CN106866806B - Protein TaVRS1-2B and its encoding gene and application - Google Patents
Protein TaVRS1-2B and its encoding gene and application Download PDFInfo
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- CN106866806B CN106866806B CN201710153220.1A CN201710153220A CN106866806B CN 106866806 B CN106866806 B CN 106866806B CN 201710153220 A CN201710153220 A CN 201710153220A CN 106866806 B CN106866806 B CN 106866806B
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- HAYADTTXNZFUDM-IHRRRGAJSA-N Ser-Tyr-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O HAYADTTXNZFUDM-IHRRRGAJSA-N 0.000 description 1
- YBXMGKCLOPDEKA-NUMRIWBASA-N Thr-Asp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YBXMGKCLOPDEKA-NUMRIWBASA-N 0.000 description 1
- KCRQEJSKXAIULJ-FJXKBIBVSA-N Thr-Gly-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O KCRQEJSKXAIULJ-FJXKBIBVSA-N 0.000 description 1
- GIAMKIPJSRZVJB-IHPCNDPISA-N Trp-Phe-Ser Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N GIAMKIPJSRZVJB-IHPCNDPISA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- LZRWTJSPTJSWDN-FKBYEOEOSA-N Val-Trp-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N LZRWTJSPTJSWDN-FKBYEOEOSA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010044940 alanylglutamine Proteins 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- 108010052670 arginyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108010063431 methionyl-aspartyl-glycine Proteins 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 108010074082 phenylalanyl-alanyl-lysine Proteins 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000026206 response to starvation Effects 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 108010017949 tyrosyl-glycyl-glycine Proteins 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- 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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- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses protein TaVRS1-2B and its encoding gene and applications.The amino acid sequence of protein TaVRS1-2B provided by the present invention is as shown in sequence 2 in sequence table.It it is demonstrated experimentally that being overexpressed TaVRS1-2B in section's agriculture 199, can significantly shorten the development time of young fringe, and then spikelet number reduction, the reduction of little Hua number and grain number per spike is caused to reduce.Therefore, protein TaVRS1-2B has in regulation wheatear type character and in the young spike development time important application value, has bright prospects in cultivating wheat breed.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to protein TaVRS1-2B and its encoding gene and application.
Background technique
Wheat is that one of main cereal crops, the population of whole world 35-40% are come with it for main food in the world
Source.Currently, breeding per unit area yield new variety of wheat outstanding is to stabilize and increase wheat yield most in the case where limited cultivated area
Cost-effective approach.Therefore, the solid sexual organ " wheatear " of wheat is always the important object of wheat breeding scholar research.
Flower arrangement mode regular on common peduncle is known as inflorescence, and inflorescence development is the important set that phytomorph is built up
At inflorescence meristem is broken up by shoot apical meristem, from development, and side organ is further budded by spire, side shoot
Colored and inflorescence (i.e. small ear).Wheat is compound spike, on cob raw several joints, each joint base portion life it is one small
Fringe.Each small ear generates 3-10 little Hua.Fringe type character includes grain number per spike, spikelet number and little Hua number.Yu Zhenwen etc. studies have shown that
There is extremely significant positive correlation between Ear weight and grain yield.Therefore, high-yield field can lay particular emphasis on the growth and development for improving single plant
Condition improves single fringe productivity.There are extremely significant positive correlation, mass of 1000 kernel and every Ear weights between number of grain per ear and every Ear weight
Between only show certain positive relationship, illustrate under certain cultivation condition, pass through increase number of grain per ear approach improve fringe grain
It is a kind of feasible approach again.
Summary of the invention
The technical problem to be solved by the present invention is to how regulate and control wheatear type character and/or young spike development time.
In order to solve the above technical problems, present invention firstly provides protein TaVRS1-2B.
Protein TaVRS1-2B provided by the present invention is derived from wheat (Triticum aestivum L.), can is
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
The obtained protein with plant fringe type character and/or reproductive development time correlation is deleted and/or added；The fringe type
Shape is d1) and/or d2) and/or d3): d1) grain number per spike；D2) spikelet number；D3) little Hua number.
Wherein, sequence 2 can be made of 238 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 and obtain.
The nucleic acid molecules of code for said proteins TaVRS1-2B also belong to protection scope of the present invention.
The nucleic acid molecules of code for said proteins TaVRS1-2B can be following b1) or b2) or b3) or b4) shown in DNA
Molecule:
B1) code area DNA molecular as shown in sequence 1 in sequence table；
B2) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encoding said proteins
The DNA molecular of matter TaVRS1-2B；
B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and code for said proteins TaVRS1-
The DNA molecular of 2B.
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.
Wherein, sequence 1 is made of 717 nucleotide in sequence table, in sequence table in the nucleotide coding sequence table of sequence 1
Amino acid sequence shown in sequence 2.
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 code for said proteins TaVRS1-2B of the invention.Those by manually modified,
The nucleosides of nucleotide sequence 75% or higher identity with the protein TaVRS1-2B isolated with the present invention
Acid is derived from nucleotide sequence of the invention and to be equal to of the invention as long as code for said proteins TaVRS1-2B
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 TaVRS1-2B of the composition of amino acid sequence shown in the sequence 2 of bright polynucleotide has
The nucleotide sequence of 75% or higher or 80% or higher or 85% or higher or 90% or higher identity.Identity can
With with the naked eye or computer software is evaluated.Using computer software, the identity between two or more sequences can be used
Percentage (%) indicates, can be used to evaluate the identity between correlated series.
The expression cassettes of nucleic acid molecules containing code for said proteins TaVRS1-2B, recombinant vector, recombinant microorganism turn
Gene cell system also belongs to protection scope of the present invention.
What the recombinant vector can obtain for the nucleic acid molecules for being inserted into code for said proteins TaVRS1-2B to expression vector
Recombinant plasmid.
The recombinant vector concretely recombinant plasmid pUbi::TaVRS1-2B-pAHC25.The recombinant plasmid pUbi::
TaVRS1-2B-pAHC25 concretely will be between the restriction enzyme BamH I of carrier pUbi-pAHC25 and Kpn I identification sequence
Small fragment replace with the DNA molecular shown in the 1st to 714 from 5 ' ends of sequence 1 in sequence table.
The recombinant microorganism can be obtained by the way that the recombinant vector is imported the microorganism that sets out.The microorganism that sets out can
For yeast, bacterium, algae or fungi.
The transgenic plant cells system does not include propagation material.The genetically modified plants are interpreted as not only including by institute
The first generation transgenosis that TaTFL1-2B gene (i.e. the encoding gene of protein TaTFL1-2B) transformation receptor plant obtains is stated to plant
Object also includes its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to which traditional breeding techniques will
The gene transfer enters other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, are cured
Injured tissue, intact plant and cell.
Following g1) or g2) also belong to protection scope of the present invention:
G1) the protein TaVRS1-2B, or, the nucleic acid molecules of code for said proteins TaVRS1-2B, or, containing compiling
Expression cassette, recombinant vector, recombinant microorganism or the transgenic cell line of the nucleic acid molecules of the code protein TaVRS1-2B,
Regulate and control plant fringe type character and/or the application in the reproductive development time；
G2) the protein TaVRS1-2B, or, the nucleic acid molecules of code for said proteins TaVRS1-2B, or, containing compiling
Expression cassette, recombinant vector, recombinant microorganism or the transgenic cell line of the nucleic acid molecules of the code protein TaVRS1-2B,
Cultivate the application in the genetically modified plants that fringe type character changes and/or the reproductive development time changes；
The fringe type character can be d1) and/or d2) and/or d3): d1) grain number per spike；D2) spikelet number；D3) little Hua number.
In above-mentioned application, the regulation plant fringe type character can be bad for plant fringe type character.The fringe type character is bad can body
Now be e1) and/or e2) and/or e3): e1) grain number per spike reduce；E2) spikelet number is reduced；E3) little Hua number is reduced.The regulation is planted
The object reproductive development time concretely shortens plant generative organ's development time.
In order to solve the above technical problems, the present invention also provides a kind of methods for cultivating genetically modified plants.
The method provided by the present invention for cultivating genetically modified plants, it may include by code for said proteins TaVRS1-2B's
The step of nucleic acid molecules import in recipient plant, obtain genetically modified plants；Compared with the recipient plant, the genetically modified plants
Fringe type character change and/or the reproductive development time shorten.
In the above method, the nucleic acid molecules of code for said proteins TaVRS1-2B can be following b1) or b2) or b3) or
B4 DNA molecular shown in):
B1) code area DNA molecular as shown in sequence 1 in sequence table；
B2) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encoding said proteins
The DNA molecular of matter TaVRS1-2B；
B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and code for said proteins TaVRS1-
The DNA molecular of 2B.
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.
Wherein, sequence 1 is made of 717 nucleotide in sequence table, in sequence table in the nucleotide coding sequence table of sequence 1
Amino acid sequence shown in sequence 2.
In the above method, described " importing the nucleic acid molecules of code for said proteins TaVRS1-2B in recipient plant " can
It is realized by importing recombinant vector into recipient plant；The recombinant vector can be to be inserted into code for said proteins to expression vector
The recombinant plasmid that the nucleic acid molecules of TaVRS1-2B obtain.
The recombinant vector concretely recombinant plasmid pUbi::TaVRS1-2B-pAHC25.
In order to solve the above technical problems, the present invention also provides a kind of plant breeding methods.
Plant breeding method provided by the present invention, it may include following steps: increase protein TaVRS1- described in plant
The content and/or activity of 2B, so that fringe type character changes and/or the reproductive development time shortens.
In any of the above-described the method, the fringe type character change into the fringe type character change into grain number per spike reduce and/
Or spikelet number reduces and/or little Hua number is reduced.The reproductive development time, which shortens, shows as the shortening of young spike development time.Institute
State the young spike development time concretely young fringe two rib phases development time and/or young fringe the floret differentiation phase development time
And/or young fringe is in the development time of Pistil And Stamen idiophase.
Any of the above-described plant can be following c1) any one of to c5): c1) dicotyledon；C2) unifacial leaf is planted
Object；C3) gramineae plant；C4) wheat；C5) wheat breed section agriculture 199.
It is demonstrated experimentally that being overexpressed protein TaVRS1-2B in section's agriculture 199, it can significantly shorten the development time of young fringe (such as
Development time in two rib phases, the development time in the floret differentiation phase, in the development time of Pistil And Stamen idiophase), and then cause
Spikelet number is reduced, little Hua number is reduced and grain number per spike is reduced.Therefore, protein TaVRS1-2B is in regulation wheatear type character and children
There is important application value in fringe development time, there are bright prospects in cultivating wheat breed.
Detailed description of the invention
Fig. 1 is the relative expression quantity of TaTFL1-2B gene and the correlation analysis of every fringe spikelet number.
Fig. 2 is the relative expression quantity of TaTFL1-2B gene and the correlation analysis of every fringe little Hua number.
Fig. 3 is the relative expression quantity of TaTFL1-2B gene and the correlation analysis of every fringe grain number per spike.
Fig. 4 is the part of test results of electron-microscope scanning.
Fig. 5 is the part statistical result that each developmental stage develops number of days.
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.
RNA Miniprep Kit is the product of Axygen company.TransScript First-Strand cDNA
Synthesis SuperMix Kit is the product of TransGen.Premix Ex TaqTMFor the production of TaKaRa company
Product.Reverse transcriptase is the product of TransGen, catalog number AT301-02.KOD-plus-DNA polymerase and 10 × PCR
Buffer for KOD-plus is the product of TOYOBO company, catalog number KOD-201.Carrier pGEM-Teasy is
The product of Promega company, catalog number A3600.Scanning electron microscope critical point drying instrument is HITACHI company
Product, product type HCP-2.Freeze the product that scanning electron microscope is HITACHI company, product type S-3000N.
Carrier pUbi-pAHC25 is recorded in following document: Jing Wang, Jinghan Sun, et al.A
phosphate starvation response regulator Ta-PHR1is involved in phosphate
signalling and increases grain yield in wheat.Ann Bot.2013Jun；111 (6): 1139-53.
The clone of embodiment 1, TaVRS1-2B gene
1, the total serum IgE that wheat breed section agriculture 199 (hereinafter referred to as section's agriculture 199) is in the young fringe tissue of two rib phases is extracted, so
The total serum IgE reverse transcriptase reverse transcription is gone out into the first chain cDNA to get to the cDNA of the young fringe tissue of section's agriculture 199 afterwards.199 children of section's agriculture
DNA content is 66ng/ μ L in the cDNA of fringe tissue.
2, the cDNA of the young fringe tissue of the section's agriculture 199 obtained using step 1 is template, using primers F: 5 '-
CAGCAAGCATGGACAAGCAGC-3 ' and primer R:5 '-TACCAGTCACCACCACCATC-3 ' carries out PCR amplification, obtains about
The pcr amplification product of 1066bp.
Reaction system is 50 μ L, by 1 μ L KOD-plus-DNA polymerase, 1 μ L template, 5 μ 10 × PCR of L buffer
For KOD-plus, 5 μ L concentration are the dNTPs aqueous solution (i.e. the concentration of dATP, dTTP, dCTP and dGTP are 2mM) of 2mM, 2
μ L concentration is the MgSO of 25mM4The primer R that primers F aqueous solution that aqueous solution, 1.5 μ L concentration are 10 μM, 1.5 μ L concentration are 10 μM
Aqueous solution and 33 μ L water composition.
Reaction condition: 98 DEG C of initial denaturation 2min；98 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 68 DEG C of extension 40s, 35 recycle.
3, the pcr amplification product that step 2 obtains is connected with carrier pGEM-Teasy, obtains recombinant plasmid pGEM-
Teasy-TaVRS1-2B。
Recombination plasmid pGEM-Teasy-TaVRS1-2B is sequenced.Sequencing result shows recombinant plasmid pGEM-
Containing DNA molecular (being named as TaVRS1-2B gene below) shown in sequence 1 in ordered list in Teasy-TaVRS1-2B.
Embodiment 2, the acquisition and its identification for turning TaVRS1-2B DNA triticum
One, construction recombination plasmid pUbi::TaVRS1-2B-pAHC25
1, the recombinant plasmid pGEM-Teasy-TaVRS1-2B constructed using embodiment 1 is template, with 5 '-GGATCCATGGACAAGCAGCAGGTCTTTGG-3 ' the restriction endonuclease recognition sequence of restriction enzyme BamH I (underscore be) and
5’-GGTACCAATTAGCCCATACAGGCTAAA-3 ' (restriction endonuclease recognition sequence that underscore is restriction enzyme Kpn I) is to draw
Object carries out PCR amplification, obtains the pcr amplification product of about 730bp.
2, the pcr amplification product obtained with restriction enzyme BamH I and I double digestion step 1 of Kpn, recycles digestion products.
3, with I double digestion carrier pUbi-pAHC25 of restriction enzyme BamH I and Kpn, the carrier bone of about 6.9kb is recycled
Frame.
4, digestion products are connected with carrier framework, obtains recombinant plasmid pUbi::TaVRS1-2B-pAHC25.
According to sequencing result, structure is carried out to recombinant plasmid pUbi::TaVRS1-2B-pAHC25 and is described as follows: by carrier
Small fragment between restriction enzyme BamH I and Kpn I the identification sequence of pUbi-pAHC25 replaces with sequence 1 from 5 ' in sequence table
DNA molecular shown in end the 1st to 714.Sequence 2 in recombinant plasmid pUbi::TaVRS1-2B-pAHC25 expressed sequence table
Shown in protein (being named as TaVRS1-2B albumen or protein TaVRS1-2B below).
Two, T0The generation quasi- acquisition for turning TaVRS1-2B DNA triticum
The genetic transforming method mediated using particle gun, by the agriculture of recombinant plasmid pUbi::TaVRS1-2B-pAHC25 conversion section
199, obtain T0In generation, intends turning TaVRS1-2B DNA triticum.
Carrier pUbi-pAHC25 conversion section agriculture 199 is obtained T by the genetic transforming method mediated using particle gun0In generation, turns
Empty carrier wheat.
Three, T0The generation quasi- real-time quantitative PCR detection for turning TaVRS1-2B DNA triticum
Randomly select 96 plants of T0In generation, the quasi- TaVRS1-2B DNA triticum that turns carried out real-time quantitative PCR detection, and specific steps are such as
Under:
1,96 plants of T are extracted using RNA Miniprep Kit respectively0Generation is quasi- to be turned TaVRS1-2B DNA triticum and was in for two rib phases
Young fringe tissue total serum IgE, then use TransScript First-Strand cDNA Synthesis SuperMix
The total serum IgE reverse transcription is gone out the first chain cDNA by Kit, obtains each T0The generation quasi- cDNA for turning TaVRS1-2B DNA triticum.Each T0
DNA content is about 74ng/ μ L in the generation quasi- cDNA for turning TaVRS1-2B DNA triticum.
2, each T is detected using RT-qPCR technology0The generation quasi- phase for turning TaVRS1-2B gene in TaVRS1-2B DNA triticum
To expression quantity (using β-TaTubulin gene as reference gene).
The primer of detection TaVRS1-2B gene is forward primer 1:5 '-CATCCTCCACAAATGCCACC-3 ' and reversely draws
Object 1:5 '-ACGCACATCATCAGGTCATC-3 '.The primer for detecting β-TaTubulin gene is positive primer 2: 5 '-
ACCGCCAGCTCTTCCACCCT-3 ' and reverse primer 2:5 '-TCACTGGGGCATAGGAGGAA-3 '.
Reaction system is 20 μ L, by 10 μ LPremix Ex TaqTM, 0.5 μ L concentration be 10 μM forward primer,
Reverse primer, 1 μ L T of the 0.5 μ L concentration for 10 μM0The generation quasi- cDNA and 8.0 μ L nuclease frees for turning TaTFL1-2B DNA triticum
Water composition.
Response procedures: 94 DEG C of initial denaturation 2min；95 DEG C of denaturation 3sec, 60 DEG C of annealing 30sec, 40 recycle.
According to the method described above, by T0Generation is quasi- to be turned TaVRS1-2B DNA triticum and replaces with section's agriculture 199, and other steps are constant,
Obtain the relative expression quantity of TaVRS1-2B gene in section's agriculture 199.
According to the method described above, by T0Generation is quasi- to be turned TaVRS1-2B DNA triticum and replaces with T0In generation, turns empty carrier wheat, Qi Tabu
It is rapid constant, obtain T0In generation, turns the relative expression quantity of TaVRS1-2B gene in empty carrier wheat.
Using the relative expression quantity of the TaVRS1-2B gene in section's agriculture 199 as 1, TaVRS1- in other each wheats is counted
The relative expression quantity of 2B gene.The result shows that compared with section's agriculture 199,65 plants of T0In generation, intends turning TaVRS1-2B DNA triticum (respectively
It is named as OE-1-T0To OE-65-T0) in the relative expression quantity of TaVRS1-2B gene dramatically increase, and T0It is small that in generation, turns empty carrier
The relative expression quantity of TaVRS1-2B gene is then without significant difference in wheat.
Four, T2In generation, turns TaVRS1-2B DNA triticum and T4In generation, turns acquisition and the real-time quantitative PCR of TaVRS1-2B DNA triticum
Detection
By OE-1-T0To OE-65-T0It is selfed through continuous two generation, obtains T2In generation, turns TaVRS1-2B DNA triticum, names respectively
For OE-1-T2To OE-65-T2。
By OE-1-T0To OE-65-T0It is selfed through continuous four generation, obtains T4In generation, turns TaVRS1-2B DNA triticum, names respectively
For OE-1-T4To OE-65-T4。
By T0In generation, turns empty carrier wheat and is selfed through continuous two generation, obtains T2Turn empty carrier wheat for homozygosis.
By T0In generation, turns empty carrier wheat and is selfed through continuous four generation, obtains T4Turn empty carrier wheat for homozygosis.
According to the method for step 3, to OE-1-T2To OE-65-T2、OE-1-T4To OE-65-T4、T2Turn empty carrier for homozygosis
Wheat, T4Turn empty carrier wheat and Ke Nong 199 for homozygosis and carries out real-time quantitative PCR detection respectively.The result shows that with section agriculture 199
It compares, OE-1-T2To OE-65-T2And OE-1-T4To OE-65-T4The relative expression quantity of middle TaVRS1-2B gene significantly increases
Add, and T2Turn empty carrier wheat and T for homozygosis4The generation homozygous relative expression quantity for turning TaVRS1-2B gene in empty carrier wheat then without
Significant difference.
Five, the correlation analysis of the relative expression quantity of TaVRS1-2B gene and wheatear type character
Wheat to be measured is OE-1-T2、OE-2-T2、OE-3-T2、OE-4-T2、OE-5-T2、OE-6-T2、OE-7-T2、OE-8-
T2、OE-9-T2、OE-10-T2、OE-11-T2、OE-12-T2、OE-13-T2、OE-14-T2、OE-15-T2、OE-16-T2、OE-17-
T2、OE-18-T2、OE-19-T2、OE-20-T2、OE-21-T2、OE-22-T2、OE-23-T2、OE-24-T2、OE-25-T2、OE-
26-T2、OE-27-T2、OE-28-T2、OE-29-T2、OE-30-T2、OE-31-T2、OE-32-T2、OE-33-T2、OE-34-T2、
OE-35-T2、OE-36-T2、OE-37-T2、OE-38-T2、OE-39-T2、OE-40-T2、OE-41-T2、OE-42-T2、OE-43-
T2、OE-44-T2、OE-45-T2、OE-46-T2、OE-47-T2、OE-48-T2、OE-49-T2、OE-50-T2、OE-51-T2、OE-
52-T2、OE-53-T2、OE-54-T2、OE-55-T2、OE-56-T2、OE-57-T2、OE-58-T2、OE-59-T2、OE-60-T2、
OE-61-T2、OE-62-T2、OE-63-T2、OE-64-T2Or OE-65-T2。
Wheat planting to be measured is investigated into every fringe spikelet number, every fringe little Hua number and every fringe fringe grain to Post flowering 20d in greenhouse
Then number carries out correlation analysis with the relative expression quantity of TaVRS1-2B gene.Condition of culture is specific as follows: 25 DEG C of temperature, light
According to 350 μm of ol photons m-2s-1, humidity 60-70%.
Experimental result is shown in Fig. 1, Fig. 2 and Fig. 3.The result shows that the relative expression quantity of TaVRS1-2B gene and every fringe small ear
Several, every fringe little Hua number and every fringe grain number per spike are negatively correlated, i.e. the relative expression quantity of TaVRS1-2B gene is higher, then every fringe small ear
The quantity of number, little Hua number and grain number per spike is fewer.
Six, the research of the relative expression quantity of TaVRS1-2B gene and wheat children tassel development time
Wheat to be measured is OE-1-T4、OE-2-T4、T4Turn empty carrier wheat or section's agriculture 199 for homozygosis.
The young fringe of 2d, 9d, 15d or 22d after taking 30 plants of wheat children tassels to be measured to enter single rib phase, are first placed in first
15min is impregnated in alcohol, then is placed in dehydrated alcohol and is impregnated 30min, it is then cold using scanning electron microscope critical point drying instrument
Dry 2h is lyophilized, electron-microscope scanning is finally carried out using freezing scanning electron microscope.According to electron-microscope scanning as a result, counting wheat to be measured
Development number of days of the young fringe in two rib phases, floret differentiation phase and Pistil And Stamen idiophase.
The part of test results of electron-microscope scanning is shown in that (A is section's agriculture 199, B OE-1-T to Fig. 44；LP is lemma former base, and GP is shield
Clever former base, number 6,7,8,9,10 are spikelet number, and white five-pointed star is small ear).The part of each period development number of days counts knot
Fruit sees Fig. 5 (WT is section's agriculture 199).
The result shows that compared with section's agriculture 199, OE-1-T4And OE-2-T4Young fringe in two rib phases, floret differentiation phase and male and female
The development time of stamen idiophase significantly shorten (single rib phase development time without significant difference), and T4Turn empty carrier for homozygosis
The young fringe of wheat single rib phase, two rib phases, floret differentiation phase and Pistil And Stamen idiophase development time without significant difference.Therefore,
In section's agriculture 199 be overexpressed TaVRS1-2B gene, can significantly shorten young fringe development time (such as the development time of two rib phases,
Development time in the floret differentiation phase and the development time in the Pistil And Stamen idiophase), and then spikelet number reduction, little Hua number is caused to subtract
Few and grain number per spike is reduced.
The above results show protein TaVRS1-2B in regulation wheatear type character (such as spikelet number, little Hua number, fringe grain
Number) and the young spike development time (as young fringe two rib phases development time and/or young fringe the floret differentiation phase development time with/
Or young fringe is in the development time of Pistil And Stamen idiophase) in there is important application value.
<110>Inst. of Genetics and Development Biology, CAS
<120>protein TaVRS1-2B and its encoding gene and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 717
<212> DNA
<213>wheat (Triticum aestivum L.)
<400> 1
atggacaagc agcaggtctt tggttcctcc tacgtggaca tgcctttctt cgcggccaat 60
ggtgcagcca cggcacaggg ggagagccgg ccgagggcgc ggcgcaggag gcggagggcc 120
gcgaggtgcg gcagagggga cggtgacggc ggggagatgg acgggggagg ggacctcaag 180
aagcggcggc tgaccgacga gcaggtgcac agtctggagc tgagcttccg ggaggagcgc 240
aagctggaga ccggccggaa ggttcatctc gctgccgagc tcgggctcga tcccaagcaa 300
gtcgcggtgt ggttccagaa ccgccgggcg cgacacaaga gcaagcttat ggaggaggag 360
ttcgccaagc tcaagcacgc ccacgacgcg gccatcctcc acaaatgcca cctcgagaac 420
gaggtgctga ggctgaagga gaggctggga gcgaccgagg aggaggtgcg acgcctcagg 480
tcggcagctg ggagccacgg agcatccggc gacggcggag acgccgctgg cgccgttggc 540
gcgtgtggcg ggagcccgag ctcgtccttc tcgacgggaa cctgccagca gcatccgggt 600
ttcagcggcg cagacgtgct ggggccggac gatgacctga tgatgtgcgt ccccgagtat 660
ggtggctacg ccgatagcag cgtggtcgag tggtttagcc tgtatgggct aatttga 717
<210> 2
<211> 238
<212> PRT
<213>wheat (Triticum aestivum L.)
<400> 2
Met Asp Lys Gln Gln Val Phe Gly Ser Ser Tyr Val Asp Met Pro Phe
1 5 10 15
Phe Ala Ala Asn Gly Ala Ala Thr Ala Gln Gly Glu Ser Arg Pro Arg
20 25 30
Ala Arg Arg Arg Arg Arg Arg Ala Ala Arg Cys Gly Arg Gly Asp Gly
35 40 45
Asp Gly Gly Glu Met Asp Gly Gly Gly Asp Leu Lys Lys Arg Arg Leu
50 55 60
Thr Asp Glu Gln Val His Ser Leu Glu Leu Ser Phe Arg Glu Glu Arg
65 70 75 80
Lys Leu Glu Thr Gly Arg Lys Val His Leu Ala Ala Glu Leu Gly Leu
85 90 95
Asp Pro Lys Gln Val Ala Val Trp Phe Gln Asn Arg Arg Ala Arg His
100 105 110
Lys Ser Lys Leu Met Glu Glu Glu Phe Ala Lys Leu Lys His Ala His
115 120 125
Asp Ala Ala Ile Leu His Lys Cys His Leu Glu Asn Glu Val Leu Arg
130 135 140
Leu Lys Glu Arg Leu Gly Ala Thr Glu Glu Glu Val Arg Arg Leu Arg
145 150 155 160
Ser Ala Ala Gly Ser His Gly Ala Ser Gly Asp Gly Gly Asp Ala Ala
165 170 175
Gly Ala Val Gly Ala Cys Gly Gly Ser Pro Ser Ser Ser Phe Ser Thr
180 185 190
Gly Thr Cys Gln Gln His Pro Gly Phe Ser Gly Ala Asp Val Leu Gly
195 200 205
Pro Asp Asp Asp Leu Met Met Cys Val Pro Glu Tyr Gly Gly Tyr Ala
210 215 220
Asp Ser Ser Val Val Glu Trp Phe Ser Leu Tyr Gly Leu Ile
225 230 235
Claims (11)
1. it is protein shown in sequence 2 in sequence table that protein TaVRS1-2B, which is amino acid sequence,.
2. encoding the nucleic acid molecules of protein TaVRS1-2B described in claim 1.
3. nucleic acid molecules as claimed in claim 2, it is characterised in that: the nucleic acid molecules be following b1) or b2) shown in
DNA molecular:
B1) code area DNA molecular as shown in sequence 1 in sequence table；
B2) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table.
4. expression cassette, recombinant vector, recombinant microorganism containing nucleic acid molecules described in Claims 2 or 3.
5.g1) or g2):
G1) protein TaVRS1-2B described in claim 1, or, nucleic acid molecules described in Claims 2 or 3, or, containing having the right to want
Expression cassette, recombinant vector, the recombinant microorganism for seeking 2 or 3 nucleic acid molecules, in regulation wheatear type character and/or genitals
Application in official's development time；
G2) protein TaVRS1-2B described in claim 1, or, nucleic acid molecules described in Claims 2 or 3, or, containing having the right to want
Expression cassette, recombinant vector, the recombinant microorganism for seeking 2 or 3 nucleic acid molecules are cultivating the change of fringe type character and/or genitals
The application in transgenic wheat that official's development time changes；
The fringe type character be d1) and/or d2) and/or d3): d1) grain number per spike；D2) spikelet number；D3) little Hua number.
6. application as claimed in claim 5, it is characterised in that: the regulation plant fringe type character is that plant fringe type character is bad；
The fringe type character is bad to be presented as e1) and/or e2) and/or e3): e1) grain number per spike reduce；E2) spikelet number is reduced；E3) little Hua number
It reduces；Regulation plant generative organ's development time is to shorten plant generative organ's development time.
7. the application as described in claim 5 or 6, it is characterised in that: the wheat is wheat breed section agriculture 199.
8. a kind of method for cultivating transgenic wheat, the nucleic acid point including protein TaVRS1-2B described in claim 1 will be encoded
The step of son imports in recipient wheat, obtains transgenic wheat；Compared with the recipient wheat, the fringe type of the transgenic wheat
Character changes and/or the reproductive development time shortens.
9. a kind of method for breeding wheat includes the following steps: to increase protein TaVRS1-2B described in claim 1 in wheat
Content, so that fringe type character changes and/or the reproductive development time shortens.
10. method as claimed in claim 8 or 9, it is characterised in that: the fringe type character change into grain number per spike reduce and/or
Spikelet number is reduced and/or little Hua number is reduced；The reproductive development time, which shortens, shows as the shortening of young spike development time.
11. method as claimed in claim 8 or 9, it is characterised in that: the wheat is wheat breed section agriculture 199.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502269A (en) * | 2011-04-29 | 2014-01-08 | 先锋国际良种公司 | Down-regulation of a homeodomain-leucine zipper i-class homeobox gene for improved plant performance |
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2017
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103502269A (en) * | 2011-04-29 | 2014-01-08 | 先锋国际良种公司 | Down-regulation of a homeodomain-leucine zipper i-class homeobox gene for improved plant performance |
Non-Patent Citations (4)
| Title |
|---|
| Molecular interactions of the c-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit;John C. Harris et al;《Plant Mol Biol》;20160123;第90卷;第435-452页 * |
| Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene;Takao Komatsuda et al;《PNAS》;20070123;第104卷(第4期);第1424-1429页 * |
| W5C3L7;Brenchley R.et al;《Uniprot数据库》;20170215;第1页 * |
| 同源域- 亮氨酸拉链蛋白ATHB6的研究进展;赵洪梅等;《中国农学通报》;20060831;第22卷(第8期);第77-82页 * |
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