CN110028563A

CN110028563A - Application of the protein OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds

Info

Publication number: CN110028563A
Application number: CN201810025524.4A
Authority: CN
Inventors: 孟征; 吴凤; 王忠妮; 林学磊
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2018-01-11
Filing date: 2018-01-11
Publication date: 2019-07-19

Abstract

The invention discloses application of the protein OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds.Protein OsMADS27 is that amino acid sequence is protein shown in sequence 2 in sequence table.The sprout time of regulation vegetable seeds is the sprouting for promoting vegetable seeds or the sprouting of delay plant seed.The suspend mode of the regulation vegetable seeds is the suspend mode for releasing vegetable seeds or the suspend mode for extending vegetable seeds.It is demonstrated experimentally that importing the encoding gene of protein OsMADS27 into rice osmads27-1 mutant, obtains seed and sprout in advance and/or the genetically modified plants of the termination of diapause of seed.Therefore, protein OsMADS27 has important theory significance and practical value in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds.

Description

Sprout time and regulation vegetable seeds of the protein OsMADS27 in regulation vegetable seeds Suspend mode in application

Technical field

The present invention relates to field of biotechnology, and in particular to sprout time of the protein OsMADS27 in regulation vegetable seeds The application in suspend mode with regulation vegetable seeds.

Background technique

Seed dormancy is the important biomolecule characteristic that plant adapts to changeable natural environment.For crops, due to Suspend mode can directly affect its yield, seed quality and agricultural production and application, therefore by hereditary and breeding scholar wide General concern.The dormancy mechanism for furtheing investigate rice paddy seed has extremely important practical meaning for improving grain yield and quality Justice.For rice as important one of crops, the sprouting of seed is to guarantee crop normal growth and then guarantee the first of yield A is also a critically important link.So far, it still knows little about it to the understanding of such as rice paddy seed dormancy mechanism.

Summary of the invention

The technical problem to be solved by the present invention is to how promote the sprouting of vegetable seeds and/or release stopping for vegetable seeds It sleeps.

In order to solve the above technical problems, present invention firstly provides protein OsMADS27 in the sprouting for regulating and controlling vegetable seeds Application in the suspend mode of time and/or regulation vegetable seeds.The Chinese of protein OsMADS27 turns for rice MADS-box Record the factor 27.

In above-mentioned application, the protein OsMADS27 can be a1) or a2) or a3):

A1) amino acid sequence is protein shown in sequence 2 in sequence table；

A2) the fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；

A3) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or Obtained protein relevant to the suspend mode of the sprout time of vegetable seeds and/or vegetable seeds is deleted and/or added.

Wherein, sequence 2 is made of 240 amino acid residues in sequence table.

In order to make a1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of 1 label of table

Above-mentioned a3) in protein, the substitution and/or deletion and/or addition of one or several amino acid residues be No more than the substitution and/or deletion and/or addition of 10 amino acid residues.

Above-mentioned a3) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.

Above-mentioned a3) in the encoding gene of protein can be by the way that one will be lacked in DNA sequence dna shown in sequence 1 in sequence table The codon of a or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.

Sprout time and/or regulation plant of the nucleic acid molecules of code for said proteins OsMADS27 in regulation vegetable seeds Application in the suspend mode of seed also belongs to protection scope of the present invention.

In above-mentioned application, the nucleic acid molecules of code for said proteins OsMADS27 can be following b1) or b2) or b3) or b4) Shown in DNA molecular:

B1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；

B2) code area DNA molecular as shown in sequence 1 in sequence table；

B3) and b1) or (b2) nucleotide sequence for limiting has 75% or 75% or more identity, and encoding said proteins The DNA molecular of matter OsMADS27；

B4) the nucleotide sequence hybridization limited under strict conditions with (b1) or (b2), and code for said proteins The DNA molecular of OsMADS27.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.

Sequence 1 is made of 723 nucleotide in sequence table, sequence 2 in the nucleotide coding sequence table of sequence 1 in sequence table Shown in amino acid sequence.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of protein OsMADS27 of the invention.Those have and this hair by manually modified The nucleotide sequence 75% of bright isolated protein OsMADS27 or the nucleotide of higher identity, as long as coding albumen Matter OsMADS27 and have the function of protein OsMADS27, is derived from nucleotide sequence of the invention and to be equal to this hair Bright sequence.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair The nucleotide sequence of the protein OsMADS27 of the composition of amino acid sequence shown in the sequence 2 of bright polynucleotide has 75% Or higher or 80% or higher or 85% or higher or 90% or higher or 95% or higher identity nucleotide sequence. Identity can with the naked eye or computer software is evaluated.It is same between two or more sequences using computer software Property can be indicated with percentage (%), can be used to evaluate identity between correlated series.

In any of the above-described application, the sprout time of the regulation vegetable seeds can be the sprouting of promotion vegetable seeds Or the sprouting of delay plant seed.

In any of the above-described application, the suspend mode of the regulation vegetable seeds can be to release the suspend mode of vegetable seeds or prolong The suspend mode of long vegetable seeds.

In any of the above-described application, the plant can be following c1) any one of to c6):

C1) dicotyledon；

C2) monocotyledon；

C3) gramineae plant；

C4) rice；

C5) rice osmads27-1 mutant；

C6) rice osmads27-3 mutant.

In above-mentioned application, rice osmads27-1 mutant and rice osmads27-3 mutant concretely U.S. UCD (network address is mutant libraryhttp://sundarlab.ucdavis.edu/rice/seedorder/instructions.htm) Product, number be respectively RGT1942_5.1 and RGT395_5.1.Osmads27-1 mutant and osmads27-3 mutant Background is rice varieties OryzasativaLcv.Nipponbare.

In order to solve the above technical problems, the present invention also provides the methods for cultivating genetically modified plants.

The method provided by the present invention for cultivating genetically modified plants concretely cultivates the method one of genetically modified plants, packet The step of including and import the nucleic acid molecules of code for said proteins OsMADS27 in the plant that sets out, obtaining genetically modified plants；It is described Genetically modified plants seed compared with the plant that sets out is sprouted in advance and/or the termination of diapause of seed.

In the method one of above-mentioned cultivation genetically modified plants, the nucleic acid molecules of code for said proteins OsMADS27 can be as follows B1) or b2) or b3) or b4) shown in DNA molecular:

B2) code area DNA molecular as shown in sequence 1 in sequence table；

Described " importing the nucleic acid molecules of code for said proteins OsMADS27 in the plant that sets out " can be by planting to setting out Recombinant vector is imported in object to realize；The recombinant vector can divide to be inserted into the nucleic acid of code for said proteins OsMADS27 to carrier The recombinant plasmid that son obtains.The recombinant vector concretely recombinant plasmid pCAMBIA23a-OsMADS27.The recombinant plasmid The building process of pCAMBIA23a-OsMADS27 is as follows: (1) using restriction enzyme BamHI and KpnI double digestion plasmid OSJNBa0079M09 recycles the segment of about 12kb；(2) restriction enzyme BamHI and KpnI double digestion carrier are used PCAMBIA23a recycles carrier framework；(3) segment is connected with carrier framework, obtains recombinant plasmid pCAMBIA23a- OsMADS27.OsMADS27 albumen shown in sequence 2 in the recombinant plasmid pCAMBIA23a-OsMADS27 expressed sequence table.

The method provided by the present invention for cultivating genetically modified plants concretely cultivates the method two of genetically modified plants, packet The step of including the expression and/or activity for inhibiting the protein OsMADS27 in the plant that sets out, obtaining genetically modified plants；It is described Sprout compared with the plant that sets out and/or the suspend mode of seed extends by seed delay for genetically modified plants.

It is described " to inhibit the protein OsMADS27 in the plant that sets out in the method two of above-mentioned cultivation genetically modified plants Expression and/or activity " can pass through T-DNA insertion, RNA interference, homologous recombination, it is gene site-directed editor etc. side well known in the art Method reaches the expression and/or active purpose for inhibiting the protein OsMADS27 in the plant that sets out.

In order to solve the above technical problems, the present invention also provides plant breeding methods.

Plant breeding method provided by the present invention, concretely plant breeding method one, includes the following steps: to increase and plant The content or activity of protein OsMADS27 described in object, so that seed is sprouted in advance and/or the termination of diapause of seed.

In above-mentioned plant breeding method one, " content or activity that increase protein OsMADS27 described in plant " can By multicopy, change the methods well known in the art such as promoter, regulatory factor, transgenosis, reaches and increase egg described in plant The content of white matter OsMADS27 or active effect.

Plant breeding method provided by the present invention, concretely plant breeding method two, include the following steps: to reduce and plant The content or activity of protein OsMADS27 described in object, so that seed delay is sprouted and/or the suspend mode of seed extends.

In above-mentioned plant breeding method two, " content or activity that reduce protein OsMADS27 described in plant " can By methods well known in the art such as RNA interference, homologous recombination, gene site-directed editors, reaching reduces protein described in plant The content of OsMADS27 or active purpose.

In any of the above-described method, the plant can be following c1) any one of to c6):

C1) dicotyledon；

C2) monocotyledon；

C3) gramineae plant；

C4) rice；

C5) rice osmads27-1 mutant；

C6) rice osmads27-3 mutant.

In the above method, rice osmads27-1 mutant and rice osmads27-3 mutant concretely U.S. UCD (network address is mutant libraryhttp://sundarlab.ucdavis.edu/rice/seedorder/instructions.htm) Product, number be respectively RGT1942_5.1 and RGT395_5.1.Osmads27-1 mutant and osmads27-3 mutant Background is rice varieties OryzasativaLcv.Nipponbare.

Above, the genetically modified plants be interpreted as not only comprising the nucleic acid molecules transformation receptor plant is obtained the Generation genetically modified plants also include its filial generation.For genetically modified plants, the nucleic acid molecules can be bred in the species, The nucleic acid molecules can be transferred into other kinds of same species with traditional breeding techniques, particularly including in commercial variety. The genetically modified plants include seed, callus, intact plant and cell.

It is demonstrated experimentally that importing protein into rice osmads27-1 mutant or rice osmads27-3 mutant The encoding gene of OsMADS27 obtains seed and sprouts in advance and/or the genetically modified plants of the termination of diapause of seed.Therefore, albumen Matter OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds with important theory significance and Practical value.

Detailed description of the invention

Fig. 1 is the Molecular Identification of osmads27-1 mutant and osmads27-3 mutant.

Fig. 2 is the Phenotypic Observation of osmads27-1 mutant and osmads27-3 mutant.

Fig. 3 is expression pattern analysis.

Fig. 4 is situ Analysis.

Fig. 5 is the correlation that OsMADS27 gene and osmads27-1 mutation type surface are verified using complementation test.

Fig. 6 is the experimental result of embodiment 4.

Fig. 7 is the experimental result of embodiment 5.

Fig. 8 is the experimental result of embodiment 6.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

Osmads27-1 mutant (number RGT1942_5.1) and osmads27-3 mutant (number RGT395_ 5.1) being purchased from U.S.'s UCD mutant library, (network address ishttp://sundarlab.ucdavis.edu/rice/seedorder/ instructions.htm).The background of osmads27-1 mutant and osmads27-3 mutant is rice varieties OryzasativaLcv.Nipponbare. Osmads27-1 mutant hereinafter referred to as 27-1.Osmads27-3 mutant hereinafter referred to as 27-3.

Rice varieties OryzasativaLcv.Nipponbare is purchased from the Chinese Academy of Agricultural Sciences's crop science research institute.Rice varieties OryzasativaLcv.Nipponbare is hereinafter named For wild rice or WT.

Trizol RNA extracts reagent and reverse transcription reagent box is Promega Products.In situ hybridization reagent is Roche Products.Agrobacterium tumefaciems EHA105 is the product of TIANGEN Biotech (Beijing) Co., Ltd..Carrier PCAMBIA23a is the product of Shanghai North Connaught biological technology CO., LTD..Plasmid OSJNBa0079M09 is country, the Chinese Academy of Sciences The product of cara gene, the nucleotide sequence containing OsMADS27 gene.

Illumination alternate culture, that is, light culture and dark culture alternating, condition are as follows: 28 DEG C；Illumination cultivation/10 hour are black within 14 hours Dark culture；Intensity of illumination when illumination cultivation is 90 μ E/m²/s。

YEB fluid nutrient medium: by beef extract 5g, yeast extract 1g, peptone 5g, sucrose 5g, MgSO₄·7H₂O 0.04g It is dissolved in 1L deionized water, adjusts pH to 7.2, autoclave sterilization 20min with 10M NaOH aqueous solution.

Induced medium: by a large amount of mother liquor 20mL of 50 × N6, the micro mother liquor 10mL of 100 × B5,200 × MS mother liquid of iron salt The organic mother liquor 1mL of 5mL, 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline 2.8g, 2,4-D 2.0mg, Sucrose 30.0g and plant gel 4.5g is dissolved in 1L distilled water, adjusts pH to 5.8,121 DEG C of sterilizing 15min.

Co-culture medium: by a large amount of mother liquor 20mL of 50 × N6, the micro mother liquor 10mL of 100 × B5,200 × MS mother liquid of iron salt The organic mother liquor 1mL of 5mL, 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline 2.8g, 2,4-D 2.0mg, Sucrose 30.0g, plant gel 4.5g, carbenicillin 100mg and cephalosporin 400mg are dissolved in 1L distilled water, adjust pH to 5.2,121 DEG C of sterilizing 15min, glucose 10g, the carboxylic benzyl being added when being cooled to 50~60 DEG C Jing Guo 0.22 μM of filtration sterilization are green Mycin 100mg and cephalosporin 400mg and acetosyringone, final concentration of 100 μM in system of acetosyringone.

Primary screening culture medium: a large amount of mother liquor 20mL of 50 × N6, the micro mother liquor 10mL of 100 × B5,200 × MS molysite is female The organic mother liquor 1mL of liquid 5mL, 1000 × B5, caseinhydrolysate 300mg, glutamine 500mg, proline 2.8g, 2,4-D 2.0mg, sucrose 30.0g, plant gel 4.5g are dissolved in 1L distilled water, adjust pH to 5.8, and 121 DEG C of sterilizing 15min are cooled to Hygromycin 30mg, carbenicillin 100mg and the cephalosporin 400mg Jing Guo 0.22 μM of filtration sterilization are added at 50~60 DEG C.

Postsearch screening culture medium: other except the hygromycin 30mg in primary screening culture medium is replaced with hygromycin 50mg Ingredient and content are constant.

Differential medium: by a large amount of mother liquor 50mL of 20 × MS, the micro mother liquor 5mL of 200 × MS, 200 × MS mother liquid of iron salt The organic mother liquor 5mL of 5mL, 200 × MS, caseinhydrolysate 1000mg, glutamine 500mg, sucrose 30.0g, sorbierite 20g, NAA 0.4mg, 6-BA 2.0mg, KT 1mg and plant gel 4.5g are dissolved in 1L distilled water, adjust pH to 5.8,121 DEG C of sterilizing 15min.

Root media: by a large amount of mother liquor 25mL of 20 × MS, the micro mother liquor 2.5mL of 200 × MS, 200 × MS mother liquid of iron salt The organic mother liquor 2.5mL of 2.5mL, 200 × MS, sucrose 15g and plant gel 4.5g are dissolved in 1L distilled water, adjust pH to 5.8,121 DEG C sterilizing 15min, is added the hygromycin 30mg Jing Guo 0.22 μM of filtration sterilization when being cooled to 50~60 DEG C.

1/2MS culture solution: by a large amount of mother liquor 25mL of 20 × MS, the micro mother liquor 2.5mL of 200 × MS, 200 × MS mother liquid of iron salt The organic mother liquor 2.5mL of 2.5mL, 200 × MS is dissolved in 1L distilled water, adjusts pH to 5.8.

The solute and its concentration of a large amount of mother liquors of 50 × N6 are as follows: 141.50g/L KNO₃、20g/L KH₂PO₄、23.15g/L (NH₄)₂SO₄、9.25g/L MgSO₄·7H₂O、8.30g/L CaCl₂·2H₂O, solvent are water, and pH is natural.

The solute and its concentration of the micro mother liquor of 100 × B5 are as follows: 0.3g/L H₃BO₃、1g/L MnSO₄·4H₂O、0.0025g/ L CoCl₂·6H₂O、0.0025g/L CuSO₄·5H₂O、0.2g/L ZnSO₄·7H₂O、0.025g/L Na₂MoO₄·2H₂O、 0.075g/L KI, solvent are water, and pH is natural.

The solute and its concentration of the organic mother liquor of 1000 × B5 are as follows: 2g/L glycine, 100g/L inositol, 1g/L niacin, 1g/L Puridoxine hydrochloride, 10g/L thiamine hydrochloride, solvent are water, and pH is natural.

The solute and its concentration of a large amount of mother liquors of 20 × MS are as follows: 38.00g/L KNO₃, 8.80g/L CaCl₂·2H₂O, 7.40g/L MgSO₄·7H₂O, 3.40g/L KH₂PO₄, 33.00g/L NH₄NO₃, solvent is water, and pH is natural.

The solute and its concentration of the micro mother liquor of 200 × MS are as follows: 4.46g/L MnSO₄·4H₂O, 0.166g/L KI, 1.24g/ L H₃BO₃, 1.72g/L ZnSO₄·7H₂O, 0.050g/L Na₂MoO₄·2H₂O, 0.005g/L CuSO₄·5H₂O, 0.005g/L CoCl₂·6H₂O, solvent are water, and pH is natural.

The solute and its concentration of 200 × MS mother liquid of iron salt are as follows: 5.56g/L FeSO₄·7H₂O, 7.46g/L Na₂· EDTA·2H₂O, solvent are water, and pH is natural.

The solute and its concentration of the organic mother liquor of 200 × MS are as follows: 20g/L inositol, 100mg/L niacin, 100mg/L hydrochloric acid pyrrole are trembled Alcohol, 100mg/L thiamine hydrochloride, 400mg/L glycine, solvent are water, and pH is natural.

Germination rate=sprouting seed number/seed sum × 100%.

Embodiment 1, the identification of osmads27-1 mutant and osmads27-3 mutant and Phenotypic Observation

One, the identification of osmads27-1 mutant and osmads27-3 mutant

1, OsMADS27 full length gene 7152bp includes 8 exons and 7 intrones (A, white rectangle representatives in Fig. 1 UTR, blue rectangle represent exon, and lines represent introne, and triangle represents Ds insertion).

The full-length genome of osmads27-1 mutant and osmads27-3 mutant is sequenced.

The sequencing result of osmads27-1 mutant are as follows: the insertion position T-DNA of osmads27-1 mutant is at second Introne；In addition to the T-DNA of insertion, the nucleotide sequence and rice product of the other positions of the genome of osmads27-1 mutant Plant the completely the same of OryzasativaLcv.Nipponbare.

The sequencing result of osmads27-3 mutant are as follows: the insertion position T-DNA of osmads27-3 mutant is in 5 ' UTR； In addition to the T-DNA of insertion, nucleotide sequence and the rice varieties Japan of the other positions of the genome of osmads27-3 mutant Fine is completely the same.

2, the genomic DNA for extracting the blade of 27-1 or 27-3, utilizes " three-primer method " PCR amplification to the gene of mutant Type is identified that (primer is respectively as follows: BP (the Ds vector borders sequence provided on U.S. UCD mutant library website), LP and RP (LP With the distinguished sequence that RP is near insertion position)).

The result shows that 27-1 and 27-3 are Mutants homozygous.

3, rice (the osmads27-1 mutant, osmads27- that reagent extracts sprouting 5-7d are extracted using Trizol RNA 3 mutant or wild rice) plant the tender tip of a root of children total serum IgE, which is gone out first Chain cDNA.

Using above-mentioned cDNA as template, RT-PCR detect OsMADS27 gene relative expression quantity (using ACTIN1 gene as Reference gene).Identify the primer of OsMADS27 gene are as follows: 5 '-AAGCACCTAGGCACTGGTTT-3 ' and 5 '- TTTCCATGGATGCTAGTGCC-3′.Identify ACTIN1 gene primer are as follows: 5 '-CCAATCGTGAGAAGATGACCCA-3 ' and 5′-CCATCAGGAAGCTCGTAGCTCT-3′。

Using above-mentioned cDNA as template, qRT-PCR detect OsMADS27 gene relative expression quantity (using ACTIN1 gene as Reference gene).Identify the primer of OsMADS27 gene are as follows: 5 '-GCCAAGCATGCACTTAATCA-3 ' and 5 '- TCAACAAGCAACAAGCAAGC-3′.Identify ACTIN1 gene primer are as follows: 5 '-CCAATCGTGAGAAGATGACCCA-3 ' and 5′-CCATCAGGAAGCTCGTAGCTCT-3′。

RT-PCR experimental result is shown in C in Fig. 1 (WT is wild rice), qRT-PCR experimental result is shown in D (WT in Fig. 1 For wild rice).The result shows that the expression of OsMADS27 gene, osmads27-1 can be detected in wild rice plant The expression of OsMADS27 gene cannot be detected in the plant of mutant and the plant of osmads27-3 mutant.As it can be seen that OsMADS27 gene is knocked out completely in osmads27-1 mutant and osmads27-3 mutant.

Two, the Phenotypic Observation of osmads27-1 mutant and osmads27-3 mutant

The seed for taking osmads27-1 mutant, osmads27-3 mutant or wild rice is placed in equipped with appropriate 1/ In the culture dish of 2MS culture solution, 28 DEG C of culture (i.e. water suction culture) 120h.Observe the sprouting feelings of the seed of different absorbent times Condition, and count germination rate (experiment is averaged in triplicate, repeats 50 seeds every time).

The sprouting situation of seed is shown in that (WT is wild rice to A in Fig. 2, and 0h is absorbent time 0h, and 36h is absorbent time 36h, 60h are absorbent time 60h, and 72h is absorbent time 72h, and 84h is absorbent time 84h).The result shows that with wild rice Seed is compared, and the sprout time of 27-1 seed and 27-3 seed postpones 12~for 24 hours.

The germination rate of seed is shown in B in Fig. 2 when different absorbent times (WT is wild rice).The result shows that absorbent time When for 72h, the germination rate of wild rice seed about 70%, and the germination rate of 27-1 seed and 27-3 seed is only 30%； When absorbent time is 120h, the germination rate of wild rice seed, 27-1 seed and 27-3 seed reaches 100%.Later Growth, 27-1 plant, 27-3 plant and wild rice plant plant height, in terms of without significant difference, it is seen that OsMADS27 gene specific functions in the seed sprouting stage.The expression quantity and table of OsMADS27 gene in 27-1 and 27-3 Type is almost the same.

The expression pattern analysis of embodiment 2, OsMADS27 gene

One, the relative expression quantity of OsMADS27 gene in different tissues is detected

It carries out repeating to test three times, it is as follows to repeat the step of testing every time:

1, sample is obtained

The root for sprouting the wild rice plant of 5-7d is taken, Liquid nitrogen storage is put into, obtains sample 1.

The blade for taking the wild rice plant in Seedling Stage, is put into Liquid nitrogen storage, obtains sample 2.

The fringe for taking the wild rice plant in heading stage, is put into Liquid nitrogen storage, obtains sample 3.

The embryo for taking the wild rice seed of fertilization the 22nd day, is put into Liquid nitrogen storage, obtains sample 4.

The embryo for taking wild rice dry seeds, is put into Liquid nitrogen storage, obtains sample 5.

The embryo for taking wild rice dry seeds is then placed in Liquid nitrogen storage with distilled water immersion 12h, obtains sample 6.

2, the expression pattern analysis of OsMADS27 gene

Using Trizol RNA extract reagent extraction step 1 in sample (sample 1, sample 2, sample 3, sample 4, sample 5 or Sample 6) total serum IgE, which is gone out into the first chain cDNA.

Using the cDNA as template, RT-PCR detects the relative expression quantity of OsMADS27 gene in sample (with ACTIN1 gene As reference gene).Identify the primer of OsMADS27 gene are as follows: 5 '-GCTTGAGACAACAACTGCAC-3 ' and 5 '- TTCAGCATACAAGGGTGTAG-3′.Identify ACTIN1 gene primer are as follows: 5 '-CCAATCGTGAGAAGATGACCCA-3 ' and 5′-CCATCAGGAAGCTCGTAGCTCT-3′。

According to the method described above, cDNA is replaced with into deionized water, other steps are constant, as negative control.

Experimental result is shown in Fig. 3, (NC is negative control, and G is sample 6, and R is sample 1, and L is sample 2, and P is sample 3, and E22 is Sample 4, DS are sample 5).The result shows that relative expression quantity highest of the OsMADS27 gene in the root of rice, in fertilization the 22nd Relative expression quantity in it embryo takes second place, and expression quantity is lower in the embryo of blade and the dry seeds through distilled water immersion 12h, in fringe It is not expressed in the embryo of dry seeds.

Two, expression of the situ Analysis OsMADS27 gene in root and embryo

The different parts of the root of wild rice plant are taken using in situ hybridization reagent respectively and in different developmental phases Embryo carry out in situ hybridization, to determine that OsMADS27 gene embodies position and period in root and embryo.

Experimental result is shown in Fig. 4 (wherein F is Sense probes as control).The result shows that OsMADS27 gene is in the tip of a root point The expression quantity highest (A in Fig. 4) in raw area；Also having a degree of expression in epidermis, cortex and vascular cylinder, (B, ep are table in Fig. 4 Skin, v are vascular cylinder)；In elongation zone, the expression of base portion is concentrated mainly on exodermis (C, ed are exodermis in Fig. 4)；Close at The elongation zone in ripe area can't detect the expression (D in Fig. 4) of OsMADS27 gene；Embryo formation stage (fertilization the 3rd day), OsMADS27 Gene does not express (E in Fig. 4) in the embryo of seed；Embryonic development period (fertilization the 8th day), OsMADS27 gene is in the embryo of seed It expresses (G in Fig. 4)；The embryo maturation phase (fertilization the 15th day), the expression of OsMA gene DS27 is concentrated mainly on skeleton (Fig. 4 Middle H)；It is fertilized the 22nd day, the expression of OsMADS27 gene concentrates at radicle, root cap, epiblast and coleorrhiza (I, Cr in Fig. 4 For coleorrhiza, Eb is epiblast)；After seed impregnates 12h, the expression of OsMADS27 gene concentrates on radicle, coleorrhiza (in Fig. 4 J, Rd are radicle, and RC is root cap root cap).

Embodiment 3 utilizes the correlation of complementation test verifying OsMADS27 gene and osmads27-1 mutation type surface

One, the building of complementing vector

1, with restriction enzyme BamHI and KpnI double digestion plasmid OSJNBa0079M09, the segment of about 12kb is recycled.

2, with restriction enzyme BamHI and KpnI double digestion carrier pCAMBIA23a, carrier framework is recycled.

3, the segment for recycling step 1 is connected with carrier framework, obtains recombinant plasmid pCAMBIA23a-OsMADS27, i.e., Complementing vector.

Two, the genetic transformation of rice

1, the recombinant plasmid pCAMBIA23a-OsMADS27 by step 1 building imports Agrobacterium tumefaciems by electric shock EHA105 obtains recombinational agrobacterium, is named as EHA105-pCAMBIA23a-OsMADS27.

2, EHA105-pCAMBIA23a-OsMADS27 monoclonal is inoculated in 20mL 50mg/L containing kanamycins and Li Fu The YEB fluid nutrient medium of flat 50mg/L, 28 DEG C, after 12~16h of 220rpm shake culture, be then inoculated in the ratio of 2% (v/v) In the YEB fluid nutrient medium containing 100 μM of acetosyringone, 28 DEG C, 220rpm shaken cultivation to OD₆₀₀Value reaches 0.5 or so, obtains To Agrobacterium infected liquid.

3, the seed decladding threshing of osmads27-1 mutant, is placed in 100mL triangular flask, and 70% (v/v) ethyl alcohol is added Aqueous solution soaking 30sec, then be placed in 25% (v/v) aqueous sodium hypochlorite solution, 120rpm concussion sterilizing 30min, sterile water punching It washes 3 times, with filter paper suck dry moisture, then seed embryo is placed in callus induction in induced medium, 30 DEG C of full exposure inductions 7 downward It, the callus grown is used for rice conversion after pinching bud.

4, after completing step 3, the preferable embryo callus of growth conditions is taken, the Agrobacterium that step 2 obtains is soaked in and invades Dye liquor, 28 DEG C, 80rpm shaking table jog callus, infects 30min, then, is placed on the co-culture medium for being covered with one layer of sterilizing filter paper On, 25 DEG C dark culture 4 days.

5, the callus for taking step 4 to obtain is placed in primary screening culture medium and more renews after illumination alternate culture 2 weeks Primary screening culture medium, callus continue illumination alternate culture 2 weeks, and the resistant calli grown on former callus is turned It moves on postsearch screening culture medium, illumination alternate culture 2 weeks.

6, after completing step 5, eugonic resistant calli is taken, differential medium, illumination alternate culture 3 are placed in Week, resistance budlet was differentiated on resistant calli.

7, after completing step 6, resistance budlet is taken, root media is placed in, illumination alternate culture obtains resistant plant.To Resistant plant it is long to 6~10cm when, carry out open Aquaponic, grow intermediate house after new root, obtain T₀In generation, intends transgenosis water Rice plants.Randomly choose 7 T obtained₀In generation, intends transgenic rice plant and (is successively named as quasi- turn of quasi- transgenic rice plant 1- Trans-genetic hybrid rice plant 7) it is identified.

Extract quasi- transgenic rice plant (quasi- transgenic rice plant 1, quasi- transgenic rice plant 2, quasi- transgenic paddy rice Plant 3, quasi- transgenic rice plant 4, quasi- transgenic rice plant 5, quasi- transgenic rice plant 6 or quasi- transgenic rice plant 7) the total serum IgE reverse transcription reagent box reverse transcription is gone out the first chain cDNA by total serum IgE.Using the cDNA as template, RT-PCR detection The relative expression quantity of OsMADS27 gene in quasi- transgenic rice plant (using ACTIN1 gene as reference gene).Identification The primer of OsMADS27 gene are as follows: 5 '-GCTTGAGACAACAACTGCAC-3 ' and 5 '-TTCAGCATACAAGGGTGTAG-3 '. Identify the primer of ACTIN1 gene are as follows: 5 '-CCAATCGTGAGAAGATGACCCA-3 ' and 5 '- CCATCAGGAAGCTCGTAGCTCT-3′。

According to the method described above, quasi- transgenic rice plant is replaced with into wild rice plant, other steps are constant, make For positive control.

Experimental result is shown in A in Fig. 5, (1 turns base for quasi- transgenic rice plant 2,3 for quasi- transgenic rice plant 1,2 to be quasi- It is to intend transgenic rice plant 5,6 as quasi- transgenic rice plant 6,7 because rice plant 3,4 is quasi- transgenic rice plant 4,5 To intend transgenic rice plant 7, WT is wild rice plant, and NC is water).Can expand and obtain size is about 480bp band Quasi- transgenic rice plant be positive T₀For transgenic rice plant.Therefore, intend transgenic rice plant 1, quasi- transgenosis Rice plant 2, quasi- transgenic rice plant 3, quasi- transgenic rice plant 4, quasi- transgenic rice plant 6 and quasi- transgenic paddy rice Plant 7 is positive T₀For transgenic rice plant and OsMADS27 gene relative expression quantity and wild rice plant without aobvious Difference is write, intending transgenic rice plant 5 is non-positive T₀For transgenic rice plant.Each plant is a strain, therefore, Above-mentioned positive T₀Complement strain L1, complement strain L2, complement strain L3, complement strain are successively named as transgenic rice plant L4, complement strain L6 and complement strain L7.

By the T of complement strain L3₂It is named as BAC for homozygote seed, carries out the experiment of subsequent step four.

Three, turn the acquisition of empty carrier plant

According to the method for step 2, recombinant plasmid pCAMBIA23a-OsMADS27 is replaced with into carrier pCAMBIA23a, is obtained To turning empty carrier plant.

Four, the phenotypic analysis of osmads27-1 mutant plants

By wild rice seed, turn the T of empty carrier plant₂For homozygote seed, osmads27-1 mutant seeds and BAC is placed in 1/2MS culture solution, 25 DEG C of cultures (i.e. water suction culture).The seed phenotypes sprouted are observed, and count culture different time Seed germination rate.Experiment is averaged in triplicate, repeats 50 seeds every time.

The result shows that turning the T of empty carrier plant₂For the germination rate of homozygote seed and osmads27-1 mutant seeds without Significant difference sprouts phenotype also without significant difference；The germination rate of BAC has largely recovery (C in Fig. 5), sprouts phenotype There is largely recovery (B in Fig. 5), it is substantially consistent with wild rice.

The result shows that the phenotype that osmads27-1 mutant sprouts delay is related with the missing of OsMADS27 gene, OsMADS27 gene plays an important role in the sprout time of adjusting and controlling rice seed.

Embodiment 4, osmads27-1 mutant externally apply the sensitivity of plant hormone

Plant hormone GA and ABA is played an important role in the germination process of seed, and wherein GA can promote to sprout, and ABA then Inhibiting germination.To probe into whether the phenomenon that 27-1 sprouts delay lacks with GA or ABA is related, tested as follows.

One, sensitivity of the osmads27-1 mutant to GA

Seed to be measured (wild rice seed or osmads27-1 mutant seeds) is placed in 1/2MS culture solution or containing 5 μ On the 1/2MS culture solution of M GA, 25 DEG C of cultures (i.e. water suction culture).The germination rate of statistics culture different time seed.Experiment weight It is multiple to be averaged three times, 30 are repeated every time.

Experimental result is shown in A in Fig. 6, (1/2MS WT is the wild rice seed for being placed in 1/2MS culture solution, 1/2MS 27-1 For the osmads27-1 mutant seeds for being placed in 1/2MS culture solution, GA WT is the open country for being placed in the 1/2MS culture solution containing 5 μM of GA Raw type rice paddy seed, GA 27-1 is the osmads27-1 mutant seeds for being placed in the 1/2MS culture solution containing 5 μM of GA).As a result table Bright, external source, which applies GA, cannot restore the phenotype of 27-1, and the phenotype of the sprouting delay of 27-1 is not due to caused by GA shortage；27-1 It is consistent with wild rice to the response of GA, illustrate that GA signal path is normal in 27-1.

Two, sensitivity of the osmads27-1 mutant to ABA

Seed to be measured (wild rice seed or osmads27-1 mutant seeds) is placed in 1/2MS culture solution, containing 2 μM On the 1/2MS culture solution of ABA, the 1/2MS culture solution containing 5 μM of ABA or the 1/2MS culture solution containing 10 μM of ABA, 25 DEG C of cultures (i.e. water suction culture) 5 days.Count the germination rate of seed.The germination rate of statistics culture different time seed.Experiment takes in triplicate Average value repeats 30 every time.

Experimental result is shown in B in Fig. 6 (WT is wild rice seed, and 27-1 is osmads27-1 mutant seeds).As a result Show the increase with ABA concentration in culture medium, germination rate gradually decreases, and the germination rate of 27-1 reduces degree and becomes apparent from.Table Bright sprouting stage 27-1 enhances the sensibility of ABA.

The measurement of ABA content in embodiment 5, Seed Germination

1, rice paddy seed to be measured (wild rice seed or osmads27-1 mutant seeds) is first impregnated with distilled water 0h or 6h, obtains sample to be tested.The content of ABA in the embryo of sample to be tested is surveyed by the centre of Beijing Forestry University Fixed, measuring method is plant hormone HPLC-Deca MAX-MSn analytic approach.

Experimental result is shown in left figure in Fig. 7 (WT is wild rice seed, and 27-1 is osmads27-1 mutant seeds).Knot Fruit shows that seed to be measured impregnates 0h or 6h, and the ABA content of osmads27-1 mutant seeds is above wild rice seed, This is consistent with the experimental result of embodiment 4, i.e. the phenotype that 27-1 sprouts delay is related with ABA content raising；6h is impregnated simultaneously, The decline degree of ABA content is close in wild type rice paddy seed and osmads27-1 mutant seeds, is 52%, this shows The metabolic process of ABA is normal in sprouting stage 27-1, causes ABA content in 27-1 raised the reason is that seed maturation The increase of middle ABA accumulation, this experimental result are higher than wild rice seed phase one with ABA content when impregnating 0h in 27-1 It causes.

It is fertilized by the centre of Beijing Forestry University to rice to be measured (wild rice or osmads27-1 mutant) 3rd day seed, the seed being fertilized the 8th day, the seed being fertilized the 15th day or the seed being fertilized the 30th day embryo in ABA content It is measured, measuring method is plant hormone HPLC-Deca MAX-MSn analytic approach.

Experimental result is shown in right figure in Fig. 7 (WT is wild rice seed, and 27-1 is osmads27-1 mutant seeds).Knot Fruit shows the ABA content in the seed that the seed that wild rice is fertilized the 3rd day and osmads27-1 mutant are fertilized the 3rd day It is close, about 300ng/g DW, this with this stage OsMADS27 gene still unexpressed experimental result (see 2 step 2 of embodiment, E in Fig. 4) it is consistent；ABA content is about 450ng/gDW in the seed that wild rice is fertilized the 8th day, and osmads27-1 is prominent The ABA content in seed that variant is fertilized the 8th day is about 700ng/g DW, hence it is evident that is higher than wild rice；In development later The content of ABA declines in wild rice seed and osmads27-1 mutant seeds in stage, but the ABA content in 27-1 It is still higher than wild rice, this experimental result expressed with OsMADS27 gene in these stages (see 2 step 2 of embodiment) It is consistent.The above results illustrate that the difference of ABA content appears in seed development in wild rice and osmads27-1 mutant Stage was continued for the sprouting stage.In addition, ABA content is about 180ng/g DW in the BAC of fertilization the 30th day, with wild type The ABA content in seed that rice is fertilized the 30th day is about that 170ng/g DW is close, and shows that ABA content increases certain in 27-1 It is due to caused by OsMADS27 gene delection.

The dormancy time extension of embodiment 6, osmads27-1 mutant seeds

1, by Post flowering 6 weeks rice paddy seeds to be measured (wild rice seed, osmads27-1 mutant seeds or BAC) It is placed in 1/2MS culture solution, 25 DEG C of cultures (i.e. water suction culture).Day by day seed germination rate, and the growth shape after observation 7 days are counted State.Experiment is averaged in triplicate, repeats 30 seeds every time.

The result shows that BAC then can partially restore the phenotype (A in Fig. 8) of osmads27-1 mutant.It is wild after culture 7 days The germination rate of raw type rice is close to the germination rate of 50%, BAC close to 40%, and the germination rate of osmads27-1 mutant is obviously low In wild rice, about 20% (B in Fig. 8).This result shows that, cause osmads27-1 mutant seeds sprout delay True cause is that the dormancy time of 27-1 seed extends.

2, Post flowering 4 weeks rice paddy seeds to be measured (wild rice seed, osmads27-1 mutant seeds or BAC), Post flowering 6 weeks rice paddy seeds to be measured (wild rice seed, osmads27-1 mutant seeds or BAC), Post flowering 8 weeks The rice paddy seed to be measured of rice paddy seed (wild rice seed, osmads27-1 mutant seeds or BAC) to be measured or after-ripening is (wild Raw type rice paddy seed, osmads27-1 mutant seeds or BAC) it is placed in 1/2MS culture solution, 25 DEG C of cultures (i.e. water suction culture) Then 7d counts seed germination rate.Experiment is averaged in triplicate, repeats 30 seeds every time.

The experimental results showed that (C in Fig. 8), Post flowering 4 weeks rice paddy seeds to be measured cannot be sprouted, with kernel maturing Raising, germination rate gradually rises, but the germination rate of osmads27-1 mutant seeds is significantly lower than wild rice, BAC's Germination rate is between osmads27-1 mutant and wild rice；The germination rate of the seed to be measured of after-ripening reaches 100%, The germination process of osmads27-1 mutant after showing breaking dormancy is normal.

After the above results show OsMADS27 gene delection, the suspend mode of seed is caused to extend, and then affect sprouting for seed Hair process.

27-1 of the embodiment 2 into embodiment 6 is replaced with into 27-3, other steps are constant, the experimental result of 27-3 with The experimental result of 27-1 is almost the same.

<110>Institute of Botany, Chinese Academy of Sciences

<120>application of the protein OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 723

<212> DNA

<213>rice (Oryza sativa L.)

<400> 1

atggggaggg ggaagattgt gatccgccgg atcgacaact cgacgagccg gcaggtgacg 60

ttctcgaagc ggaggaacgg gatcttcaag aaggccaagg agctggccat cctctgcgac 120

gccgaggtcg gcctcatgat cttctccagc accggccgcc tctacgagta ctccagcacc 180

agcatgaagt cagttataga tcggtatggc aagtccaagg atgagcagca agccgtcgca 240

aatcccaact cggagcttaa gttttggcaa agggaggcag caagcttgag acaacaactg 300

cacaacttgc aagaaaatca tcggcagttg atgggcgaag atctatctgg gctgaatgtt 360

aaggaattgc aatctctaga gaatcagctg gaaataagtc tacgtagtgt ccgtacaaag 420

aaggaccacg tcttgattga tgaaattcat gaactgaatc ggaagggaag tctagttcac 480

caagaaaaca tggaattata caagaagatc agtttaattc gtcaagaaaa tgctgagtta 540

tataagaaga tctacgagac tgaaggacca agtgaagtca atcgggattc accaactcct 600

tacaattttg cagtaattga aaaaacaaat gttcctgtgc aacttggact cagcacacta 660

ccacaacata gtgacgccga acaatcaact gctcctaagc tagggttaca gttgaatcca 720

tga 723

<210> 2

<211> 240

<212> PRT

<213>rice (Oryza sativa L.)

<400> 2

Met Gly Arg Gly Lys Ile Val Ile Arg Arg Ile Asp Asn Ser Thr Ser

1 5 10 15

Arg Gln Val Thr Phe Ser Lys Arg Arg Asn Gly Ile Phe Lys Lys Ala

20 25 30

Lys Glu Leu Ala Ile Leu Cys Asp Ala Glu Val Gly Leu Met Ile Phe

35 40 45

Ser Ser Thr Gly Arg Leu Tyr Glu Tyr Ser Ser Thr Ser Met Lys Ser

50 55 60

Val Ile Asp Arg Tyr Gly Lys Ser Lys Asp Glu Gln Gln Ala Val Ala

65 70 75 80

Asn Pro Asn Ser Glu Leu Lys Phe Trp Gln Arg Glu Ala Ala Ser Leu

85 90 95

Arg Gln Gln Leu His Asn Leu Gln Glu Asn His Arg Gln Leu Met Gly

100 105 110

Glu Asp Leu Ser Gly Leu Asn Val Lys Glu Leu Gln Ser Leu Glu Asn

115 120 125

Gln Leu Glu Ile Ser Leu Arg Ser Val Arg Thr Lys Lys Asp His Val

130 135 140

Leu Ile Asp Glu Ile His Glu Leu Asn Arg Lys Gly Ser Leu Val His

145 150 155 160

Gln Glu Asn Met Glu Leu Tyr Lys Lys Ile Ser Leu Ile Arg Gln Glu

165 170 175

Asn Ala Glu Leu Tyr Lys Lys Ile Tyr Glu Thr Glu Gly Pro Ser Glu

180 185 190

Val Asn Arg Asp Ser Pro Thr Pro Tyr Asn Phe Ala Val Ile Glu Lys

195 200 205

Thr Asn Val Pro Val Gln Leu Gly Leu Ser Thr Leu Pro Gln His Ser

210 215 220

Asp Ala Glu Gln Ser Thr Ala Pro Lys Leu Gly Leu Gln Leu Asn Pro

225 230 235 240

Claims

1. application of the protein OsMADS27 in the suspend mode of the sprout time and/or regulation vegetable seeds of regulation vegetable seeds.

2. application as described in claim 1, it is characterised in that: the protein OsMADS27 is a1) or a2) or a3):

A1) amino acid sequence is protein shown in sequence 2 in sequence table；

A3) amino acid sequence shown in sequence 2 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues And/or the protein relevant to the suspend mode of the sprout time of vegetable seeds and/or vegetable seeds that addition obtains.

3. encoding sprout time of the nucleic acid molecules in regulation vegetable seeds of protein OsMADS27 described in claims 1 or 2 And/or the application in the suspend mode of regulation vegetable seeds.

4. application as claimed in claim 3, it is characterised in that: protein described in described coding claims 1 or 2 The nucleic acid molecules of OsMADS27 be following b1) b2) or b3) or b4) shown in DNA molecular:

B2) code area DNA molecular as shown in sequence 1 in sequence table；

B3) and b1) or (b2) nucleotide sequence for limiting has 75% or 75% or more identity, and encodes claims 1 or 2 Described in protein OsMADS27 DNA molecular；

B4) the nucleotide sequence hybridization limited under strict conditions with (b1) or (b2), and encode described in claims 1 or 2 The DNA molecular of protein OsMADS27.

5. the application as described in Claims 1-4 is any, it is characterised in that: the sprout time of the regulation vegetable seeds is to promote Into the sprouting of vegetable seeds or the sprouting of delay plant seed.

6. the application as described in Claims 1-4 is any, it is characterised in that: the suspend mode of the regulation vegetable seeds is to release to plant The suspend mode of species or the suspend mode for extending vegetable seeds.

7. the application as described in claim 1 to 6 is any, it is characterised in that: any of the plant for following c1) into c6) Kind:

C1) dicotyledon；

C2) monocotyledon；

C3) gramineae plant；

C4) rice；

C5) rice osmads27-1 mutant；

C6) rice osmads27-3 mutant.

8. cultivating the method one of genetically modified plants or cultivating the method two of genetically modified plants:

The method one for cultivating genetically modified plants, including the core of protein OsMADS27 described in claims 1 or 2 will be encoded The step of acid molecule importing is set out in plant, and genetically modified plants are obtained；Genetically modified plants kind compared with the plant that sets out Son is sprouted in advance and/or the termination of diapause of seed；

The method two for cultivating genetically modified plants, is included in the plant that sets out and inhibits protein described in claims 1 or 2 The expression and/or activity of OsMADS27, the step of obtaining genetically modified plants；The genetically modified plants are compared with the plant that sets out Seed delay is sprouted and/or the suspend mode of seed extends.

9. plant breeding method one or plant breeding method two:

The plant breeding method one includes the following steps: to increase protein described in claims 1 or 2 in plant The content or activity of OsMADS27, so that seed is sprouted in advance and/or the termination of diapause of seed；

The plant breeding method two includes the following steps: to reduce protein described in claims 1 or 2 in plant The content or activity of OsMADS27, so that seed delay is sprouted and/or the suspend mode of seed extends.

10. the method as described in claim 7 to 9 is any, it is characterised in that: any of the plant for following c1) into c6) Kind:

C1) dicotyledon；

C2) monocotyledon；

C3) gramineae plant；

C4) rice；

C5) rice osmads27-1 mutant；

C6) rice osmads27-3 mutant.