)).BLAST calculates It is minimum and probability (P (N)) that the similarity that method provides measures, and it provides between two nucleotide or aminoacid sequence accidental The instruction of the probability of coupling occurs.Such as, if minimum and probability that detection nucleotide sequence compares with reference to nucleotide sequence are less than About 0.1, more preferably less than about 0.01, most preferably less than about 0.001, then think that detection nucleotide sequence is similar to canonical sequence.

Essentially identical another index of two nucleotide sequences is that two molecules hybridize the most mutually.Phrase " specific hybrid " refers to when this sequence is present in complex mixture (such as, total cell) DNA or RNA, at stringent condition Under, molecule is only combined with specific nucleotide sequence, forms Double helix or hybridization." substantially combine " and refer between probe nucleic acid and target nucleic acid Complementary hybridization, and comprise less mispairing, can tolerate described mispairing, to realize target by the stringency reducing hybridization medium The expectation detection of nucleotide sequence.

In nucleic acid hybridization assay such as Southern and Northern hybridization context " stringent hybridization condition " and " the most miscellaneous Hand over rinsing condition " it is sequence dependent, and be different under varying environment parameter.Longer sequence is special at higher temperature Specific hybridization.At Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part i the 2nd chapter " Overview of principles of hybridization and the strategy of nucleic acid probe assays″ It appeared that a large amount of guides of nucleic acid hybridization in Elsevier, New York.Typically, under limiting ionic strength and pH Particular sequence, high stringency hybridization and rinsing condition are selected below heat fusion joint (T_m) about 5 DEG C.Typically, at " strict bar Part " under, probe will hybridize with its target subsequence, and not with other sequence hybridization.

T_mIt is (under the conditions of limiting ionic strength and pH) 50% target sequence and the temperature during probe hybridization mated completely. For specific probe, the strictest condition is selected equal to T_m.In Southern or Northern trace on filter membrane The example having a stringent hybridization condition of the complementary nucleic acid hybridization more than 100 complementary residues is at 42 DEG C, has 1mg heparin 50% Methanamide, overnight carry out this hybridization.The example of high stringency wash conditions is 72 DEG C, 0.15MNaCl about 15 minutes.Sternly The example of lattice rinsing condition is at 65 DEG C, 0.2x SSC rinse 15 minutes (see, Sambrook, hereafter, retouching of SSC buffer State).Generally, before high stringency wash, low stringency wash is carried out to remove background probe signal.For such as more than 100 For the Double helix of nucleotide, the example of middle stringency wash is 45 DEG C, and 1x SSC rinses 15 minutes.For such as more than 100 For the Double helix of individual nucleotide, the example of low stringency wash is 40 DEG C, and 4-6x SSC rinses 15 minutes.For short probe (such as, about 10 to 50 nucleotide), stringent condition is typically included in the salt of the less than about 1.0M Na ion of pH7.0 to 8.3 Concentration, normally about 0.01 arrives 1.0Na ion concentration (or other salt), typical temperature at least about 30 DEG C.Gone stable by interpolation Agent such as Methanamide can also obtain stringent condition.Usually, in specific cross measures, noise signal to noise ratio is with regard to observed by unrelated probe To value high by 2 × (or higher) show the detection of specific hybridization.If they volumes of the nucleic acid hybridized the most mutually The protein of code is essentially identical, then they are still essentially identical.Such as, close when the maximum allowed with genetic code When numeral degeneracy creates copy nucleic acid, arise that this situation.

Example that hybridization/rinsing condition arranged is presented herein below, and described condition may be used for clone with the present invention with reference to nucleotide The homologous nucleotide sequence that sequence is essentially identical: with reference to nucleotide sequence with reference nucleotide sequence preferably at 50 DEG C, 7% 12 Alkyl sodium sulfate (SDS), 0.5M NaPO₄, 1mM EDTA hybridizes, at 50 DEG C, 2X SSC, 0.1%SDS rinses, it more desirable to At 50 DEG C, 7% sodium lauryl sulphate (SDS), 0.5M NaPO₄, 1mM EDTA hybridizes, at 50 DEG C, 1X SSC, 0.1% SDS rinses, it more desirable at 50 DEG C, 7% sodium lauryl sulphate (SDS), 0.5M NaPO₄, 1mM EDTA hybridizes, 50 DEG C, 0.5X SSC, 0.1%SDS rinses, it is preferable that at 50 DEG C, 7% sodium lauryl sulphate (SDS), 0.5MNaPO₄, 1mM EDTA hybridizes, at 50 DEG C, 0.1X SSC, 0.1%SDS rinses, it is highly preferred that at 50 DEG C, 7% sodium lauryl sulphate (SDS), 0.5M NaPO₄, 1mMEDTA hybridizes, at 65 DEG C, 0.1X SSC, 0.1%SDS rinses.

Two nucleotide sequences or essentially identical another index of protein are the protein and second of the first nucleic acid coding The Western Immuno cross reaction of nucleic acid coding or specific bond.Therefore, protein is the most essentially identical with the second protein, example Such as, two of which protein only because preservative replacement and different.

" synthesis " refers to comprise the nucleotide sequence of non-existent architectural feature in native sequences.Such as, claim more closely The artificial sequence of similar dicotyledonous and/or monocot genes G+C content and normal codon distribution is synthesis.

" converting " process being to introduce heterologous nucleic acids in host cell or organism, especially, " conversion " means that DNA divides Sub-stable integration enters in purpose organism genome.

" conversion/transgenic/restructuring " refer to have been incorporated into the host organisms of exogenous nucleic acid molecule, such as antibacterial or plant Thing.Nucleic acid molecules can stably be integrated into host genome or nucleic acid molecules can also be deposited as extrachromosomal molecule ?.This extrachromosomal molecule can be autonomous duplication.The cell converted, tissue, or plant are interpreted as not only comprising conversion The end product of process, also comprises its transgenic progeny." non-transformed ", " not genetically modified ", or " nonrecombinant " host refer to Do not contain the wild-type organisms of exogenous nucleic acid molecule, such as antibacterial or plant.

Term used herein " polynucleotide ", " polynucleotide molecule ", " polynucleotide sequence ", " coded sequence ", " open Put reading frame (ORF) " etc. include strand or the DNA of double-strand and RNA molecule, one or more protokaryon sequence, cDNA sequence can be comprised Row, comprise the genomic dna sequence of exon and intron, the DNA of chemosynthesis and RNA sequence, and have justice with corresponding Antisense strand.

The method producing and operating polynucleotide molecule disclosed herein and oligonucleotide molecules is those skilled in the art Known, and can according to have described that recombinant technique (see Maniatis etc., 1989,Molecular cloning, laboratory manual, cold spring Publishing house of Cold Spring Harbor Laboratory, Cold SpringHarbor, New York；Ausubel etc., 1989,Molecular biology current techniques, Greene Publishing Associates & Wiley Interscience, NY；Sambrook etc., 1989,Molecular cloning, laboratory Handbook, second edition, CSH Press, Cold SpringHarbor, New York；Innis etc. (compile), and 1995,PCR strategy, Academic Press, Inc., San Diego；With Erlich (volume), 1992, round pcr, Oxford University Press, New York) complete.

Accompanying drawing is sketched

That the following picture having white scale is not illustrated is 20em.

Fig. 1 shows the plant type of a pair NIL (WD44-SDT and WD44-sdt) under Anhui rice 44 background and yield Shape compares.A () plant type compares；B length that () stem stalk respectively saves compares；C () ganglion cell's longitudinal section compares, white scale: 0.1mm；Lodging index, flowering time, tiller number, spike length, Primary branch number, Secondary branch between (d)～(m) NIL Number, number of grain per ear, mass of 1000 kernel, single plant yield and harvest index compare.

Fig. 2 shows the map based cloning to sdt gene.A () builds two parents used by genetical population for map based cloning Evening, three and sdt mutant plant types compared；Contain under (b) 9311 and 9311 background between the CSSL population of sdt gene Plant type compares；(c) map based cloning schematic diagram, final section screens at No. six chromosome long arm of Oryza sativa L. between 3554P2 and 5453P1 Between the region of 26.3-kb, this microRNA of Pre-mRNA of LOC_Os06g44034 gene loci coding OsmiR156h Coding site is positioned at First Exon, and Second Exon order-checking finds the disappearance of 131bp and from mitochondrial ccmB gene Insertion mutation；D () its own promoter drives the OsmiR156h precursor cDNA from sdt mutant isolated can suppress Anhui The phenotype of rice 44-SDT height stalk；E () SDT gene outcome is more longer at 3 '-UTR ends than sdt gene outcome；F () is at different tissues Location detection OsmiR156h expression；(g) with Northern blot detect a pair near isogene based material stem stalk and The expression of tiller maturation body OsmiR156h；(h) WD44-sdt with carry OsSPL14 gene excellent allelic variation site OsSPL14^WFPWhat paddy rice cross breeding was separated contains and without OsSPL14^WFPThe phenotype of the WD44-sdt background plant in site compares.

The DNA that Fig. 3 is shown that being separated to from WD44-SDT and WD44-sdt respectively is at this base of LOC_Os06g44034 Sequence alignment because of site, it can be seen that have lacking of 131bp at Second Exon compared with DNA with WD44-SDT in WD44-sdt Lose and add and reversely insert from mitochondrial ccmB genetic fragment.

Fig. 4 is shown that the Pre-mRNA of OsmiR156h sequence alignment in a pair near isogene based material, Ke Yifa In existing WD44-sdt, after there is the Second Exon of disappearance and insertion, mRNA sequence is derived from mitochondrion ccmB by a bit of The fragment of gene is substituted；Special primer amplification cDNA fragment prediction mRNA length is utilized to shorten to from original 1552nt 987nt.See the e of Fig. 2.

Fig. 5 is shown that the different allelic variation heredity reciprocal effects of sd1 from sdt gene under 9311 backgrounds and to hybridization The impact of rice yield.(a) under 9311 backgrounds, four NIL 9311-SD1/SDT, 9311-SD1/sdt, 9311- Sd1/SDT and 9311-sd1/sdt plant type contrasts；The tiller number comparative result of material described in (b) (a)；Material in (c) (a) Lodging index comparative result；D () NIL peace selects 6 (Anxuan6) and peace to select 6-sdt (Anxuan6-sdt) as male parent Compare as the plant type of maternal sterile line first familiar generation with newly pacifying S (XinanS) respectively；E ()～(k) each economical character compares: open Take time (e), plant height (f), tiller number (g), mass of 1000 kernel (h), number of grain per ear (i), lodging index (j), single plant yield (k).

Fig. 6 shows excellent allelic variation OsSPL14^WFPCarry out being polymerized the near isogene based material NIL-of selection-breeding with sdt OsSPL14^WFP/ SDT and OsSPL14^WFP/ sdt character pair ratio.A () plant type compares；B ()～(f) Correlated Yield Characters contrasts: fall Volt index (b), tiller number (c), mass of 1000 kernel (d), number of grain per ear (e), single plant yield (f).

Fig. 7 show the carrier of the Oryza sativa L. sdt gene driven containing 35S promoter proceeded to Semen Tritici aestivi section agriculture 199 after obtain Transgenic positive wheat plant and non-transgenic section agriculture 199 contrast ratio are downgraded substantially, and tiller number showed increased.

Sequence table describes

In sequence table, for convenience of description, no matter DNA or mRNA sequence base t therein or corresponding u use t Representing, t can be considered as and u equivalence here.

SEQ ID NO:1:SDTcDNA sequence (from Anhui rice 44-SDT)；

SEQ ID NO:2:sdt cDNA sequence (from Anhui rice 44-sdt)；

SEQ ID NO:3:SDTgDNA sequence (from Anhui rice 44-SDT)；

SEQ ID NO:4:sdt gDNA sequence (from Anhui rice 44-sdt)；

SEQ ID NO:5:OsmiR156h loop-stem structure sequence (from Anhui rice 44-sdt)；

SEQ ID NO:6:sdt gDNA insertion sequence (part, from Anhui rice 44-sdt)；

SEQ ID NO:7:SDT promoter sequence (from Anhui rice 44-SDT)；

SEQ ID NO:8:sdt promoter sequence (from Anhui rice 44-sdt)；

SEQ ID NO.9:SDT cDNA3 '-UTR sequence (from Anhui rice 44-SDT)；

SEQ ID NO.10:sdt cDNA3 '-UTR sequence (from Anhui rice 44-sdt)；

SEQ ID NO:11:sdt gene loci special primer F；

SEQ ID NO:12:sdt gene loci special primer R；

SEQ ID NO:13:OsSPL14^WFPGene promoter sequence (5kb).

Detailed description of the invention

Through extensively in-depth study, present invention determine that one can change Plant Height of Rice, lodging index, effectively divides The gene SDT of tiller number and yield, this gene is positioned at No. six chromosome long arm of Oryza sativa L., and this gene expression amount suitably rises and may result in Plant half is downgraded, lodging index reduces, available tillering increases, single plant yield improves.The present inventor by this control Plant Height of Rice, The unnamed gene improving lodging tolerance, increase tiller number and yield is SDT.

Plant Transformation

In a particularly preferred embodiment, in higher organisms such as plant, express the control strain of at least one present invention The high mRNA/cDNA/microRNA with tiller number.Can be by the nucleotide sequence of the gene controlling plant height and tiller number of the present invention It is inserted in expression cassette, the most preferably, by this expression cassette stable integration in described Plant Genome.The most real at another Execute in mode, the nucleotide sequence of described control plant height and the gene of tiller number is included in the virus of non-pathogenic self replication In.The plant converted according to the present invention can be monocotyledon or dicotyledon, includes but not limited to Semen Maydis, Semen Tritici aestivi, greatly Wheat, rye (Secale cereale L.), Rhizoma Dioscoreae esculentae, bean, Semen Pisi sativi, Herba Cichorii, Caulis et Folium Lactucae sativae, Caulis et Folium Brassicae capitatae, Brassica oleracea L. var. botrytis L., Broccoli, Radix Brassicae rapae, Radix Raphani, Herba Spinaciae, Germinatus Phragmitis, Bulbus Allii Cepae, Bulbus Allii, Fructus Piperis, Herba Apii graveolentis, Cucurbita maxima, Fructus Cucurbitae moschatae, Fructus Cannabis, zucchini, Fructus Mali pumilae, pears, temperature, melon, Fructus Pruni salicinae, Fructus Pruni pseudocerasi, Fructus Persicae, Prunus persicanucipersica Schneider, Fructus Pruni, Fructus Fragariae Ananssae, Fructus Vitis viniferae, rasp berry, blackberry, Fructus Ananadis comosi, American Avocado Tree, papaya, Fructus Mangifera Indicae, Fructus Musae, Semen sojae atricolor, Fructus Lycopersici esculenti, Sorghum vulgare Pers., Caulis Sacchari sinensis, Radix Betae, Xiang Certain herbaceous plants with big flowers, oil seed rape, Herba Trifolii Pratentis, Nicotiana tabacum L., Radix Dauci Sativae, Cotton Gossypii, Herba Medicaginis, rice, Rhizoma Solani tuber osi, Fructus Solani melongenae, Fructus Cucumidis sativi, Arabidopsis and woody plant Thing such as coniferous tree and deciduous tree.Particularly preferably Oryza sativa L., Semen Tritici aestivi, Fructus Hordei Vulgaris, Semen Maydis, Herba bromi japonici or rye (Secale cereale L.).

The most desired nucleotide sequence has been converted and has entered in specified plant species, can breed in these species it or With traditional breeding method, it is transitioned into other kind of same species, in commercial variety.

Preferably, transgenic plant is expressed the nucleotide sequence of the present invention, in transgenic plant, thus causes phase Answer the biosynthesis of the microRNA of plant height and tiller number change.By this way, can produce there is the transgenic of Ameliorative character Plant.In order to express nucleotide sequence of the present invention in transgenic plant, nucleotide sequence of the present invention may need to modify and excellent Change.Its real bioactive sequence is present in a relatively small region, can be referring to SEQ ID NO:5.Additionally, nucleotide can be screened Sequence is to find the existence of the unconventional splice site causing message truncation.Utilize Patent Application Publication EP0385962 (Monsanto), the method described in EP0359472 (Lubrizol) and WO93/07278 (Ciba-Geigy), ripe with this area The site-directed induced-mutation technique known, PCR and synthetic gene build carry out needing in these nucleotide sequences carrying out all and change Become, as those described above changes.

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that the following example is only used for further illustrating The present invention, and it is not limited to the spirit and scope of the present invention.

It should be noted that it should be appreciated by those skilled in the art that in following embodiment that reagent used, enzyme etc. are except spy Do not mentionlet alone bright outside, be reagent or the enzyme of the analytical pure rank can being purchased from Reagent Company.

The structure of embodiment 1:SDT NIL

The present inventor is wheel with conventional japonica rice kind Anhui rice 44 (Anhui authorization in 1997, numbering: Anhui product examine 97010204) Return parent, downgrade More-tiller mutant sdt with the half of natural mutation and carry out backcrossing of continuous multi-generation for donor parents.At BC_sF₂ In colony, follow the tracks of according to the PCR of plant height reduction, the phenotype of tiller number increase and screens target zone and select individual plant.Except right Outside target zone selects, also other chromosome segments beyond objective trait are carried out background scans, final acquisition and Anhui NIL Anhui rice 44-sdt (WD44-sdt) that rice 44 genetic background is sufficiently close to and Anhui rice 44-SDT (WD44-SDT) material Material is for follow-up research.NIL WD44-sdt and WD44-SDT phenotype refer to Fig. 1.According to NIL phenotype ratio Relatively analyzing discovery, sdt is little on impact at heading stage, but significantly reduce plant height, improve lodging tolerance, increase available tillering and Improve yield.

The molecular marker of institute is the labelling based on PCR, mainly SSR marker (SDT gene mapping and cloning Primer used and sequence thereof refer to table 2).SSR marker is all from sequence alignment and finds that InDel section uses online Primer3 again Instrument carries out design of primers to these target sequences.

Table 2:sdt gene map position cloning primer

Primer	5 '-3 ' sequence
		4008P3F	ttaaggccctttcgaacgta
4008P3R	ccacagccagggtgtatttc
		3517P1F	ggcgtgtgtggagaaagaat
3517P1R	gaccgtaatgcatgcaagtg
		3517P2F	aacctcgcatttggattttg
3517P2R	ctgacctggtctccgtgatt
		3554p2F	gattttcgcgtcaacagagg
3554p2R	cagccaaacatacgcacagt
		5453P1F	caagaagccaagaagcaagaa
5453P1R	ggggaagactccagtgaagg
		5453P2F	gaaaacggagagacgcattt
5453P2R	gcgagaaggaaaacgaatga
		5453P3F	ctagctgcagcacggactc
5453P3R	cagtcagcgtgtgagagagg
		5453P4F	tcgatgaagtccctgtaccc
5453P4R	tggatcttgttgctgctctg
		3565P1F	gctttttgtgatcgggtcat
3565P1R	ccagcttcgtgttcatagca
		3579P1F	gacacgagcattgttttgct
3579P1R	acactcggcattcctgattt

PCR program is revised a little according to the method for (1996) such as Panaud and is carried out, and is specially often pipe 20 μ l amplified reaction body System, including: 0.15 μM of SSR primer, 200 μMs of dNTP, 1 × PCR reaction buffer (50mM KCL, 10mM Tris-HCL PH8.3,1.5mM MgCL2,0.01% gelatin), 50-100ng template DNA, 1U Taq enzyme；Response procedures is: 94 DEG C of DNA degeneration 5 minutes, circulation (94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 minute) re-extend 5 minutes 35 times, 72 DEG C.The PCR primer amplified Carrying out electrophoresis with 6% polyacrylamide denaturant gel, voltage is adjusted to 300V, electrophoresis about 3 hours under room temperature.After electrophoresis, EB Stained gel imaging.

Illustrate: these primers are that template expands with the genomic DNA (gDNA) of two parents, and the size of PCR primer is deposited In difference, can be according to PCR primer difference in size, it is determined that whether filial generation carries the sdt gene of sudden change.These primers are permissible It is used as polymorphic molecular marker, for molecular mark.

The comparative study of the economical character of embodiment 2:SDT NIL

The present inventor is respectively in Hefei City, Anhui Province science island Experimental Base and Experimental Base experimental plot, Lingshui county of Hainan Province Sowing WD44-SDT and WD44-sdt (introduction building detailed in Example 1 of NIL WD44-SDT and WD44-sdt, even In continuous 7 generations that backcrossed, obtain NIL), after plant to be planted maturation, add up plant height (plant height), available tillering respectively (number oftillers per plant), lodging index (lodging index), Primary branch number (number of Primary branches), Secondary branch number (number of secondary branches), spike length (panicle Length), mass of 1000 kernel (1,000grain weight) and number of grain per ear (number of grains per panicle) and single The character such as strain yield, are specifically shown in Fig. 1.

Concrete statistical method: after Oryza sativa L. maturation, takes the fringe in the 120 main tillers of strain respectively in field, and statistics is every respectively Grain number per spike (number of grain per panicle) on fringe, Primary branch number (number of primary Branches), Secondary branch number (number of secondary branches), spike length (panicle length) etc., Directly counting record.

Grain type, the measurement of grain weight.After grain natural drying, swim to wash away blighted grain, after 37 DEG C of freeze-day with constant temperature, at room temperature condition Under deposit 3 months and more than, it is ensured that being fully dried and water content relatively uniform between each strain of grain.From each strain with 100 normal seeds of form chosen by machine, carry out weighing its gross weight on electronic balance, randomly select the meansigma methods of 120 times i.e. For grain weight.

The method of significance of difference detection: 1. set up null hypothesis, the most first think that both do not have difference；2. transported by statistics Calculate, determine the probability P assuming to set up；3. according to the size of P, it is judged that assume whether set up.Standard P in our experiment < 0.05.Although statistics shows that WD44-sdt with WD44-SDT compares spike length, Primary branch number, Secondary branch number, mass of 1000 kernel Slightly reduce (Fig. 1) with on every fringe grain number per spike, but dramatically increase (Fig. 1 f) on every strain number of productive ear, and lodging tolerance and Single plant yield and harvest index are all significantly increased (Fig. 1 d and Fig. 1 m), though and flowering time statistically there were significant differences, But the two absolute value is more or less the same (only existing 1-2 days difference) (Fig. 1 e).

Embodiment 3: control Plant Height of Rice, improve lodging tolerance, increase tiller number and the acquisition of yield gene

The present invention utilizes Oryza sativa L. evening 3 (plant height is higher, average 108cm), and (plant height is relatively with partly downgrading many tillers sdt mutant Low, average 78cm) construct BC₁F₂Colony and BC₁F₇Recombinant inbred lines, developed polymorphism mark (markers), Utilizing map-based cloning to clone sdt gene, the genomic dna sequence (WD44-sdt gDNA) of its gene is such as SEQ ID Shown in NO:4.This gene contains 4 introns, and the mRNA sequence length transcribing generation is 987nt, as shown in SEQ ID NO:2, The mRNA of long 1552nt is produced, as shown in SEQ ID NO:1 at Anhui rice 44 transcription.Between relatively WD44-sdt and WD44-SDT DNA sequence difference understand, in WD44-sdt in the middle part of the Second Exon of SDT gene to intron 2 (1281～1411) send out It is grown to the disappearance of 131bp, and deletion sites is derived from 5 ' one section of sequence of end of the ccmB gene of rice mitochondria genome Inserting and replaced, the change of this segment DNA reflects on transcription product, it is simply that the sdt gene transcription product than SDT is in 3 '-UTR length Degree shortens, as shown in Fig. 2 e, Fig. 3 and Fig. 4.

Sequence comparing analysis shows, this SDT gene loci is one of member of Oryza sativa L. miR156 gene family, directly transcribes Product is OsmiR156h precursor pre-mRNA；According to the description to this member such as Xie (2006), it is classified as OsmiR156h gene. But MIRBase data base (www.mirbase.org) this site is classified as osa-miR156j (accession MI0000662). Prediction finds, is sheared the loop-stem structure (stem-loop that could form final ripe body OsmiR156h by secondary structure Sequence) be positioned at First Exon (sequence is as shown in SEQ ID NO.5), with disappearance, insertion point the most overlapping, such as Fig. 1 Shown in.And two kinds of mRNA have the complete 5 ' end structures comprising First Exon, simply the 3 '-UTR length of mRNA have change Change sdt gene outcome shorter than SDT gene outcome.Utilize qRT-PCR to detect this site mrna expression amount to find, shorten Sdt mRNA expresses higher, as shown in figure 2f than SDTmRNA at different corresponding tissue sites.

The difference of this pre-mRNA expression reflects that on ripe body OsmiR156h be exactly Northern blot result Show ripe body OsmiR156h in the tissue of WD44-sdt (here with the stem stalk of nascent secondary tiller and jointing stage as representative) Content is also obviously improved relative to WD44-SDT, sees Fig. 2 g.So, we establish OsmiR156h gene expression and The content of OsmiR156h maturation body microRNA is with the association of Dwarfing phenotypes, and further functional verification needs by transgenic real Test and prove.

Sequencing result is as follows:

SEQ ID NO:1:SDT cDNA sequence (from Anhui rice 44-SDT)；

SEQ ID NO:2:sdt cDNA sequence (from Anhui rice 44-sdt)；

SEQ ID NO:3:SDT gDNA sequence (from Anhui rice 44-SDT)；

SEQ ID NO:4:sdt gDNA sequence (from Anhui rice 44-sdt)；

SEQ ID NO:5:OsmiR156h loop-stem structure sequence (from Anhui rice 44-sdt)；

SEQ ID NO:6:sdt gDNA insertion sequence (part, from Anhui rice 44-sdt).

Illustrate: in follow-up function confirmatory experiment, make use of following correlated series:

SEQ IDNO:1:SDT cDNA sequence (from Anhui rice 44-SDT)；

SEQ ID NO:2:sdt cDNA sequence (from Anhui rice 44-sdt).

Embodiment 4:sdt reduces Plant Height of Rice and increases the transgenic checking of tiller number

The present inventor constructs the sdt cDNA fragment containing the driving of SDT gene its own promoter respectively, and (its sequence is SEQ ID NO:2, sdt cDNA sequence is from Anhui rice 44-sdt) and SDT gene (its sequence containing Oryza sativa L. Actin promoters driven Being SEQ ID NO:1, SDT cDNA sequence is from Anhui rice 44-SDT) the Transgenic Rice carrier of full-length cDNA fragment (with double Based on unit plant expression vector pCAMBIA2300, carry out vector construction；PCAMBIA2300 is purchased from CAMBIA company), by it The method infecting WD44-SDT rice callus with Agrobacterium converts, and utilizes on carrier (with pCAMBIA2300 as skeleton) Own resistant gene carries out the screening of positive plant.

PCR identifies that positive transgenic plant phenotype observed result finds: under WD44-SDT background, and its own promoter drives The positive transgenic plant of sudden change sdt gene occurs in that with plant height similar for WD44-sdt becomes the short and phenotype of tiller number increase； Under WD44-SDT background, the positive transgenic plant of the wild type SDT gene of Oryza sativa L. Actin promoters driven occurs too Along with the expression of SDT gene is gradually increased and respective degrees gradually plant height becomes the phenotype that short and tiller number increases, such as Fig. 2 d Shown in.These transgenic experiments demonstrate the high level expression of SDT (OsmiR156h) gene and cause being similar to sdt mutant Downgrade many tillers phenotype.

Embodiment 5:sdt gene loci is in the effect improved in lodging resistance in rice ability

We are used as the assessment ginseng of the lodging tolerance to rice varieties with lodging index (lodging index, LI) According to, the computing formula of lodging index is as follows: the center of Oryza sativa L. stem internode third from the bottom is node to the length (L) of fringe point, takes advantage of With the fresh weight (M) of all biomaterials on node top, then add, divided by the stem stalk of this node, the maximum that sheath can bear and fracture Power (F), utilizes formula LI=L × M/F × 0.98 to calculate, and takes the average of the result of calculation of 60 experiments.Measured and meter Calculating, the lodging index of WD44-sdt significantly reduces than WD44-SDT, shows that lodging tolerance further improves.

Because the effect wherein importance of Green revolution gene sd1 just includes improving lodging tolerance, so the present invention The lodging index of two genes of sd1 and sdt under 9311 backgrounds is compared by people, and Fig. 5 shows: with what expection was consistent be The plant lodging index of 9311-SD1/SDT is higher than 9311-sd1/SDT, and corresponding 9311-SD1/sdt compares 9311-sd1/sdt Lodging index wants height, say, that the introducing of sdt gene loci can't affect the effect of sd1 gene and play.Our experiment Result also finds that the 9311-sd1/SDT lodging index than 9311-sd1/sdt is significantly higher.This explanation is in existing high product Under the background of the sd1 gene being widely present in kind, importing half new dwarfing sdt gene still has the existing high-yield rice of raising further The effect of kind lodging tolerance.

Embodiment 6:sdt improves capacity for the resistance to lodging and the yield of Hybrid Rice Combinations

Utilize peace select No. 6 (Anxuan6, Anhui product examine 04010409, restorer) and many generations backcross build containing sdt base Selecting No. 6-sdt (Anxuan6-sdt) because of the NIL peace in site is male parent, and (XinanS, Anhui product are examined with newly pacifying S respectively 05010459, sterile line) as maternal structure F1 cross combination.Carry out F1 generation hybrid rice in Hefei and survey product contrast experiment, And some important economical characters of F1 generation hybrid rice are measured and compare, as shown in Fig. 5 e～k.Result shows, In two kinds of hybridization F1 Oryza sativa L. that the two pairs of cross combination produces, heading stage, number of grain per ear, mass of 1000 kernel difference are little；Lodging index is bright Aobvious decline, every strain tiller number has pole significant difference, causes cross combination new on single plant yield than original new two excellent 6 hybridization groups Close yield and be obviously improved (volume increase about 20%).Show that sdt gene is improving hybrid rice lodging tolerance and yield further On there is application prospect.

Embodiment 7:sdt and OsSPL14^WFPExcellent allelic variation polymerization can cultivate tiller number and grain number per spike improved coordination High-yield variety

OsSPL14^WFPIt it is an excellent allelic variation that can improve grain number per spike and yield of rice Os SPL14 gene；With Japan is fine to be compared, OsSPL14^WFPThere are 5 SNP change (SEQ ID NO.13) in gene, cause this base in its 5kb promoter region Because of up-regulated (Miura etc., 2010).Although OsSPL14^WFPGrain number per spike can be increased, but in actual breeding, there is tiller number relatively Less, the shortcoming (Miura etc., 2010) that plant is higher.Numerous studies show, rice Os SPL14 gene is OsmiR156 family Directly action target spot (as shown in Figure 2 e), the expression of OsmiR156 gene can directly determine the mRNA abundance of OsSPL14. SDT gene code OsmiR156h, this is just for sdt allelic variation and OsSPL14^WFPAllelic variation combination jointly regulates plant height, divides Tiller number and grain number per spike provide experiment basis.

Under long-grained nonglutinous rice NP174 background, we construct a pair NIL NIL-OsSPL14 by continuous backcross^WFP/ SDT and NIL-OsSPL14^WFP/sdt.Agronomic characteristic compares discovery, sdt and OsSPL14^WFPAfter polymerization, plant shows Tiller number improves, plant height suitably reduces, the phenotype that plant lodging tolerance improves and rice yield improves further, sees Fig. 6.

With NIL-OsSPL14^WFP/ SDT compares, and importing sdt gene can cause plant height decline 20%, and (plant height declines about 25em), lodging index is decreased obviously；Although every fringe grain number per spike declines about 34%, but available tillering increases about 55%, leads Cause average single plant yield and improve more than 18%.These researchs show that sdt gene and OsmiR156h-OsSPL14 module are in regulation and control Rice tillering number and every fringe grain number per spike have critical function, sdt and OsSPL14^WFPTwo excellent allelic variation are aggregated in further Improve, on rice yield, there is application prospect.

Embodiment 8: utilize specific primer to detect sdt gene loci

The present inventor devises the primer of a pair specific detection sdt mutant allele, available PCR amplification side Method, just can identify according to PCR primer size and whether there is sudden change sdt gene, sequence is shown in SEQ ID NOs:11 and 12.

Embodiment 9: proceed to sdt gene to obtain the transgenic wheat downgrading many tillers in Semen Tritici aestivi

The present inventor is by the vector introduction wheat breed section of the Oryza sativa L. sdt genetic fragment containing CaMV35S promoters driven Agriculture 199, it is thus achieved that transgenic regenerated plant.Transgenic plant of wheat shows as dwarfing, the performance of many tillers, sees Fig. 7.These experiments Illustrate that overexpression Oryza sativa L. SDT (OsmiR156h) gene can be used for controlling plant height and the tiller number of Semen Tritici aestivi.

The qualification of the promoter of embodiment 10:SDT gene

The carrier of the sdt gene containing the driving by SDT gene its own promoter (SEQ ID NO:7) is turned by the present inventor Changing Anhui rice 44-SDT rice material, transgenic positive plant produces similar Anhui rice 44-sdt phenotype: plant height reduces, tiller number increases Many, illustrate that SDT gene promoter produces particularly significant, as shown in Fig. 2 d to downgrading many tillers phenotype.

It addition, utilize Anhui rice 44-SDT different tissues to extract mRNA, qRT-PCR analysis SDT gene expression research show SDT Gene mainly high expressed (Fig. 2 f) in tiller (tiller), illustrates that this promoter has tissue specific expression feature.

List of references

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Claims (10)

1. controlling Plant Height of Rice, improve lodging tolerance, increase Oryza sativa L. available tillering and the gene of yield, it is a kind of separation Polynucleotide, its nucleotide sequence is as shown in any one in SEQ ID NOs:1-4.

2. a recombinant precursor, it is characterised in that comprise the polynucleotide sequence of the gene of claim 1, described construct institute Carrier be cloning vehicle or for expressing the expression vector of described polynucleotide.

3. a recombinant host cell, it is characterised in that comprise polynucleotide sequence or the claim 2 of the gene of claim 1 Recombinant precursor, or its genome is integrated the polynucleotide sequence of gene of requirement 1 of having the right, wherein said cell is Microbial cell.

4. the recombinant host cell described in claim 3, wherein said cell is Bacillus coli cells.

5. the recombinant host cell described in claim 3, wherein said cell is agrobatcerium cell.

6. the method cultivating the crop of output increased, described method includes: the gene described in claim 1 is transfected into work Thing cell obtains genetically modified crops plant so that in described genetically modified crops control Plant Height of Rice described in claim 1, The expression of the gene of lodging tolerance, tiller number and yield increases, thus obtains plant height reduction, lodging tolerance enhancing, divides The crop that tiller number and yield increase, wherein said crop is Oryza sativa L. or Semen Tritici aestivi.

7. the method cultivating the Oryza sativa L. of output increased, described method includes: by containing the control Oryza sativa L. described in claim 1 Plant height, the rice plant of the gene improving lodging tolerance, increase tiller number and yield obtain miscellaneous with the hybridization of another rice plants Hand over rice plant so that control Plant Height of Rice described in claim 1, lodging tolerance, tiller number in described hybrid rice Increase with the expression of the gene of yield, thus obtain the water that plant height reduction, lodging tolerance enhancing, tiller number and yield increase Rice.

8. the method cultivating the Oryza sativa L. of output increased, described method includes: comprising OsSPLl4^WFPUnder the background of gene, Import control Plant Height of Rice, lodging tolerance, tiller number and the gene of yield shown in SEQ ID NO:2 or 4.

9. the method cultivating the Oryza sativa L. of output increased, described method includes: under the background comprising sdl gene, imports SEQ Control Plant Height of Rice, lodging tolerance, tiller number and the gene of yield shown in ID NO:2 or 4.

10. controlling Plant Height of Rice, improve lodging tolerance, increase Oryza sativa L. available tillering and yield described in claim 1 The promoter sequence of gene, its nucleotide sequence is as shown in SEQ ID NO:7.