CN110386968B - Application of TaYgl protein and coding gene thereof in wheat leaf color regulation - Google Patents

Abstract

The invention discloses a TaYgl protein and application of a coding gene thereof in regulating and controlling wheat leaf color. The invention discloses application of any protein shown in A1) or A2) or A3) in regulation and control of plant leaf color: A1) a protein consisting of an amino acid sequence shown in sequence 2; A2) the N end or/and the C end of the protein shown in the sequence 2 is connected with a protein label to obtain a fusion protein; A3) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2, has more than 90% of identity with the protein shown in A1), and has the same function. The invention performs site-specific mutagenesis on the coding gene of TaYgl protein by gene knockout technology to obtain a wheat mutant with yellow-green leaf color, which shows that the protein can regulate and control the leaf color of wheat, and has important significance for increasing the yield of wheat and improving the quality of wheat when being applied to wheat breeding.

Description

Application of TaYgl protein and coding gene thereof in wheat leaf color regulation

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to application of TaYgl protein and a coding gene thereof in wheat leaf color regulation.

Background

China is the largest wheat producing and consuming country in the world and is also one of the major imports. As important commercial grains and strategic reserve grain varieties in China, the high and stable yield of wheat plays an important role in guaranteeing the national grain safety. The leaf is the main organ of the plant for photosynthesis, contains various enzymes and tissue structures related to photosynthesis, and the chloroplast is the main organelle of the plant for photosynthesis, and the structure and the function of the chloroplast are closely related to the photosynthesis of green plants. Research shows that photosynthesis contributes 95% of crop yield, and efficient photosynthesis depends on normal development of chloroplasts and normal synthesis of chlorophyll. The leaf color is the comprehensive expression of various pigments in the chloroplast, so the leaf color mutation is also called chloroplast mutation.

The division standard of the leaf color mutants is various, the common standard is divided according to the phenotype of the leaf color, and the phenotype of the abnormal leaf color is obvious in the seedling stage, so that the types of the mutants can be distinguished according to the phenotype of the leaf color of the mutants in the seedling stage. Among the classification methods, Gustafsson et al (Gustafsson)

The plastic deformation in The various types of chlorinated masses Hereditas, 1942, 28 (3): 483-492.) the classification method proposed in 1942 classified the leaf color mutants into 5 types of yellowing, whitening, light green, mottling and streaking. Awan et al (Awan M.A. Konzak C.F. Rutger J.N. Nilan R.A. Mutagenic effect of Sodium Azide in rice.crop Science, 1980, 20, 663-668.) more specifically classify leaf color mutants into etiolated, yellowish-green, greenish-yellow, greenish-white, albino, light green, emerald and streak 8 types according to their seedling stage leaf color and phenotype. In addition to the relatively organoleptic classification methods, leaf color mutants can also be classified according to the change and proportion of pigment content: total chlorophyll deficient type, total chlorophyll increased type, chlorophyll deficient a type and chlorophyll deficient b type.

Although leaf color mutants often show a decrease in chlorophyll content and a decrease in photosynthetic efficiency, research on such mutants has helped provide a more thorough understanding of the processes of chlorophyll synthesis and photosynthesis. In addition, with the research of wheat functional genomics and the development of molecular design breeding, the leaf color mutant also shows important research and utilization values.

Disclosure of Invention

The technical problem to be solved by the invention is how to obtain the wheat leaf color mutant.

In order to solve the technical problems, the invention provides a protein, wherein the protein is TaYgl protein, is derived from wheat (Triticum aestivum L.), and is a protein represented by any one of A1), A2) or A3):

A1) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

A2) the N end or/and the C end of the protein shown in the sequence 2 in the sequence table is connected with a protein label to obtain a fusion protein;

A3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1), and has the same function.

In the protein, the sequence 2 in the sequence table is composed of 421 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

Among the above proteins, protein-tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate the expression, detection, tracking and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, among others.

In the above proteins, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

The invention also provides application of the TaYgl protein in regulation and control of plant leaf color.

The biological material related to the TaYgl protein also belongs to the protection scope of the invention, and the invention also provides a new application of the biological material related to the TaYgl protein.

The invention provides application of biological materials related to TaYgl protein in regulation and control of plant leaf color.

In the above application, the biomaterial is any one of the following B1) -B10):

B1) a nucleic acid molecule encoding a TaYgl protein;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;

B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector;

B8) a transgenic plant containing B1) the nucleic acid molecule, or a transgenic plant containing B2) the expression cassette, or a transgenic plant containing B3) the recombinant vector;

B9) a tissue culture produced from regenerable cells of said transgenic plant of B8);

B10) protoplasts produced by the tissue culture of B9).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, B1) the nucleic acid molecule encoding the TaYgl protein may specifically be any of the following C1) or C2) or C3):

C1) DNA molecule shown in sequence 1 in the sequence table;

C2) the coding sequence is a DNA molecule shown in 72 th-1337 th site of a sequence 1 in a sequence table;

C3) a DNA molecule which hybridizes with a DNA molecule defined by C1) or C2) under stringent conditions and encodes a TaYgl protein.

Wherein, the sequence 1 in the sequence table consists of 1550 nucleotides, the coding sequence consists of 1266 nucleotides, and the protein shown in the sequence 2 in the sequence table is coded.

The stringent conditions are hybridization and washing of the membrane 2 times 5min at 68 ℃ in a solution of 2 XSSC, 0.1% SDS and 2 times 15min at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS.

In the above-mentioned biological materials, the expression cassette described in B2) refers to a DNA capable of expressing TaYgl protein in a host cell, which DNA may include not only a promoter for initiating transcription of TaYgl gene but also a terminator for terminating transcription of TaYgl. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-; chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (us patent 5,187,267); tetracycline inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053.) they can be used alone or in combination withOther plant promoters are used in combination. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene, 56: 125; guerineau et al (1991) mol.gen.genet, 262: 141, a solvent; proudfoot (1991) Cell, 64: 671; sanfacon et al Genes dev., 5: 141, a solvent; mogen et al (1990) Plant Cell, 2: 1261; munroe et al (1990) Gene, 91: 151, and (b); ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) nucleic acid res, 15: 9627).

A plant expression vector can be used for constructing a recombinant vector containing the TaYgl coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When TaYgl is used for constructing a recombinant vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as cauliflower mosaic virus (CAMV)35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like, can be added in front of the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

In the above biological material, the recombinant vector of B3) may contain a DNA sequence for encoding TaYgl protein shown at positions 72-1337 of sequence 1 in the sequence table.

In order to facilitate the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the above biological material, the recombinant microorganism of B4) can be specifically yeast, bacteria, algae and fungi.

In the above biological material, the transgenic plant organ of B7) may be a root, stem, leaf, flower, fruit and seed of the transgenic plant.

In the above biological material, B9) the tissue culture may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the application, the regulation and control of the leaf color of the plant is green or yellow-green.

The invention further provides the application of the TaYgl protein or the biological material related to the TaYgl protein in cultivating transgenic plants with yellow-green leaves; or, the TaYgl protein or the biological material related to the TaYgl protein is applied to plant breeding.

The invention also provides a method for changing the leaf color of the plant.

The method for changing the leaf color of the plant comprises the step of changing the structure and/or the function of the TaYgl protein in the target plant so as to change the leaf color of the target plant.

In the above method, the leaf color change is manifested by a change of the leaves of the plant from green to yellow-green.

The invention further provides a method for cultivating the transgenic plant with yellow-green leaf color.

The invention relates to a method for cultivating transgenic plants with yellow-green leaf color, which comprises the steps of changing the structure and/or function of TaYgl protein in target plants to obtain transgenic plants; compared with the target plant, the leaf color of the transgenic plant is yellow green.

In the above method, the method for modifying the structure and/or function of the TaYgl protein in the plant of interest is to inhibit the expression and/or activity of the TaYgl protein in the plant of interest. Specifically, the method for changing the structure and/or function of the TaYgl protein in the target plant is realized by knocking out or mutating a coding gene of the TaYgl protein in the target plant.

In the above method, the method for modifying the structure and/or function of the TaYgl protein in the target plant is achieved by knocking out a coding gene of the TaYgl protein in the target plant by using a gene editing method.

Specifically, the target sequence of the gene editing is shown as the 325 th-343 th site of the sequence 1 and the 713 th-732 th site of the sequence 1 in the sequence table.

In the above application or method, the plant is a monocotyledon or dicotyledon; the monocotyledon is a gramineous plant; the gramineous plant is a triticum plant, the triticum plant is triticum aestivum, and the triticum plant can be wheat Fielder.

The gene TaYgl for regulating and controlling the wheat leaf color is cloned from the wheat leaf color mutant by a BSR-Seq technology. Transmission electron microscope observation shows that the mutant leaf color is changed due to the fact that a large number of chloroplast outer membrane structures are broken, chloroplast matrixes and thylakoids in the mutant leaf color are exposed outside, and the number of normal chloroplasts is small, so that the gene can influence development of chloroplasts, the leaf color of wheat can be regulated and controlled, and TaYgl is proved to have important research values in the aspects of researching a chloroplast development process and improving photosynthesis efficiency. The invention obtains the wheat mutant with yellow-green leaf color by site-directed mutagenesis of the TaYgl protein coding gene through a gene knockout technology, can more deeply understand the synthesis and photosynthesis process of chlorophyll, is applied to wheat breeding, and has important significance for increasing the yield of wheat and improving the quality of wheat.

Drawings

FIG. 1 shows the seedling and field phenotypes of wild type YZ4110 and mutant ygl. Wherein a is a seedling stage phenotype; b is a field phenotype.

FIG. 2 shows the content determination of chlorophyll a, chlorophyll b and carotene in the leaf of wild type YZ4110 and mutant ygl seedling stage.

FIG. 3 shows transmission electron microscopy of wild-type YZ4110 and mutant ygl chloroplast. Wherein, a and b are transmission electron micrographs of chloroplast of wild type YZ 4110; c. d is a transmission electron microscope picture of mutant ygl chloroplast; in the figure cp represents chloroplast, PG represents starch granule, G represents thylakoid, S represents stroma, CW represents cell wall, and ST represents stromal thylakoid.

FIG. 4 shows the phenotype of transgenic plants of the gene-edited T0 generation. Wherein TW181-18 is a plant with an unedited target site; TW181-36 and TW181-3 are plants with edited target sites.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative experiments in the following examples, all set up in triplicate, and the results averaged.

BsaI restriction enzyme and T4 DNA ligase were purchased from NEB (Beijing) Inc. under the catalog numbers # R0535V and # M0202V, respectively.

Intermediate vector pCBC-MT1T 2: in the literature "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, 14: 327, was stored by the national academy of agricultural sciences crop science institute, Youging teacher, Kouchun, China. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

The pBUE411-TaU3p vector was disclosed in the "doctor Wangzhei (2017) thesis," creation and functional verification of plant genome and base editing toolbox ", and was maintained by Xiuxing teacher, the institute of crop science, China academy of agricultural sciences. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

High fidelity PCR enzyme (KOD FXneo) was purchased from TOYOBO under catalog number KFX-201.

AxyPrep plasmid DNA minikits were purchased from Axygen, catalog No.: AP-MN-P-250.

AxyPrep DNA gel recovery kit was purchased from Axygen, catalog No.: AP-GX-250.

Coli TOP10 competent cells were purchased from Beijing Jianzhu alliance Biotechnology, Inc., catalog number ZC 104-2.

Agrobacterium EHA105 competent cells were purchased from Beijing Jianzhu alliance Biotechnology, Inc., catalog number ZC 142.

Triticale repentium 4110 (abbreviated as YZ4110) is described in the following documents: the comprehensive performance and utilization prospect analysis of the new wheat variety elytrigia 4110 is examined in China, the agricultural bulletin is 2004, 20 (5): 77-78, preserved by the Kouchun Saxiu English teacher of the Chinese academy of agricultural sciences crop science research institute. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

Bainong 3217 is described in the following documents: huangguang, Zhumingzi, Wangshenge, Hurui, Ruzheng, Bainong 3217 wheat tillering and heading characteristics and statistical analysis thereof Baiquan university newspaper, 1983, 1: 1-9, preserved by the Koxiuxin teacher of the institute of crop science, academy of agricultural sciences, China. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

Wheat Fielder describes the following documents: ke Wang, Huiyun Liu, Lipu Du, Xingguo Ye.Generation of marker-free transgenic soybean via an Agrobacterium-mediated co-transformation strategy in commercial Chinese soybean varieties plant Biotechnology journal, 2017, 15, 614-. The public can obtain from the research institute of crop science of Chinese academy of agricultural sciences to repeat the experiment of the application, and can not be used for other purposes.

Example 1 phenotypic identification of wheat leaf color mutant ygl (yellow green leaf) and discovery of encoding Gene

Phenotype of wheat leaf color mutant ygl

In a mutant library constructed by EMS mutagenesis of Elytrigia tritici 4110(YZ4110), a mutant with yellow-green leaf color is screened, the leaf color phenotype can be stably inherited, and the mutant is named ygl (yellow green leaf). In contrast to YZ4110 plants, ygl plants were yellow-green, showing a greenish-yellow trait, as shown in FIG. 1 a. As shown in b in FIG. 1, the ygl plant leaves show more obvious yellow-green leaf color from seedling stage to mature stage, the growth is delayed under the influence of mutation, the plant height is short, the heading stage is late, but the growth cycle can be completed. By examining the contents of chlorophyll a (chlorophyl a), chlorophyll b (chlorophyl b), and Carotene (Carotene) in ygl 4110 and ygl leaves, as shown in fig. 2, it was found that the contents of all three of the ygl leaves were lower than that of YZ 4110.

When observing YZ4110 and ygl leaf blades using transmission electron microscopy, as shown in fig. 3, it was found that the shapes and sizes of chloroplasts of YZ4110 and ygl were not greatly different, but in ygl, a large amount of chloroplast outer membrane structures were broken, the inner chloroplast stroma and thylakoid body were exposed, and the number of normal chloroplasts was small.

Genetic analysis of wheat leaf color mutant ygl and candidate gene acquisition

F derived from 1204 strain YZ4110 Xygl₂The generation group analysis shows that F₂The generation yellow-green leaf plants are 274 plants, the normal leaf plants are 930 plants, and the genetic patterns of a pair of recessive genes are met (X2 is 2.53 < X)² _0.053.84; p ═ 0.11 > 0.05), therefore, the leaf color mutation of ygl is controlled by a pair of recessive genes.

In order to obtain the gene for regulating leaf color in the mutant, the F of 3217 Xygl of Bainong is taken at the seedling stage₃Mixed pools containing about 30 individuals were constructed from phenotypically correct material leaves in the population, and 2 biological replicates were submitted to Kyoto York City York City future technologies, Inc. for BSR-Seq experiments and bioinformatics analysis. Sequencing of each phenotype pool yielded approximately 20Gb data by comparison to the Chinese spring reference sequence, a pool of mixed normal leaf colors and a yellow-green colorThe leaf color mixed pool has 792839 and 777780 genome locus variations respectively, the distribution of the variations on a chromosome is further analyzed, the 671Mb position on the chromosome 7A is very obviously different, and the 745 th nucleotide of the coding region of the gene of the Traes CS7A02G480700 is mutated from G to A by the genomic variations and the gene analysis near the locus, so that the coding protein is changed.

Primers (ygl-F1 and ygl-R1) are designed according to a Chinese spring reference genome sequence (https:// urgi. versalles. inra. fr/blast /) published on the web and are used for detecting the genotype of the site in the parent material and the 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'

The PCR reaction (20. mu.L) was as follows:

the PCR reaction was performed in an ABI veriti 96-well PCR instrument and the reaction procedure was as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 98 ℃ for 5s, annealing at 62 ℃ for 30s, extension at 68 ℃ for 1min, and 35 cycles; extension at 68 ℃ for 5 min.

And recovering and purifying the PCR product by using an AxyPrep DNA gel recovery kit, and sending the PCR product to Beijing Optimalaceae New Biotechnology Limited company for sequencing, wherein a sequencing primer is ygl-SeqF 1: 5'-TGAGAAGGCGCTCACCGAAG-3' are provided. The results showed that the genotype at position 745 of the coding region of the TracescS 7A02G480700 gene in YZ4110 was GG, and the genotype at position 745 of the coding region of the TracescS 7A02G480700 gene in ygl was AA.

Further sequencing analysis of the backcross population shows that the 745 th genotype of the coding region of the gene of the TramesCS 7A02G480700 of 43 homozygous individuals with green leaves is GG, and the 745 th genotype of the coding region of the gene of the TramesCS 7A02G480700 of 34 homozygous individuals with yellow-green leaves is AA. Then, the Traes CS7A02G480700 gene is determined as a candidate gene of wheat leaf color and named TaYgl, the nucleotide sequence of the gene is shown as a sequence 1 in a sequence table, and the sequence of a gene coding region is shown as DNA molecules shown as 72 th-1337 th sites of the sequence 1 in the sequence table; the protein coded by the gene is named TaYgl, and the amino acid sequence of the protein is shown as a sequence 2 in a sequence table.

Example 2 acquisition and characterization of transgenic plants

Construction of recombinant plasmid

Firstly, selecting two Target sites of gene editing, namely Target1 and Target1, according to the sequence characteristics of TraveCS 7A02G480700, wherein the sequences of the two Target sites are as follows:

target 1: 5'-ACCCGTTCCCGGCGATCGT-3' (sequence 1, position 325-

Target 2: 5'-CAACAGGGGGATACTGTATG-3' (sequence 1 at position 713 and 732)

(II) constructing the two target sites in the step (I) into an expression vector pBUE411-TaU3 p:

amplifying a PCR reaction system (20 mu L) and a PCR reaction program in the embodiment 1 by using an intermediate vector pCBC-MT1T2 as a template and ygl-MT1T2-F and ygl-MT1T2-R as primers, and recovering and purifying the obtained PCR product;

wherein, the primer is:

ygl-MT1T2-F：

5′-aataatggtctcaagcgACCCGTTCCCGGCGATCGTgttttagagctagaaatagc-3′；

ygl-MT1T2-R：

5′-attattggtctctaaacCATACAGTATCCCCCTGTTGTcgcttcttggtgcc-3′。

recovering the purified PCR product, and establishing an enzyme digestion-connection system to obtain a product:

and (III) transforming the product obtained in the step (II) into escherichia coli TOP10 competent cells, selecting positive clones, and sending the positive clones to Beijing Ongji scientific New Biotechnology Limited company for sequencing, wherein sequencing primers are as follows:

pBUE411-TaU3p-SeqF1：5′-TTTCCCAGTCACGACGTTGT-3′

pBUE411-TaU3p-SeqR1：5′-ATCTCTAGAGAGGGGCACGA-3′

and (3) selecting positive clones with target site sequences all correctly constructed on a pBUE411-TaU3p vector, and extracting plasmids by using an AxyPrep plasmid DNA small-scale kit to obtain recombinant plasmids.

Second, recombinant Agrobacterium obtaining

The recombinant plasmid is transformed into an agrobacterium EHA105 competent cell to obtain recombinant agrobacterium, and the specific experimental steps are described according to the specification of an escherichia coli TOP10 competent cell (product of union biotechnology, Beijing village).

Meanwhile, the empty vector pBUE411-TaU3p is transformed into agrobacterium EHA105 competent cells to obtain control agrobacterium.

Third, agroinfection

Taking seeds of wheat Fielder, shelling, sterilizing, spreading on a callus induction culture medium for two weeks, picking out callus for subculture for two weeks until callus particles with the diameter of about 2mm grow out.

(II) culturing the recombinant agrobacterium obtained in the step two in LB liquid medium (containing rifampicin 50mg/L and kanamycin 50mg/L) overnight to enable OD to be achieved₆₀₀The value is about 0.6-0.8, and a bacterial liquid is obtained.

And thirdly, taking about 3mL of bacterial liquid 4000rmp, centrifuging for 3 minutes, removing supernatant, suspending the bacterial liquid in about 20mL of AMM (containing 100 mu mol/L acetosyringone) liquid culture medium, and shaking at 28 ℃ and 150rpm for two hours to obtain the target bacterial liquid.

(IV) methods for padding are detailed in the literature Yuji Ishida, Masako Tsunashima, Yukoh Hiei, Toshihiko Komari.Wheat (Triticum aestivum L.) Transformation Using Imformation organisms. methods in Molecular Biology, 2015, 1223, 189-198: soaking the callus particles obtained in the step (I) in the target bacterial liquid for 20 minutes, and then placing the callus particles on a co-culture medium with a layer of filter paper. Three days later, the callus is washed with sterilized distilled water for three times, each time lasts for about 20 minutes, then washed with sterilized water containing 500mg/L of carboxybenzyl twice, each time lasts for 30 minutes, the washing liquid is repeated for many times until the washing liquid is clear, the callus is put on sterile filter paper to be dried in the air, and then the callus is put on a sieve culture medium. After two weeks, the calli were transferred to a two-sieve medium, and after another two weeks, the calli were transferred to a three-sieve medium to obtain resistant calli.

And (V) transferring the resistant callus to a differentiation culture medium to obtain a differentiated seedling.

Sixthly, transferring the differentiated plantlets to 1/2MS culture medium for rooting, and then transferring the plantlets to a flowerpot of an incubator to obtain T of an experimental group₀And (5) plant generation.

Simultaneously carrying out the experiment on the control agrobacterium obtained in the step two to obtain T of a control group₀And (5) plant generation.

Fourth, identification of transgenic plants

(I) PCR molecular characterization

Extracting T of experimental group and control group₀The genomic DNA of the plant leaf was amplified by PCR reaction system (20. mu.L) and PCR reaction program in example 1 using ygl-CRISPR-F1 (5'-ACATCGTCCGGCCTATCCAAGTC-3') and ygl-CRISPR-R1 (5'-GTCACGATGTCCCTTCCCTTTAG-3') as primers, the PCR product was recovered and purified, sent to Beijing Pongku New technology Co., Ltd for sequencing, and the sequencing result was analyzed by DSDecodeM (http:// skl.scau.edu.cn/dsdecode /) to confirm that the target site gave the edited plant. Through detection, 22T strains exist in the experimental group₀The target sites of the generation plants are edited, 20T plants₀The target sites of the generations were not edited.

(II) phenotypic characterization

The plants with the edited 2 target sites and the plants with the unedited 1 target sites were cultured under parallel conditions, and the color of the leaves was observed, as shown in fig. 4, the leaves of the plants TW181-36 (both chromosomal target sites obtained 4bp deletion variation) and TW181-3 (both chromosomal target sites obtained 2bp deletion and 4bp deletion variation, respectively) with the edited target sites appeared yellow-green, while the leaves of the plants (TW181-18) with the unedited target sites still showed green of normal plants, thus demonstrating that the mutant phenotype in ygl is caused by the mutation of TaYgl.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> institute of crop science of Chinese academy of agricultural sciences

<120> TaYgl protein and application of coding gene thereof in wheat leaf color regulation

<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 (4)

1. A method for changing the leaf color of a plant, characterized by: knocking out or mutating a coding gene of a protein with an amino acid sequence shown as a sequence 2 in a sequence table in a target plant to change the leaf color of the target plant;

the mutation is to mutate the 745 th site of the coding region of the coding gene of the protein from 'G' to 'A';

the leaf color change is represented by the change of leaves of the plant from green to yellow-green;

the plant is a wheat plant.

2. A method for cultivating transgenic plants with yellow-green leaf color is characterized in that: knocking out a coding gene of a protein with an amino acid sequence shown as a sequence 2 in a sequence table in a target plant to obtain a transgenic plant; compared with the target plant, the leaf color of the transgenic plant is yellow green;

the plant is a wheat plant.

3. The method according to claim 1 or 2, characterized in that: the method for knocking out the coding gene of the protein with the amino acid sequence shown as the sequence 2 in the sequence table in the target plant is realized by knocking out the coding gene of the protein in the target plant by using a gene editing method.

4. The method of claim 3, wherein: the target sequences of the gene editing are DNA molecules shown in the 325 th-343 bit of the sequence 1 and the 713 th-732 bit of the sequence 1 in the sequence table.

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