Wheat taCKS1 protein, coding gene and mutant sequence and application thereof in regulating wheat kernel development

Abstract

The application discloses a wheat TaCKS1 protein, a coding gene and application of a mutant sequence thereof in regulation of wheat kernel development, belongs to the technical field of biology, and the amino acid sequence of the TaCKS1 protein is shown as SEQ ID NO. 7. The application clones SUC1 / CKS1 homologous gene TaCKS1 from Arabidopsis thaliana to wheat, and a tacks1 mutant is screened in a EMS mutagenized population of Jimai 20, and it is found through analysis of kernel phenotypes of the tacks1 mutant and the wild type JM20 that the kernel length, kernel width, 1000-grain weight and yield per plant of the mutant are all increased compared with the wild type. The TaCKS1 gene of wheat is identified for the first time, the function of the TaCKS1 gene in negatively regulating the kernel length, kernel width, 1000-grain weight and yield per plant of wheat is verified through the EMS mutant, and new gene resources are provided for wheat breeding.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of wheat TaCKS1 protein, its encoding gene and its mutant sequences in regulating wheat grain development. Background Technology

[0002] Wheat (Triticum aestivum L.) belongs to the genus Triticum in the family Gramineae. Originating in western Asia, it is distributed worldwide, adapting to different latitudes and climatic conditions. Wheat grains are rich in carbohydrates, and their protein content is significantly higher than other cereal crops. Simultaneously improving wheat yield and quality is crucial for ensuring national food security and public health. However, given the trends of global climate change, population growth, changing dietary habits, and increased biofuel consumption, a significant gap remains between total wheat production and demand. Therefore, cultivating and developing high-yielding and high-quality wheat varieties is of great importance for ensuring safe wheat production and national and even global food security.

[0003] Wheat yield is primarily determined by the number of spikes per unit area, thousand-grain weight, and number of grains per spike. High-yield breeding aims to gradually improve the levels of these three factors and coordinate their relationship to achieve an optimal combination effect under specific ecological conditions. Among these, thousand-grain weight is the most stable and heritable trait, mainly dependent on morphogenesis (volume) and grain filling (fullness). A higher thousand-grain weight indicates fuller wheat grains, greater dry matter accumulation, higher starch content, and better flour quality. The thousand-grain weight of wheat is not only directly related to yield and milling quality but also affects the vigor and growth of wheat seedlings, thus indirectly influencing plant growth and final wheat yield. Grain weight and grain size are major components of wheat yield, and improving grain size and weight is an effective measure to increase yield at certain stages of breeding. Therefore, in-depth analysis of grain-related traits is of great significance for variety improvement to enhance wheat yield and quality.

[0004] The cell cycle plays a crucial role in cell development. Cell cycle progression is primarily regulated by cyclin-dependent kinases (CDKs), cyclins, and endogenous CDK inhibitors (CKIs). CDKs are the main cell cycle regulators, binding to cyclins to form the cyclin-CDK complex, which phosphorylates hundreds of substrates, regulating interphase and mitosis. Cks1 (cyclin-dependent kinase subunit 1) is the structural subunit of the CDK complex. It is highly conserved in sequence, approximately 18 kDa in size, and belongs to the Cks / Suc1 family. Cks1 plays a vital role in cell cycle progression, transcriptional regulation, and protein ubiquitination and degradation by binding to CDKs and other phosphorylated proteins, making it a key gene in cell cycle regulation. The Saccharomyces cerevisiae CDK complex comprises the catalytic subunit CDK1, the cycle-specific regulatory subunit cyclin, and the structural subunit Cks1. Saccharomyces cerevisiae uses Cks1, and Schizosaccharomyces pombe uses Suc1; both are homologous proteins belonging to the Cks / Suc1 family of cell cycle regulatory proteins. Numerous studies have shown that Cks1 is highly expressed in the pathological tissues of malignant tumors such as gastric and hepatocellular carcinoma, and is associated with various human malignant tumor diseases. In Arabidopsis thaliana, Cks1 has been found to bind to CDK, thereby regulating the cell cycle; overexpression of CKS1At reduces leaf size and root growth rate, and also significantly reduces the size of meristematic tissue. However, whether Cks1 is related to wheat grain-related traits has not been reported in current technology. Summary of the Invention

[0005] The purpose of this invention is to provide the application of wheat TaCKS1 protein, its encoding gene and its mutant sequences in regulating wheat grain development, so as to solve the problems existing in the prior art. TaCKS1 protein can negatively regulate wheat grain length, grain width and thousand-grain weight, providing new gene resources for wheat breeding.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of TaCKS1 protein in regulating wheat grain development, the amino acid sequence of which is shown in SEQ ID NO.7.

[0008] This invention also provides the application of biomaterials associated with the TaCKS1 protein in regulating wheat grain development, said biomaterials comprising any one of the following:

[0009] The TaCKS1 gene encoding the TaCKS1 protein;

[0010] The mutant sequence of the TaCKS1 gene is tacks1;

[0011] Recombinant vectors containing the TaCKS1 gene or tacks1;

[0012] Recombinant microorganisms containing the recombinant vector;

[0013] Mutants containing the aforementioned tacks1;

[0014] The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6;

[0015] The nucleotide sequence of tacks1 is shown in any one of SEQ ID NO.19-21.

[0016] Preferably, the regulation is negative regulation.

[0017] Preferably, the wheat grain development includes grain length, grain width, and thousand-grain weight.

[0018] The present invention also provides the use of the TaCKS1 protein or the biomaterials described herein in promoting longer and / or wider wheat grains and / or increased thousand-grain weight.

[0019] The present invention also provides a method for promoting longer grain length and / or wider grain width and / or increased thousand-grain weight in wheat, including the step of reducing the transcriptional level of the TaCKS1 gene in wheat;

[0020] The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6.

[0021] The present invention also provides the use of the TaCKS1 protein or the biological material described herein in the cultivation of transgenic wheat with increased grain length and / or increased grain width and / or increased thousand-grain weight.

[0022] The present invention also provides a method for breeding transgenic wheat with longer grain length and / or wider grain width and / or increased thousand-grain weight, comprising the step of reducing the transcription level of the TaCKS1 gene in wheat to obtain transgenic wheat;

[0023] The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6.

[0024] The present invention discloses the following technical effects:

[0025] This invention cloned the SUC1 / CKS1 homologous gene TaCKS1 from Arabidopsis thaliana into wheat. A mutant of TaCKS1 was screened in the Jimai 20 EMS mutagenesis population. Analysis of the grain phenotype of the TaCKS1 mutant and the wild-type JM20 revealed that the mutant exhibited increased grain length, grain width, thousand-grain weight, and yield per plant compared to the wild type. This invention is the first to identify the TaCKS1 gene in wheat and, through EMS mutant analysis, validated the function of the TaCKS1 gene in negatively regulating grain length, grain width, thousand-grain weight, and yield per plant in wheat, providing a new genetic resource for wheat breeding. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 For TaCKS1 amino acid sequence alignment;

[0028] Figure 2 For subcellular localization analysis of TaCKS1 protein;

[0029] Figure 3 For screening and identification of tacks1 mutations; A: Location of tacks1 mutation; B: Comparison of tacks1 mutant and wild-type DNA; C: Comparison of tacks1-a1 mutant and wild-type CDS.

[0030] Figure 4 Analysis of tacks1 mutant expression levels;

[0031] Figure 5 The following charts show the grain phenotype and yield per plant of the tacks1 EMS mutant: A: Comparison of grain length between tacks1 mutant and wild-type wheat; B: Comparison of grain width between tacks1 mutant and wild-type wheat; C: Statistical chart of grain length between tacks1 mutant and wild-type wheat; D: Statistical chart of grain width between tacks1 mutant and wild-type wheat; E: Statistical chart of thousand-grain weight between tacks1 mutant and wild-type wheat; F: Statistical chart of yield per plant between tacks1 mutant and wild-type wheat. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] The wheat varieties Kenong 199 and Jimai 20 (JM20) used in this invention were preserved by the Crop Genomics and Molecular Breeding Center Laboratory of Henan Agricultural University.

[0038] Example 1: Cloning of a gene

[0039] The Arabidopsis gene CKS1At(TAIR10) sequence was submitted to the Plant Genome Database (http: / / plants.ensembl.org / index.html) for comparison. The search revealed that there are three copies of CKS1 in wheat: TraesCS4A02G410900, TraesCS4B02G311000, and TraesCS4D02G308900 (nucleotide sequences are shown in SEQ ID NO.1-3, CDS sequences are shown in SEQ ID NO.4-6, and the amino acid sequence encoding the protein is shown in SEQ ID NO.7). The identity was 88.9%, so it was named TaCKS1 and used for subsequent research.

[0040] SEQ ID NO.1 (TaCKS1-4A DNA):

[0041] ATTCGTCGGCTGCTGATCGATTGGTCGGTGGTTTTACTCGGATTTTTCTGAAATGGTTTGGGGATTTGTTCTTGTCGCAGGGCCTCGATCGAGAAGCGGCGGCGGCGGACATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGTTGGTGCCCAAACCCTAGCCCCTCCCGATCTTATCCCCCGGTGCGCCGCGGATCGCGAAACCCTAACTAACTAACTAACACGGCCGATGTCCCCGCGCGCAGGCACGTCGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGTCCGCATTCCCGTAAGATCGCGTTTTCTTGCCGGGTCCGTTCTCTGATCTTGCGAAATTTTGGGTTTTTTTTCCCTTGCAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGTGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGATCGGCCCCAGCGAGGGCAGTGGCGACCGGGGTTGGAACTCCTGAAAACCCCAGGCCCTTTTCATCTCTAAAAGGGCGGTCGCACTGGTTTGTCTGATCAGGATCTAGTTATGTTGGTGGTGTCCACCATGTGTTTGTGGAAATGCCTGGGAGAAACTTGCAGTGTGTTAGTAGTCTGTGTTATTATCCTGGATACTCTTGAATGCTACCTGTTATGTTATGCTGGTACTGTCTGTAGATCTTCCTGTGATGATCTGTTAGTGTTGATGCTTGCTTTACTGTTATCTGGTTCCTTATGATTAGGAGTATAGAGTTCATGTGATAGTGAATCTTATGTTTGCAAGAGGGCATGGAGCATTTGTTATGAATGA;

[0042] SEQ ID NO.2(TaCKS1-4B DNA):

[0043]

[0044] SEQ ID NO.3(TaCKS1-4D DNA):

[0045]

[0046] SEQ ID NO.4(TaCKS1-4A CDS):

[0047] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGCACGTCGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGTGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0048] SEQ ID NO.5(TaCKS1-4B CDS):

[0049] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTGCTCCCCAAGAACCGCCTCCTCGCCGAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCACTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0050] SEQ ID NO.6(TaCKS1-4D CDS):

[0051] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCCTCGCCGAGAACGAGTGGCGCGCGCTGGGCGT GCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCTGCTGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0052] SEQ ID NO.7 (amino acid sequences of proteins encoded by TaCKS1-4A, TaCKS1-4B, and TaCKS1-4D):

[0053] MGQIQYSEKYFDDTFEYRHVVLPPEVAKLLPKNRLLAENEWRALGVQQSRGWVHYA VHRPEPHIMLFRRPLNYQQQQDAAAAAAAQMMPK*.

[0054] Total RNA was extracted from seeds of Kenong 199 at different post-flowering stages according to the instructions of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (catalog number: DP441) from Tiangen Biotech (Beijing) Co., Ltd. To prevent RNA degradation, the entire extraction process was carried out on ice.

[0055] The first-strand cDNA was synthesized by reverse transcription using the extracted total RNA as a template, following the procedures provided by the TaKaRa PrimeScriper 1st strand cDNA Synthesis Kit (Code No. 6110A).

[0056] Specific primers TaCKS1-cds-F and TaCKS1-cds-R were designed using Primer Premier 5 software. Their nucleotide sequences are as follows:

[0057] TaCKS1-cds-F: 5'-ATGGGCCAGATCCAGTACTCC-3' (SEQ ID NO. 8);

[0058] TaCKS1-cds-R: 5'-TCACTTGGGCATCATCTGCG-3' (SEQ ID NO. 9).

[0059] Using the above cDNA as a template, the target gene was amplified by PCR and ligated into a cloning vector. Sequencing analysis was then performed to obtain the TaCKS1 gene sequence.

[0060] Example 2: TaCKS1 Bioinformatics Analysis

[0061] The TaCKS1 gene and protein sequence of Arabidopsis thaliana were used to search for the wheat TaCKS1 gene in the Plant Genome Database (http: / / plants.ensembl.org / index.html).

[0062] Cks1 sequences of several species, including *Saccharomyces cerevisiae*, *Arabidopsis thaliana*, *Homo sapiens*, barley (*Hordeum vulgare* L.), rice (*Oryzasativa* L.), maize (*Zea mays* L.), and sorghum (*Sorghum bicolor* L.), were searched on NCBI (https: / / www.ncbi.nlm.nih.gov / ), and their amino acid sequences were compared using DNAMAN software. The comparison results are shown below. Figure 1 ,Depend on Figure 1 It can be seen that Cks1 contains a specific CDK-binding amino acid site "HxPEPH" (His-any-Pro-Glu-ProHis) in wheat, rice, Arabidopsis thaliana, barley, maize, sorghum, humans, and Saccharomyces cerevisiae. This indicates that Cks1 is conserved during evolution.

[0063] Example 3: Subcellular localization analysis of TaCKS1 protein

[0064] Grain development-related genes are typically located in the cell nucleus, where they perform their transcriptional regulatory functions. Therefore, the subcellular localization of the wheat TaCKS1 protein in tobacco leaves was analyzed using a fusion protein expression vector.

[0065] 1. Carrier Construction

[0066] The primers used are shown below:

[0067] TaCKS1-GFP-F: 5'-AGTCCGGAGCTAGCTCTAGAATGGGCCAGATCCAGTACTCC-3' (SEQ IDNO.10);

[0068] TaCKS1-GFP-R: 5'-TGCTCACCATGGATCCTCACTTGGGCATCATCTGCG-3' (SEQ ID NO. 11).

[0069] The TaCKS1 coding region fragment was amplified, and TaCKS1 with homologous arms was recombined into the GFP vector using seamless cloning technology to form a fusion protein with GFP, which was expressed by the 35S promoter.

[0070] 2. Injecting tobacco

[0071] (1) Transformation into Agrobacterium: The expression plasmid was transformed into GV3101 Agrobacterium.

[0072] (2) Small-scale shaking: Select Agrobacterium clones (GV3101) transformed with expression plasmids and place them in 1 mL of LB liquid medium containing the corresponding antibiotics, and culture them at 28°C and 200 rpm for 24 h.

[0073] (3) Large-scale culture: Transfer 500 μL of Agrobacterium culture to 5 mL of LB medium containing the corresponding antibiotic, and culture at 28°C and 220 rpm until the logarithmic growth phase (OD50) of Agrobacterium. 600 =0.5-0.6).

[0074] (4) Treatment of bacterial strains: Collect bacterial cells by centrifugation at 4000 rpm for 10 min at room temperature, and suspend Agrobacterium cells in a staining buffer (containing 10 mM MgCl2, 10 mM MES, 200 μM As, pH = 5.6) until OD200. 600 =1.0. Let stand at room temperature for 2-3 hours.

[0075] Mix equal volumes of the above-mentioned Agrobacterium with Agrobacterium containing the endoplasmic reticulum marker protein (KDEL-mCherry) expression vector. Gently make a small incision on the back of a tobacco leaf with a 1 mL needle (be careful not to puncture it). Then, use a syringe without the needle to draw up the bacterial solution and inject it into the leaf through the wound. Mark the water-stained areas on the tobacco leaf with a marker.

[0076] 48 hours after tobacco injection, observe whether there is fluorescence in the area injected with Agrobacterium tumefaciens and image the fluorescence using a laser confocal microscope.

[0077] 3. Observation of GFP expression

[0078] GFP expression was observed under a laser confocal microscope, and the results are as follows: Figure 2 As shown, the fluorescence of TaCKS1-GFP protein is mainly distributed in the nucleus and cytoplasm surrounded by the endoplasmic reticulum marker KDEL-mCherry, indicating that TaCKS1 protein mainly functions in the nucleus and cytoplasm.

[0079] Example 4: Screening of TaCKS1 mutants

[0080] To analyze the biological function of TaCKS1, mutants of TaCKS1 were screened from a total of 629 JM20EMS mutagenesis populations previously grown in Zhaoxian County, Hebei Province, in our laboratory.

[0081] Based on the TaCKS1 genome sequence, specific primers for genomes A, B, and D were designed using SnapGene 6.0.2 software and synthesized by Sangon Biotech (Shanghai) Technology Service Co., Ltd. The nucleotide sequences are shown in Table 1.

[0082] Table 1 PCR primers

[0083]

[0084] The target gene was amplified by PCR reaction, followed by sequencing analysis and sequence alignment to screen for tacks1 mutants.

[0085] The specific steps are as follows:

[0086] 1. Extraction of DNA from wheat grains

[0087] (1) Crush wheat seeds (about 1g) with a hammer, put them into a 2mL centrifuge tube containing a 5mm steel ball, mark the seed number on the tube cap, add 700μL of seed DNA extraction solution (formula as shown in Table 2), and place in a shaker at 37℃ for 30min.

[0088] Table 2 Formula for Wheat Grain DNA Extraction Solution

[0089]

[0090] (2) Add the same volume of phenol:chloroform (1:1) reagent to the centrifuge tube, shake on a shaker for 10-20 min, and then centrifuge at 12000 rpm for 10 min.

[0091] (3) Transfer the supernatant (about 500 μL) obtained by centrifugation to a 1.5 mL tube, add the same volume of chloroform:isoamyl alcohol (24:1), shake on a shaker for 10-20 min, and centrifuge at 12,000 rpm for 10 min.

[0092] (4) Transfer the supernatant obtained by centrifugation to a new 1.5 mL tube, add 0.7 times the volume of pre-cooled isopropanol, mix well and place at -20℃ for 30 min.

[0093] (5) Centrifuge at 12,000 rpm for 10 min. Discard the supernatant and wash the precipitate with 70% alcohol.

[0094] (6) Repeat step (5).

[0095] (7) Discard the alcohol, place it in a ventilated place for 5-10 minutes to dry the alcohol, and finally dissolve the DNA with an appropriate amount of sterile water.

[0096] Using the extracted DNA as a template, PCR amplification was performed using the primers listed in Table 1. The PCR reaction volume was 25 μL, including 1 μL template DNA, 13 μL KOD One, 1.5 μL each of forward and reverse primers, and sterile water was added to bring the volume to 25 μL. The PCR reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 58℃ annealing for 5 s, 68℃ extension for 5 s (5 kb / s), 35 cycles; 68℃ extension for 7 min, and storage at 4℃.

[0097] 2. Mutant screening

[0098] PCR products were separated by agarose gel electrophoresis, excised, and sent for sequencing. Sequencing was performed by Sangon Biotech (Shanghai) Technology Service Co., Ltd. Sequence alignment was performed using SnapGene, and the results are shown below.

[0099] Depend on Figure 3 It is known that a base substitution occurs at the junction of the first exon and the first intron in the TaCKS1-4A gene, resulting in a frameshift mutation in the amino acid sequence, tentatively named tacks1-a1. Its nucleotide sequence is shown in SEQ ID NO.18, and its mutated CDS sequence is shown in SEQ ID NO.19, in which four ATTG bases are inserted.

[0100] The TaCKS1-4D gene undergoes a base substitution in its second exon, changing from AAC to AAT, and is tentatively named tacks1-d1. Its nucleotide sequence is shown in SEQ ID NO.20;

[0101] The TaCKS1-4D gene undergoes a base substitution in its third exon, changing from GTG to TTG, and is tentatively named tacks1-d2. Its nucleotide sequence is shown in SEQ ID NO.21.

[0102] SEQ ID NO.18(tacks1-a1):

[0103] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGATTGGTGCCCAAACCCTAGCCCCTCCCGATCTTATCCCCCGGTGCGCCGCGGATCGCGAAACCCTAACTAACTAACTAACACGGCCGATGTCCCCGCGCGCAGGCACGTCGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGTCCGCATTCCCGTAAGATCGCGTTTTCTTGCCGGGTCCGTTCTCTGATCTTGCGAAATTTTGGGTTTTTTTTCCCTTGCAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGTGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0104] Note: The positions marked with shadows are the sites of base substitutions.

[0105] SEQ ID NO.19 (tacks1-a1 CDS):

[0106] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGATTGGCACGTCGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGTGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0107] Note: The four inserted bases are marked with shadows.

[0108] SEQ ID NO.20 (tacks1-d1):

[0109] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGTTCGTGCCCAAACCCTAGCCCCTCCCGATCTCATCCCCTCGGTGCGCCGCGGATGGCGAAACCCTAAGTAACCAACTAACACGGCCGATGTCGCCGCGCGCAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAATCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGTCCGCATTCCCGTAAGATCGCGTTTTCTCGCCGGATCGGCTCTCTGATGTTGCGAAATTGGGGTTTTTTTCCCCTTGCAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCTGCTGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0110] Note: The positions marked with shadows are where base substitutions occurred.

[0111] SEQ ID NO.21 (tacks1-d2):

[0112] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGTTCGTGCCCAAACCCTAGCCCCTCCCGATCTCATCCCTCGGTGCGCCGCGGATGGCGAAACCCTAAGTAACCAACTAACACGGCCGATGTCGCCGCGCAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGT CCGCATTCCCGTAAGATCGCGTTTTCTCGCCGGATCGGCTCTCTGATGTTGCGAAATTGGGGTTTTTTTCCCCTTGCAGAACGAGTGGCGCGCTGGGCTTGCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGTCCAACTACCAGCAGCAGCAGGACGCGGCTGCTGCCGCCGCGGCGCAGATGATGCCCAAGTGA.

[0113] Note: Shaded areas indicate locations where base substitutions occur.

[0114] 3. The transcriptional level of the mutant is reduced compared to the wild type.

[0115] To detect whether the transcriptional level of the mutant has changed, total RNA was extracted from the leaves of the tacks1 mutant according to the instructions of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (catalog number: DP441) from Tiangen Biotech (Beijing) Co., Ltd.

[0116] The total RNA extracted was reverse transcribed into cDNA using the procedures provided by the TaKaRa PrimeScriper 1st strand cDNA Synthesis Kit (Code No. 6110A).

[0117] The expression of tacks1 mutant and wild type in leaves was detected by qRT-PCR. The results showed that compared with wild type, the transcriptional level of tacks1-a1 was reduced, and the transcriptional levels of tacks1-d1 and tacks1-d2 were significantly reduced. Figure 4 ).

[0118] 4. Investigation of agronomic traits of mutants

[0119] Jimai 20 and the screened mutant tacks1 were planted in Maozhuang, Zhengzhou. Each mutant was planted in 5 rows with a row length of 2m, a plant spacing of 10cm, and a row spacing of 20cm.

[0120] Under normal field management, plant height, ear length, number of grains per ear, number of spikelets, and number of tillers were measured 15 days after flowering.

[0121] A field agronomic trait survey of the tacks1 mutant and the wild-type JM20 revealed that, compared with the wild-type, the mutant showed no significant changes in plant height, panicle length, number of spikelets, and number of tillers. The number of grains per panicle was slightly lower than that of the wild-type, but the difference was not significant (Table 3).

[0122] Table 3. Agronomic traits of the tacks1 mutant.

[0123]

[0124] After harvesting and threshing, the grain phenotypes of the tacks1 mutant and the wild-type JM20 were analyzed. It was found that compared with the wild-type, the mutant grain length and width were increased to varying degrees. Figure 5 ).

[0125] The Wansen SC-C automated seed analysis and thousand-grain weight system were used to statistically analyze grain length, grain width, and thousand-grain weight. The results showed that compared to the wild type, the tacks1 mutant exhibited significantly increased grain length, grain width, thousand-grain weight, and yield per plant. Figure 5 ).

[0126] This invention utilizes EMS mutant technology to verify the function of the TaCKS1 gene in negatively regulating wheat grain length, grain width, and thousand-grain weight, providing new gene resources for wheat breeding.

[0127] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of reducing TaCKS1 protein expression in promoting longer and / or wider wheat grains and / or increased thousand-grain weight, characterized in that, The amino acid sequence of the TaCKS1 protein is shown in SEQ ID NO.

7.

2. The use of biomaterials related to the TaCKS1 protein described in claim 1 in promoting increased grain length and / or increased grain width and / or increased thousand-grain weight in wheat, characterized in that, The biomaterial includes any one of the following: The TaCKS1 gene encoding the TaCKS1 protein; The mutant sequence of the TaCKS1 gene is tacks1; Recombinant vectors containing the TaCKS1 gene or tacks1; Recombinant microorganisms containing the recombinant vector; Mutants containing the aforementioned tacks1; The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6; The nucleotide sequence of the tacks1 is shown in any one of SEQ ID NO.19-21; By reducing the expression of TaCKS1 protein or the transcription level of the TaCKS1 gene in wheat, it is possible to promote longer grain length and / or wider grain width and / or increased thousand-grain weight in wheat.

3. A method for promoting longer grain length and / or wider grain width and / or increased thousand-grain weight in wheat, characterized in that, This includes steps to reduce the transcriptional level of the TaCKS1 gene in wheat; The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6.

4. The application of the TaCKS1 protein as described in claim 1 or the biomaterial as described in claim 2 in the cultivation of transgenic wheat with increased grain length and / or increased grain width and / or increased thousand-grain weight, characterized in that, By reducing the expression of TaCKS1 protein or the transcription level of the TaCKS1 gene in wheat, it is possible to promote longer grain length and / or wider grain width and / or increased thousand-grain weight in wheat.

5. A method for breeding transgenic wheat with increased grain length and / or increased grain width and / or increased thousand-grain weight, characterized in that, This includes the step of reducing the transcription level of the TaCKS1 gene in wheat to obtain transgenic wheat; The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6.

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