CN102268080B - Plant blossom related protein GmFTLa, coding gene thereof and application thereof - Google Patents

Abstract

The invention discloses plant blossom related protein GmFTLa, a coding gene thereof and an application thereof. The protein (GmFTLa) provided by the present invention is the protein selected from the following (a) or (b): (a) the protein comprises amino acid sequence represented by SEQ ID NO.1 in a sequence list; (b) the protein comprises the amino acid sequence represented by the SEQ ID NO.1, and is related to the plant blossom, and is derived from the SEQ ID NO.1, wherein the amino acid sequence represented by the SEQ ID NO.1 is processed from substituting and/or deleting and/or adding one or a plurality of amino acid residues. The invention further provides the coding gene of the plant blossom related protein GmFTLa and the application of the plant blossom related protein GmFTLa. The GmFTLa is an enhancing factor for the plant blossom, and has significant correlation with the photoperiodic reaction of the plant, and can change the sensitivity of the photoperiodic reaction of the plant so as to change the flowering stage and maturing stage of the plant. With the present invention, the conditions for cultivating wide-adaptability crop varieties by using the molecular mean are created, and the plant blossom related protein GmFTLa and the coding gene thereof will play important roles in the genetic improvement of the plants.

Description

Flowering of plant associated protein GmFTLa and encoding gene thereof and application

Technical field

The present invention relates to a kind of flowering of plant associated protein GmFTLa and encoding gene 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, the Wide-adaptive soybean varieties of cultivating relative insensitiveness of photoperiod is the important goal of soybean breeder always.Conventional breeding is the main method of soybean varieties improvement, cultivate the soybean varieties that is fit to the different areas plantation by hybridization and strain selection etc., but progress is comparatively slow aspect the improvement of photoperiodic reaction susceptibility, at present, in the urgent need to adopting the molecular breeding means, introducing by foreign gene or to the genetic manipulation of inherent gene, the photoperiodic reaction susceptibility of orientation adjustment soybean varieties is accelerated the seed selection process of Wide-adaptive kind.

In recent years, the research of Arabidopis thaliana and paddy rice isotype flowering of plant approach has been disclosed the mechanism of Photoperiod flowering of plant.Correlative study shows, the molecular pathways that long day plant (as Arabidopis thaliana) and short day plant (as paddy rice) are bloomed is guarded on evolving.Therefore, can clone, screen the key gene of photoperiodic reaction from dicotyledonous short day plant soybean by the bloom conservative property of genes involved of model plant, and analyze its phraseology by changing the photoperiod condition, further verify the function of gene by genetic transformation, and then obtain the different soybean varieties of photoperiodic reaction susceptibility by the genetic manipulation means.

In Arabidopis thaliana, after Photoreceptors receives external optical signals, the optical signal that will receive with the clock gene interaction is delivered to downstream gene FT (FLOWERINGLOCUST) through CO (CONSTANCE), activates the expression of FT, and then the expression of inducing floral meristem characterizing gene AP1.In paddy rice, hd3a is the homologous gene of FT, also has the effect of Accelerate bloom.To studies show that of Arabidopis thaliana and paddy rice, FT and Hd3a are the conformity genes of optical signal, are the key genes that promotes flowering of plant.

Summary of the invention

The purpose of this invention is to provide a kind of flowering of plant associated protein GmFTLa and encoding gene and application.

Protein name provided by the invention is called GmFTLa (Glycine max FLOWER LOCUS T LIKE), derives from pulse family Glycine the cultivated soybean (Glycine max (L.) Merrill), is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in sequence in sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to the flowering of plant protein that is derived by sequence 1.

Protein shown in sequence 1 is comprised of 198 amino-acid residues, is the RKIP structural domain from N-terminal 30-187 position.

In order to make the GmFTLa in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in sequence 1 forms in by sequence table connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the GmFTLa synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of GmFTLa in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in table 1.

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

Described gene can be following 1) or 2) or 3) or 4) DNA molecular:

1) in sequence table sequence 2 from the DNA molecular shown in the 1st to 597 Nucleotide of 5 ' end;

2) DNA molecular shown in sequence 2 in sequence table;

3) under stringent condition with 1) or 2) the bloom DNA molecular of associated protein of the DNA sequence dna hybridization that limits and coded plant;

4) with 1) or 2) or the DNA sequence dna that limits have 90% above homology, and the bloom DNA molecular of associated protein of coded plant.

Above-mentioned stringent condition can be at 6 * SSC, and in the solution of 0.5%SDS, hybridization, then use 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively to wash film once under 65 ℃.

Sequence 1 in sequence table is comprised of 658 Nucleotide, and its open reading frame (ORF) is from the 1st to 597 Nucleotide of 5 ' end.

The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described gene all 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 the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' of foreign gene and hold untranslated regional, namely comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised, but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, as add the coding that to express in plant can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described recombinant expression vector specifically can be p3301m-FTL.The recombinant plasmid that described p3301m-FTL obtains for the multiple clone site of described gene being inserted p3301m.Described p3301m-FTL is preferably the sequence 2 of sequence table is cut from XbalI and SacI enzyme that the DNA fragmentation shown in the 1st to 597 Nucleotide of 5 ' end inserts p3301m the recombinant plasmid that obtains between recognition site.

The preparation method of described p3301m is as follows:

(1) with HindIII and EcoR I double digestion pBI221, reclaim the DNA fragmentation of 3kb;

(2) with HindIII and EcoR I double digestion pCAMBIA3301, reclaim the DNA fragmentation of 11256bp;

(3) DNA fragmentation that step (1) is reclaimed is connected 2 with step) DNA fragmentation that reclaims connects, and obtains p3301m.

The increase total length of described gene or the primer pair of its any fragment also belongs to protection scope of the present invention.

The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in the purpose plant, obtains flowering period early than the transgenic plant of described purpose plant.Described gene specifically can import in described purpose plant by described recombinant expression vector.Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that transforms is cultivated into plant.Purpose plant (plant host that is converted) can be both that monocotyledons can be also dicotyledons.Described dicotyledons can be Arabidopis thaliana, Arabidopis thaliana as environmental in Colombia.

Described gene can be used for the breeding of plant.

Experiment showed, that GmFTLa is the promotion factor of flowering of plant, with the photoperiodic reaction of plant, obvious dependency is arranged, can change the photoperiodic reaction susceptibility of plant, thereby the flowering period of plant, ripening stage are changed.The present invention has created condition for cultivating the Wide-adaptive crop varieties by Molecular tools, will play a significant role in the genetic improvement of plant.

Description of drawings

Fig. 1 is the pcr amplification product electrophoretogram of GmFTLa sequence fragment.

Fig. 2 is the pcr amplification product electrophoretogram of GmFTLa full-length cDNA.

Fig. 3 is the expression pattern of GmFTLa in photoperiod-sensitive kind Zi Gong winter beans leaf.

Fig. 4 is the collection of illustrative plates schematic diagram of recombinant plasmid p3301m-FTL.

Fig. 5 is that the PCR of transgenic arabidopsis plant identifies electrophoretogram.

Fig. 6 is the T of wild-type plant and 2 strains ₃In generation, turn the photo of GmFTLa gene plant.

Fig. 7 is the T of wild-type plant and 2 strains ₃In generation, turn the RT-PCR electrophoretogram of GmFTLa gene plant.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.% in following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges repeated experiments three times, results averaged.Primer in following embodiment synthesizes and examining order is completed by the rich company in Beijing three.

Zi Gong winter beans (photoperiod-sensitive kind): reference: Han Tianfu, Wang Jinling. the research of photoperiodic reaction after soybean blossoming. Botany Gazette, 1995,37:863-869.; Offer the applicant by Sichuan Province's Zigong City Institute of agricultural sciences Yang Huawei; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public; The public also can obtain this plant variety from national Germplasm Resources of Farm Crop storehouse, Zi Gong winter beans be numbered ZDD13278.

The environmental Arabidopis thaliana (Col0) of Colombia: reference: Hsu CY, Liu Y, Luthe DS, Yuceera C.Poplar FT2 Shortens the Juvenile Phase and Promotes Seasonal Flowering.Plant Cell, 2006,18:1846-1861.; Institute of Crop Science, Chinese Academy of Agricultural Science guarantees to provide to the public.

Embodiment 1, the Protein G mFTLa relevant to plant blossom time and the acquisition of encoding gene thereof

Utilize reverse transcription-polymerase chain reaction (RT-PCR) method, obtain the fragment of soybean GmFTLa, the aminoacid sequence of its coding has the part conserved domain of RKIP.Take the reverse transcription cDNA of the total RNA of Zi Gong winter beans as template, utilize RACE technology (Rapid amplification cDNA ends, cDNA end rapid amplifying technology) to obtain 3 ' end and 5 ' end fragment, and clone the full-length cDNA that obtains GmFTLa.

1, the acquisition of GmFTLa sequence fragment and confirmation

In the GenBank database, the amino acid of soybean est sequence (CA936882) coding has the subregion of conservative RKIP, the amino acid of the cone production factor FT genes encoding of Arabidopis thaliana contains conservative RKIP structural domain, and this est sequence may be FT homogenic fragment in soybean.Take est sequence as template design primer M1 with M2 (sequence 9 in sequence table and sequence 10).

Take to the soybean varieties Zi Gong winter beans of photoperiod-sensitive as experiment material, two kinds of photoperiod processing modes of long day (16h illumination/8h is dark) and short day (12h illumination/12h dark) are set.Zi Gong winter beans are not bloomed under long day condition (LD), bloom under short day (SD).Extract total RNA of Zi Gong winter beans blade under SD, and reverse transcription is cDNA, the primer pair take it as template with M1 and M2 composition carries out RT-PCR.

The PCR reaction system:

H ₂O 18.3μl

10 * PCR damping fluid, 2.5 μ l

The cDNA 1.0 μ l of reverse transcription

M1(10.0μM) 0.5μl

M2(10.0μM) 0.5μl

dNTP mix(2.5mM) 2.0μl

Taq enzyme (5U/ μ l) 0.2 μ l

Amount to 25.0 μ l

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

The PCR product is carried out 1% agarose gel electrophoresis.Result as shown in Figure 1, wherein, M is the DNA molecular amount standard of DL2000,1 and 2 is the PCR product of GmFTLa fragment.Result shows: the purpose fragment that obtains 217bp.

Use the multifunctional dna purifying and reclaim test kit glue recovery PCR product, reclaim product and be connected to the pMD18-T carrier, connect at last product and transform DH5 α competent cell.Homology through order-checking gained sequence fragment and est sequence is 90.65%, and the amino acid of coding has part RKIP conserved domain.

2, the amplification of 5 ' RACE

At 5 ' end design nested primers M3 and M4 (sequence 3 in sequence table and sequence 4) of GmFTLa sequence fragment, with RACE test kit (FirstChoice ^TMRLM-RACE Catalog # 1700) the outside primer that provides (5 '-GCTGATGGCGATGAATGAACACTG-3 ') and inner primer (5 '-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3 ') primer that partners respectively carries out pcr amplification.First round PCR product dilutes 500 times and takes turns template for second afterwards.

First round reaction mixture is as follows:

H ₂O 18.3μl

10 * PCR damping fluid, 2.5 μ l

The cDNA 1.0 μ l of reverse transcription

The 5 ' outside primer (10.0 μ M) 0.5 μ l

M3(10.0μM) 0.5μl

dNTP mix(2.5mM) 2.0μl

Taq enzyme (5U/ μ l) 0.2 μ l

Amount to 25.0 μ l

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

Second to take turns the PCR response procedures identical with the first round, and in reaction mixture, primer changes the 5 ' inner primer and M4, all the other components unchanged into.Second takes turns the PCR reaction is adjusted into 62 ℃ with annealing temperature, and the remaining reaction program is identical with the first round.

First round amplified production is the disperse shape through the result of 1.0% agarose gel electrophoresis.Second takes turns amplified production detects through 1.0% agarose gel electrophoresis, and result obtains the band of about 400bp.Reclaim this fragment, be connected on the pMD18-T carrier, check order.Sequencing result shows: this fragment contains the 5 ' terminal sequence of the former EST of the 35bp that has an appointment, also amplifies the approximately new segment of 375bp.

3, the amplification of 3 ' RACE

For obtaining the 3 ' terminal sequence of GmFTLa, at 3 ' end design primer M5 and M6 (sequence 5 in sequence table and sequence 6) of GmFTLa sequence fragment, respectively with 3 ' RACE test kit (FirstChoice ^TMRLM-RACE Catalog#1700) the outside primer that provides (5 '-GCGAGCACAGAATTAATACGACT-3 ') and inner primer (5 '-CGCGGATCCGAATTAATACGACTCACTATAGG-3 ') primer that partners carries out pcr amplification.First round PCR product dilutes 500 times and takes turns template for second afterwards.

First round reaction mixture is as follows:

H ₂O 18.3μl

10 * PCR buffered soln, 2.5 μ l

The cDNA 1.0 μ l of reverse transcription

The 3 ' outside primer (10.0 μ M) 0.5 μ l

M5(10.0μM) 0.5μl

dNTPmix(2.5mM) 2.0μl

Taq enzyme (5U/ μ l) 0.2 μ l

Amount to 25.0 μ l

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

Second to take turns the PCR response procedures identical with the first round, and in reaction mixture, primer changes the 3 ' inner primer and M6, all the other components unchanged into.Second takes turns the PCR reaction is adjusted into 64 ℃ with annealing temperature, and the remaining reaction program is identical with the first round.

First round amplified production is the disperse shape through the result of 1% agarose gel electrophoresis, and second takes turns the band that amplified production is about 209bp.Reclaim this fragment, be connected on the pMD18-T carrier, check order.Sequencing result shows: this fragment contains the 3 ' terminal sequence of the former EST of the 143bp that has an appointment, also amplifies the new segment of 66bp.

4, obtain the total length of GmFTLa gene

For obtaining the total length of GmFTLa gene, the sequence that GmFTLa fragment, 5 ' RACE and 3 ' RACE obtain is spliced, design respectively primer M7 and M8 (sequence 7 in sequence table and sequence 8) at the two ends of 5 ' and the 3 ' sequence of splicing sequence.

Be cDNA with total RNA reverse transcription of the Zi Gong winter beans leaf of growing under the short day condition, with RT-PCR method amplification total length GmFTLa, reaction mixture is as follows:

H ₂O 18.3μl

10 * PCR damping fluid, 2.5 μ l

The cDNA 1.0 μ l of reverse transcription

M7(10.0μM) 0.5μl

M8(10.0μM) 0.5μl

dNTPmix(2.5mM) 2.0μl

Taq enzyme (5U/ μ l) 0.2 μ l

Amount to 25.0 μ l

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

The PCR reaction product is carried out 1.0% agarose gel electrophoresis detection.Result as shown in Figure 2, wherein, M is GeneRuler ^TMThe DNA molecular amount standard of DNA ladder Mix (MBI-SM0331), 1-2 is the pcr amplification result of total length GmFTLa gene cDNA.

Reclaim the PCR product of 658bp, be connected on the pMD18-T carrier, obtain recombinant plasmid pMD18-T, check order.Sequencing result shows: the nucleotide sequence of PCR product is as shown in the sequence 2 of sequence table.Albumen shown in DNA encoding sequence 1 shown in sequence 2.With the GmFTLa of albumen called after shown in sequence 1 albumen, formed by 198 amino-acid residues.Encoding gene name GmFTLa gene with GmFTLa albumen.The cDNA of GmFTLa albumen is comprised of 658 Nucleotide, and as shown in the sequence 2 of sequence table, open reading frame is that sequence 2 is from the 1st to 597 Nucleotide of 5 ' end.

Embodiment 2, the GmFTLa expression in soybean different development stage leaf

Zi Gong winter beans are planted under the condition of 16h illumination/8h dark until single leaf launches, choose the consistent seedling of growth and carry out respectively short day (SD; 12h illumination/12h is dark) and long day (LD; 16h illumination/8h is dark) process.

From carrying out the processing of difference photoperiod, extract the total RNA in Soybean Leaves every day, reverse transcription is cDNA, use Real-time RT-PCR method, take GmACTIN as internal standard gene, detect the relative expression quantity of GmFTLa in soybean different development stage leaf (take the numerical value of the minimum time point of expression amount as 1, the relative expression quantity that remaining numerical value is all compared and drawn with lower-most point).Detect GmFTLa primer pair used and form (sequence 11 of sequence table and sequence 12) by M11 and M12.Detect GmACTIN primer pair used and form (sequence 13 of sequence table and sequence 14) by M13 and M14.

The reaction system of Real-time pcr amplification:

Premix Ex TaqTM(2×) 10.0μl

M11 (or M13) (10.0 μ M) 0.4 μ l

M12 (or M14) (10.0 μ M) 0.4 μ l

ROX Reference Dye(50×) 0.4μl

The cDNA template 2.0 μ l of reverse transcription

H ₂O 6.8μl

Amount to 20.0 μ l.

The reaction conditions of Real-time pcr amplification:

Denaturation:

95℃ 30s

The PCR reaction:

Solubility curve is analyzed: 60 ℃.

The results are shown in Figure 3.Under the short day condition, along with the carrying out of reproductive development, the expression of GmFTLa in leaf shows as the trend that raises gradually, reaches the peak of expression when 25d, by nourishing and growing in the switching process of reproductive development, the expression of GmFTLa has the trend that increases gradually soybean in this explanation.Under the long day condition, the expression pattern of GmFTLa in whole vegetative period do not have to change substantially, low expression level, and expression amount all the time always lower than under short day with the expression of time point.Above result shows: short day is induced the expression of GmFTLa, and its expression amount that carries out of expressing along with reproductive development improves.

Embodiment 3, the acquisition that turns the GmFTLa gene plant and evaluation

One, turn the acquisition of GmFTLa gene plant

1, the structure of recombinant plasmid p3301m-FTL

(1) structure of plasmid p3301m

1. use HindIII and EcoR I double digestion pBI221 (available from CLONTECH company, to reclaim the approximately DNA fragmentation of 3kb;

2. use HindIII and EcoR I double digestion pCAMBIA3301 (Australian CAMBIA), reclaim the DNA fragmentation of 11256bp;

3. use T ₄The DNA fragmentation that 1. DNA ligase reclaims step is connected the DNA fragmentation that reclaims and is connected with step, obtain recombinant plasmid p3301m.

(2) GmFTLa gene cloning

Single leaf is launched the Zi Gong winter beans of phase lower cultivation of short day condition (12h illumination/12h is dark), in samplings in 19 days of short day processing, total RNA and the reverse transcription of then extracting blade are cDNA.Carry out RT-PCR take cDNA as template, obtain PCR product (GmFTLa gene).

RT-PCR primer upstream and downstream introduces respectively XbalI and the SacI enzyme is cut recognition site, and sequence is as follows:

5 '-CGTCTAGAATGAAAGTCATAACAAA-3 ' (sequence 15);

5 '-ATGAGCTCTTAACATAGCCTTCTTC-3 ' (sequence 16).

The PCR reaction system:

H ₂O 18.3μl

10 * PCR damping fluid, 2.5 μ l

Plasmid DNA 1.0 μ l

M11(10.0μM) 0.5μl

M12(10.0μM) 0.5μl

dNTPmix(2.5mM) 2.0μl

Taq enzyme (5U/ μ l) 0.2 μ l

Amount to 25.0 μ l

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

(3) structure of recombinant plasmid p3301m-FTL

1. use the PCR product of restriction enzyme XbalI and SacI double digestion step (2), reclaim enzyme and cut product.

2. use restriction enzyme XbalI and SacI double digestion plasmid p3301m, reclaim carrier framework.

3. step enzyme is 1. cut product and step carrier framework T 2. ₄16 ℃ of connections of DNA ligase are spent the night.

4. will connect product and transform intestinal bacteria (E.coli) DH5 α (available from sky root biochemical technology company limited) by the heat shock method, contain screening positive clone on the LB substratum of 50mg/L kantlex; Extract the plasmid DNA of positive colony, carry out PCR take M11, M12 as primer and identify, PCR identifies the evaluation of checking order of positive plasmid.Sequencing result shows, obtained recombinant plasmid p330lm-FTL (having inserted the sequence 2 of sequence table from the 1st to 597 Nucleotide of 5 ' end between the XbalI of p3301m and SacI restriction enzyme site).The structural representation of recombinant plasmid p3301m-FTL is seen Fig. 4.

2, preparation restructuring Agrobacterium

Transform Agrobacterium Gv3101 (available from sky root biochemical technology company limited) competent cell with recombinant plasmid p3301m-FTL, cultivated two days upper 28 ℃ of LB substratum (containing Rifampin 50mg/L, gentamicin 25mg/L, kantlex 50mg/L).Select positive colony, do the evaluation of bacterium liquid PCR as primer take M15 and M16, all amplify the target stripe that size is about 597bp, namely obtained containing the restructuring Agrobacterium of recombinant plasmid p3301m-FTL.The Agrobacterium of recombinating is inoculated in the LB substratum and (contains Rifampin 50mg/L, gentamicin 25mg/L, kantlex 50mg/L), it is 0.6-0.8 that 28 ℃, 250rpm are cultured to OD600, collect thalline at the centrifugal 5min of 5000rpm, with MS liquid nutrient medium suspension thalline (the OD value of bacterium liquid is about 0.6), be restructuring Agrobacterium suspension.

3, the acquisition of transgenic arabidopsis

Add Silwet-L77 in restructuring Agrobacterium suspension, adopt and be stained with colored dip method arabidopsis thaliana transformation Col0, results T ₁For seed.At 4 ℃ with T ₁For the seed vernalization 3d on substratum that tiles, then directly be planted in Nutrition Soil, after growth 20d, (the careless fourth phosphine of the weedicide with 0.5 ‰; PPT) spray transformed plant.Because pCAMBIA3301 is with the gene of antiweed, during gene transformation, anti-herbicide gene and goal gene will be incorporated in Plant Genome, and the genetically modified plant of success is not with death after spraying herbicide, and genetically modified positive plant can normal growth.

T ₂T is shown in representative ₁The seed that produces for selfing reaches the plant that is grown up to by it, T ₃T is shown in representative ₂The seed that produces for selfing reaches the plant that is grown up to by it.Divide strain results T ₂For seed.Then the T that obtains isozygotying through identical screening process ₃For the Arabidopis thaliana seed.With T ₃The seed in generation directly is seeded in compost, and the plant that obtains is T ₃In generation, turn the GmFTLa plant.

Two, turn the acquisition of empty carrier control plant

Transform Agrobacterium Gv3101 with plasmid p3301m, obtain the Agrobacterium of recombinating, with restructuring Agrobacterium-mediated Transformation Arabidopis thaliana Col0, obtain turning the empty carrier adjoining tree, the same step 1 of method.

Three, the PCR that turns the GmFTLa gene plant identifies

Extract respectively T ₃In generation, turn GmFTLa gene plant and T ₃In generation, turn the genomic dna of empty carrier adjoining tree.Take genomic dna as template, carry out PCR with primer M17 and M18 (sequence 17 in sequence table and sequence 18).Take p3301m-FTL as positive control, water is as blank.

The PCR reaction conditions:

94℃，5min

72℃，10min

4 ℃, preserve.

Result as shown in Figure 5.In Fig. 5, M:Marker; 1: water; 2,3: transfer-gen plant; 4: turn the empty carrier adjoining tree; 5: positive control.Positive control and T ₃In generation, turn the goal gene band that the GmFTLa gene plant all shows 317bp, turns not occur this goal gene band in the empty carrier adjoining tree.

Four, turn the phenotypic evaluation of GmFTLa gene plant

T with 10 strains ₃In generation, turn the T of GmFTLa gene plant, 10 strains ₃The seed (10 seeds of each strain) that generation turns empty carrier adjoining tree and wild-type plant (Arabidopis thaliana Col0) directly is seeded in compost, makes its sprouting; Then cultivate under the same conditions (16h illumination/8h is dark).

Sprout after 21 days the T of wild-type plant and 2 strains ₃In generation, turns the photo of GmFTLa gene plant and sees Fig. 6.In Fig. 6, Col0: wild-type plant; The T of 1-2:2 strain ₃In generation, turn the GmFTLa gene plant.Compare with the wild-type plant, obviously shift to an earlier date the flowering period that turns the GmFTLa gene plant.The phenotype that turns the GmFTLa gene plant of other strain turns the phenotype of empty carrier adjoining tree with the wild-type plant with two strains in figure.

Sprout after 21 days, extract respectively the T of wild-type plant, 2 strains ₃In generation, turn GmFTLa gene plant, T ₃In generation, turn total RNA of empty carrier adjoining tree, and reverse transcription is cDNA, take cDNA as template, carries out RT-PCR with primer 17 and primer 18.Take the TUBLIN gene of Arabidopis thaliana as internal standard gene (primer pair that primer 19 and primer 20 form).The results are shown in Figure 7.In Fig. 7, Col0: wild-type plant; The T of 1-2:2 strain ₃In generation, turn the GmFTLa gene plant.There is no GmFTLa genetic expression in the wild-type plant, and turn the GmFTLa gene plant in, GmFTLa gene strongly expressed.

Recording seed germination is flowering period (in the sky) to the time that bud occurs for the first time, the basal leaf quantity (in sheet) after flowering period and stem leaf quantity (in sheet).The results are shown in Table 2.

The statistics of the flowering time of the Arabidopis thaliana of each strain of table 2, basal leaf and stem leaf

As can be seen from Table 2, genetically modified plant blossom time average is 22.0d, than wild-type with turn p3301m plasmid the adjoining tree respectively about 7.3d of Blooming and 7.5d; The basal leaf of transfer-gen plant and the quantity of stem leaf are 9.9, bloom when lacking respectively 2 and 1.6 than wild-type and the adjoining tree that turns the p3301m plasmid, illustrate that GmFTLa crosses expression and promotes its prematurity in Arabidopis thaliana, may have the function similar to FT: GmFTLa is the promotion factor of soybean blossoming.

Sequence table

＜110〉Institute of Crop Science, Chinese Academy of Agricultural Science

＜120〉the relevant egg GmFTLa of flowering of plant and encoding gene and application

<130>CGGNARY 102356

<160>20

<210>1

<211>198

<212>PRT

<213>

<400>1

Met Lys Val Ile Thr Lys His Lys Lys Thr Asn Cys Lys Val Val Ser

1 5 10 15

Thr Val Ser Leu Leu Ile Met Pro Arg Gly Ser Arg Asp Pro Leu Val

20 25 30

Val Gly Arg Val Ile Gly Asp Val Leu Asp Pro Phe Glu Cys Ser Ile

35 40 45

Pro Met Arg Val Thr Tyr Asn Asn Lys Asp Val Ser Asn Gly Cys Glu

50 55 60

Phe Lys Pro Ser Gln Val Val Asn Gln Pro Arg Ile Asn Ile Gly Gly

65 70 75 80

Asp Asp Phe Arg Asn Phe Tyr Thr Leu Ile Ala Val Asp Pro Asp Ala

85 90 95

Pro Ser Pro Ser Asp Pro Asn Phe Arg Glu Tyr Leu His Trp Leu Val

100 105 110

Thr Asp Ile Pro Ala Thr Thr Gly Pro Thr Phe Gly His Glu Val Val

115 120 125

Thr Tyr Glu Asn Pro Arg Pro Met Met Gly Ile His Arg Ile Val Phe

130 135 140

Val Leu Phe Arg Gln Gln Gly Arg Glu Thr Val Tyr Ala Pro Gly Trp

145 150 155 160

Arg Gln Asn Phe Ile Thr Arg Glu Phe Ala Glu Leu Tyr Asn Leu Gly

165 170 175

Leu Pro Val Ala Ala Val Tyr Phe Asn Ile Gln Arg Glu Ser Gly Cys

180 185 190

Gly Gly Arg Arg Leu Cys

195

<210>2

<211>658

<212>DNA

<213>2

<400>2

atgaaagtca taacaaagca taagaaaacc aattgtaaag tagtttccac tgtctcctta 60

ttgatcatgc ctcgtggaag tagggaccct ctagttgttg ggcgtgtgat tggggatgta 120

ttggaccctt ttgaatgttc tattcctatg agggtcacct acaataacaa agatgtcagc 180

aatggatgtg aattcaaacc ctcacaagtt gtcaaccaac caagaataaa tatcggtggt 240

gatgatttca ggaacttcta cactttgatc gcggttgatc ctgatgcacc tagcccaagt 300

gatcccaatt tcagagaata cctccattgg ttagtaactg acattccagc aacaacgggg 360

cctactttcg gtcatgaggt tgtaacatat gaaaatccac gacccatgat ggggatccat 420

cgtatagtct ttgtgttatt tcgtcaacag ggtagagaga cagtgtatgc accaggatgg 480

cgccaaaatt tcattactag agaatttgct gaactttaca atcttggatt gccagttgct 540

gctgtctatt ttaacatcca gagagaatct ggttgtggtg gaagaaggct atgttaataa 600

taaggtttaa ataaattttt gatctttata aaataaggga cttttgaatg tgttttta 658

<210>3

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223><400>3

tgttatttcg tcaacagggt agag 24

<210>4

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

atatgaaaat ccacgaccca tgat 24

<210>5

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>5

tctctctacc ctgttgacga aataa 25

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>6

tggtgcatac actgtctctc taccc 25

<210>7

<211>26

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>7

atgaaagtca taacaaagca taagaa 26

<210>8

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>8

taaaaacaca ttcaaaagtc cctta 25

<210>9

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>9

gaggttgtaa catatgaaaa tccac 25

<210>10

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>10

atagccttct tccaccacaa cc 22

<210>11

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>11

tgaaagtcat aacaaagcat aagaa 25

<210>12

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>12

ggtgaccctc ataggaatag aac 23

<210>13

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>13

gagcaagaac tcgagactgc aa 22

<210>14

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>14

ttccagcagc ttccatttca 20

<210>15

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>15

cgtctagaat gaaagtcata acaaa 25

<210>16

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>16

atgagctctt aacatagcct tcttc 25

<210>17

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>17

aacaaagcat aagaaaacca attgt 25

<210>18

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>18

aatggaggta ttctctgaaa ttg 23

<210>19

<211>22

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>19

ctgtctccaa gggttccagg tt 22

<210>20

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>20

gaagaagtga agacggggga a 21

Claims (10)

1. protein, the protein that is formed by the aminoacid sequence shown in sequence in sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene as claimed in claim 2 is characterized in that: it is following 1) or 2) DNA molecular:

1) in sequence table sequence 2 from the DNA molecular shown in the 1st to 597 Nucleotide of 5 ' end;

2) DNA molecular shown in sequence 2 in sequence table.

4. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described genes.

5. recombinant expression vector as claimed in claim 4 is characterized in that:

Described recombinant expression vector is p3301m-FTL; The recombinant plasmid that described p3301m-FTL obtains for the multiple clone site of the described gene of claim 2 or 3 being inserted p3301m; The preparation method of described p3301m is as follows:

(1) with HindIII and EcoR I double digestion pBI221, reclaim the DNA fragmentation of 3kb;

(2) with HindIII and EcoR I double digestion pCAMBIA3301, reclaim the DNA fragmentation of 11256bp;

(3) DNA fragmentation that step (1) is reclaimed is connected 2 with step) DNA fragmentation that reclaims connects, and obtains p3301m.

6. a method of cultivating transgenic plant, be that the described gene of claim 2 or 3 is imported in the purpose plant, obtains flowering period early than the transgenic plant of described purpose plant.

7. method as claimed in claim 6 is characterized in that: the described gene of claim 2 or 3 imports in described purpose plant by the described recombinant expression vector of claim 4 or 5.

8. method as described in claim 6 or 7, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.

9. method as claimed in claim 8, it is characterized in that: described dicotyledons is Arabidopis thaliana.

10. the application of the described gene of claim 2 or 3 in plant breeding.

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