Wheat transcription factor MYB106 gene and its application
Genetically engineered the gene editing material of wheat transcription factor MYB106, which solved the problem of low resistance to wheat stripe rust in traditional breeding methods, achieved efficient improvement of disease-resistant varieties, and broadened the research scope of disease-resistant materials.
Patent Information
- Application Number
- CN202411819718.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-11
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2044-12-11
AI Technical Summary
The prior art is difficult to effectively utilize the wheat transcription factor MYB106 gene to improve wheat resistance to stripe rust, and traditional disease-resistant breeding methods have problems of long time and low efficiency.
Through genetic engineering, the MYB106 gene of wheat transcription factor was screened and analyzed, the MYB106 gene editing material was created, the MYB106 gene was knocked out to enhance the resistance of wheat to striped rust bacteria, and the disease-resistant varieties were cultivated using Agrobacterium-mediated genetic transformation technology.
In a short period of time, the stable genetic resistance of wheat to stripe rust has been effectively improved, new ways to breed plants with disease resistance, breakthroughs in interspecies reproductive isolation, and provides theoretical guidance for disease resistance materials.
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Figure CN119685336B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant genetic 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 grass plant, 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 yields. 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 way, we not only continue to explore key genes for wheat stripe rust resistance, but also continuously explore and knock out wheat susceptible genes to create disease-resistant varieties. This is a long-term battle faced by wheat disease-resistant breeders.
[0003] Transcription factors are proteins that recognize and bind to specific regions of DNA, using them as transcription start sites to initiate or repress transcription of associated genes. The MYB family of transcription factors is one of the largest and most multifunctional transcription factor families in plants. MYB transcription factors contain a typical MYB domain at their N-termini, and are classified into different subgroups based on the number of R-repeat sequences within the MYB domain. Their C-terminal domains, on the other hand, vary significantly, resulting in diverse functions. Upon activation by environmental signals, MYB transcription factors can bind to cis-acting elements (MYBCORE) and AC-boxes in the promoter regions of downstream target genes, either independently or through interactions with other proteins, to regulate the expression of downstream stress-response genes, thereby modulating plant tolerance to adverse stresses. MYB responds to abiotic stresses (such as salt, drought, extreme temperatures, nutrient deficiency, and heavy metals) and biotic stresses (such as pathogens) by participating in signaling pathways involving abscisic acid (ABA), brassinosteroids (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 cultivating new wheat materials resistant to stripe rust. Summary of the Invention
[0005] The present invention screened out a wheat transcription factor MYB106 gene through genetic engineering means, and revealed its mechanism of action by functional analysis of the wheat transcription factor MYB106 gene and its encoded protein. This has important implications for 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 the 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 from the amino acid sequence in 1), said derivative amino acid sequence having the activity of the amino acid sequence shown in 1); or
[0013] 3) An amino acid sequence having at least 80% homology to 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 host cell of the wheat transcription factor MYB106 gene of the present invention is provided.
[0017] In a fourth aspect, the present invention provides an application of the wheat transcription factor MYB106 gene in creating wheat transcription factor MYB106 gene editing materials.
[0018] In a fifth aspect, the present invention provides the use of the wheat transcription factor MYB106 gene in breeding wheat stripe rust-resistant varieties.
[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 resistance to stripe rust.
[0021] Preferably, the wheat transcription factor MYB106 gene plays a negative regulatory role in wheat stripe rust resistance.
[0022] In a sixth aspect, a recombinant vector or recombinant bacteria of the wheat transcription factor MYB106 gene of 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 will be understood by those skilled in the art that nucleotide sequences are generally presented in 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 in which amino acids are arranged in a protein. This sequence is determined by genetic information, specifically, by genes in DNA or RNA through the processes 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 a 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 (Lys) are Lys and K, respectively.
[0026] In this application, the term "gene" refers to the primary mechanism of genetic variation. It is the fundamental unit of genetic variation, consisting of the sequence of numerous base pairs within a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule that determine biological traits. Genes express the genetic information they carry by directing protein synthesis, thereby controlling the expression of traits in an individual organism. Specifically, genes are located on chromosomes, arranged linearly and occupying a specific position within the chromosome. Genes possess a dual nature: they can faithfully replicate themselves to maintain the basic characteristics of an organism, while also potentially mutating during reproduction. Mutated genes can also continue to replicate and be passed on to future generations. Genes not only transmit genetic information to the next generation through replication but also enable this information to be expressed in the molecular structure of proteins. Therefore, genes are the fundamental unit of genetic variation. A gene consists of only one DNA molecule, but a single DNA molecule contains multiple genes. Genes are segments of DNA molecules with heritable effects that determine the expression of traits.
[0027] As used herein, the term "protein" refers to protein, which is the material basis of life. Protein is an organic macromolecule, the fundamental organic substance that constitutes cells, and the primary driver of life activities. Without protein, there would be no life. Amino acids are the basic building blocks of protein. Protein is a substance that is closely linked to life and all forms of life activities.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[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 Figure 2 shows the expression profile of the wheat transcription factor MYB106 gene after the second leaf of the wheat variety Fielder was inoculated with the avirulent stripe rust race CYR23 (incompatible interaction) and the virulent stripe rust race CYR32 (compatible interaction); ** indicates P value < 0.01, and * indicates P value < 0.05.
[0034] Figure 2 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; and 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 on chromosome 5 of wild-type wheat Fielder; 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 represent the editing conditions of mutant plants. DETAILED DESCRIPTION
[0037] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also 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 stripe rust race CYR23 and the stripe rust race CYR32 were inoculated on the second leaf of wheat at the two-leaf and one-heart stage by the smearing method and kept in the dark and moist for 24 hours. A water control group was set up. The inoculated leaves were collected 24 hours, 48 hours, 72 hours, 96 hours and 120 hours after inoculation and frozen at -80℃.
[0042] Total RNA was extracted from the collected samples and reverse transcribed into cDNA first strand using reverse transcriptase. Real-time quantitative PCR was performed using cDNA as template and wheat elongation factor TaEF as internal reference gene and MYB106 specific fragment. 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 stripe rust race CYR23 and the stripe rust race CYR32 were inoculated on the second leaf of wheat 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 leaves inoculated with fungi were collected at 24h, 48h, 72h, 96h and 120h after inoculation and stored at -80℃.
[0056] Total RNA from the sample was extracted and reverse transcribed into the first strand of cDNA using reverse transcriptase. Using cDNA as a template, specific primers for the wheat transcription factor MYB106 gene were designed and amplified and sequenced using a conventional PCR instrument. If sequencing was correct, a DNA fragment containing the open reading frame of the wheat transcription factor MYB106 gene was obtained. The nucleotide sequence of the wheat transcription factor MYB106 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the wheat transcription factor MYB106 gene is shown in SEQ ID NO: 2.
[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 the wheat transcription factor MYB106 gene were designed in the wheat genome, and fragments target-1 and target-2 were synthesized and annealed. The intermediate vector sgRNA was simultaneously digested with BtgZ1, and the digested vector was ligated with the synthesized target-1 fragment using T4 ligase. The ligation product was transformed into Escherichia coli using the DH5α strain. When the plaques were grown to the point where plaques could be picked, 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 digested with Bsa1, and the digested vector was ligated with the target-2 fragment using T4 ligase. The ligation product was transformed into E. coli as described 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 then ligated with the final vector Cas9 using LR reaction. The above steps of 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 tumefaciens strain EHA105 and used to infect immature embryos of wheat (Fielder) as the recipient. Using Agrobacterium-mediated genetic transformation, the wheat transcription factor MYB106 gene-edited material was created.
[0066] Using NOS primers, L35, L38, and L55 were tested, and all three lines were positive. Positive plants from L35, L38, and L55 were cultured to the T2 generation. DNA was extracted from the T2 generation wheat to determine the extent of gene editing.
[0067] T2 seeds of L35, L38, and L55 were sown in 7×7×8 cm pots, with 9 seeds per pot. Wild-type material was also sown at the same time. When the T2 materials of L35, L38, and L55 had grown to two leaves and one heart, they were inoculated with the compatibility race CYR32 on the second leaf. The plants were incubated in the dark at 12°C for 24 hours, then transferred to normal growth conditions (16 hours of light, 14°C; 8 hours of darkness, 10°C as one growth cycle). The phenotypes were 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 were 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 hypersensitive 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 embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concepts of the present invention are within the scope of protection of the present invention.
Claims
1. Wheat transcription factors MYB106 The application of the gene in breeding wheat stripe rust-resistant varieties is characterized in that: The wheat transcription factor MYB106 The gene encodes the wheat transcription factor MYB106 protein, which MYB106 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The amino acid sequence of the wheat transcription factor MYB106 protein is shown in SEQ ID NO: 2; Knockout of the wheat transcription factor MYB106 Genetically enhanced wheat resistance to stripe rust; The wheat transcription factor MYB106 The gene plays a negative regulatory role in wheat stripe rust resistance.
2. The use according to claim 1, characterized in that The wheat transcription factor MYB106 The gene expression was induced by wheat stripe rust infection.
3. A method for cultivating transgenic wheat with enhanced resistance to stripe rust, characterized in that: Including knockout of wheat transcription factor MYB106 The steps to genetically modify wheat; The wheat transcription factor MYB106 The gene encodes the wheat transcription factor MYB106 protein, which MYB106 The nucleotide sequence of the gene is shown in SEQ ID NO: 1; The amino acid sequence of the wheat transcription factor MYB106 protein is shown in SEQ ID NO: 2.