Wheat transcription factor MYB106 gene and application thereof

Abstract

The invention belongs to the technical field of plant genetic engineering, and relates to a wheat transcription factor MYB106 gene and application thereof. The wheat transcription factor MYB106 gene provided by the invention comprises the following steps: a) encoding a wheat transcription factor MYB106 protein; and b) a nucleotide sequence as shown in SEQ ID NO: 1. The wheat transcription factor MYB106 gene is utilized to improve and cultivate a new wheat stripe rust resistant variety, and the research range of new wheat stripe rust resistant materials is widened.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering and relates to a wheat transcription factor MYB106 gene and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the main sources of food for people. As a plant of the Poaceae family, wheat is an annual or biennial herb, mainly divided into three species: common wheat (Triticum aestivum), cold wheat (Triticum spelta) and durum wheat (Triticum durum). However, wheat stripe rust is one of the important reasons for reducing wheat production. Wheat stripe rust is caused by the wheat-specific type of Puccinia striiformis f.sp.tritici. Wheat stripe rust is a type of wheat fungal disease that spreads quickly, is widely distributed, and has frequent toxicity mutations. In order to prevent and control wheat stripe rust in a long-term, effective, green and economical manner, we not only continue to explore the key genes for wheat stripe rust resistance, but also continue to explore and knock out wheat susceptible genes to create disease-resistant varieties. This is a protracted battle faced by wheat disease-resistant breeders.

[0003] Transcription factors are a class of proteins that can recognize and bind to specific regional structures of DNA, making them serve as transcription start sites, and start or inhibit the transcription process of genes associated with them. The MYB transcription factor family is one of the largest multifunctional transcription factor families in plants. The N-terminus of MYB transcription factors contains a typical MYB domain, which is divided into different subgroups according to the number of R repeat sequences in the MYB domain; while the C-terminal domains vary greatly and have functional diversity. After being activated by external environmental signals, MYB transcription factors can bind to the cis-acting elements MYBCORE and AC-box in the promoter region of downstream target genes alone or through interactions with other proteins, regulate the expression of downstream stress response-related genes, and thus regulate the tolerance of plants to adverse stress. MYB responds to abiotic stresses (such as salt, drought, extreme temperature, nutrient deficiency, heavy metals) and biotic stresses (such as pathogens) by participating in signaling pathways such as abscisic acid (ABA), brassinolide (BR), jasmonic acid (JA), and reactive oxygen species (ROS).

[0004] Therefore, studying how MYB transcription factors regulate disease resistance signaling pathways during the interaction between wheat and stripe rust and revealing the molecular mechanism of transcription factors in resistance to stripe rust are of great significance for improving and breeding new wheat materials resistant to stripe rust. Summary of the invention

[0005] The present invention screens out a wheat transcription factor MYB106 gene by means of genetic engineering, and reveals its mechanism of action by functional analysis of the wheat transcription factor MYB106 gene and its encoded protein, which plays an important role in improving wheat breeding efficiency, using the wheat transcription factor MYB106 gene to improve and cultivate new wheat stripe rust-resistant varieties, and broadening the research scope of new wheat stripe rust-resistant materials.

[0006] To achieve this technical purpose, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a wheat transcription factor MYB106 gene, wherein the wheat transcription factor MYB106 gene:

[0008] a) Encodes wheat transcription factor MYB106 protein;

[0009] b) having the nucleotide sequence shown in SEQ ID NO: 1.

[0010] In a second aspect, the present invention provides a wheat transcription factor MYB106 protein, wherein the wheat transcription factor MYB106 protein:

[0011] 1) having the amino acid sequence shown in SEQ ID NO: 2;

[0012] 2) a derivative amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence in 1), wherein the derivative amino acid sequence has the activity of the amino acid sequence shown in 1); or

[0013] 3) An amino acid sequence having at least 80% homology with the amino acid sequence in 1).

[0014] Preferably, the amino acid sequence has at least 90% homology with the amino acid sequence in 1).

[0015] More preferably, the amino acid sequence has at least 95% homology with the amino acid sequence in 1).

[0016] In a third aspect, a polynucleotide construct or a host cell of the wheat transcription factor MYB106 gene of the present invention is provided.

[0017] In a fourth aspect, the invention provides an application of the wheat transcription factor MYB106 gene in creating wheat transcription factor MYB106 gene editing material.

[0018] In a fifth aspect, the invention provides the use of the wheat transcription factor MYB106 gene in breeding wheat varieties resistant to stripe rust.

[0019] Preferably, the wheat transcription factor MYB106 gene is induced to express by infection with wheat stripe rust.

[0020] Preferably, gene editing of the wheat transcription factor MYB106 gene enhances wheat's resistance to stripe rust.

[0021] Preferably, the wheat transcription factor MYB106 gene plays a negative regulatory role in wheat resistance to stripe rust.

[0022] In a sixth aspect, a recombinant vector or recombinant bacteria of the wheat transcription factor MYB106 gene described in the present invention is provided.

[0023] In a seventh aspect, a method for cultivating transgenic wheat with enhanced resistance to stripe rust is provided, comprising the step of knocking out the wheat transcription factor MYB106 gene of the present invention in wheat to obtain transgenic wheat.

[0024] In the present invention, the term "nucleotide sequence" or "polynucleotide" refers to a linear polymer of natural or synthetic nucleotide residues linked by phosphodiester bonds or their analogs. It can be single-stranded or double-stranded, and includes RNA, DNA (e.g., genomic, cDNA or synthetic), analogs thereof, or combinations thereof. It should be understood by those skilled in the art that the order of representation of nucleotide sequences is usually 5'-3' order from left to right, and, unless otherwise indicated, "A" refers to deoxyadenosine, "C" refers to deoxycytidine, "G" refers to deoxyguanosine, "T" refers to deoxythymidine, and "U" refers to deoxyuridine. Typically, nucleotide sequences contain four natural deoxynucleotides or four natural ribonucleotides; however, they may also contain non-natural nucleotide analogs.

[0025] In the present invention, the term "amino acid sequence" refers to the order of arrangement of amino acids in a protein. This sequence is determined by genetic information, specifically, by genes in DNA or RNA through the process of transcription and translation. Amino acid sequences are usually represented by a string of letters, each letter representing a specific amino acid. These sequences are arranged in the direction from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus). In biology, there are 20 different standard amino acids, each of which has a specific single-letter or three-letter abbreviation. For example, the three-letter and single-letter abbreviations for methionine (Lysine) are Lys and K, respectively.

[0026] In the present invention, the term "gene" is the main substance of genetic variation. It is the basic unit that controls traits and is composed of many different base pair arrangements on deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules that determine biological traits. Genes express the genetic information they carry by guiding the synthesis of proteins, thereby controlling the expression of traits of individual organisms. Specifically, genes are located on chromosomes and are arranged linearly. They are genetic units that occupy a certain position on chromosomes. Genes have duality, that is, they can faithfully replicate themselves to maintain the basic characteristics of organisms; at the same time, they may mutate during reproduction. Mutated genes can also continue to replicate and pass on to offspring. Genes can not only pass genetic information to the next generation through replication, but also express genetic information on the molecular structure of proteins. Therefore, genes are the basic units that control traits. A gene has only one DNA molecule, but a DNA molecule contains multiple genes. Genes are fragments on DNA molecules that have genetic effects, and they determine the expression of traits.

[0027] In the present invention, the term "protein" refers to protein, which is the material basis of life, an organic macromolecule, the basic organic matter constituting cells, and the main bearer of life activities. Without protein, there is no life. Amino acids are the basic building blocks of protein. It is a substance closely associated with life and various forms of life activities.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] (1) The present invention analyzes the expression pattern of the wheat transcription factor MYB106 gene and determines that the wheat transcription factor MYB106 gene is induced to express by stripe rust infection during the interaction between wheat and stripe rust.

[0030] (2) The present invention created a wheat transcription factor MYB106 gene editing material, and clarified that knocking out the wheat transcription factor MYB106 gene enhanced wheat resistance to stripe rust, indicating that the wheat transcription factor MYB106 gene plays a negative regulatory role in the wheat resistance to stripe rust.

[0031] (3) Compared with traditional disease-resistant breeding technology, plant disease-resistant genetic engineering technology based on molecular biology has opened up a new path for plant disease-resistant breeding. It has made certain breakthroughs in reproductive isolation between species and incompatibility in distant hybridization, and can efficiently achieve targeted improvement of target traits in a relatively short period of time, providing theoretical guidance for the cultivation of stable genetic disease-resistant materials.

[0032] The invention has the characteristics of high operability and wide application, and provides an effective and stable method for plant genetic improvement and quality breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is an analysis diagram of the expression profile of the wheat transcription factor MYB106 gene after the second leaf of the wheat variety Fielder was inoculated with the non-toxic race CYR23 of stripe rust (non-compatible interaction) and the virulent race CYR32 of stripe rust (compatible interaction); among them, ** indicates P value < 0.01, and * indicates P value < 0.05.

[0034] Figure 2 This is a schematic diagram of the PCR positive plant detection results of the T2 generation wheat transcription factor MYB106 gene-edited plants; among them, L35, L38 and L55 are gene-edited plants Line35, Line38 and Line55 respectively; M is a DNA Marker.

[0035] Figure 3 Schematic diagram of the phenotypic results of T2 generation wheat transcription factor MYB106 gene-edited plants inoculated with stripe rust fungus CYR32; among them, Fielder is a wild-type wheat variety; CYR32 is the stripe rust physiological race CYR32 inoculated with wild-type wheat varieties and wheat transcription factor MYB106 gene-edited plants L35, L38, and L55; TaMYB106-KO is a wheat transcription factor MYB106 gene-edited plant; L35 is a wheat transcription factor MYB106 gene-edited plant L35; L38 is a wheat transcription factor MYB106 gene-edited plant L38; L55 is a wheat transcription factor MYB106 gene-edited plant L55.

[0036] Figure 4 Schematic diagram of the editing results of T2 generation wheat transcription factor MYB106 gene-edited plants; among them, target-1 and target-2 are the target sequences of the wheat transcription factor MYB106 gene; WT-5A, WT-5B and WT-5D are the three copy sequence fragments A, B, and D of wild-type wheat Fielder on chromosome 5; TaMYB106-KO-5A, TaMYB106-KO-5B and TaMYB106-KO-5D are the three copy sequence fragments A, B, and D on chromosome 5 of mutant plants; -2bp, -4bp, and -4bp are the editing conditions of mutant plants. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] All experimental methods and detection methods in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0039] Example 1

[0040] This example describes and analyzes the expression profile of the wheat transcription factor MYB106 gene (MYB106).

[0041] Fielder wheat varieties grown in normal soil were used as materials. The second leaf was inoculated with stripe rust race CYR23 and race CYR32 respectively at the two-leaf and one-heart stage by the smearing method and kept in the dark for 24 hours. A water control group was set up. The inoculated leaves were collected at 24h, 48h, 72h, 96h and 120h after inoculation and frozen at -80℃.

[0042] Total RNA of the collected samples was extracted and reverse transcriptase was used to reverse transcribe the total RNA into the first strand of cDNA. Real-time quantitative PCR was performed using cDNA as a template and wheat elongation factor TaEF as an internal reference gene, using the MYB106 specific fragment. The results are shown in Figure 1 As shown by Figure 1 It can be seen that the wheat transcription factor MYB106 gene is induced to express by stripe rust infection during the interaction between wheat and stripe rust.

[0043] TaEF internal reference gene primers:

[0044] TaEF-F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3';

[0045] TaEF-R: 5'-ACTCATGGTGCATCTCAACGGACT-3'.

[0046] Wheat transcription factor MYB106 gene (MYB106) specific fragment primers:

[0047] MYB106-qRT-F: 5'-GGGCTTCGAATCCTGGTGAT-3';

[0048] MYB106-qRT-R: 5'-ACAGCGGAAAGAGCAACTACT-3'.

[0049] Reverse transcription reaction conditions:

[0050] Incubate at 42°C for 1 hour; heat at 95°C for 5 minutes.

[0051] Real-time fluorescence quantitative PCR reaction conditions:

[0052] Pre-denaturation: 95°C, 3 min; denaturation: 95°C, 30 sec; annealing: 60°C, 30 sec; extension: 72°C, 30 sec; 40 cycles.

[0053] Example 2

[0054] This example describes the isolation and cloning of the wheat transcription factor MYB106 gene.

[0055] Fielder wheat varieties grown in normal soil were used as materials. The second leaf was inoculated with stripe rust species CYR23 and CYR32 respectively at the two-leaf and one-heart stage of wheat by the smearing method and kept in the dark for 24 hours. A water control group was set up. The leaves inoculated with bacteria were collected at 24h, 48h, 72h, 96h, and 120h after inoculation and stored at -80℃.

[0056] Total RNA of the sample was extracted, and reverse transcriptase was used to reverse transcribe the total RNA into the first strand of cDNA. Using cDNA as a template, specific primers of wheat transcription factor MYB106 gene were designed and amplified and sequenced using a common PCR instrument. If the sequencing was correct, a DNA fragment containing the open reading frame of wheat transcription factor MYB106 gene was obtained. The nucleotide sequence of wheat transcription factor MYB106 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by wheat transcription factor MYB106 gene is shown in SEQ ID NO: 2. Among them,

[0057] Wheat transcription factor MYB106 gene (MYB106) specific primers:

[0058] MYB106-F: 5'-ATGGGGAGGCCTCCGTGC-3';

[0059] MYB106-R: 5'-TTAGAAGAACTCACTGGGGTCCCCT-3'.

[0060] Common PCR reaction conditions:

[0061] Pre-denaturation: 95°C, 5 min; denaturation: 95°C, 30 sec; annealing: 56°C, 30 sec; extension: 72°C, 2 min; 35 cycles.

[0062] Example 3

[0063] This example describes the creation, editing analysis and disease resistance identification of mutant plants of the wheat transcription factor MYB106 gene.

[0064] Two editing targets of wheat transcription factor MYB106 gene were designed in the wheat genome, and fragments target-1 and target-2 were synthesized and annealed. At the same time, the intermediate vector sgRNA was cut with BtgZ1, and then the cut vector was connected with the synthetic fragment target-1 with T4 ligase, and the connection product was transformed into Escherichia coli with DH5α as the strain. When it grew to the point where plaques could be picked, the monoclonal positive colonies were detected and sent for sequencing by shaking. The monoclonal plaques with correct sequencing were cultured and the plasmids were extracted. The vector sgRNA-target-1 was cut with Bsa1, and then the cut vector was connected with the fragment target-2 with T4 ligase. The connection product was transformed into E. coli as above, sent for sequencing by shaking, and the plasmid was extracted to obtain the intermediate vector sgRNA-target-1-target-2. The intermediate vector sgRNA-target-1-target-2 was connected with the final vector Cas9 by LR reaction, and the above steps such as E. coli transformation and plasmid extraction were continued to obtain the final vector MYB106-Cas9.

[0065] The final vector MYB106-Cas9 was introduced into Agrobacterium with strain EHA105 to infect wheat embryos with Fielder as the recipient variety. The genetic transformation technology mediated by Agrobacterium was used to create the wheat transcription factor MYB106 gene editing material.

[0066] Using NOS primers, L35, L38, and L55 were tested, and all three lines were positive. The positive plants of L35, L38, and L55 were cultured and planted to the T2 generation. DNA was extracted from the T2 generation wheat to determine the gene editing situation.

[0067] The T2 generation seeds of L35, L38, and L55 were sown in 7×7×8cm pots, with 9 seeds in each pot, and wild-type materials were sown at the same time. When the T2 generation materials of L35, L38, and L55 grew to two leaves and one heart, the affinity race CYR32 was inoculated on the second leaf, and cultured in the dark at 12℃ for 24h, and then moved to normal growth conditions (16h of light, 14℃; 8h of darkness, 10℃ for one growth cycle) for culture, and the phenotype was observed after 14 days.

[0068] The wheat transcription factor MYB106 gene provided in the embodiment of the present invention is used for improving wheat rust-resistant varieties. The transgenic plants obtained by Agrobacterium-mediated genetic transformation are subjected to PCR detection. The results are as follows: Figure 2 As shown, L35, L38, and L55 are all positive plants. Figure 4 As shown, the specific editing conditions of L35, L38, and L55 mutant plants are -2bp, -4bp, and -4bp. Figure 3As shown, compared with the control, the mutant plants had significantly reduced spore production and increased allergic necrosis, indicating that knocking out the wheat transcription factor MYB106 gene enhanced wheat resistance to stripe rust.

[0069] Gene editing target design:

[0070] MYB106-target-1-F: 5'-ACTTCATGCTCGTCTCCTACATCC-3';

[0071] MYB106-target-1-R: 5'-AAACGGATGTAGGAGACGAGCATG-3';

[0072] MYB106-target-2-F: 5'-ACTCGCAAGAGCTGCCGGCTTCGG-3';

[0073] MYB106-target-2-R: 5'-AAACCCGAAGCCGGCAGCTCTTGC-3'.

[0074] Primers for detecting gene-edited positive plants:

[0075] NOS-F: 5'-AAGCACATACGTCAGAAACATTAT-3';

[0076] NOS-R: 5'-TGGGTGAGATTCCTTGAAGTTGAGTA-3'.

[0077] The above-described embodiments are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A wheat transcription factor MYB106 gene, characterized in that: The wheat transcription factor MYB106 gene: a) Encodes wheat transcription factor MYB106 protein; b) having the nucleotide sequence shown in SEQ ID NO:

1.

2. Wheat transcription factor MYB106 protein, characterized in that The wheat transcription factor MYB106 protein: 1) having the amino acid sequence shown in SEQ ID NO: 2; 2) a derivative amino acid sequence obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence in 1), wherein the derivative amino acid sequence has the activity of the amino acid sequence shown in 1); or 3) An amino acid sequence having at least 80% homology with the amino acid sequence in 1).

3. A polynucleotide construct or host cell containing the wheat transcription factor MYB106 gene as claimed in claim 1.

4. Use of the wheat transcription factor MYB106 gene according to claim 1 in creating wheat transcription factor MYB106 gene editing materials.

5. Use of the wheat transcription factor MYB106 gene according to claim 1 in breeding wheat varieties resistant to stripe rust.

6. The use according to claim 5, characterized in that: The wheat transcription factor MYB106 gene is induced to express by wheat stripe rust infection.

7. The use according to claim 5, characterized in that: Gene editing of the wheat transcription factor MYB106 gene enhanced wheat resistance to stripe rust.

8. The use according to claim 5, characterized in that: The wheat transcription factor MYB106 gene plays a negative regulatory role in wheat resistance to stripe rust.

9. A recombinant vector or recombinant bacterium containing the wheat transcription factor MYB106 gene as claimed in claim 1.

10. A method for cultivating transgenic wheat with enhanced resistance to stripe rust, characterized in that: The method comprises the step of knocking out the wheat transcription factor MYB106 gene according to claim 1 in wheat to obtain transgenic wheat.

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