CN102690341A - Plant tillering related protein and coding gene thereof - Google Patents

Abstract

The invention discloses plant tillering related protein and a coding gene thereof. The protein is protein (a) or protein (b), wherein the protein (a) has amino acid sequences shown as a sequence 2 in a sequence table; and the protein (b) has an amino acid residue sequence which is derived from the sequence 2 in the sequence table by substituting and/or losing and/or adding one or more amino acid residues and has the same function. The experiment proves that expression vectors are inserted into an OsMADS57 gene which is cloned in japonica rice zhonghua 10 in forward and reverse directions so as to obtain plus sense recombinant plasmid and antisense recombinant plasmid; the plus sense recombinant plasmid and the antisense recombinant plasmid are imported into the japonica rice zhonghua 10 so as to obtain plus sense transgenic rice strains and antisense transgenic rice strains; and compared with that of wild-type rice, the tillering of the plus sense transgenic rice strains is increased, and the tillering of the plus sense transgenic rice strains is decreased.

Description

A kind of and plant tillering associated protein and encoding sox thereof

Technical field

The present invention relates to biological technical field, relate in particular to a kind of and plant tillering associated protein and encoding sox thereof.

Background technology

The plant plant type is one of important factor that influences crop yield, especially as far as rice crop.Know that at present tillering number, tillering angle, plant height, the leaf angle of paddy rice, the size and the paddy rice small ear branch of paddy rice tassel have determined plant type of rice jointly, and the shape of tiller number of paddy rice and tassel is the most important factor of decision rice yield.For rice tillering was grown, be the main period of rice nutrition growth 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 influence the growth of tillering.Long generally short than the growth cycle rice tillering of rice varieties of growth cycle lacks.Rice tillering comprises that one-level is tillered and secondary is tillered.According to the blocky situation of tillering, tiller and be divided into two kinds of effective tillering and ineffective tillerings.Effective tillering how much be one of decision rice yield important factor, can educate tillering quantity so we increase through various means as much as possible.

Along with the genomics of the progress of scientific research, particularly paddy rice and developing rapidly of information biology.At present, some genes relevant with tiller development are come out by the clone successively in the paddy rice.Directly influence the tillering number of paddy rice like the foreign laboratory clone's of Chinese scholar Lee family MOC1 gene, after this transgenation, show the phenotype of tillering and reducing, 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 this proteic transgenation of encoding.Research shows in the paddy rice that D17/HTD1 and Arabidopis thaliana homologous gene MAX3 and D10 and the Arabidopis thaliana homologous gene MAX4 carrotenoid of all encoding cuts dioxygenase albumen, and they are called for short carotenoid cleavage deoxygenate protein 7 (CCD7) and carotenoid cleavage deoxygenate protein 8 (CCD8) respectively.These two genes of MAX3 and MAX4 have been participated in the biosynthesizing of plant branch hormone (witchweed lactone); Find in this hormone synthetic approach that there is the other key gene in the downstream that are positioned at CCD7 and CCD8; A kind of cytochrome P 450 enzymes MAX1 of coding in the Arabidopis thaliana for example, the also relevant homogenic report of paddy rice.Foreign seminar of Lee family in 2009 reported one with the d10 classics similar two mutants d27 of mutation type surface of tillering, iron ion of D27 coding comprises albumen, Subcellular Localization shows that this gene and chloroplast(id) locate altogether, mainly in the microtubule cell of stem and root, expresses.Final experimental evidence shows that D27 possibly be a newcomer of witchweed lactone biosynthetic pathway.Another gene D3 that influences the rice tillering growth then encodes one and contains the LRR albumen of F-box, also shows the phenotype of tillering and increasing after this transgenation.Several seminars had reported another and the two mutants d14/htd2/d88 of tillering relevant in succession in 2009; The phenotype that has classical two mutants in the witchweed lactone approach equally; Be that tiller number increases and the plant dwarfing, also show the phenotype that tassel shortens and seed diminishes.Seminar of Japan in 2003 is according to TEOSINTEBRANCHED1 (TB1) the high homology gene OsTB1/FC1 of homology analysis discovery corn, and the albumen of this genes encoding TCP transcription factor family is the negative regulatory factor of tiller development.The two mutants of this gene shows tiller number and increases the phenotype of downgrading with half, and this gene overexpression transfer-gen plant shows the phenotype of tillering and reducing.Japanese another one seminars in 2009 have found another two mutants fc1 of this gene; The Physiological Experiment proof fc1 that the witchweed lactone is handled this two mutants shows the insensitive phenotype of this hormone; The author infers that OsTB1/FC1 also possibly be positioned at the downstream of MAX/RMS/D genetic approach, subparticipation the signal conduction of witchweed lactone.A rice dwarf and low-tillering (dlt) mutant in 2009 comes to light and studies; This two mutants shows the phenotype of downgrading and tillering and reduce; Corresponding D LT transgenic has confirmed the phenotype of this two mutants, and the result shows that the dlt two mutants shows the phenotype that is similar to brassinolide defective and sign mutation body in the paddy rice.

Composing type overexpression technology and antisense transgene are technology that has developed the research gene function of comparative maturity.It is that gene is inserted into the downstream of strong promoter with forward and reverse mode, can make the gene transcripts of expression obtain strong expression in vivo and expression amount reduces, thereby the target protein expression amount is increased and the expression amount minimizing.When some interested genes are carried out Function Identification; Adopt composing type overexpression and antisense transgene technology that we are understood in this expression of gene and strengthen the expression amount minimizing with native gene significantly; Under the situation of enhancing of research destination gene expression and expression decreased; Whether processes such as growth and development 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 a kind of and plant tillering associated protein and encoding sox thereof.

Protein provided by the invention, name is called OsMADS57, derives from and spends (Oryza sativaL.cv Zhonghua 10) in the paddy rice No. ten, is (a) or (b) as follows:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by sequence 2 deutero-protein.

Sequence 2 in the above-mentioned sequence table is made up of 241 amino-acid residues.The replacement of said one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

The gene of encoding said proteins (OsMADS57) also belongs to protection scope of the present invention.

The gene of encoding said proteins (OsMADS57) is following 1)-4) in arbitrary described dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) sequence 1 is held the dna molecular shown in the Nucleotide of 1-726 position from 5 ' in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding proteic dna molecular with identical function;

4) with 1) or 2) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the proteic dna molecular that 99% homology and coding have identical function at least at least.

Sequence 1 in the above-mentioned sequence table is made up of 762 Nucleotide, and its coding region is from 5 ' terminal 1-726 position Nucleotide.

Said stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain said gene also are the scopes that the present invention protects.

Said recombinant vectors obtains expressing above-mentioned proteic recombinant vectors for said gene is inserted in the expression vector; Said recombinant vectors specific as follows 1) or 2):

1) for the sequence in the said sequence table 1 is inserted the carrier that obtains between BamHI and the KpnI restriction enzyme site of expression vector pUN1301 from 5 ' terminal 1-726 position Nucleotide; And the sequence in the said sequence table 1 holds the BamHI restriction enzyme site through said pUN1301 to be connected with it from 5 ' of 5 ' terminal 1-726 position Nucleotide, and the sequence 1 in the said sequence table holds the KpnI restriction enzyme site through said pUN1301 to be connected with it from 3 ' of 5 ' terminal 1-726 position Nucleotide;

2) for the sequence in the said sequence table 1 is inserted the carrier that obtains between KpnI and the BamHI restriction enzyme site of expression vector pUN1301 from 5 ' terminal 1-726 position Nucleotide; And the sequence in the said sequence table 1 holds the KpnI restriction enzyme site through said pUN1301 to be connected with it from 5 ' of 5 ' terminal 1-726 position Nucleotide, and the sequence 1 in the said sequence table holds the BamHI restriction enzyme site through said pUN1301 to be connected with it from 3 ' of 5 ' terminal 1-726 position Nucleotide.

Above-mentioned expression vector pUN1301 obtains through following steps: the first step: the about 0.2g corn seedling of clip places liquid nitrogen to grind; Add the extraction damping fluid (containing 0.1M Tris-HCl pH8.0,50mMEDTA, 0.5M NaCl, 1%SDS and 1% beta-mercaptoethanol) of the new preparation of 800 μ L then, thermal agitation makes its whole suspensions; 65 ℃ of water-baths 30 minutes were put upside down mixing once in per 5 minutes; Add the 5M potassium acetate solution of 250 μ L precoolings then, put upside down mixing immediately, ice bath 5 minutes; Add equal amounts of phenolic/chloroform, extracting once, centrifugal 5 minutes of 12000rpm; Collect supernatant, add the isopropanol precipitating DNA of 0.6 times of volume, room temperature was placed 40 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm abandon supernatant; Deposition is respectively washed once with 70%, 100% ethanol; After the drying, be dissolved among the ddH2O that 20 μ L contain 100 μ g/mL RNaseA, obtain corn gene group DNA.

Second step: get above-mentioned corn gene group DNA solution 2 μ L as template, to have 5 ' primer (GG of Hind III recognition site AAGCTTCTGCAGTGCAGCGTGACCCGG) and have 3 ' primer (CG of BamHI recognition site GGATCCAAGTAACACCAAACAACAGGG) be primer, carry out pcr amplification, the PCR reaction conditions is: earlier 94 ℃ 3 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ 10 minutes.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis detect, show to obtain the amplified fragments that length is about 2kb; Conform to expected results; Reclaim this purpose fragment,, obtain fragment through order-checking with reclaiming behind restriction enzyme Hind III and the BamH I double digestion; This fragment is that sequence 7 in the sequence table is from 5 ' terminal 1-1986 position Nucleotide, for having the corn ubiquitin promoter (UbiPro) of sticky end.(corn ubiquitin promoter (UbiPro) also can obtain by synthetic.）

The 3rd step: with restriction enzyme Sac I and EcoR I with Noster poly A terminator sequence from plasmid vector pBI 121 (Beijing Baeyer enlightening Bioisystech Co., Ltd catalog number (Cat.No.): MP-091) cutting-out; 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 restriction enzyme HindIII and BamHI double digestion pUC19-Noster again; After agarose gel electrophoresis detects; Reclaim the big fragment of linearizing carrier; And the corn ubiquitin promoter (UbiPro) that has sticky end that will reclaim fragment and cut acquisition through enzyme in second step links to each other, and obtains recombinant vectors, called after pUN19.

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

Amplification said gene total length or its any segmental primer are to also being the scope that the present invention protects.

Above-mentioned primer is to being following 1) or 2):

The nucleotides sequence of a primer of the primer centering 1) is classified the sequence 3 in the sequence table as, and the nucleotides sequence of another primer of said primer centering is classified the sequence 4 in the sequence table as;

The nucleotides sequence of a primer of the primer centering 2) is classified the sequence 5 in the sequence table as, and the nucleotides sequence of another primer of said primer centering is classified the sequence 6 in the sequence table as.

Of the present invention experiment showed, in japonica rice, to spend be cloned into an OsMADS57 gene in No. ten, with its forward and the reverse expression vector that inserts, obtain sense expression vector and antisense expression vector; Utilizing sense expression vector and antisense expression vector to import in the paddy rice respectively spends No. ten; Obtain just transgenic paddy rice strain system and antisense transgene rice strain; Compare with the wild-type paddy rice, tillering that just transgenic paddy rice strain is increases, and the tiller number of antisense transgene rice strain reduces.

Description of drawings

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

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

Fig. 3 is the physical map of antisense expression vector pUN-OsMADS57 (antisense)

Fig. 4 is that the quantitative PCR of overexpression transgenic paddy rice (justice) is identified

Fig. 5 is that the quantitative PCR of antisense transgene paddy rice is identified

The phenotype that Fig. 6 is tillered for OsMADS57 overexpression transgenic paddy rice (justice)

Fig. 7 is the phenotype of OsMADS57 antisense transgene rice tillering

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

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

Consequence devised primer according to database analysis:

Overexpression vector makes up and uses primer: 5 ' end primer: 5 '-CG GGATCCATGGGGAGGGGGAAGATAGT-3 ' (the underscore sequence is the BamHI site), 3 ' end primer: 5 '-GG GGTACCTTAAGGCAGATGAAGTCCCAGT-3 ' (the underscore sequence is the KpnI site).

Antisense expression vector makes up and uses primer: 5 ' end primer: 5 '-CG GGATCCTTAAGGCAGATGAAGTCCCAGT-3 ' (the underscore sequence is the BamHI site), 3 ' end primer: 5 '-GG GGTACCATGGGGAGGGGGAAGATAGT-3 ' (the underscore sequence is the KpnI site).

Extract in the 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 following:

1) extraction of the total RNA of plant: choose the seedling of spending No. ten (Oryza sativa L.cvZhonghua 10) in the 100mg paddy rice in tri-leaf period (Li Meifang, paddy rice training kind-in spend agricultural science and technology communication, the 26th page of 1998 the 1st phase No. 10.The public can obtain from Chinese Academy of Sciences's plant research.) be material, in liquid nitrogen, grind, the lyophilized powder that grinds in the liquid nitrogen is transferred in the 1.5ml centrifuge tube that contains 1ml Trizol reagent (Invitrogen), fully mixing; Placed 5 minutes for 25 ℃; Add the fresh chloroform of 0.2ml in every pipe, shaken 15 seconds, 25 ℃ incubation 2-3 minute; 12,000rpm, 4 ℃, centrifugal 15 minutes; Transfer to the water 0.5ml of supernatant in the new 1.5ml centrifuge tube, add the 0.5ml Virahol, place for 25 ℃ and made the RNA deposition in 10 minutes; 12,000rpm, 4 ℃, centrifugal 10 minutes; Remove supernatant, the RNA deposition is cleaned 2 times with 1ml 75% ethanol, super clean bench blows to half-dried; With 50 μ l DEPC-ddH ₂The resuspended deposition of O, 60 ℃ of water-baths 10 minutes obtain RNA solution with dissolving RNA deposition.With-70 ℃ of preservations after this RNA solution packing, do the template of reverse transcription fully.

2) RT-PCR: get the above-mentioned RNA solution of 1 μ l, use DEPC-ddH ₂100 times of O dilutions are with 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 gOligo dT primers, use DEPC-ddH ₂O is supplemented to 15 μ l, and 70 ℃ of sex change are 5 minutes behind the mixing, 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 lDEPC-ddH ₂O).Behind the mixing, transcriptive process,reversed was accomplished in 42 ℃ of water-baths in 1 hour; 75 ℃ of water-baths made the ThermoScript II inactivation in 10 minutes, obtained containing the mixture of the first chain cDNA.

Get the template of the above-mentioned first chain cDNA of 1 μ l as PCR; Carry out the PCR reaction by following system: 0.2 μ lLATaq (5U/ μ l), 10 μ l, 2 * GC buffer, 1.8 μ l dNTPs, 0.5 μ l 5 ' holds primer (10 μ M); 0.5 μ l 3 ' holds primer (10 μ M), adds ddH ₂O final volume 20 μ l.

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

Make up primer sequence 5 ' end primer: 5 '-CG with overexpression GGATCCATGGGGAGGGGGAAGATAGT-3 ' (the underscore sequence is the BamHI site, sequence 3), 3 ' end primer: 5 '-GG GGTACCTTAAGGCAGATGAAGTCCCAGT-3 ' (the underscore sequence is the KpnI site, sequence 4) is a primer, carries out pcr amplification, obtains PCR product (justice).

Make up primer sequence 5 ' end primer: 5 '-CG with antisense expression GGATCCTTAAGGCAGATGAAGTCCCAGT-3 ' (the underscore sequence is the BamHI site, sequence 5), 3 ' end primer: 5 '-GG GGTACCATGGGGAGGGGGAAGATAGT-3 ' (the underscore sequence is the KpnI site, sequence 6) is a primer, carries out pcr amplification, obtains PCR product (antisense).

Above-mentioned PCR program is: get into the PCR circulation behind 94 ° of preparatory sex change 30s of C, loop parameter is that 15 seconds renaturation → 72 ° C of 10 seconds sex change → 55 ° C of 98 ° of C extended in 40 seconds, continues synthetic 10 minutes at 72 ° of C after 35 circulations.

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

Reclaim PCR product (justice) and PCR product (antisense) respectively; Check order respectively; The result all has in the sequence table sequence 1 from 5 ' terminal 1-726 position Nucleotide for the nucleotide sequence of PCR product (justice) and PCR product (antisense), the OFR of sequence 1 be in the sequence table sequence 1 from 5 ' terminal 1-726 position Nucleotide.The unnamed gene OsMADS57 of this PCR product, the OsMADS57 encoded protein is OsMADS57, this proteic aminoacid sequence is the sequence 2 in the sequence table.

The application of embodiment 2, OsMAOS57 albumen and encoding sox thereof

One, crosses the acquisition of expression vector pUN-MADS57 (justice) and antisense expression vector pUN-MADS57 (antisense)

1, the acquisition of pUN1301 plasmid

The first step: the about 0.2g corn seedling of clip places liquid nitrogen to grind; Add the extraction damping fluid (containing 0.1M Tris-HCl pH8.0,50mM EDTA, 0.5M NaCl, 1%SDS and 1% beta-mercaptoethanol) of the new preparation of 800 μ L then, thermal agitation makes its whole suspensions; 65 ℃ of water-baths 30 minutes were put upside down mixing once in per 5 minutes; Add the 5M potassium acetate solution of 250 μ L precoolings then, put upside down mixing immediately, ice bath 5 minutes; Add equal amounts of phenolic/chloroform, extracting once, centrifugal 5 minutes of 12000rpm; Collect supernatant, add the isopropanol precipitating DNA of 0.6 times of volume, room temperature was placed 40 minutes; Centrifugal 15 minutes of 4 ℃ of 12000rpm abandon supernatant; Deposition is respectively washed once with 70%, 100% ethanol; After the drying, be dissolved among the ddH2O that 20 μ L contain 100 μ g/mL RNaseA, obtain corn gene group DNA.

Second step: get above-mentioned corn gene group DNA solution 2 μ L as template, to have 5 ' primer (GG of Hind III recognition site AAGCTTCTGCAGTGCAGCGTGACCCGG) and have 3 ' primer (CG of BamHI recognition site GGATCCAAGTAACACCAAACAACAGGG) be primer, carry out pcr amplification, the PCR reaction conditions is: earlier 94 ℃ 3 minutes; Again 94 ℃ 45 seconds, 62 ℃ 45 seconds, 72 ℃ 2 minutes, totally 35 circulations, last 72 ℃ 10 minutes.After reaction finishes, the PCR product is carried out 0.8% agarose gel electrophoresis detect, show to obtain the amplified fragments that length is about 2kb; Conform to expected results; Reclaim this purpose fragment,, obtain fragment through order-checking with reclaiming behind restriction enzyme Hind III and the BamH I double digestion; This fragment is that sequence 7 in the sequence table is from 5 ' terminal 1-1986 position Nucleotide, for having the corn ubiquitin promoter (UbiPro) of sticky end.(corn ubiquitin promoter (UbiPro) also can obtain by synthetic.）

The 3rd step: with restriction enzyme Sac I and EcoR I with Noster poly A terminator sequence from plasmid vector pBI 121 (Beijing Baeyer enlightening Bioisystech Co., Ltd catalog number (Cat.No.): MP-091) cutting-out; 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 restriction enzyme HindIII and NamHI double digestion pUC19-Noster again; After agarose gel electrophoresis detects; Reclaim the big fragment of linearizing carrier; And the corn ubiquitin promoter (UbiPro) that has sticky end that will reclaim fragment and cut acquisition through enzyme in second step links to each other, and obtains recombinant vectors, called after pUN19.

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

2, cross the acquisition of expression vector pUN-MADS57 (justice)

With restriction enzyme KpnI and BamHI the plasmid pUN1301 that 1 step obtains is carried 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 were cut 4 hours.With agarose gel electrophoresis enzyme is cut product and separate, reclaim the big fragment of linearizing pUN1301, be dissolved in 20 μ l ddH ₂Among the O.

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

The OsMADS57 (justice) of the 726bp that 10 μ l are reclaimed, the big fragment solution of carrier pUN1301,2 μ l (3U/ μ l) T4DNA ligase enzyme and the 2 μ l 10x ligase enzyme damping fluids that 6 μ l reclaim are mixed; 16 ℃ connect 16 hours; The connection product transformed into escherichia coli DH5 α competent cell that obtains obtains positive colony through the resistant panel screening that contains kantlex.

Extract the recombinant plasmid in the positive colony; Carry out sequence verification; This plasmid is for inserting the carrier that obtains between BamHI and the KpnI restriction enzyme site of pUN1301 with the sequence in the sequence table 1 from 5 ' terminal 1-726 position Nucleotide; With this plasmid called after pUN-OsMADS57 (justice); And the sequence in the sequence table 1 holds the BamHI restriction enzyme site through said pUN1301 to be connected with it from 5 ' of 5 ' terminal 1-726 position Nucleotide, and the sequence 1 in the sequence table holds the KpnI restriction enzyme site through said pUN1301 to be connected with it from 3 ' of 5 ' terminal 1-726 position Nucleotide, and the structure of promotor, gene and terminator is correct among the pUN-OsMADS57.In this expression vector, adopt corn ubiquitin promoter (UbiPro) to start purpose fragment OsMADS57 overexpression in plant, its physical map synoptic diagram is as shown in Figure 2.

3, the acquisition of expression vector pUN-MADS57 (antisense)

With restriction enzyme KpnI and BamHI the 726bp PCR product (antisense) that is obtained by embodiment 1 is carried out double digestion, the enzyme that obtains is cut product and is connected with the plasmid pUN1301 that 1 step of cutting through same enzyme obtains, and obtains recombinant plasmid.

Recombinant plasmid is sent to order-checking; The result for this plasmid for the sequence in the sequence table 1 is inserted the carrier that obtains between KpnI and the BamHI restriction enzyme site of pUN1301 from 5 ' terminal 1-726 position Nucleotide; With this plasmid called after pUN-OsMADS57 (antisense); And the sequence in the sequence table 1 holds the KpnI restriction enzyme site through said pUN1301 to be connected with it from 5 ' of 5 ' terminal 1-726 position Nucleotide; Sequence 1 in the sequence table holds the BamHI restriction enzyme site through said pUN1301 to be connected with it from 3 ' of 5 ' terminal 1-726 position Nucleotide, and its physical map synoptic diagram is as shown in Figure 3.

Two, change acquisition and the evaluation of OsMADS57 paddy rice (justice) and commentaries on classics OsMADS57 paddy rice (antisense)

1, changes the acquisition of OsMADS57 paddy rice (justice) and commentaries on classics OsMADS57 paddy rice (antisense)

With reference to electric exciter (EasyJecT Plus electric exciter; Britain EquiBio company) operational guidance; Respectively pUN-MADS57 (justice) and pUN-MADS57 (antisense) are transformed Agrobacterium EHA105 (Biovector Co. with electric shocking method; The catalog number (Cat.No.) Biovec-11 of LTD company), screen the overexpression engineering bacteria that obtains positive colony, called after EHA105/pUN-OsMADS57 (justice) and EHA105/pUN-OsMADS57 (antisense) through the resistant panel that contains kantlex.

Respectively with spending paddy rice (Oryza sativa L.cv Zhonghua 10 No. ten in EHA105/pUN-OsMADS57 (justice) and EHA105/pUN-OsMADS57 (antisense) importing; Hereinafter to be referred as the wild-type paddy rice) callus in; With the sterilized water washing that contains 300mg/L cephamycin 4-5 time, after blotting, aseptic filter paper goes to N again ₆D ₂S ₁On the substratum, a screening generation; After two weeks, be transferred to N ₆D ₂S ₂Screening two generations (2 week/generation) on the substratum; Taking-up is screened eugonic resistant calli through 3 generations, is transferred to division culture medium (1), on, in differentiation culture case (12 hour photoperiod, 28 ℃ of daytimes, 25 ℃ of nights), cultivated 7 days; Be transferred to division culture medium (2) then, on, in the differentiation culture case, be cultured to the generation regrowth.The regenerated plant is strong plantlets and rootage on the strong plantlets and rootage substratum; When treating that seedling grows to 10 centimetres of left and right sides; Open the container closure film; Refining seedling 2-3 days moves into the phytotron cultivation with seedling then, obtain respectively 10 strains systems totally 60 T0 for change OsMADS57 paddy rice (justice) and 10 strain systems totally 100 T0 for commentaries on classics OsMADS57 paddy rice (antisense).

Used substratum is following:

2, change the evaluation of OsMADS57 paddy rice (justice) and commentaries on classics OsMADS57 paddy rice (antisense)

1), GUS histochemical stain:

To be put into respectively in the GUS staining fluid for the long root segment of the 2-3mm that changes OsMADS57 paddy rice (justice) by 70 T0 that embodiment 1 obtains, the several minutes of bleeding places 37 ℃ to be incubated overnight then, and the tissue after the dyeing is used 70% ethanol decolorization.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.The result identifies 6 strains system altogether and adds up to 50 positive T0 for changeing OsMADS57 paddy rice (justice), and this seedling is moved to greenhouse production, receives kind according to homophyletic system not, obtains T1 for transgenic seed, on this basis through the numerous excessively kind of T2 that obtains isozygotying for seed.Choose in the experiment afterwards that to change OsMADS57 paddy rice (justice) strain be that the homozyous seed T2 of 1 (OE1) and 2 (OE2) is a material.

To be put into respectively in the GUS staining fluid for the long root segment of the 2-3mm that changes OsMADS57 paddy rice (antisense) by 100 T0 that embodiment 1 obtains, the several minutes of bleeding places 37 ℃ to be incubated overnight then, and the tissue after the dyeing is used 70% ethanol decolorization.It is positive transgenic line that root is blue plant.GUS staining fluid (pH 7.0) component is: 100mMNa 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.The result identifies 8 strains system altogether and adds up to 80 positive T0 for changeing OsMADS57 paddy rice (antisense), and this seedling is moved to greenhouse production, receives kind according to homophyletic system not, obtains T1 for transgenic seed, on this basis through the numerous excessively kind of T2 that obtains isozygotying for seed.Choosing the homozyous seed T2 that changes OsMADS57 paddy rice (antisense) 1 (AS1) and 2 (AS3) in the experiment afterwards is material.

2), quantitative PCR is identified:

(1) T2 changes OsMADS57 paddy rice (justice) evaluation

From T2 commentaries on classics OsMADS57 paddy rice (justice) seedling of OE1 and OE2, extracting mRNA, and transcribe acquisition cDNA respectively, is contrast with wild-type paddy rice (spending in the paddy rice No. ten).Utilize the fluorescence real-time quantitative PCR method; With cDNA is 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 ') be primer, the expression abundance of positive T2 being changeed OsMADS57 paddy rice (justice) detects.The reagent that is used for quantitative analysis is SYBR Green Realtime PCR Master Mix (TOYOBO).Used instrument is the real-time fluorescence quantitative PCR appearance Mx3000P of U.S. Stratagene company.Draw 1 μ l, the first chain cDNA solution, dilute 50 times, carry out the PCR reaction by following system: 10 μ l SYBR Green Realtime PCR Master Mix as template; 4 μ l templates; 1 μ l 5 ' holds primer 1 (10 μ M), and 1 μ l 3 ' holds primer 1 (10 μ M), adds ddH2O final volume 20 μ l.

The result is as shown in Figure 4; Under the situation of Actin gene as confidential reference items; Compare with the wild-type paddy rice, the expression abundance of OsMADS57 has had rise in various degree in the T2 seedling of OE1 and OE2, and illustration purpose gene (OsMADS57) is transcriptional level successful expression again.

(2) T2 changes OsMADS57 paddy rice (antisense) evaluation

From T2 commentaries on classics OsMADS57 paddy rice (antisense) seedling of AS1 and AS3, extracting mRNA, and transcribe acquisition cDNA respectively, is contrast with wild-type paddy rice (spending in the paddy rice No. ten).Utilize the fluorescence real-time quantitative PCR method; With cDNA is template; With 1 μ l 5 ' end primer 1 (10 μ M) (5 '-TTGAGCCAATCACAGCAAAGAG-3 '); 1 μ l 3 ' end primer 1 (10 μ M) (5 '-TCGTCCAGCTTGTACGGCATAG-3 ') be primer, positive T2 is detected for the expression abundance of changeing OsMADS57 in the OsMADS57 paddy rice.The reagent that is used for quantitative analysis is SYBR Green Realtime PCR Master Mix (TOYOBO).Used instrument is the real-time fluorescence quantitative PCR appearance Mx3000P of U.S. Stratagene company.Draw 1 μ l, the first chain cDNA solution, dilute 50 times, carry out the PCR reaction by following system: 10 μ lSYBR Green Realtime PCR Master Mix as template; 4 μ l templates; 1 μ l 5 ' holds primer 1 (10 μ M), and 1 μ l 3 ' holds primer 1 (10 μ M), adds ddH2O final volume 20 μ l.

As confidential reference items, 5 of Actin ' holds primer with Actin: 5 '-CGTATGAGCAAGGAGATCAC-3 ', 3 ' end primer is: 5 '-CACATCTGTTGGAAGGTGCT-3 '.The PCR program is: in advance sex change is 2 minutes, gets into the PCR circulation, and loop parameter is 94 ° of C 15 seconds → 15 seconds → 72 ° C of 56 ° of C 15 seconds, totally 40 circulations.

The result is as shown in Figure 5; Under the situation of Actin gene as confidential reference items; Compare with the wild-type paddy rice; The expression abundance of OsMADS57 has had downward modulation in various degree in the T2 seedling of AS1 and AS3, and illustration purpose gene (OsMADS57) is transcriptional level successful expression and endogenous OsMADS57 transcript expression amount is reduced again.

Adopt and use the same method obtaining T0 for changeing the empty carrier paddy rice in the empty carrier pUN1301 importing wild-type paddy rice; Gather in the crops T1 for changeing the pUN1301 rice paddy seed from T0 for changeing the pUN1301 paddy rice; Sowing is gathered in the crops T2 for changeing the pUN1301 rice paddy seed from T1 for changeing the pUN1301 paddy rice.

The phenotype of three, changeing OsMADS57 paddy rice (justice) and commentaries on classics OsMADS57 paddy rice (antisense) is observed

The growth phenotype of 1, changeing OsMADS57 paddy rice (justice) mature period is observed

With T2 for the OE1 that changes OsMADS57 paddy rice (justice) and OE2 seed, in spend No. ten wild-type rice paddy seeds (ZH10) and T2 for commentaries on classics pUN1301 rice paddy seed; All sow in the mixture of flower nutrition soil and vermiculite (both blending ratios are 4:1); Be placed on after 30 ℃ of sproutings that (32 ℃) were cultured to for 3 leaf phases in the greenhouse, then the seedling cultivation of growth in rice terrace.Each strain is 30, experiment triplicate, results averaged.

The plant tillering number is observed, and the result is following:

Take pictures shown in Fig. 6 A, can find out, compare with the wild-type paddy rice, T2 increases for the tiller number that changes OsMADS57 paddy rice (justice) OE1.

After planting about the 70th day statistics T2 for the OE1 that changes OsMADS57 paddy rice (justice) and OE2 seed, in spend No. ten wild-type rice paddy seeds (ZH10) and T2 for commentaries on classics pUN1301 rice tillering number; The result shown in Fig. 6 B, T2 for change OsMADS57 paddy rice (justice) OE1, T2 for change OsMADS57 paddy rice (justice) OE2, in spend the tiller number of No. ten wild-type paddy rice (ZH10) to be respectively 14,17 and 26.

T2 does not have significant difference for changeing pUN1301 paddy rice and wild-type paddy rice result.

The growth phenotype of 2, changeing OsMADS57 paddy rice (antisense) mature period is observed

With T2 for the AS1 that changes OsMADS57 paddy rice (antisense) and AS3 seed, in spend No. ten wild-type rice paddy seeds (ZH10) and T2 for commentaries on classics pUN1301 rice paddy seed; All sow in the mixture of flower nutrition soil and vermiculite (both blending ratios are 4:1); Be placed on after 30 ℃ of sproutings that (32 ℃) were cultured to for 3 leaf phases in the greenhouse, then the seedling cultivation of growth in rice terrace.Each strain is 40, experiment triplicate, results averaged.

The plant tillering number is observed, and the result is following:

Take pictures shown in Fig. 7 A, can find out, compare with the wild-type paddy rice, T2 reduces for the tiller number that changes OsMADS57 paddy rice (justice) OE1.

After planting about the 70th day statistics T2 for the AS1 that changes OsMADS57 paddy rice (antisense) and AS3 seed, in spend No. ten wild-type rice paddy seeds (ZH10) and T2 for commentaries on classics pUN1301 rice tillering number; The result shown in Fig. 7 B, T2 for change OsMADS57 paddy rice (antisense) AS1, T2 for change OsMADS57 paddy rice (antisense) AS3, in spend the tiller number of No. ten wild-type paddy rice (ZH10) to be respectively 14,11 and 8.T2 does not have significant difference for changeing pUN1301 paddy rice and wild-type paddy rice result.

Claims (7)

1. albumen is (a) or (b) as follows:

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by sequence 2 deutero-protein.

2. coding claim 1 said proteic gene.

3. gene as claimed in claim 2 is characterized in that: said gene is following 1)-4) in any dna molecular:

1) dna molecular shown in the sequence 1 in the sequence table;

2) sequence 1 is held the dna molecular shown in the Nucleotide of 1-726 position from 5 ' in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and coding proteic dna molecular with identical function;

4) with 1) or 2) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the proteic dna molecular that 99% homology and coding have identical function at least at least.

4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said genes.

5. recombinant vectors according to claim 4, its spy is: said recombinant vectors obtains expressing the said proteic recombinant vectors of claim 1 for claim 2 or 3 said genes are inserted in the expression vector.

6. amplification claim 2 or 3 said full length genes or its any segmental primer are right.

7. primer according to claim 6 is right, it is characterized in that: said primer is to being following 1) or 2):

The nucleotides sequence of a primer of the primer centering 1) is classified the sequence 3 in the sequence table as, and the nucleotides sequence of another primer of said primer centering is classified the sequence 4 in the sequence table as;

The nucleotides sequence of a primer of the primer centering 2) is classified the sequence 5 in the sequence table as, and the nucleotides sequence of another primer of said primer centering is classified the sequence 6 in the sequence table as.

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