CN103788187B - Flowering of plant associated protein GmSOC1-like and encoding gene thereof and application - Google Patents

Abstract

The invention discloses a kind of flowering of plant associated protein GmSOC1-like and encoding gene thereof and application.Experiment proves, by the positive transgenic plant transforming Root or stem of Littleleaf Indianmulberry containing the 277th recombinant expression vector pGFPGUS-GmSOC1-like to the 912nd shown DNA molecular in sequence 2 and obtain, under long day condition (16 h light/8 h dark) with turn empty vector control plant and to compare with wild-type Root or stem of Littleleaf Indianmulberry and bloom 32 days in advance.The present invention is significant in the photoperiod of orientation adjustment plant.

Description

Flowering of plant associated protein GmSOC1-like and encoding gene thereof and application

Technical field

The present invention relates to a kind of flowering of plant associated protein GmSOC1-like and encoding gene thereof and application.

Background technology

Soybean is the short day crop of photoperiodic reaction sensitivity, and the subject range of single kind is comparatively narrow.For a long time, cultivate the Wide-adaptive soybean varieties of photoperiod relative insensitiveness is the important goal of soybean breeder always.Conventional breeding is the main method of soybean varieties improvement, the soybean varieties of applicable different areas plantation is cultivated by hybridization and strain selection etc., but progress is comparatively slow in the improvement of photoperiodic reaction susceptibility, at present, in the urgent need to adopting molecular breeding means, by the introducing of foreign gene or the genetic manipulation to inherent gene, the photoperiodic reaction susceptibility of orientation adjustment soybean varieties, accelerates the seed selection process of Wide-adaptive soybean varieties.

Summary of the invention

The object of this invention is to provide a kind of flowering of plant associated protein GmSOC1-like and encoding gene thereof and application.

Flowering of plant associated protein provided by the present invention derives from soybean (Glycinemax(L.) Merrill), name is called GmSOC1-like, and this protein is following protein a) or b):

A) protein be made up of the aminoacid sequence shown in sequence 1;

B) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and by (a) the derivative protein relevant to flowering of plant.

Aminoacid sequence shown in sequence 1 is made up of 211 amino-acid residues.

In order to make the albumen in above-mentioned (a) be convenient to purifying, label as shown in table 1 can be connected at the N-terminal of the protein be made up of the aminoacid sequence shown in sequence 1 or C-terminal.

The sequence of table 1 label

Albumen in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) is by the codon by lacking one or several amino-acid residue in the 277th of sequence 2 the to the DNA sequence dna shown in the 912nd, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.

The gene of code for said proteins also belongs to protection scope of the present invention.

The gene of code for said proteins is following 1) or 2) or 3) or 4) gene:

1) its nucleotide sequence is the DNA molecular shown in the 277th to the 912nd in sequence 2;

2) its nucleotide sequence is the DNA molecular shown in sequence 2;

3) with 1) or 2) DNA sequence dna that limits at least 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 at least have the DNA molecular of 99% homology and code for said proteins;

4) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and the DNA molecular of code for said proteins.

Sequence 2 is made up of 1081 deoxynucleotides, is the full length cDNA sequence of soybean protein GmSOC1-like, and wherein the 277th is open reading frame to the 912nd, the albumen shown in polynucleotide sequence 1.

Described stringent condition can be as follows: 50 DEG C, at 7% sodium lauryl sulphate (SDS), 0.5MNa ₃pO ₄hybridize with in the mixing solutions of 1mMEDTA, at 50 DEG C, rinsing in 2 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5MNa ₃pO ₄hybridize with in the mixing solutions of 1mMEDTA, at 50 DEG C, rinsing in 1 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5MNa ₃pO ₄hybridize with in the mixing solutions of 1mMEDTA, at 50 DEG C, rinsing in 0.5 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5MNa ₃pO ₄hybridize with in the mixing solutions of 1mMEDTA, at 50 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be: 50 DEG C, at 7%SDS, 0.5MNa ₃pO ₄hybridize with in the mixing solutions of 1mMEDTA, at 65 DEG C, rinsing in 0.1 × SSC, 0.1%SDS; Also can be: in the solution of 6 × SSC, 0.5%SDS, hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vectors containing described gene, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus also belong to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.As pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb(CAMBIA company) etc.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.When using described gene constructed recombinant plant expression vector, can add any one enhancement type promotor (ubiquitin promoter (Ubiquitin) as cauliflower mosaic virus (CAMV) 35S promoter, corn), constitutive promoter or organizing specific expression promotor (promotor as seed specific expression) before its transcription initiation Nucleotide, they can be used alone or are combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce the enzyme of colour-change or the gene (gus gene of luminophor, luciferase genes etc.), antibiotic marker gene is (as given the nptII gene to kantlex and associated antibiotic resistance, give the bar gene to herbicide phosphinothricin resistance, give the hph gene to microbiotic hygromycin resistance, with the dhfr gene given methatrexate resistance, give EPSPS gene to glyphosate) or chemical resistance reagent marker gene etc. (as anti-weedkiller gene), the mannose-6-phosphate isomerase gene of metabolism seminose ability is provided.

Recombinant vectors containing described gene is the recombinant expression vector obtaining described gene insertion vector pGFPGUSplus to express described albumen; Be specially and xbalI and the sacI enzyme of the DNA molecular insertion vector pGFPGUSplus shown in the 277th to the 912nd in sequence 2 is cut the recombinant expression vector obtained between recognition site.

The present invention protects the application in the Characteristics in florescence of regulation and control object plant of described albumen and described gene, or after regulation and control, the material of described albumen or described gene expression amount or method can be used for regulation and control object plant blossom time proterties.

The present invention also provides a kind of method that object flowering of plant is shifted to an earlier date, and comprises the step that described gene is expressed in described object plant.

A kind of method that the present invention also provides cultivation to bloom transgenic plant in advance, comprises described channel genes object plant, obtains the step of the transgenic plant of expressing said gene; Described transgenic plant bloom in advance compared with described object plant; The step described transgenic plant and other plant hybridization being obtained the filial generation containing described gene also can be comprised in the method; Described transgenic plant are interpreted as the first-generation transgenic plant not only comprising and obtained by described gene transformation object plant, also comprise its filial generation.For transgenic plant, this gene can be bred in these species, also with traditional breeding method, this transgenosis can be entered other kind of same species, particularly including in commercial variety.Described transgenic plant comprise seed, callus, whole plant and cell.

In the above-mentioned methods, described object plant is monocotyledons or dicotyledons, and described dicotyledons specifically can be Root or stem of Littleleaf Indianmulberry (LotuscorniculatusL.) or soybean (Glycinemax(L.) Merrill).

Experiment proves, by the positive transgenic plant transforming Root or stem of Littleleaf Indianmulberry containing the 277th recombinant expression vector pGFPGUS-GmSOC1-like to the 912nd shown DNA molecular in sequence 2 and obtain, under long day condition (16 h light/8 h dark) with turn empty vector control plant and to compare with wild-type Root or stem of Littleleaf Indianmulberry and bloom 32 days in advance.The present invention is significant in the photoperiod of orientation adjustment plant.

Accompanying drawing explanation

Fig. 1 is the pcr amplification electrophorogram of GmSOC1-like gene.Wherein, swimming lane M is molecular weight standard; The amplified band of gene GmSOC1-like for the purpose of swimming lane 1 and 2.

Fig. 2 is the soybean that grows under fluorescence real-time quantitative PCR the detects short day condition relative expression quantity at different time different sites GmSOC1-like gene.Wherein, the number of days of continued growth after 7,13,19,23,27 expression soybean germinations, F represents colored, and P represents pod.

Fig. 3 is the relative expression quantity change that fluorescence real-time quantitative PCR detects GmSOC1-like gene in day when soybean grows under different sunshine condition.Wherein, figure A is short day, and figure B is the long day.

Fig. 4 is the relative expression quantity change that fluorescence real-time quantitative PCR detects soybean gene GmSOC1-like and GmFT2a after conversion at sunshine.Wherein, figure A is gene GmFT2a, and figure B is gene GmSOC1-like.

Fig. 5 is the structural representation of recombinant vectors pGFPGUS-GmSOC1-like.

Fig. 6 is the electrophorogram that PCR detects transfer-gen plant.Wherein, M is marker, and 1 is plasmid pGFPGUS-GmSOC1-like positive control, and 2 is wild-type Root or stem of Littleleaf Indianmulberry negative control, and 3,4,5 for turning the Root or stem of Littleleaf Indianmulberry plant of pGFPGUS-GmSOC1-like, and 6,7 for turning empty vector control plant.

Fig. 7 is the electrophorogram that RT-PCR detects transfer-gen plant.Wherein, 1-5 is the Root or stem of Littleleaf Indianmulberry plant turning pGFPGUS-GmSOC1-like, and 6 is wild-type Root or stem of Littleleaf Indianmulberry plant.

Fig. 8 is the bloom situation of transfer-gen plant under long-day conditions.Wherein, TL is the Root or stem of Littleleaf Indianmulberry plant turning pGFPGUS-GmSOC1-like, and CK is wild-type Root or stem of Littleleaf Indianmulberry plant.

Embodiment

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

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

Root or stem of Littleleaf Indianmulberry (Lotuscorniculatus) (long day plant), carrier pGFPGUSplus and Agrobacterium rhyzogenesK599 (AgrobacteriumrhizogenesK599): reference: BoJian, WenshengHou, CunxiangWu, BinLiu, WeiLiu, ShikuiSong, YurongBi, TianfuHan.Agrobacteriumrhizogenes-mediatedtransformation ofSuperroot-derivedLotuscorniculatusplants:avaluabletool forfunctionalgenomics.BMCplantBiology.2009,9:78; Institute of Crop Science, Chinese Academy of Agricultural Science ensures to provide to the public.

The acquisition of embodiment 1, plant blossom time associated protein GmSOC1-like and encoding gene thereof

The aminoacid sequence of Arabidopis thaliana AtSOC1 gene is carried out homology comparison in Genebank database, find the gene that homology in soybean is higher, and according to the full length cDNA sequence obtained further, design PCR primer F1:5 '-ATGGTGAGAGGAAAGACTCAGCT-3 ' and R1:5 '-ATAGCTAGACCTGAGTAGTCCAATGA-3 '.

(Glycinemax(L.) Merrill is selected in the soybean varieties tribute of cultivating three weeks under getting short day (every day 8 h light and 16 h dark) condition) seedling, with its top of the careful clip of scissors (removing young tender compound leaf) liquid nitrogen cryopreservation afterwards as far as possible, and in-80 DEG C of Refrigerator stores.Trizol method is utilized to extract total serum IgE, reverse transcription is cDNA, again with this cDNA for template, above-mentioned primers F 1 and R1 is utilized to carry out pcr amplification, the amplified production obtained is carried out 1% agarose gel electrophoresis (as shown in Figure 1), the fragment reclaiming about 0.6kb checks order, and result is as the 277th of sequence 2 the to shown in the 912nd.

The long 1081bp of sequence 2 sequence is the full length cDNA sequence of GmSOC1-like gene, is open reading frame from the 277th of sequence 2 to the 912nd, the Protein G mSOC1-like shown in polynucleotide sequence 1.

Embodiment 2, the expression pattern of GmSOC1-like gene in soybean different development stage, different tissues

No. one soybean varieties tribute is selected to plant under short day (every day 8 h light and 16 h dark) condition, the root of plant, stem, leaf and apical meristem (i.e. branch apical meristem within 7,13,19,23,27 days, is got respectively at continued growth after sprouting, also comprise flower and the beanpod in later stage) sample, preserve in-80 DEG C after liquid nitrogen freezing.Trizol method extracts the total serum IgE of each sample respectively, reverse transcription is cDNA again, with this cDNA for template, 5 '-AGGCTGTGTGAGCAGTATGGTATC-3 ' and 5 '-TAGATAGACCTGGGTAGTCCAATGA-3 ' is primer, the relative expression quantity that fluorescence real-time quantitative PCR detects GmSOC1-like gene (sets the expression amount numerical value of expression amount lower-most point in an apical meristem as 1, the expression amount of all the other time points all compares with the relative expression quantity of lower-most point, draw the relative expression quantity of different time points), wherein, reference gene is GMG _gdPH, primer is 5 '-ACTCCTTGATACCGTTGTCCAT-3 ' and 5 '-GTCTGTTATCCGCCTACAGCCT-3 '.Result as shown in Figure 4.

Result shows, under short day condition, along with soybean is by the carrying out changed to reproductive growth that nourish and grow, GmSOC1-like shows as the trend raised gradually at the expression amount of soybean apical meristem, and it is early stage that its peak value appears at that tribute selects No. one to bloom and change.In addition, in stem, root, flower, leaf and pod, the expression of a small amount of GmSOC1-like gene only detected.This shows that GmSOC1-like may be a kind of soybean blossoming inducible factor.

The daily rhythms phenomenon of embodiment 3, GmSOC1-like genetic expression and the expression pattern under different duration of day process thereof

1, the daily rhythms phenomenon of GmSOC1-like genetic expression under different sunshine

Soybean varieties tribute selected a seed to plant respectively and sprout rear growth after 9 days under short day (8h illumination and 16h dark) and long day (16h illumination and 8h dark) condition, got the apical meristem sample of plant every one hour, preserve in-80 DEG C after liquid nitrogen freezing.Trizol method extracts the total serum IgE of each sample respectively, then reverse transcription is cDNA, and with this cDNA for template, detect the relative expression quantity of GmSOC1-like gene in each sample according to the fluorescence real-time quantitative PCR method of embodiment 2, result is as shown in A and B in Fig. 3.Result shows, under short day condition, in one day 24 hours, in soybean there is peak value at midnight (15 hours) in the expression of GmSOC1-like gene, soybean under long-day conditions does not then have such peak value to occur, illustrate GmSOC1-like gene can induce by short day condition.

2, the expression pattern of GmSOC1-like gene under the different duration of day

1) two kinds of process are established

Process 1: soybean varieties tribute select a seed to plant to sprout under short day (8h illumination every day dark 16h in) condition after growth 13 days time go to continued growth under long day (16h illumination every day dark in 8h) condition;

Process 2: soybean varieties tribute is selected a seed to plant to sprout under short day (8h illumination every day dark 16h in) condition and to grow.

2) acquisition of sampling and cDNA

After the soybean germination of step 1), grow 9 days start, get apical meristem sample every three days, preserve in-80 DEG C after liquid nitrogen freezing.Trizol method extracts the total serum IgE of each sample respectively, then reverse transcription is cDNA.

3) fluorescence real-time quantitative PCR detects

With step 2) cDNA be template, No. GmFT2a(Genbank, the gene detecting GmSOC1-like gene and known control soybean bloom in each sample according to the fluorescence real-time quantitative PCR method of embodiment 2 is respectively AB550122) relative expression quantity, the primer wherein detecting GmFT2a is 5 '-TAATTCATAACAAAGCAAACGAGTA-3 ' and 5 '-GCTGACATCTCTGTTATTGTAGGTA-3 '.Result is as shown in A and B in Fig. 4.

Result shows: under short day condition, GmSOC1-like gene latter 19 days of sprouting by a large amount of abduction delivering, induce required time (sprouting latter 23 days) shorter than gene GmFT2a, and long-day conditions can not the expression of induced gene GmSOC1-like and GmFT2a.This illustrates compared with GmFT2a gene, and GmSOC1-like gene is a kind of induction of flowering factor more early formed, and not by the regulation and control of GmFT2a.

The acquisition of embodiment 4, the transgenosis Root or stem of Littleleaf Indianmulberry plant in advance that blooms

1, the recombinant expression vector of goal gene GmSOC1-like builds

The primers F 1-X of XbalI and SacI restriction endonuclease recognition sequence is contained respectively with 5 ' end:

5 '-GC tCTAGAaTGGTGAGAGGAAAGACTCAGCT-3 ' and R1-S:

5 '-C gAGCTCin ATAGCTAGACCTGAGTAGTCCAATGA-3 ' pcr amplification embodiment 1, the cDNA of a GmSOC1-like gene is selected in soybean varieties tribute, acquisition PCR primer is carried out 1% agarose gel electrophoresis, reclaim the fragment of purifying 0.65kb, with xbalI and SacI double digestion, between the site being connected into xbalI and SacI of carrier pGFPGUSplus, obtain recombinant vectors pGFPGUS-GmSOC1-like.Confirm through order-checking, recombinant vectors pGFPGUS-GmSOC1-like(is as shown in Figure 5) be between the site of xbalI and SacI of carrier pGFPGUSplus, insert the DNA fragmentation shown in sequence 2 the 277th to the 912nd.

2, the acquisition of restructuring Agrobacterium rhizogenes

Get the recombinant vectors pGFPGUS-GmSOC1-like of step 1, electric shocking method transforming agrobacterium rhizogenes K599, obtain the restructuring Agrobacterium rhyzogenesK599/pGFPGUS-GmSOC1-like containing recombinant vectors pGFPGUS-GmSOC1-like.

After the same method, by electroporated for empty carrier pGFPGUSplus Agrobacterium rhyzogenesK599, obtain the restructuring Agrobacterium rhyzogenesK599/pGFPGUSplus containing empty carrier pGFPGUSplus.

3, the acquisition of transgenosis Root or stem of Littleleaf Indianmulberry plant

Reference literature " BoJian, WenshengHou, CunxiangWu, BinLiu, WeiLiu, ShikuiSong, YurongBi, TianfuHan.Agrobacteriumrhizogenes-mediatedtransformation ofSuperroot-derivedLotuscorniculatusplants:atoolforfunct ionalgenomicsinlegumes.BMCplantBiology.2009, 9:78 " in method, infect Root or stem of Littleleaf Indianmulberry (LotuscorniculatusL.) with restructuring Agrobacterium rhyzogenesK599/pGFPGUS-GmSOC1-like that step 2 obtains and obtain the Root or stem of Littleleaf Indianmulberry plant that resistance turns pGFPGUS-GmSOC1-like, concrete steps are as follows:

1) carry out streak culture on the LB solid medium containing kantlex 50mg/L by the Agrobacterium rhyzogenesK599/pGFPGUS-GmSOC1-like that recombinates, picking list bacterium colony is in the LB liquid nutrient medium containing kantlex 50mg/L, 28 DEG C of incubated overnight.Get bacterium liquid and shake bacterium to OD according to the ratio of 1:1000 ₆₀₀liquid is infected in=0.6 acquisition.

2) cutting shoots of Root or stem of Littleleaf Indianmulberry (LotuscorniculatusL.) seedling will cultivated under long day condition, every section containing an internode, be soaked in step one obtain infect in liquid, 25 DEG C, 30min is infected in 50rpm vibration, outwell liquid, unnecessary bacterium liquid is drawn again with aseptic filter paper, put on the CCM substratum of surface paving filter paper, Dual culture uses aseptic water washing three times after 2 days, then 50rpm vibration 1h in washing substratum is placed on, outwell liquid, liquid is blotted with aseptic filter paper, be placed on Hairy root inducing culture in 25 DEG C, the generation of induction Hairy root is cultivated under 16h illumination every day 8h dark condition.

The formula of above-mentioned substratum is as follows:

CCM substratum: add MES sodium (MES) 3.9mg/L, Syringylethanone (As) 200mM, dithiothreitol (DTT) (DTT) 150mg/L, halfcystine (Cys) 100mg/L, sucrose 30g/L and agar 7.5g/L, pH5.4 in 1/10MS liquid nutrient medium;

Washing substratum: add cephamycin (Cef) 250mg/L, Pyocianil (Cb) 250mg/L and sucrose 30g/L, pH6.8 in 1/2MS liquid nutrient medium;

Hairy root inducing culture: add cephamycin (Cef) 250mg/L, Pyocianil (Cb) 250mg/L, sucrose 30g/L and agar 7.5g/L, pH6.8 in 1/2MS liquid nutrient medium.

3) after two weeks, the Hairy root grown is cut, be placed on induced bud differentiation on regeneration culture medium.Be positioned on root media by the bud grown and cultivate, final acquisition 20 strain resistances turn the Root or stem of Littleleaf Indianmulberry plant of pGFPGUS-GmSOC1-like.

The formula of above-mentioned substratum is as follows:

Regeneration culture medium: add 6-BA0.5mg/L, Cef250mg/L, Cb250mg/L, agar 7.5g/L and sucrose 30g/L, pH6.8 in MS liquid nutrient medium.

Root media: add Cef250mg/L, Cb250mg/L, agar 7.5g/L and sucrose 30g/L, pH6.8 in MS liquid nutrient medium.

Meanwhile, according to above-mentioned steps 1)-3) method restructuring Agrobacterium rhyzogenesK599/pGFPGUSplus conversion Root or stem of Littleleaf Indianmulberry (LotuscorniculatusL.), obtain 20 strain resistances and turn empty vector control plant.

4, the PCR qualification of transfer-gen plant

Get resistance that step 3 obtains and turn the Root or stem of Littleleaf Indianmulberry plant of pGFPGUS-GmSOC1-like and resistance turns empty vector control plant, extract genomic dna respectively, with gus gene fragment for target gene, PCR detection is carried out with upstream primer 5 '-GATGATAGTTACAGAACCGACG-3 ' and downstream primer 5 '-CATTCGGAATCTCCACGTTAC-3 ', the size 750bp of prediction product, partial results as shown in Figure 6.Plant 20 strain turning pGFPGUS-GmSOC1-like is all positive, and turns empty vector control plant 20 strain and is all positive.

5, the RT-PCR of transfer-gen plant detects

Step 4PCR test positive of learning from else's experience turns the blade of the Root or stem of Littleleaf Indianmulberry strain plant of pGFPGUS-GmSOC1-like, Trizol method extracts total serum IgE, reverse transcription is after cDNA, with this cDNA for template, pcr amplification goal gene GmSOC1-like, with β-tubulin for reference gene, what simultaneously turn goal gene GmSOC1-like in empty vector control strain plant and wild-type Root or stem of Littleleaf Indianmulberry through step 4PCR test positive is expressed as contrast, and partial results as shown in Figure 7.

The primer sequence detecting GmSOC1-like is as follows:

Upstream primer: 5 '-TTCAAAGCGGCGTAATGGGT-3 ' (the 339-358 position corresponding to sequence 2);

Downstream primer: 5 '-CACTCCTGTTATGCCTGCGG-3 ' (the 483-502 position corresponding to sequence 2).

The primer sequence detecting β-tubulin is as follows:

Upstream primer: 5 '-TCTGATACTGTTGTGGAGCCT-3 ';

Downstream primer: 5 '-TGGGATCAGATTCACTGCTAG-3 ';

Product size is 252bp.

Result: the Root or stem of Littleleaf Indianmulberry plant equal stably express gene GmSOC1-like turning pGFPGUS-GmSOC1-like, and turn in empty vector control strain plant and wild-type Root or stem of Littleleaf Indianmulberry and do not express GmSOC1-like.

6, transfer-gen plant is bloomed in advance

Observe positive the bloom situation of Root or stem of Littleleaf Indianmulberry plant under greenhouse long day (h light/8 h dark every day 16) culture condition turning pGFPGUS-GmSOC1-like of 20 strains that step 3 obtains, the 20 strain positives obtained with the step 3 of same seedling age (namely the number of blade is identical) turn empty vector control strain plant and 20 strain wild-type Root or stem of Littleleaf Indianmulberry are contrast.

Result: under same culture conditions, the Root or stem of Littleleaf Indianmulberry plant turning pGFPGUS-GmSOC1-like all blooms, and turn empty vector control strain plant and wild-type Root or stem of Littleleaf Indianmulberry plant during this period always without bud differentiation (Fig. 8) with seedling age, started to bloom after 32 ± 2 days.

The above results shows, Protein G mSOC1-like and encoding gene GmSOC1-lilke thereof has regulating plant and blooms or the function of Characteristics in florescence.

Claims (5)

1. the application of protein in the Characteristics in florescence of regulation and control object plant of the aminoacid sequence composition shown in sequence 1; Described plant is Root or stem of Littleleaf Indianmulberry LotuscorniculatusL. or soybean Glycinemax (L.) Merrill.

2. application according to claim 1, is characterized in that: the encoding gene of described protein is following 1) or 2) gene:

1) its nucleotide sequence is the DNA molecular shown in the 277th to the 912nd in sequence 2;

2) its nucleotide sequence is the DNA molecular shown in sequence 2.

3. the method making object flowering of plant shift to an earlier date, the step that the encoding gene comprising the protein that the aminoacid sequence shown in sequence 1 is formed is expressed in described object plant; Described plant is Root or stem of Littleleaf Indianmulberry LotuscorniculatusL. or soybean Glycinemax (L.) Merrill.

4. cultivation is bloomed a method for transgenic plant in advance, comprises and the encoding gene of the protein of the aminoacid sequence composition shown in sequence 1 is imported object plant, obtain the transgenic plant of expressing said gene; Described plant is Root or stem of Littleleaf Indianmulberry LotuscorniculatusL. or soybean Glycinemax (L.) Merrill.

5. the method according to claim 3 and 4, is characterized in that: the encoding gene of the protein of aminoacid sequence shown in described sequence 1 composition is following 1) or 2) gene:

1) its nucleotide sequence is the DNA molecular shown in the 277th to the 912nd in sequence 2;

2) its nucleotide sequence is the DNA molecular shown in sequence 2.