CN110386968A - The application of TaYgl albumen and its encoding gene in regulation wheat leaf color - Google Patents
The application of TaYgl albumen and its encoding gene in regulation wheat leaf color Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
Abstract
The invention discloses a kind of application of TaYgl albumen and its encoding gene in regulation wheat leaf color.The invention discloses following A1) or A2) or A3) it is any shown in application of the protein in regulation plant leaf color: A1) protein that forms of the amino acid sequence shown in sequence 2；A2) the fusion protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection protein tag obtain；A3) by amino acid sequence shown in sequence 2 by one or several amino acid residues substitution and/or deletion and/or addition obtain with protein shown in A1) have the function of 90% or more identity and identical protein.The present invention passes through the encoding gene of gene Knockout rite-directed mutagenesis TaYgl albumen, obtain the Wheat Mutant that leaf color shows as yellow green, show that the albumen can regulate and control the leaf color of wheat, be applied in wheat breeding, is of great significance to increase wheat yield, improving wheat quality.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to TaYgl albumen and its encoding gene are in regulation wheat leaf color
Application.
Background technique
China is maximum Wheat Production state and country of consumption in the world, while being also one of main body entrance state.As China
Important commodity food, strategic reserves grain kind, the stable high yield of wheat have important work in terms of ensureing national food security
With.Blade is that plant carries out photosynthetic major organs, contains various enzymes related with photosynthesis and institutional framework, Ye Lv
Body is that plant carries out photosynthetic main cell device, and the photosynthesis of structure and function and green plants contacts very close
It cuts.Studies have shown that photosynthesis contributes to the 95% of crop yield, efficient photosynthesis depends on the normal development of chloroplaset
With the normal synthesis of chlorophyll.Leaf color is then the general performance of various pigments in chloroplaset, so Leaf color mutant is also referred to as leaf
Green body mutation.
The criteria for classifying of leaf color mutant has very much, and common standard is divided according to the phenotype of leaf color, Er Qieyi
As in seedling stage when this leaf color exception phenotype with regard to obvious, so can be to prominent according to mutant seedling leaf color phenotype
The type of variant is distinguish.In this kind of classification method, (the Gustafsson such as Gustafsson The plastid development in various types of chlorophyll
Mutations.Hereditas, 1942,28 (3): 483-492.) leaf color mutant is divided in the classification method that nineteen forty-two proposes
For yellow, 5 seed type of albefaction, light green, spot and striped.And (the Awan M.A.Konzak C.F.Rutger such as Awan
J.N.Nilan R.A.Mutagenic effect of Sodium Azide in rice.Crop Science, 1980,20,
663-668.) according to the seedling leaf color of leaf color mutant and phenotype, it is more detailed leaf color mutant be divided into yellow, it is yellowish green,
8 seed type of greenish-yellow, green white, albefaction, light green, Bai Cui and striped.It, can also be by pigment in addition to the opposite classification method by sense organ
The variation of content and ratio are divided into leaf color mutant: lack Chlorophyll type, Chlorophyll increment type, lack chlorophyll a type and
Lack chlorophyll b type.
Although leaf color mutant often shows as the decline of chlorophyll content and the reduction of photosynthetic efficiency, to such mutation
The research of body facilitates the synthesis for more in depth understanding chlorophyll and photosynthetic process.In addition, with wheat function base
Because of a group development for the research learned and Molecular design breeding, leaf color mutant also highlights important research and utility value.
Summary of the invention
The technical problem to be solved by the present invention is to how obtain wheat leaf color mutant.
In order to solve the above technical problems, the protein is TaYgl albumen, source the present invention provides a kind of protein
Following A1 in wheat (Triticum aestivum L.)) or A2) or A3) it is any shown in protein:
A1) the protein that the amino acid sequence shown in sequence 2 in sequence table forms；
A2) the fusion protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection protein tag 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
Be deleted and/or added obtain with protein shown in A1) have the function of 90% or more identity and identical protein.
In above-mentioned protein, the sequence 2 in sequence table is made of 421 amino acid residues.
Above-mentioned protein can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.
In above-mentioned protein, protein tag (protein-tag) refers to using DNA extracorporeal recombination, with destination protein
A kind of polypeptide or albumen of amalgamation and expression together, in order to the expression of destination protein, detection, tracer and/or purifying.The egg
White label can be Flag label, His label, MBP label, HA label, myc label, GST label and/or SUMO label etc..
In above-mentioned protein, identity refers to the identity of amino acid sequence.The homology on Internet can be used
The identity for retrieving website measurement amino acid sequence, such as the BLAST webpage of NCBI homepage website.For example, can be advanced
In BLAST2.1, by using blastp as program, 10 are set by Expect value, OFF is set by all Filter, makes
Use BLOSUM62 as Matrix, by Gap existence cost, Per residue gap cost and Lambda ratio
It is respectively set to 11,1 and 0.85 (default values) and retrieve the identity of a pair of of amino acid sequence to be calculated, then
Obtain the value (%) of identity.
In above-mentioned protein, described 90% or more identity can be at least 91%, 92%, 95%, 96%, 98%,
99% or 100% identity.
The present invention also provides application of the TaYgl albumen in regulation plant leaf color.
Biomaterial relevant to TaYgl albumen also belongs to protection scope of the present invention, the present invention also provides with TaYgl
The new application of the relevant biomaterial of albumen.
The present invention provides application of the biomaterial relevant to TaYgl albumen in regulation plant leaf color.
Any one of in above-mentioned application, the biomaterial is following B1)-B10):
B1 the nucleic acid molecules of TaYgl albumen) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or
Contain B3) recombinant microorganism of the recombinant vector；
B5) contain B1) the transgenic plant cells systems of the nucleic acid molecules or contain B2) transgenosis of the expression cassette
Plant cell contains B3) the transgenic plant cells system of the recombinant vector；
B6) contain B1) Transgenic plant tissues of the nucleic acid molecules or contain B2) transgenosis of the expression cassette plants
Object tissue contains B3) Transgenic plant tissue of the recombinant vector；
B7) contain B1) the genetically modified plants organs of the nucleic acid molecules or contain B2) transgenosis of the expression cassette plants
Sundries official contains B3) the genetically modified plants organ of the recombinant vector；
B8) contain B1) transgenic plants of the nucleic acid molecules or contain B2) transgenic plant of the expression cassette or
Contain B3) transgenic plant of the recombinant vector；
B9) by B8) tissue culture that generates of the regenerable cell of the transgenic plant；
B10) by B9) protoplast that generates of the tissue culture.
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.
In above-mentioned biomaterial, B1) coding TaYgl albumen nucleic acid molecules concretely following C1) C2) or C3) in
Shown in any:
C1) DNA molecular shown in sequence 1 in sequence table；
C2) coded sequence is DNA molecular shown in sequence 1 72-1337 in sequence table；
C3) DNA molecular limited under strict conditions with C1) or C2) hybridizes, and encodes the DNA molecular of TaYgl albumen.
Wherein, the sequence 1 in sequence table is made of 1550 nucleotide, and coded sequence is made of 1266 nucleotide, is compiled
Protein shown in sequence 2 in code sequence table.
The stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film 2 times, every time
5min, but in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and washes film 2 times, each 15min.
In above-mentioned biomaterial, B2) described in expression cassette be the DNA for referring to express TaYgl albumen in host cell,
The DNA not only may include the promoter for starting TaYgl genetic transcription, may also include the terminator for terminating TaYgl transcription.Into one
Step, the expression cassette may also include enhancer sequence.Promoter for use in the present invention includes but is not limited to: composing type starting
Son is organized, the promoter and inducible promoter that organ and development are special.The example of promoter includes but is not limited to: cauliflower
The constitutive promoter 35S of mosaic virus；Wound-inducible promoter from tomato, leucine aminopeptidase (" LAP ",
Chao et al. (1999) Plant Physiol 120:979-992)；Chemical inducible promoter from tobacco, pathogenesis
1 (PR1) of correlation (is induced) by salicylic acid and BTH (diazosulfide -7- carbothioic acid S-methyl ester)；Tomato protease inhibitors
II promoter (PIN2) or LAP promoter (available methyl jasmonate induction)；Heat-shock promoters (United States Patent (USP) 5,187,
267)；Tetracycline inducible promoter (United States Patent (USP) 5,057,422)；Seed specific promoters, such as Millet Seed specificity
Promoter pF128 (CN101063139B (Chinese patent 2,007 1 0099169.7)), the special starting of seed storage protein matter
Son is (for example, promoter (Beachy et al. (1985) of phaseolin, napin, oleosin and soybean beta conglycin
EMBO is J.4:3047-3053).They can be used alone or are used in combination with other plant promoters.It is cited herein all
Bibliography is cited in full text.Suitable transcription terminator includes but is not limited to: Agrobacterium nopaline syntase terminator (NOS
Terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and
Octopine synthase terminator (see, e.g.: Odell et al. (I985) Nature 313:810；Rosenberg et al. (1987)
Gene, 56:125；Guerineau et al. (1991) Mol.Gen.Genet, 262:141；Proudfoot (1991) Cell, 64:
671；Sanfacon et al. Genes Dev., 5:141；Mogen et al. (1990) Plant Cell, 2:1261；Munroe et al.
(1990) Gene, 91:151；Ballad et al. (1989) Nucleic Acids Res.17:7891；Joshi et al. (1987)
NucleicAcid Res., 15:9627).
The recombinant vector of the TaYgl encoding gene expression cassette can be contained with plant expression vector construction.The plant table
Up to carrier can be Gateway systemic vectors or double base agrobacterium vector etc., as pGWB411, pGWB412, pGWB405,
pBin438、pCAMBIA1302、pCAMBIA2300、pCAMBIA2301、pCAMBIA1301、pCAMBIA1300、pBI121、
PCAMBIA1391-Xa or pCAMBIA1391-Xb.It, can before its transcription initiation nucleotide when constructing recombinant vector using TaYgl
In addition any enhanced, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S
Promoter, general raw plain gene Ubiqutin promoter (pUbi) etc., they can be used alone or with other plant promoter knots
It closes and uses；In addition, enhancer, including translational enhancer also can be used when using gene constructed plant expression vector of the invention
Or transcriptional enhancer, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be with
The reading frame of coded sequence is identical, to guarantee the correct translation of entire sequence.The translation control signal and initiation codon
Source be it is extensive, can be natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or knot
Structure gene.
In above-mentioned biomaterial, B3) recombinant vector can contain in ordered list and use shown in sequence 1 72-1337
In the DNA sequence dna of coding TaYgl albumen.
For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out
Processing, as be added the coding that can be expressed in plant can produce color change enzyme or luminophor gene (gus gene,
Luciferase genes etc.), resistant antibiotic marker (gentamicin marker, kanamycins marker etc.) or anti-
Chemical reagent marker gene (such as anti-herbicide gene).
In above-mentioned biomaterial, B4) recombinant microorganism concretely yeast, bacterium, algae and fungi.
In above-mentioned biomaterial, B7) the genetically modified plants organ can be genetically modified plants root, stem, leaf, flower, fruit
And seed.
In above-mentioned biomaterial, B9) tissue culture can derive from root, stem, leaf, flower, fruit, seed, pollen, embryo
And anther.
In above-mentioned application, the regulation plant leaf color is that regulation plant leaf color is green or yellow green.
Invention further provides above-mentioned TaYgl albumen or the relevant biomaterial of TaYgl albumen to cultivate blade table
It is now the application in the genetically modified plants of yellow green；Or, above-mentioned TaYgl albumen or the relevant biomaterial of TaYgl albumen are being planted
Application in object breeding.
The present invention also provides a kind of methods for changing plant leaf color.
The method that the present invention changes plant leaf color, structure and/or function including changing TaYgl albumen in purpose plant,
Change the leaf color of purpose plant.
In the above method, the blade that the leaf color change shows as plant changes into yellow green by green.
The present invention further additionally provides a kind of method for cultivating the genetically modified plants that leaf color is yellow green.
A kind of method for cultivating the genetically modified plants that leaf color is yellow green of the present invention, including change TaYgl in purpose plant
The structure and/or function of albumen, obtain genetically modified plants；The genetically modified plants are compared with the purpose plant, leaf color performance
For yellow green.
In the above method, the method for changing the structure and/or function of TaYgl albumen in purpose plant is to inhibit purpose
The expression and/or activity of TaYgl albumen in plant.Specifically, it is described change purpose plant in TaYgl albumen structure and/or
The method of function is to carry out knockout or mutagenesis by the encoding gene to TaYgl albumen in the purpose plant to realize.
In the above method, the method for changing the structure and/or function of TaYgl albumen in purpose plant be using base
It is realized because encoding gene of the edit methods to TaYgl albumen in the purpose plant is knocked out.
Specifically, sequence 1 325-343 and 1 713-732 of sequence in the target sequence of the gene editing such as sequence table
DNA molecular shown in position.
In above-mentioned application or method, the plant is monocotyledon or dicotyledon；The monocotyledon is standing grain
Graminaceous plant；The gramineae plant is wheat platymiscium, and it specifically can be wheat that the wheat platymiscium, which is wheat,
Fielder。
The present invention is cloned into the gene TaYgl of regulation wheat leaf color by BSR-Seq technology from wheat leaf color mutant.
The change of transmission electron microscope observing discovery mutant leaf color is the chloroplaset base broken, internal due to a large amount of outer chloroplast membrane structure
Outside matter and thylakoid are exposed to, normal chloroplast quantity is less, illustrates that the gene can influence the development of chloroplaset, Jin Erke
To regulate and control the leaf color of wheat, it is important to show that TaYgl has in research Development of Chloroplasts process and in terms of improving photosynthetic efficiency
Researching value.The present invention is shown as yellowish green by gene Knockout rite-directed mutagenesis TaYgl protein coding gene acquisition leaf color
The Wheat Mutant of color can more in depth understand the synthesis and photosynthetic process of chlorophyll, be applied in wheat breeding,
It is of great significance to increase wheat yield, improving wheat quality.
Detailed description of the invention
Fig. 1 is wild type YZ4110 and mutant ygl seedling stage and field phenotype.Wherein, a is seedling stage phenotype；B is field table
Type.
Fig. 2 is wild type YZ4110 and mutant ygl seedling leaf chlorophyll a, chlorophyll b, carotene carotene content measure.
Fig. 3 is wild type YZ4110 and mutant ygl chloroplaset transmission electron microscope observing.Wherein, a, b are wild type YZ4110
Chloroplaset transmission electron microscope picture；C, d is mutant ygl chloroplaset transmission electron microscope picture；Cp represents chloroplaset in figure, PG represents starch
Grain, G represents thylakoid, S represents matrix, CW represents cell wall, ST represents stroma-thylakoid.
Fig. 4 is phenotype of the gene editing T0 for transgenic plant.Wherein, TW181-18 is target site plant not to be edited
Strain；TW181-36, TW181-3 are target site plant to be edited.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Used in following embodiments
Experimental method unless otherwise specified, be conventional method.The materials, reagents and the like used in the following examples, such as without special theory
It is bright, it is commercially available.
Quantitative experiment in following embodiment, is respectively provided with and repeats three times, and results are averaged.
BsaI restriction enzyme and T4 DNA ligase are purchased from NEB (Beijing) Co., Ltd, and catalog number is respectively #
R0535V and #M0202V.
Intermediate vector pCBC-MT1T2: in document " Hui-Li Xing, Li Dong, Zhi-Ping Wang, Hai-Yan
Zhang, Chun-Yan Han, Bing Liu, Xue-Chen Wang, Qi-Jun Chen.A CRISPR/Cas9 toolkit
For multiplex genome editing in plants.BMC Plant Biology, 2014, it discloses in 14:327. "
It crosses, is saved by Institute of Crop Science, Chinese Academy of Agricultural Science teacher Kong Xiuying.The public can be from Chinese Academy of Agricultural Sciences crop
Science Institute obtains, and to repeat the application experiment, not can be used as other purposes and uses.
PBUE411-TaU3p carrier is in document " Wang Zhiping (2017) doctoral thesis, " Plant Genome and base editor's work
Have case initiative and functional verification " " in be disclosed, by Institute of Crop Science, Chinese Academy of Agricultural Science teacher Kong Xiuying save.
The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application experiment, not can be used as other purposes
It uses.
Hi-fi PCR enzyme (KOD FX Neo) is purchased from TOYOBO, catalog number KFX-201.
AxyPrep Plasmid DNA small volume of reagent box is purchased from Axygen company, catalog number: AP-MN-P-250.
AxyPrep DNA gel QIAquick Gel Extraction Kit is purchased from Axygen company, catalog number: AP-GX-250.
Escherichia coli TOP10 competent cell is purchased from Beijing Zhuan Meng Bioisystech Co., Ltd, catalog number ZC104-
2。
Agrobacterium EHA105 competent cell is purchased from Beijing Zhuan Meng Bioisystech Co., Ltd, catalog number ZC142.
Wheat Yanzhan4110 (referred to as YZ4110) is documented in following document: Cao Tingjie, Zhao Hong, Wang Xicheng, Yang Hui state examine
General performance and Utilization prospects the analysis China agronomy notification of new variety of wheat Yanzhan4110,2004,20 (5): 77-78. is in
Academy of Agricultural Sciences, state crop science research institute teacher Kong Xiuying saves.The public can study from Chinese Academy of Agricultural Sciences's crop science
It is obtained, to repeat the application experiment, not can be used as other purposes and use.
Hundred agricultures 3217 are documented in following document: just, Zhu Mingzhe, Wang Shijie, hundred agriculture 3217 of Hu Ruifa, Ru Zhengang is small for yellow light
Wheat Tiller ears characteristic and its statistical analysis hundred special journal of spring agriculture of, 1983,1:1-9. are ground by Chinese Academy of Agricultural Sciences's crop science
Study carefully institute teacher Kong Xiuying preservation.The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, real to repeat the application
It tests, not can be used as other purposes and use.
Wheat Fielder is documented in following document: Ke Wang, Huiyun Liu, Lipu Du, Xingguo
Ye.Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-
mediated co-transformation strategy in commercial Chinese wheat
Varieties.Plant BiotechnologyJournal, 2017,15,614-623. by Chinese Academy of Agricultural Sciences's crop science
Research institute teacher Kong Xiuying saves.The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, to repeat the application
Experiment not can be used as other purposes and use.
The phenotypic evaluation of 1 wheat leaf color mutant ygl of embodiment (yellow green leaf) and the discovery of encoding gene
One, the phenotype of wheat leaf color mutant ygl
In the mutant library of wheat Yanzhan4110 (YZ4110) EMS mutagenesis building, a leaf color is screened in Huang
The mutant of green, leaf color phenotype can stablize heredity, and mutant is named as ygl (yellow green leaf).With YZ4110
Plant is compared, and as shown in figure 1 shown in a, ygl plant is in yellow green, shows the characteristic of minus green.As shown in figure 1 shown in b, ygl Plant Leaf
Piece all shows obvious yellowish green leaf color from seedling stage to the maturity period, is influenced delayed growth by mutation, and plant height is shorter, heading stage compared with
Evening, but breeding cycle can be completed.By detecting YZ4110 and ygl blade Determination of Chlorophyll a (Chlorophyll a), chlorophyll b
(Chlorophyll b), carrotene (Carotene) content, as shown in Fig. 2, the content of three is low in discovery ygl blade
In YZ4110.
YZ4110 and ygl blade is observed using transmission electron microscope observing, as shown in fig. 3, it was found that the chloroplaset of YZ4110 and ygl
Shapes and sizes there is no very big difference, but in ygl, a large amount of outer chloroplast membrane structure is broken, internal chloroplaset base
Outside matter and thylakoid are exposed to, normal chloroplast quantity is less.
Two, the genetic analysis of wheat leaf color mutant ygl and candidate gene obtain
To F derived from 1204 plants of YZ4110 × ygl2Show F for population analysis2Yellow green leaf plants are 274 plants in generation,
Normal leaf plants are 930 plants, meet hereditary pattern (the χ 2=2.53 < χ of single recessive gene2 0.05=3.84；P=0.11 >
0.05), therefore, the Leaf color mutant of ygl is controlled by single recessive gene.
In order to obtain the gene for regulating and controlling leaf color in mutant, seedling stage takes the F of hundred 3217 × ygl of agriculture3Phenotype is correct in group
Material blade construct mixing pit, each mixing pit include about 30 single plants, 2 secondary pollutants repeat, transfer to Chengdu day at future
Science and Technology Ltd. carries out BSR-Seq experiment and bioinformatic analysis.Each phenotype pond is sequenced to obtain about 20Gb data, passes through
Compared with China spring reference sequences, there are 792839 and 777780 bases in normal leaf color mixing pit and yellow-green mixture of colours pond respectively
Because of a group Mutation, these distributions of variation on chromosome are further analyzed, discovery has extremely aobvious on chromosome 7A at 671Mb
Difference is write, by finding the gene coding region TraesCS7A02G480700 to the location proximate genome mutation and genetic analysis
745th nucleotide sports A by G, causes it to encode albumen and changes.
Genome sequence (https: //urgi.versailles.inra.fr/ is referred to according to the China spring announced on the net
Blast/), design primer (ygl-F1 and ygl-R1), for detecting the genotype in the site in parent material and backcross population.
Wherein, the sequence of the primer is as follows:
Ygl-F1 (primer 1): 5 '-TCCACTCCCCTCGTCCTCGTC-3 '
Ygl-R1 (primer 2): 5 '-CGACGGCTGCACAAGTGCATA-3 '
PCR reaction system (20 μ L) is as follows:
PCR reaction carries out in 96 hole PCR instrument of ABI veriti, and response procedures are as follows: 95 DEG C of initial denaturation 5min；98℃
It is denaturalized 5s, 62 DEG C of annealing 30s, 68 DEG C of extension 1min, 35 recycle；68 DEG C of extension 5min.
After PCR product is utilized AxyPrep DNA gel QIAquick Gel Extraction Kit recovery purifying, send to Beijing and hold up the new industry biology of section
Technology Co., Ltd.'s sequencing, sequencing primer are ygl-SeqF1:5 '-TGAGAAGGCGCTCACCGAAG-3 '.As the result is shown
The genotype of the gene coding region TraesCS7A02G480700 the 745th of YZ4110 is GG, ygl's
The genotype that the gene coding region TraesCS7A02G480700 is the 745th is AA.
Greeny 43 plants of blade homozygous single plants further are shown to backcross population sequencing analysis
The genotype that the gene coding region TraesCS7A02G480700 is the 745th is GG, and blade is 34 plants of homozygous single plants of yellow green
The genotype of the gene coding region TraesCS7A02G480700 the 745th be AA.Then by TraesCS7A02G480700
Gene is determined as the candidate gene of wheat leaf color, is named as TaYgl, the nucleotide sequence such as 1 institute of sequence in sequence table of the gene
Show, coding sequence DNA molecular as shown in sequence 1 72-1337 in sequence table；The albumen name of gene coding
For TaYgl, the amino acid sequence of above-mentioned albumen is as shown in sequence 2 in sequence table.
The acquisition and identification of 2 genetically modified plants of embodiment
One, the building of recombinant plasmid
(1) according to TraesCS7A02G480700 sequence signature, select two gene editings target site Target1 and
Target1, two target sequences are as follows:
Target1:5 '-ACCCGTTCCCGGCGATCGT-3 ' (sequence 1 325-343)
Target2:5 '-CAACAGGGGGATACTGTATG-3 ' (sequence 1 713-732)
(2) two target sites in step (1) are building up to expression vector pBUE411-TaU3p:
Using intermediate vector pCBC-MT1T2 as template, using ygl-MT1T2-F and ygl-MT1T2-R as primer, according to implementation
PCR reaction system (20 μ L) and the amplification of PCR response procedures, obtained PCR product carry out recovery purifying in example 1；
Wherein, the primer are as follows:
Ygl-MT1T2-F:
5′-aataatggtctcaagcgACCCGTTCCCGGCGATCGTgttttagagctagaaatagc-3′；
Ygl-MT1T2-R:
5′-attattggtctctaaacCATACAGTATCCCCCTGTTGTcgcttcttggtgcc-3′。
The PCR product of recovery purifying establishes following digestion-linked system, obtains product:
(3) product obtained step (2) converts Escherichia coli TOP10 competent cell, selects positive colony, send to
The sequencing of Beijing Qing Kexin industry Bioisystech Co., Ltd, sequencing primer are as follows:
PBUE411-TaU3p-SeqF1:5 '-TTTCCCAGTCACGACGTTGT-3 '
PBUE411-TaU3p-SeqR1:5 '-ATCTCTAGAGAGGGGCACGA-3 '
Selection target site sequence correctly constructs the positive colony on pBUE411-TaU3p carrier, uses AxyPrep matter
Grain DNA small volume of reagent box extracts plasmid, obtains recombinant plasmid.
Two, recombinational agrobacterium obtains
Above-mentioned recombinant plasmid transformed is entered into Agrobacterium EHA105 competent cell, obtains recombinational agrobacterium, specific experiment step
Suddenly it is described according to the specification of Escherichia coli TOP10 competent cell (for Beijing Zhuan Meng Bioisystech Co., Ltd product).
Empty carrier pBUE411-TaU3p is converted into Agrobacterium EHA105 competent cell simultaneously, obtains control Agrobacterium.
Three, Agrobacterium is infected
(1) the seed decladding of wheat Fielder is taken to sterilize, tiling to calli induction media induces two weeks, chooses callus
Squamous subculture two weeks is to the callus particle for growing diameter 2mm or so.
(2) recombinational agrobacterium for the obtaining step 2 LB liquid medium (kanamycins of 50mg/L+ containing rifampin
50mg/L), overnight incubation makes OD600Value is 0.6-0.8 or so, obtains bacterium solution.
(3) it takes the bacterium solution 4000rmp of 3mL or so to be centrifuged 3 minutes, removes supernatant, bacterium solution is resuspended in the AMM of 20mL or so
In (containing 100 μm of ol/L acetosyringones) fluid nutrient medium, in 28 DEG C, 150rpm shakes two hours, obtains purpose bacterium solution.
(4) method disseminated is detailed in document Yuji Ishida, Masako Tsunashima, Yukoh Hiei,
Toshihiko Komari.Wheat(Triticum aestivum L.)Transformation Using Immature
Embryos.Methods in Molecular Biology, 2015,1223,189-198.: the callus that step (1) is obtained
Particle impregnates 20 minutes in purpose bacterium solution, then is put on the co-cultivation base of one layer of filter paper.Callus is sterilized after three days
Distillation is washed three times, and 20 minutes or so every time, then washed twice with the sterilizing of the benzyl of carboxylic containing 500mg/L, it 30 minutes every time, repeats
Repeatedly until washing lotion is very limpid, callus is placed on aseptic filter paper and is dried, then is put it on a sieve culture medium.It after two weeks will be more
Wound is gone on two sieve culture mediums, then goes to three sieve culture mediums after two weeks, obtains kanamycin-resistant callus tissue.
(5) kanamycin-resistant callus tissue is gone on differential medium, obtains differentiation seedling.
(6) differentiation seedling is transferred on 1/2MS culture medium and is taken root, then gone in incubator flowerpot, obtain experimental group
T0For plant.
The control Agrobacterium that step 2 obtains, which is carried out above-mentioned experiment, obtains the T of control group simultaneously0For plant.
Four, transgenic plant is identified
(1) PCR Molecular Identification
Extract the T of experimental group and control group0For the genomic DNA of plant leaf, with ygl-CRISPR-F1 (5 '-
ACATCGTCCGGCCTATCCAAGTC-3 ') and ygl-CRISPR-R1 (5 '-GTCACGATGTCCCTTCCCTTTAG-3 ') be draw
Object is expanded with PCR reaction system in embodiment 1 (20 μ L) and PCR response procedures, by PCR product recovery purifying, send to Beijing and hold up
The sequencing of Ke Xin industry Bioisystech Co., Ltd, it is right using DSDecodeM (http://skl.scau.edu.cn/dsdecode/)
Sequencing result is analyzed, and determines the plant that target site is edited.There are 22 plants of T through detecting, in experimental group0For plant target site
It is edited, 20 plants of T0It is not edited for plant target site.
(2) phenotypic evaluation
The plant that plant that 2 plants of target sites are edited, 1 plant of target site are not edited is cultivated under parallel condition,
The color for observing blade, as shown in figure 4, (two chromosomal target sites obtain the plant TW181-36 that target site is edited
4bp deletes variation) and TW181-3 (two chromosomal target sites obtain respectively 2bp is deleted, 4bp is deleted and made a variation) blade shows as
Yellow green, and target site plant (TW181-18) blade not to be edited still shows the green of normal plant, therefore, it was demonstrated that
Mutant phenotype in ygl is as caused by the mutation of TaYgl.
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims,
It can carry out the application of some essential characteristics.
SEQUENCE LISTING
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>application of TaYgl albumen and its encoding gene in regulation wheat leaf color
<130> GNCFY191160
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1550
<212> DNA
<213>wheat (Triticum aestivum L.)
<400> 1
aacccgtgcc tcccaatccc tctcttatcc cctcgccccc tccatccatc cactcccctc 60
gtcctcgtcc catggccatg gcctccccgt tctcccaggc gtcggccgcc gccgcctcgc 120
cggccctccc cttctccgtc tccacctccc gccctctctc cctcaccacc gccgcaaccg 180
ccgccgtctc agcccgggct ccgtgcaggg gcagcagagg attccgccgc ggccgcttcg 240
ccgtctgcaa tgtcgctgcc ccctccgccg ccgagctgga gaccaagccg gcggcggccg 300
cgaaggagag ccagcggccg gtgtacccgt tcccggcgat cgtggggcag gacgagatga 360
agctctgcct gctgctcaac gtcatcgacc ccaagatcgg cggcgtcatg ataatgggcg 420
accggggcac cggcaagtcc accaccgtcc gctccctcgt cgacctgctc ccggacatca 480
gcgtcgtggt cggcgacccg ttcaactccg accccttcga ccccgaggtc atgggccccg 540
aggtccgcga ccgcctcctc aagggcgagg accttcccgt caccaccacc aagatcacca 600
tggtcgacct gcccctcggc gccaccgagg acagggtgtg cggcaccatc gacattgaga 660
aggcgctcac cgaaggtgtc aaggcgttcg agccaggcct gctcgccaag gccaacaggg 720
ggatactgta tgtggacgag gtcaatctgc tggacgacca tctggtggat gttctgctgg 780
attccgcggc ttccgggtgg aacacggtgg agagggaggg catctccatc tcccaccctg 840
cgcggttcat cctcattggg tccggtaacc cggaggaagg cgagctccgg ccgcagctgc 900
tggaccggtt cgggatgcac gcgcaggtcg gcacggtcag ggacgcggag ctgagggtga 960
agattgtgga ggagagggct cggttcgaca aggacccgaa aacgttccgg cagtcctact 1020
tggaggagca agggaagctc caggaccaga tcacatccgc tcggagcaac ctcggttctg 1080
tgcagctcga ccatgatctc cgggttaaga tatcccaggt gtgttctgag ctgaatgtgg 1140
atgggctgag aggagacatt gtcactaaca gggctgccaa ggcgttggct gccctaaagg 1200
gaagggacat cgtgacagtg gaggacattg ccaccgtgat tcccaactgt ttgaggcatc 1260
ggctccgtaa agacccactc gaatcgatcg actcgggctt gcttgtagtt gagaagtttt 1320
atgaagtctt tggctagatt gctctcgagg taaatgtttc tctgtcacaa tgtaaggcag 1380
aaggcttttt gttcggctgt aaacttttat agcacctttc gtaaatcatc ttttatcgaa 1440
ctatattctg cattgtatag aaaacagtgg taccgagcgt taattatggt gaactttgtt 1500
atgttgttct gagcttgggc tcatattagc actttccccg aaaaaaaaaa 1550
<210> 2
<211> 421
<212> PRT
<213>wheat (Triticum aestivum L.)
<400> 2
Met Ala Met Ala Ser Pro Phe Ser Gln Ala Ser Ala Ala Ala Ala Ser
1 5 10 15
Pro Ala Leu Pro Phe Ser Val Ser Thr Ser Arg Pro Leu Ser Leu Thr
20 25 30
Thr Ala Ala Thr Ala Ala Val Ser Ala Arg Ala Pro Cys Arg Gly Ser
35 40 45
Arg Gly Phe Arg Arg Gly Arg Phe Ala Val Cys Asn Val Ala Ala Pro
50 55 60
Ser Ala Ala Glu Leu Glu Thr Lys Pro Ala Ala Ala Ala Lys Glu Ser
65 70 75 80
Gln Arg Pro Val Tyr Pro Phe Pro Ala Ile Val Gly Gln Asp Glu Met
85 90 95
Lys Leu Cys Leu Leu Leu Asn Val Ile Asp Pro Lys Ile Gly Gly Val
100 105 110
Met Ile Met Gly Asp Arg Gly Thr Gly Lys Ser Thr Thr Val Arg Ser
115 120 125
Leu Val Asp Leu Leu Pro Asp Ile Ser Val Val Val Gly Asp Pro Phe
130 135 140
Asn Ser Asp Pro Phe Asp Pro Glu Val Met Gly Pro Glu Val Arg Asp
145 150 155 160
Arg Leu Leu Lys Gly Glu Asp Leu Pro Val Thr Thr Thr Lys Ile Thr
165 170 175
Met Val Asp Leu Pro Leu Gly Ala Thr Glu Asp Arg Val Cys Gly Thr
180 185 190
Ile Asp Ile Glu Lys Ala Leu Thr Glu Gly Val Lys Ala Phe Glu Pro
195 200 205
Gly Leu Leu Ala Lys Ala Asn Arg Gly Ile Leu Tyr Val Asp Glu Val
210 215 220
Asn Leu Leu Asp Asp His Leu Val Asp Val Leu Leu Asp Ser Ala Ala
225 230 235 240
Ser Gly Trp Asn Thr Val Glu Arg Glu Gly Ile Ser Ile Ser His Pro
245 250 255
Ala Arg Phe Ile Leu Ile Gly Ser Gly Asn Pro Glu Glu Gly Glu Leu
260 265 270
Arg Pro Gln Leu Leu Asp Arg Phe Gly Met His Ala Gln Val Gly Thr
275 280 285
Val Arg Asp Ala Glu Leu Arg Val Lys Ile Val Glu Glu Arg Ala Arg
290 295 300
Phe Asp Lys Asp Pro Lys Thr Phe Arg Gln Ser Tyr Leu Glu Glu Gln
305 310 315 320
Gly Lys Leu Gln Asp Gln Ile Thr Ser Ala Arg Ser Asn Leu Gly Ser
325 330 335
Val Gln Leu Asp His Asp Leu Arg Val Lys Ile Ser Gln Val Cys Ser
340 345 350
Glu Leu Asn Val Asp Gly Leu Arg Gly Asp Ile Val Thr Asn Arg Ala
355 360 365
Ala Lys Ala Leu Ala Ala Leu Lys Gly Arg Asp Ile Val Thr Val Glu
370 375 380
Asp Ile Ala Thr Val Ile Pro Asn Cys Leu Arg His Arg Leu Arg Lys
385 390 395 400
Asp Pro Leu Glu Ser Ile Asp Ser Gly Leu Leu Val Val Glu Lys Phe
405 410 415
Tyr Glu Val Phe Gly
420
Claims (10)
1. following A1) or A2) or A3) it is any shown in application of the protein in regulation plant leaf color:
A1) the protein that the amino acid sequence shown in sequence 2 in sequence table forms；
A2) the fusion protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection protein tag obtain 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 addition obtain with protein shown in A1) have the function of 90% or more identity and identical protein.
2. application of the biomaterial relevant to the protein described in claim 1 in regulation plant leaf color；
Any one of the biomaterial is following B1)-B10):
B1 the nucleic acid molecules of protein described in claim 1) are encoded；
B2) contain B1) expression cassettes of the nucleic acid molecules；
B3) contain B1) recombinant vectors of the nucleic acid molecules or contain B2) recombinant vector of the expression cassette；
B4) contain B1) recombinant microorganisms of the nucleic acid molecules or contain B2) recombinant microorganism of the expression cassette or contain
B3) the recombinant microorganism of the recombinant vector；
B5) contain B1) the transgenic plant cells systems of the nucleic acid molecules or contain B2) genetically modified plants of the expression cassette
Cell line contains B3) the transgenic plant cells system of the recombinant vector；
B6) contain B1) Transgenic plant tissues of the nucleic acid molecules or contain B2) the genetically modified plants group of the expression cassette
Knit or contain B3) Transgenic plant tissue of the recombinant vector；
B7) contain B1) the genetically modified plants organs of the nucleic acid molecules or contain B2) the genetically modified plants device of the expression cassette
Official contains B3) the genetically modified plants organ of the recombinant vector；
B8) contain B1) transgenic plants of the nucleic acid molecules or contain B2) transgenic plant of the expression cassette or contain
B3) the transgenic plant of the recombinant vector；
B9) by B8) tissue culture that generates of the regenerable cell of the transgenic plant；
B10) by B9) protoplast that generates of the tissue culture.
3. application according to claim 2, it is characterised in that:
B1) nucleic acid molecules be following C1)-C3) and in it is any shown in:
C1) DNA molecular shown in sequence 1 in sequence table；
C2) coded sequence is DNA molecular shown in sequence 1 72-1337 in sequence table；
C3) DNA molecular limited under strict conditions with C1) or C2) hybridizes, and encodes protein described in claim 1
DNA molecular.
4. biomaterial described in protein described in claim 1 or Claims 2 or 3 shows as Huang in cultivation blade
Application in the genetically modified plants of green；Or, biology described in protein described in claim 1 or Claims 2 or 3
Application of the material in plant breeding.
5. changing the method for plant leaf color, it is characterised in that: including changing protein described in claim 1 in purpose plant
Structure and/or function, change the leaf color of purpose plant.
6. according to the method described in claim 5, it is characterized by: the blade that leaf color change shows as plant is changed by green
Become yellow green.
7. a kind of method for cultivating the genetically modified plants that leaf color is yellow green, it is characterised in that: weighed including changing in purpose plant
Benefit requires the structure and/or function of protein described in 1, obtains genetically modified plants；The genetically modified plants and the purpose
Plant is compared, and leaf color shows as yellow green.
8. the method according to claim 5 or 7, it is characterised in that: egg described in claim 1 in the change purpose plant
The structure of white matter and/or the method for function are to pass through the encoding gene to protein described in claim 1 in the purpose plant
Knockout or mutagenesis is carried out to realize.
9. according to the method described in claim 8, it is characterized by: albumen described in claim 1 in the change purpose plant
The structure of matter and/or the method for function are to be using gene editing method to albumen described in claim 1 in the purpose plant
The encoding gene of matter is knocked out to realize；
Specifically, the target sequence of the gene editing such as sequence 1 325-343 and the 713-732 institutes of sequence 1 in sequence table
The DNA molecular shown.
10. according to any method of claim 5-9, it is characterised in that: the plant is monocotyledon or dicotyledonous
Plant；The monocotyledon is gramineae plant.
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| CN112094838A (en) * | 2020-09-28 | 2020-12-18 | 中国科学院遗传与发育生物学研究所 | Application of glucose-6-phosphate isomerase in regulation and control of plant starch content and biomass |
| NL2032897B1 (en) * | 2022-08-30 | 2024-03-15 | Inst Of Crop Sciences Chinese Academy Of Agricultural Sciences | GENE Gmygl2 RELATED TO SOYBEAN PLANT HEIGHT AND LEAF COLOR, INSERTION-DELETION (InDel) MARKER OF GENE Gmygl2, AND USE THEREOF |
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| CN111876433A (en) * | 2020-08-07 | 2020-11-03 | 石家庄市农林科学研究院 | Wheat staged color-changing mutant gene with both marker recognition and ornamental values and application thereof |
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| CN112094838B (en) * | 2020-09-28 | 2022-12-20 | 中国科学院遗传与发育生物学研究所 | Application of glucose-6-phosphate isomerase in regulation and control of plant starch content and biomass |
| NL2032897B1 (en) * | 2022-08-30 | 2024-03-15 | Inst Of Crop Sciences Chinese Academy Of Agricultural Sciences | GENE Gmygl2 RELATED TO SOYBEAN PLANT HEIGHT AND LEAF COLOR, INSERTION-DELETION (InDel) MARKER OF GENE Gmygl2, AND USE THEREOF |
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| CN110386968B (en) | 2020-12-25 |
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