CN102690838B - Application of OsMADS57 protein or coding gene thereof in promotion of rice tillering - Google Patents

Abstract

The invention discloses application of OsMADS57 protein or coding gene thereof in promotion of rice tillering, and provides application of OsMADS57 protein or coding gene thereof or recombinant vector for expressing OsMADS57 in promotion of plant tillering. Experiments prove that an OsMADS57 gene is cloned in japonica rice Zhonghua 10, and is inserted into an expression vector in the forward direction to form a sense expression vector; and the sense expression vector is imported into rice Zhonghua 10 to form a sense transgenic rice line. Compared with wild rice, the sense transgenic rice line has more tillers.

Description

The application in promoting rice tillering of OsMADS57 albumen or its encoding gene

Technical field

The present invention relates to biological technical field, relate in particular to the application in promoting rice tillering of a kind of OsMADS57 albumen or its encoding gene.

Background technology

Plant plant type is one of important factor affecting crop yield, especially for rice crop.Know at present tillering number, tillering angle, plant height, the leaf angle of paddy rice, how many size and paddy rice small ear branches of paddy rice tassel have determined plant type of rice jointly, and the tiller number of paddy rice and the shape of tassel are the greatest factor that determines rice yield.For rice tillering is grown, be the main period of vegetative growth of rice plants tillering phase, comprises the formation from the differentiation of tiller bud to young fringe.General rice varieties, growing environment condition (temperature, moisture, nutritional status) and planting density thereof also can affect the growth of tillering.The long rice varieties of the growth cycle generally rice tillering shorter than growth cycle is fewer.Rice tillering comprises that one-level is tillered and secondary is tillered.According to the solid situation of tillering, tiller and be divided into two kinds of effective tillering and ineffective tillerings.Effective tillering number be to determine one of rice yield important factor, so we are increased as much as possible and can be educated tillering quantity by various means.

Along with the genomics of the progress of scientific research, particularly paddy rice and developing rapidly of information biology.At present, in paddy rice, some genes relevant to tiller development are cloned out successively.Laboratory clone's as foreign in Chinese scholar Lee family MOC1 gene directly affects the tillering number of paddy rice, shows the phenotype of tillering and reducing after this transgenation, and the overexpression transfer-gen plant of this gene shows the phenotype of tillering and increasing.This laboratory current research is found the interactional albumen TAD1 of MOC1, shows the phenotype of tillering and increasing after the transgenation of this albumen of encoding.Research shows in paddy rice D17/HTD1 and Arabidopis thaliana homologous gene MAX3 and D10 and the Arabidopis thaliana homologous gene MAX4 carotenoid cutting dioxygenase albumen of all encoding, and they are called for short respectively carotenoid cleavage deoxygenate protein 7 (CCD7) and carotenoid cleavage deoxygenate protein 8 (CCD8).These two genes of MAX3 and MAX4 have participated in the biosynthesizing of plant branch hormone (witchweed lactone), in the synthetic approach of this hormone, find that the downstream that is positioned at CCD7 and CCD8 exists other key gene, a kind of cytochrome P 450 enzymes MAX1 that for example encodes in Arabidopis thaliana, paddy rice is relevant homogenic report also.2009 Nian Lijiayang seminars have reported one and the d10 classics similar mutant d27 of mutation type surface of tillering, and an iron ion of D27 coding comprises albumen, and Subcellular Localization shows that this gene and chloroplast(id) locate altogether, mainly at the microtubule cells of stem and root.Final experimental evidence shows that D27 may be a newcomer of witchweed lactone biosynthetic pathway.Another affects gene D3 that rice tillering grows LRR albumen containing F-box of encoding, and also shows the phenotype of tillering and increasing after this transgenation.Within 2009, several seminars have reported another and the mutant d14/htd2/d88 of tillering relevant in succession, the phenotype equally with classical mutant in witchweed lactone approach, be that tiller number increases and plant dwarfing, also show the phenotype that tassel shortens and seed diminishes.Within 2003, Japanese Yi Ge seminar finds TEOSINTE BRANCHED1 (TB1) the high homology gene OsTB1/FC1 of corn according to homology analysis, and the albumen of this genes encoding TCP transcription factor family, is the negative regulatory factor of tiller development.The mutant of this gene shows the phenotype that tiller number increases and partly downgrade, and this gene overexpression transfer-gen plant shows the phenotype of tillering and reducing.Japanese another one seminars in 2009 have found another mutant fc1 of this gene, the Physiological Experiment proof fc1 that witchweed lactone is processed this mutant shows the insensitive phenotype of this hormone, author infers that OsTB1/FC1 also may be positioned at the downstream of MAX/RMS/D genetic approach, subparticipation the signal conduction of witchweed lactone.Within 2009, a rice dwarf and low-tillering (dlt) mutant is found and studies, this mutant shows the phenotype of downgrading and tillering and reduce, corresponding DLT transgenosis has confirmed the phenotype of this mutant, and result shows that dlt mutant shows the phenotype that is similar to brassinolide defect and sign mutation body in paddy rice.

Composing type overexpression technology and antisense transgene are technology that has developed the research gene function of comparative maturity.It is gene to be inserted into the downstream of strong promoter with forward and reverse mode, can make the gene transcripts of expressing obtain in vivo strong expression and expression amount reduction, thereby target protein expression amount be increased and expression amount minimizing.When some interested genes are carried out to Function Identification, adopting composing type overexpression and antisense transgene technology can make us understand expression at this gene strengthens significantly with the expression amount of native gene and reduces, research purpose genetic expression strengthens and expresses in situation about reducing, whether the process such as grow of plant can be affected, and never can infer the possible biological function of this gene.

Summary of the invention

An object of the present invention is to provide OsMADS57 albumen or its encoding gene or express the new purposes of the recombinant vectors of OsMADS57.

The application of the recombinant vectors that the invention provides OsMADS57 albumen or its encoding gene or express OsMADS57 in promoting plant tillering;

The aminoacid sequence of described OsMADS57 albumen is the sequence 2 in sequence table.

In above-mentioned application, the nucleotides sequence of the encoding gene of described OsMADS57 albumen is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 1-726 position Nucleotide;

The recombinant vectors of described expression OsMADS57 is that the encoding gene of described OsMADS57 is inserted in expression vector, obtains expressing the carrier of OsMADS57;

The recombinant vectors of described expression OsMADS57 is specially and the sequence in described sequence table 1 is inserted to the carrier obtaining between the BamHI of expression vector pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, and the sequence in described sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in described sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.

Above-mentioned being applied as imports the encoding gene of described OsMADS57 albumen in object plant, obtains the transgenic plant that tiller number is greater than described object plant.

In above-mentioned application, the encoding gene of described OsMADS57 albumen imports in object plant by the recombinant vectors of described expression OsMADS57.

In above-mentioned application, described object plant is monocotyledons or dicotyledons, and described monocotyledons is specially paddy rice.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

Method provided by the invention, for the encoding gene of the described OsMADS57 albumen in above-mentioned application is imported in object plant, obtains the transgenic plant that tiller number is greater than described object plant.

In aforesaid method, the encoding gene of described OsMADS57 albumen imports in object plant by recombinant vectors.

In aforesaid method, described recombinant vectors is specially and the sequence in described sequence table 1 is inserted to the carrier obtaining between the BamHI of expression vector pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, and the sequence in described sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in described sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.

The 3rd object of the present invention is to provide a kind of recombinant vectors.

Recombinant vectors provided by the invention, for the encoding gene of the described OsMADS57 in above-mentioned application is inserted in expression vector, obtains expressing the carrier of OsMADS57.

Described recombinant vectors is for inserting the sequence in described sequence table 1 carrier obtaining between the BamHI of expression vector pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, and the sequence in described sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in described sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.

Of the present invention experiment showed, to spend in japonica rice in No. ten, be cloned into an OsMADS57 gene, its forward is inserted to expression vector, obtain sense expression vector; Utilize in sense expression vector Introduced into Rice and spend No. ten, obtain just transgenic paddy rice strain, compare with wild-type paddy rice, tillering of just transgenic paddy rice strain increases.

Accompanying drawing explanation

Fig. 1 is the increase total length of OsMADS57 of RT-PCR method

Fig. 2 is overexpression vector pUN-OsMADS57(justice) physical map

Fig. 3 is that the quantitative PCR of overexpression transgenic paddy rice (justice) is identified

Fig. 4 is the phenotype that OsMADS57 overexpression transgenic paddy rice (justice) is tillered

Embodiment

The experimental technique using in following embodiment if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

The acquisition of embodiment 1, OsMADS57 albumen and encoding gene thereof

According to the consequence devised primer of database analysis:

Overexpression vector builds and uses primer: 5 ' end primer: 5 '-CG gGATCCaTGGGGAGGGGGAAGATAGT-3 ' (underscore sequence is BamHI site, sequence 3), 3 ' end primer: 5 '-GG gGTACCtTAAGGCAGATGAAGTCCCAGT-3 ' (underscore sequence is KpnI site, sequence 4).

Extract in japonica rice and spend the total RNA of seedling in tri-leaf period No. ten, adopt the increase 726bp full-length cDNA of OsMADS57 of RT-PCR method.Specific operation process is as follows:

1) extraction of the total RNA of plant: choose the seedling of spending No. ten (Oryza sativa L.cv Zhonghua 10) in 100mg paddy rice in tri-leaf period (Li Meifang, Anther Culture kind-in spend No. 10, agricultural science and technology communication, the 26th page of the 1st phase in 1998.Public Ke Cong Institute of Botany, Chinese Academy of Sciences obtains.) be material, in liquid nitrogen, grind, the lyophilized powder grinding in liquid nitrogen is transferred in the 1.5ml centrifuge tube containing 1ml Trizol reagent (Invitrogen), fully mix; Place 5 minutes for 25 ℃; In every pipe, add the fresh chloroform of 0.2ml, violent jolting 15 seconds, 25 ℃ of incubation 2-3 minute; 12,000rpm, 4 ℃, centrifugal 15 minutes; The water 0.5ml of supernatant is transferred in a new 1.5ml centrifuge tube, add 0.5ml Virahol, place for 25 ℃ and within 10 minutes, make RNA precipitation; 12,000rpm, 4 ℃, centrifugal 10 minutes; Remove supernatant, RNA precipitation is cleaned 2 times with 1ml 75% ethanol, super clean bench blows to half-dried; With 50 μ l DEPC-ddH ₂the resuspended precipitation of O, 60 ℃ of water-baths 10 minutes, obtain RNA solution to dissolve RNA precipitation.By-70 ℃ of preservations after this RNA solution packing, the standby template of doing reverse transcription.

2) RT-PCR: get the above-mentioned RNA solution of 1 μ l, use DEPC-ddH ₂100 times of O dilutions, by spectrophotometric determination RNA concentration.With reference to RT-PCR test kit (Promega) specification sheets, according to the quantitative result of RNA, get the above-mentioned RNA solution that contains 2 μ gRNA, add 1.0 μ g Oligo dT primers, use DEPC-ddH ₂o is supplemented to 15 μ l, mixes rear 70 ℃ of sex change 5 minutes, ice bath 5 minutes.Of short duration centrifugal after, add 25 μ l reverse transcription mixtures (5 μ lM-MLV 5 * Reaction Buffer, 6 μ l dNTP Mixture (2.5mM), 1 μ l M-MLV Reverse Transcriptase, 0.5 μ l RNase Inhibitor, 12.5 μ l DEPC-ddH ₂o).After mixing, 42 ℃ of water-baths complete transcriptive process,reversed for 1 hour; 75 ℃ of water-baths make ThermoScript II inactivation in 10 minutes, obtain the mixture that contains the first chain cDNA.

Get above-mentioned the first chain cDNA of 1 μ l as the template of PCR, by following system, carry out PCR reaction: 0.2 μ l LATaq(5U/ μ l), 10 μ l 2 * GC buffer, 1.8 μ l dNTPs, 0.5 μ l 5 ' end primer (10 μ M), 0.5 μ l3 ' end primer (10 μ M), adds ddH ₂o final volume 20 μ l.

5 ' end primer and 3 ' end primer is as follows respectively:

With overexpression, build primer sequence 5 ' end primer: 5 '-CG gGATCCaTGGGGAGGGGGAAGATAGT-3 ' (underscore sequence is BamHI site, sequence 3), 3 ' end primer: 5 '-GG gGTACCtTAAGGCAGATGAAGTCCCAGT-3 ' (underscore sequence is KpnI site, sequence 4) is primer, carries out pcr amplification, obtains PCR product (justice).

Above-mentioned PCR program is: after 94 ° of C denaturation 30s, enter PCR circulation, loop parameter is that 15 seconds renaturation → 72 ° C of 10 seconds sex change → 55 ° C of 98 ° of C extend for 40 seconds, after 35 circulations, at 72 ° of C, continues synthetic 10 minutes.

Above-mentioned PCR product (justice) is separated through 0.8% agarose gel electrophoresis, and as shown in Figure 1, the stripe size of PCR product (justice) is 726bp to result.

Reclaim PCR product (justice) and check order, result is that the nucleotide sequence of PCR product (justice) has in sequence table sequence 1 from 5 ' end 1-726 position Nucleotide, the OFR of sequence 1 be in sequence table sequence 1 from 5 ' end 1-726 position Nucleotide.The unnamed gene OsMADS57 of this PCR product, the albumen of OsMADS57 coding is OsMADS57, the aminoacid sequence of this albumen is the sequence 2 in sequence table.

The application of embodiment 2, OsMADS57 albumen and encoding gene thereof

One, over-express vector pUN-MADS57(justice)

1, the acquisition of pUN1301 plasmid

The first step: the about 0.2g corn seedling of clip, is placed in liquid nitrogen and grinds; Then the Extraction buffer (containing 0.1M Tris-HCl pH8.0,50mM EDTA, 0.5M NaCl, 1%SDS and 1% beta-mercaptoethanol) that adds the new preparation of 800 μ L, thermal agitation all suspends it; 65 ℃ of water-baths 30 minutes, put upside down and mix once for every 5 minutes; Then add the 5M potassium acetate solution of 250 μ L precoolings, put upside down and mix immediately, ice bath 5 minutes; Add equal amounts of phenolic/chloroform, extracting once, centrifugal 5 minutes of 12000rpm; Collect supernatant liquor, add the isopropanol precipitating DNA of 0.6 times of volume, room temperature is placed 40 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm, abandon supernatant; Precipitation is respectively washed once with 70%, 100% ethanol; After dry, be dissolved in 20 μ L containing the ddH of 100 μ g/mL RNaseA ₂in O, obtain corn gene group DNA.

Second step: get above-mentioned corn gene group DNA solution 2 μ L as template, with the 5 ' primer (GG with Hind III recognition site aAGCTTcTGCAGTGCAGCGTGACCCGG) with 3 ' primer (CG of BamHI recognition site gGATCCaAGTAACACCAAACAACAGGG) be primer, carry out pcr amplification, PCR reaction conditions is: first 94 ℃ 3 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ 10 minutes.After reaction finishes, PCR product is carried out to 0.8% agarose gel electrophoresis detection, show to obtain the amplified fragments that length is about 2kb, conform to expected results, reclaim this object fragment, with reclaiming after restriction enzyme Hind III and BamHI double digestion, obtain fragment through order-checking, this fragment be sequence 5 in sequence table from the 1-1986 position of 5 ' end Nucleotide, be the corn ubiquitin promoter (UbiPro) with sticky end.(corn ubiquitin promoter (UbiPro) also can obtain by synthetic.）

The 3rd step: with restriction enzyme Sac I and EcoR I by Noster poly A terminator sequence from plasmid vector pBI 121(Beijing Baeyer enlightening Bioisystech Co., Ltd catalog number (Cat.No.): MP-091) cut, be connected to carrier pUC19(Beijing hundred Tyke Bioisystech Co., Ltd catalog number (Cat.No.)s: between Sac I DP7801) and EcoR I site, obtain recombinant vectors, called after pUC19-Noster.Use again restriction enzyme Hind III and BamHI double digestion pUC19-Noster, after agarose gel electrophoresis detects, reclaim linearizing carrier large fragment, and this recovery fragment is connected with the corn ubiquitin promoter (UbiPro) with sticky end of cutting acquisition through enzyme in second step, obtain recombinant vectors, called after pUN19.

The 4th step: purchase from the 3rd step the recombinant vectors pUN19 building with the partially digested and Hind III complete degestion of restriction enzyme EcoR I and cut the fragment that the length that comprises UbiPro and Noster is about 2.3kb, this fragment is cloned into plasmid vector pCAMBIA1301 (Biovector Co., the catalog number (Cat.No.) Biovec-11 of LTD company) EcoR I and Hind III site, obtain recombinant vectors, called after pUN1301.

2, acquisition over-express vector pUN-MADS57(justice)

With the plasmid pUN1301 that restriction enzyme KpnI and BamHI obtain 1 step, carry out double digestion, the enzyme system of cutting is: plasmid 10 μ l, 10x enzyme cutting buffering liquid 5 μ l, BglII1 μ l(10U/ μ l), SacI 0.8 μ l(10U/ μ l), add ddH ₂o postreaction system to 50 μ l, 37 ℃ of enzymes are cut 4 hours.With agarose gel electrophoresis, enzyme is cut to product and carry out separation, reclaim linearizing pUN1301 large fragment, be dissolved in 20 μ l ddH ₂in O.

With restriction enzyme KpnI and BamHI, the 726bp PCR product (justice) being obtained by embodiment 1 is carried out to double digestion, the enzyme system of cutting is: plasmid 10 μ l, enzyme cutting buffering liquid 5 μ l, BamHI1 μ l(10U/ μ l), add ddH ₂o postreaction system to 50 μ l, 37 ℃ of enzymes are cut 4 hours.Add again KpnI 0.2 μ l(10U/ μ l), 37 ℃ of enzymes are cut 20 minutes.With 0.8% agarose gel electrophoresis, enzyme is cut to product and carry out separation, with the DNA gel recovery test kit of AxyPrep company, reclaim this segment, reclaim the OsMADS57 fragment (justice) of 726bp.

The OsMADS57(justice of the 726bp that 10 μ l are reclaimed), 6 μ l reclaim carrier pUN1301 large fragment solution, 2 μ l (3U/ μ l) T4 DNA ligase and 2 μ l 10x ligase enzyme damping fluids are mixed, 16 ℃ connect 16 hours, the connection product obtaining transforms bacillus coli DH 5 alpha competent cell, through the resistant panel screening containing kantlex, obtains positive colony.

Extract the recombinant plasmid in positive colony, carry out sequence verification, this plasmid is for inserting the sequence in sequence table 1 carrier obtaining between the BamHI of pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, by this plasmid called after pUN-OsMADS57(justice), and the sequence in sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.In pUN-OsMADS57, the structure of promotor, gene and terminator is correct.In this expression vector, adopt corn ubiquitin promoter (UbiPro) to start object fragment OsMADS57 overexpression in plant, its physical map schematic diagram as shown in Figure 2.

Two, turn acquisition and the evaluation of OsMADS57 paddy rice (justice)

1, turn the acquisition of OsMADS57 paddy rice (justice)

With reference to electric exciter (Easy JecT Plus electric exciter, Britain EquiBio company) operational guidance, by pUN-MADS57(justice) with electric shocking method, transform Agrobacterium EHA105(Biovector Co., the catalog number (Cat.No.) Biovec-11 of LTD company), through the resistant panel screening containing kantlex, obtain the overexpression engineering bacteria of positive colony, called after EHA105/pUN-OsMADS57(justice).

By EHA105/pUN-OsMADS57(justice) spend paddy rice (Oryza sativaL.cv Zhonghua10 No. ten in importing, hereinafter to be referred as wild-type paddy rice) callus in, with the sterilized water washing 4-5 time containing 300mg/L cephamycin, after blotting, aseptic filter paper goes to N again ₆d ₂s ₁on substratum, a screening generation; After two weeks, be transferred to N ₆d ₂s ₂on substratum, screened for two generations (2 weeks/generation); Taking-up is screened eugonic resistant calli through 3 generations, is transferred to division culture medium (1), upper, in differentiation culture case (12 hour photoperiod, 28 ℃ of daytimes, 25 ℃ of nights), cultivates 7 days; Then be transferred to division culture medium (2), upper, in differentiation culture case, be cultured to generation regrowth.Plant strong plantlets and rootage on Rooting and hardening-off culture base of regeneration; When seedling grows to 10 centimetres of left and right, open container closure film, hardening 2-3 days, then moves into seedling phytotron cultivation, obtains 10 strains totally 60 T0 for turning OsMADS57 paddy rice (justice).

Used medium is as follows:

2, turn the evaluation of OsMADS57 paddy rice (justice)

1), GUS histochemical stain:

70 T0 that obtained by embodiment 1 are put into respectively in GUS staining fluid for the long root segment of 2-3mm that turns OsMADS57 paddy rice (justice), and the several minutes of bleeding, is then placed in 37 ℃ and is incubated overnight, 70% ethanol decolorization of the tissue after dyeing.It is positive transgenic line that root is blue plant.GUS staining fluid (pH 7.0) component is: 100mM Na ₃pO ₄(pH 7.0), 0.1%Triton X-100,10mM EDTA, 0.5mM yellow prussiate of potash, the 0.5mM Tripotassium iron hexacyanide, 1mg/ml X-Gluc.Result identifies altogether 6 strains and adds up to 50 positive T0 for turning OsMADS57 paddy rice (justice), and this seedling is moved to greenhouse production, according to different strain sowings, obtains T1 for transgenic seed, obtains isozygotying T2 for seed on this basis through breeding.In experiment afterwards, choose and turn OsMADS57 paddy rice (justice) strain 1(OE1) and homozyous seed T2 2(OE2) be material.

2), quantitative PCR is identified:

From the T2 of OE1 and OE2, turn OsMADS57 paddy rice (justice) seedling and extract mRNA, and transcribe respectively and obtain cDNA, take wild-type paddy rice (spending in paddy rice No. ten) for contrasting.Utilize fluorescence real-time quantitative PCR method, take cDNA as template, with 1 μ l 5 ' end primer 1 (10 μ M) (5 '-GCACCAACATGAAAACTGTGA-3 '), 1 μ l 3 ' end primer 1 (10 μ M) (5 '-CTCCCTCTGCCAAATCTTAATT-3 ') is primer, and the gene expression abundance that positive T2 is turned to OsMADS57 paddy rice (justice) detects.Reagent for quantitative analysis is SYBR Green Realtime PCR Master Mix(TOYOBO).Instrument is the real-time fluorescence quantitative PCR instrument Mx3000P of U.S. Stratagene company.Draw 1 μ l the first chain cDNA solution, dilute 50 times as template, by following system, carry out PCR reaction: 10 μ l SYBR Green Realtime PCR Master Mix, 4 μ l templates, 1 μ l 5 ' end primer 1 (10 μ M), 1 μ l 3 ' end primer 1 (10 μ M), adds ddH2O final volume 20 μ l.

Result as shown in Figure 3, at Actin gene as internal reference in the situation that, compare with wild-type paddy rice, in the T2 seedling of OE1 and OE2, the gene expression abundance of OsMADS57 has had rise in various degree, and illustration purpose gene (OsMADS57) is transcriptional level successful expression again.

Adopting uses the same method imports empty carrier pUN1301 in wild-type paddy rice and obtains T0 for turning empty carrier paddy rice, from T0, for turning pUN1301 paddy rice, gather in the crops T1 for turning pUN1301 rice paddy seed, sowing, gathers in the crops T2 for turning pUN1301 rice paddy seed from T1 for turning pUN1301 paddy rice.

Three, turn the Phenotypic Observation of OsMADS57 paddy rice (justice)

In T2 generation, is turned to the OE1 of OsMADS57 paddy rice (justice) and OE2 seed, middle No. ten wild-type rice paddy seeds (ZH10) and the T2 of spending for turning pUN1301 rice paddy seed, all sow in the mixture of flower nutrition soil and vermiculite (both blending ratios are 4:1), after 30 ℃ of sproutings, be placed on (32 ℃) in greenhouse and be cultured to for 3 leaf phases, follow the seedling cultivation of growth in rice terrace.30 of each strains, test in triplicate results averaged.

Plant tillering number is observed, and result is as follows:

Take pictures as shown in Figure 4 A, can find out, compare with wild-type paddy rice, T2 increases for the tiller number that turns OsMADS57 paddy rice (justice) OE1.

After planting the approximately the 70th day statistics T2 generation, turn the OE1 of OsMADS57 paddy rice (justice) and OE2 seed, middle No. ten wild-type rice paddy seeds (ZH10) and the T2 of spending for turning pUN1301 rice tillering number, result as shown in Figure 4 B, T2 generation turn OsMADS57 paddy rice (justice) OE1, T2 generation turn OsMADS57 paddy rice (justice) OE2, the middle tiller number of No. ten wild-type paddy rice (ZH10) of spending is respectively 14,17 and 26.

T2 is for turning pUN1301 paddy rice and wild-type paddy rice result without significant difference.

Claims (7)

1. The application of the recombinant vectors of 1.OsMADS57 albumen or its encoding gene or expression OsMADS57 in promoting plant tillering;

   The aminoacid sequence of described OsMADS57 albumen is the sequence 2 in sequence table;

   Described plant is paddy rice.
2. 2. application according to claim 1, is characterized in that: the nucleotides sequence of the encoding gene of described OsMADS57 albumen is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 1-726 position Nucleotide;

   The recombinant vectors of described expression OsMADS57 is that the encoding gene of described OsMADS57 is inserted in expression vector, obtains expressing the carrier of OsMADS57;

   The recombinant vectors of described expression OsMADS57 is specially and the sequence in described sequence table 1 is inserted to the carrier obtaining between the BamHI of expression vector pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, and the sequence in described sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in described sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.
3. 3. application according to claim 1 and 2, is characterized in that: described in be applied as the encoding gene of described OsMADS57 albumen imported in object plant, the tiller number of the transgenic plant that obtain is greater than described object plant.
4. 4. application according to claim 3, is characterized in that: the encoding gene of described OsMADS57 albumen imports in object plant by the recombinant vectors of described expression OsMADS57.
5. 5. cultivate a method for transgenic plant, for the encoding gene of the described OsMADS57 albumen in arbitrary described application in claim 1-4 is imported in object plant, the tiller number of the transgenic plant that obtain is greater than described object plant;

   Described object plant is paddy rice;

   The nucleotides sequence of the encoding gene of described OsMADS57 albumen is classified sequence 1 in sequence table or the sequence 1 in sequence table as from 5 ' end 1-726 position Nucleotide.
6. 6. method according to claim 5, is characterized in that: the encoding gene of described OsMADS57 albumen imports in object plant by recombinant vectors.
7. 7. according to the method described in claim 5 or 6, it is characterized in that: described recombinant vectors, for the encoding gene of described OsMADS57 is inserted in expression vector, obtains expressing the carrier of OsMADS57;

   Described recombinant vectors is specially and the sequence in described sequence table 1 is inserted to the carrier obtaining between the BamHI of expression vector pUN1301 and KpnI restriction enzyme site from 5 ' end 1-726 position Nucleotide, and the sequence in described sequence table 1 holds the BamHI restriction enzyme site by described pUN1301 to be connected with it from 5 ' of 5 ' end 1-726 position Nucleotide, the sequence 1 in described sequence table holds the KpnI restriction enzyme site by described pUN1301 to be connected with it from 3 ' of 5 ' end 1-726 position Nucleotide.

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