CN105949295A - Protein relevant to plant flowering time and encoding gene and application thereof - Google Patents
Protein relevant to plant flowering time and encoding gene and application thereof Download PDFInfo
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- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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Abstract
The invention discloses protein relevant to plant flowering time and an encoding gene and application thereof. The protein is named GmNMHC5 protein and is obtained from Glycine max (L.) Merrill. The protein is a or b, wherein a is composed of an amino acid sequence as shown in sequence 1 in the sequence table, and b is derived from sequence 1, obtained by conducting substitution and/or deficiency and/or addition of one or more amino acid residues on the amino acid sequence as shown in sequence 1, and relevant to plant flowering time. The gene encoding the GmNMHC5 protein (GmNMHC5 gene) is also within the protection range of the invention. The GmNMHC5 protein and the encoding gene thereof can make plant flowering time earlier and shorten the juvenile phase. The protein relevant to plant flowering time and the encoding gene thereof have high application value in plant breeding.
Description
Technical field
Biological technical field of the present invention, particularly relate to a kind of albumen with flowering of plant time correlation and encoding gene thereof with
Application.
Background technology
Semen sojae atricolor is the most of paramount importance oil crop and high protein cereal crops, in human lives and international agriculture
Trade occupies very important status, plays not in terms of culture fertility, the improvement farming such as soil, crop rotation
Alternative effect.Blooming is the plant significant process that turned to reproductive growth by nourishing and growing, is one of plant growing
The topmost stage.As typical short day crop, the process of blooming of Semen sojae atricolor is by the strict regulation and control of photoperiod.Greatly
The bloom clone of related gene and functional study of bean, to instructing Semen sojae atricolor to grow normally, improves crop yield and has important
Meaning.
Research ontogenetic to model plant shows, the process of blooming of plant is mainly regulated and controled by four approach: the photoperiod
Approach (photoperiod pathway), vernalization approach (vernalization pathway), spontaneous approach
(autonomous pathway) and gibberellin pathway (GA pathway), the regulated and control network pair that these four approach are constituted
The expression of the Blossoms Correlation Gene in Plants strictly controls.Correlational study shows, long day plant (such as arabidopsis) and short day plant
The molecular pathways that thing (such as Oryza sativa L.) is bloomed is conservative on evolving.
Summary of the invention
It is an object of the invention to provide a kind of albumen with flowering of plant time correlation and encoding gene thereof and application.
Present protein, named GmNMHC5 albumen, available from Semen sojae atricolor (Glycine max (L.) Merrill),
It is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1；
B the aminoacid sequence of sequence 1 through replacement and/or the disappearance of one or several amino acid residue and/or is added by ()
Add and with the protein derived by sequence 1 of flowering of plant time correlation.
In order to make the protein in (a) be easy to purification, can in by sequence table the aminoacid sequence shown in sequence 1
The amino terminal of the protein of row composition or carboxyl terminal connect upper label as shown in table 1.
The sequence of table 1 label
Protein in above-mentioned (b) can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of the protein in above-mentioned (b) can be by lacking one in the DNA sequence shown in sequence in sequence table 2
Or the codon of several amino acid residue, and/or carry out the missense mutation of one or several base pair, and/or its 5 '
End and/or 3 ' ends connect the coded sequence of the label shown in table 1 and obtain.
The gene (GmNMHC5 gene) encoding described GmNMHC5 albumen falls within protection scope of the present invention.
Described GmNMHC5 gene following 1) or 2) or 3) DNA molecular:
1) coding region DNA molecular as shown in sequence 2 in sequence table；
2) under strict conditions with 1) the DNA sequence hybridization that limits and the DNA of coded plant flowering time associated protein
Molecule；
3) with 1) or 2) DNA sequence that limits at least has 70%, at least has 75%, at least has 80%, extremely
Have 85% less, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have
98% or at least there is more than 99% homology and the DNA molecular of coded plant flowering time associated protein.
Above-mentioned stringent condition can be 6 × SSC, 0.5%SDS solution in, at 65 DEG C hybridize, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing described GmNMHC5 gene belong to
Protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression carrier of GmNMHC5 gene.Described plant is expressed
Carrier includes double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can wrap
3 ' the end untranslated regions containing exogenous gene, i.e. comprise polyadenylation signals and any other participates in mRNA processing or base
Because of the DNA fragmentation expressed.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' ends of mRNA precursor.Make
During recombinant expression carrier gene constructed with GmNMHC5, can be plus any enhancement mode before its transcription initiation nucleotide
Promoter or constitutive promoter, they can be used alone or be used in combination with other plant promoter；Additionally, make
During recombinant expression carrier gene constructed with GmNMHC5, it be also possible to use enhancer, including translational enhancer or transcribe enhancing
Son, but must be identical with the reading frame of coded sequence, to ensure the correct translation of whole sequence.Described translation controls letter
Number and the source of start codon be widely, can be natural, it is also possible to be synthesis.Translation initiation region can
Since from transcription initiation region or structural gene.For the ease of transgenic plant cells or plant being identified and screening,
Plant expression vector used can be processed, the enzyme of color change can be produced as added the coding can expressed in plant
Or the gene of luminophor, the antibiotic marker thing with resistance or anti-chemical reagent marker gene etc..From turning base
Because of the security consideration of plant, any selected marker can be not added with, directly with character screening transformed plant.
In described expression cassette, the expression of GmNMHC5 gene can be started by constitutive promoter.Described constitutive promoter
Concretely 35S promoter.
Described recombinant expression carrier concretely inserts GmNMHC5 gene in the multiple clone site of carrier pGFPGUSplus
The recombiant plasmid obtained.Described recombinant expression carrier is concretely by Xba I and Sac I enzyme of carrier pGFPGUSplus
Cut the small fragment between site and be substituted by the recombiant plasmid that GmNMHC5 gene obtains.
The present invention also protects the application of GmNMHC5 albumen, at least one in following (c1) to (c4):
(c1) florescence of plant is regulated and controled；
(c2) advance flowering period of plant is promoted；
(c3) flowering time of plant is regulated and controled；
(c4) flowering time of plant is promoted in advance.
Described plant is monocotyledon or dicotyledon.
Described dicotyledon can be arabidopsis, such as Columbia ecotype arabidopsis.
The present invention also protects a kind of method cultivating transgenic plant, is by GmNMHC5 channel genes purpose plant,
To transgenic plant；At least one phenotype in described transgenic plant satisfied following (d1) to (d4):
(d1) bloom early than described purpose plant；
(d2) florescence is early than described purpose plant；
(d3) vegetative growth phase is shorter than described purpose plant；
(d4) generative growth phase is entered early than described purpose plant.
GmNMHC5 gene specifically can import described purpose plant by described recombinant expression carrier.In described method, institute
Stating recombinant expression carrier can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, micro-
Injection, conductance, the conventional biology methods such as agriculture bacillus mediated convert plant cell or tissue, and the plant group that will convert
Knit and cultivate into plant.
Described purpose plant is monocotyledon or dicotyledon.
Described dicotyledon can be arabidopsis, such as Columbia ecotype arabidopsis.
The present invention also protects a kind of plant breeding method, comprises the steps (e1) and/or (e1):
(e1) by increasing the activity of GmNMHC5 albumen in purpose plant, it is thus achieved that there is the plant of following character: open
Flower is shorter than described purpose early than described purpose plant and/or florescence early than described purpose plant and/or vegetative growth phase and plants
Thing and/or entrance generative growth phase are early than described purpose plant；
(e2) by promoting the expression of GmNMHC5 gene in purpose plant, it is thus achieved that there is the plant of following character: open
Flower is shorter than described purpose early than described purpose plant and/or florescence early than described purpose plant and/or vegetative growth phase and plants
Thing and/or entrance generative growth phase are early than described purpose plant.
Described purpose plant is monocotyledon or dicotyledon.
Described dicotyledon can be arabidopsis, such as Columbia ecotype arabidopsis.
The implementation " promoting the expression of GmNMHC5 gene in purpose plant " can be as follows (f1) or (f2) or
(f3):
(f1) by GmNMHC5 channel genes purpose plant；
(f2) strong promoter and/or enhancer are introduced；
(f3) other common methods in this area.
The present invention also protects recombinant expression carrier or expression described in GmNMHC5 albumen, GmNMHC5 gene or any of the above
Box application in plant breeding.The purpose of described plant breeding is to cultivate the plant of advance flowering period.Described plant is single
Cotyledon plant or dicotyledon.Described dicotyledon can be arabidopsis, such as Columbia ecotype arabidopsis.
The present invention is found that GmNMHC5 albumen and encoding gene thereof from Semen sojae atricolor, is conducted into the plant that sets out, and is turned
Gene plant, the flowering time of this transgenic plant shifts to an earlier date than Wild plant.Therefore, GmNMHC5 albumen and coding thereof
Gene has the effect promoting flowering of plant time advance.The present invention has great using value for plant breeding.
Accompanying drawing explanation
Fig. 1 is V3 phase and the R3 phase GmNMHC5 gene expression at different tissues.
Fig. 2 is the plant photo in character identification.
Fig. 3 is the flowering time in character identification and lotus throne number of blade when blooming.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.
Carrier pGFPGUSplus: list of references: Claudia E.Vickers.pGFPGUSPlus, a new binary
vector for gene expression studies and optimising transformation systems in
Plants.Biotechnol Lett, 29 phases in 2007.
Agrobacterium tumefaciems GV3101: Tian Gen biochemical technology company limited.
Columbia ecotype wildtype Arabidopsis thaliana (Col-0): south intended by list of references: Guo Xiao beautiful .Columbia type
Mustard callus culture is studied. Henan Agricultural Sciences, 01 phase in 2009.
Embodiment 1, GmNMHC5 albumen and the acquisition of encoding gene thereof
Verified by a large amount of sequence analysis, comparison, reading frame identification and preliminary experiment, from Semen sojae atricolor, find a new albumen,
By its named GmNMHC5 albumen.
Shown in the sequence 1 of GmNMHC5 albumen such as sequence table (being made up of 241 amino acid residues).
It is GmNMHC5 gene by the unnamed gene of coding GmNMHC5 albumen, the sequence of its open reading frame such as sequence table
Shown in 2 (726bp).
Embodiment 2, the express spectra of GmNMHC5 albumen
Selecting Zi Gong winter bean (photoperiod-sensitive kind) is experiment material, and short-day sunshine treatment is chosen representative
Vegetative growth phase (V3 phase) and the blade of generative growth phase (R3 phase), (stem apex of V3 phase is in the R3 phase for stem apex
Be changed into pod), root and root nodule be material.
Extracting the total serum IgE of each material respectively, reverse transcription is cDNA.Application Real-time RT-PCR method, with
GmActin is internal standard gene, detects GmNMHC5 gene tissue specific expression situation in Semen sojae atricolor body.
Tissue expression analysis result is as shown in Figure 1.Vegetative growth phase, GmNMHC5 gene is mainly in root and root nodule
Expressing, the especially expression in root is the highest.Generative growth phase, GmNMHC5 gene also has certain in soybean pod
Express.Result shows, GmNMHC5 gene is not only relevant with root system development, and grows have with reproductive growth and flower pod
Close.
Embodiment 3, the acquisition of transgenic plant and qualification
One, the structure of recombiant plasmid
1, the double chain DNA molecule shown in sequence 2 of composition sequence table.
2, with step 1 synthesis double chain DNA molecule as template, use F1 and R1 composition primer to carrying out PCR
Amplification, obtains pcr amplification product.
F1:5 '-TCTAGAATGGGGAGAGGTAAGATTGCG-3’；
R1:5 '-GAGCTCCTAATGCAGCTGCAATCCGAGTTT-3’。
3, take carrier pGFPGUSplus, with restricted enzyme Xba I and Sac I double digestion, reclaim about 13000bp
Carrier framework.
4, take the pcr amplification product that step 2 obtains, with restricted enzyme Xba I and Sac I double digestion, reclaim
Digestion products.
5, the carrier framework of step 3 and the digestion products of step 4 are connected, obtain recombiant plasmid pGUS-GmNMHC5.
According to sequencing result, recombiant plasmid pGUS-GmNMHC5 is carried out structure and is described as follows: by carrier pGFPGUSplus
Xba I and Sac I restriction enzyme site between small fragment replace for the double chain DNA molecule shown in the sequence 2 of sequence table.
In recombiant plasmid pGUS-GmNMHC5,35S promoter start the expression of GmNMHC5 gene, by GUS as the positive
The selection markers of transformation event.
Two, the acquisition of transgenic plant
1, recombiant plasmid pGUS-GmNMHC5 is imported Agrobacterium tumefaciems GV3101, obtain recombinational agrobacterium.
2, take the recombinational agrobacterium that step 1 obtains, suspend with the aqueous sucrose solution of 5g/100ml, obtain OD600nmValue
Being the bacterium solution of 0.5, last every 100ml bacterium solution adds 50 μ l Silwet L-77, obtains infecting liquid.
3, take the liquid that infects that step 2 obtains, use and be stained with colored dip method and Columbia ecotype arabidopsis is carried out heredity turn
Change, the most normally cultivate and gather in the crops seed (T1 is for seed).
4, take the seed of step 3 results, be seeded in containing on the 1/2MS solid medium containing 50mg/L hygromycin
Cultivate, the resistant plant (T1 is for plant) screen 10 strain mounted blades, growing fine.Results resistant plant
Seed (T2 is for seed).
5, take the seed of step 4 results, be seeded in containing on the 1/2MS solid medium containing 50mg/L hygromycin
Cultivate, screening mounted blade, the resistant plant (T2 is for plant) grown fine.The seed of results resistant plant
(T3 is for seed).
6, take the seed of step 5 results, normally sow and cultivate, obtaining T3 for plant.
7, sample for plant for plant and T3 for plant, T2 from T1 respectively, extract total serum IgE and reverse transcription is cDNA,
To carrying out PCR qualification, (if obtaining the amplified production of 441bp, qualification result is the primer using F2 and R2 to form
Positive；Otherwise, qualification result is negative).In T3 generation, obtain stable heredity to turn GmNMHC5 gene arabidopsis pure
Close strain.
F2:5 '-ACCCATCACCTCATGAATCCG-3 '；
R2:5 '-AGACTGTGGCTGACTTAGCTG-3 '.
Three, the acquisition of empty carrier plant is turned
Replace recombiant plasmid pGUS-GmNMHC5 with carrier pGFPGUSplus, carry out step 2, obtain turning empty carrier and intend
South mustard.
Four, character identification
Sample to be tested is as follows: the T3 of strain 1 (turning GmNMHC5 gene arabidopsis homozygous lines) is for seed, strain
Be 2 (turning GmNMHC5 gene arabidopsis homozygous lines) T3 for seed, turn the T3 of empty carrier arabidopsis for seed,
Columbia ecotype arabidopsis.
1, taking seed, in sterilized water, 4 DEG C are soaked 72h.
2, packet transaction
Long-day process group: after completing step 1, take seed, is seeded in equipped with in the nursery cave basin of seedling medium,
21 DEG C, cultivate under conditions of 16h illumination/8h dark；
Short-day sunshine treatment group: after completing step 1, takes seed, is seeded in equipped with in the nursery cave basin of seedling medium,
21 DEG C, cultivate under conditions of 8h illumination/16h dark；
Isopyknic soil and Vermiculitum are mixed to get by seedling medium.The size of nursery cave basin is 7cm × 7cm × 8cm.
Persistently carry out Phenotypic Observation, record flowering time (with the time of arabidopsis inflorescence seen from naked eyes as flowering time)
And lotus throne number of blade when blooming.
Carry out three times repeating test, repeat every kind of sample to be tested in test every time and add up 15 strain plant, results averaged.
Under long-day processes, after sowing 30 days, Fig. 2 A is shown in by the photo of plant.
Under short-day sunshine treatment, after sowing 40 days, Fig. 2 B is shown in by the photo of plant.
The flowering time of every kind of sample to be tested and lotus throne number of blade when blooming are shown in Fig. 3.
Under long-day conditions: the flowering time of strain 1 is the 25th day after planting, and when blooming, the lotus throne number of blade is 8
Sheet；The flowering time of strain 2 is the 26th day after planting, and when blooming, the lotus throne number of blade is 9；Colombia is ecological
The flowering time of type arabidopsis is the 33rd day after planting, and when blooming, the lotus throne number of blade is 14；Turn empty carrier and intend south
The flowering time of mustard is after planting the 34th day, average 15 of lotus throne number of blade when blooming.Under the conditions of short-day: strain
Be 1 flowering time be the 33rd day after planting, when blooming, the lotus throne number of blade is 9；The flowering time of strain 2 is
The 34th day after planting, when blooming, the lotus throne number of blade is 10；The flowering time of Columbia ecotype arabidopsis is
The 48th day after planting, when blooming, the lotus throne number of blade is 23；Turn the flowering time of empty carrier arabidopsis for after planting
49th day, average 24 of lotus throne number of blade when blooming.
Result above shows, compared with Columbia ecotype arabidopsis, turns blooming of GmNMHC5 gene arabidopsis bright
Show in advance, i.e. GmNMHC5 albumen has the function promoting flowering of plant.
Claims (10)
1. a protein, is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1；
B the aminoacid sequence of sequence 1 through replacement and/or the disappearance of one or several amino acid residue and/or is added by ()
Add and with the protein derived by sequence 1 of flowering of plant time correlation.
2. the gene of protein described in coding claim 1.
3. gene as claimed in claim 2, it is characterised in that: described gene is following 1) or 2) or 3)
DNA molecular:
1) coding region DNA molecular as shown in sequence 2 in sequence table；
2) under strict conditions with 1) the DNA sequence hybridization that limits and the DNA of coded plant flowering time associated protein
Molecule；
3) with 1) or 2) DNA sequence that limits at least has 70%, at least has 75%, at least has 80%, extremely
Have 85% less, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have
98% or at least there is more than 99% homology and the DNA molecular of coded plant flowering time associated protein.
4. contain the recombinant expression carrier of gene described in Claims 2 or 3, expression cassette, transgenic cell line or restructuring
Bacterium.
5. the application of protein described in claim 1, at least one in following (c1) to (c4):
(c1) florescence of plant is regulated and controled；
(c2) advance flowering period of plant is promoted；
(c3) flowering time of plant is regulated and controled；
(c4) flowering time of plant is promoted in advance.
Apply the most as claimed in claim 5, it is characterised in that: described plant is monocotyledon or dicotyledon.
7. the method cultivating transgenic plant, is by channel genes purpose plant described in Claims 2 or 3,
To transgenic plant；At least one phenotype in described transgenic plant satisfied following (d1) to (d4):
(d1) bloom early than described purpose plant；
(d2) florescence is early than described purpose plant；
(d3) vegetative growth phase is shorter than described purpose plant；
(d4) generative growth phase is entered early than described purpose plant.
8. a plant breeding method, comprises the steps (e1) and/or (e1):
(e1) by increasing the activity of protein described in claim 1 in purpose plant, it is thus achieved that have following character
Plant: bloom and be shorter than institute early than described purpose plant and/or florescence early than described purpose plant and/or vegetative growth phase
State purpose plant and/or enter generative growth phase early than described purpose plant；
(e2) by promoting the expression of gene described in Claims 2 or 3 in purpose plant, it is thus achieved that have following character
Plant: bloom and be shorter than early than described purpose plant and/or vegetative growth phase early than described purpose plant and/or florescence
Described purpose plant and/or entrance generative growth phase are early than described purpose plant.
9. described in gene, claim 2 described in protein, claim 2 described in claim 1, gene, right are wanted
Ask expression cassette application in plant breeding described in recombinant expression carrier described in 4 or claim 4.
Apply the most as claimed in claim 9, it is characterised in that: the purpose of described plant breeding carries for cultivating the florescence
Front plant.
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| CN112341529A (en) * | 2020-11-17 | 2021-02-09 | 西南大学 | Chimonanthus praecox CpFPA gene and protein coded by same and application of gene |
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| CN111072760B (en) * | 2019-12-17 | 2021-03-26 | 西南大学 | EjFRI gene for delaying loquat flowering time and encoding protein and application thereof |
| CN111171127A (en) * | 2020-02-26 | 2020-05-19 | 浙江省农业科学院 | Astragalus sinicus LHY gene and application thereof |
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