Wheat TaCKS1 protein, coding gene and application of mutant sequence of wheat TaCKS1 protein in regulation and control of wheat grain development

Abstract

The invention discloses application of a wheat TaCKS1 protein, a coding gene and a mutant sequence of the wheat TaCKS1 protein in regulation and control of wheat grain development, and belongs to the technical field of biology, and the amino acid sequence of the TaCKS1 protein is as shown in SEQ ID NO.7. According to the invention, an SUC1 / CKS1 homologous gene TaCKS1 is obtained from homologous cloning of arabidopsis thaliana into wheat, a tacks1 mutant is screened from Jimai 20EMS mutagenesis population, and analysis on grain phenotypes of the tacks1 mutant and a wild type JM20 shows that the grain length, the grain width, the thousand grain weight and the single plant yield of the mutant are increased compared with those of the wild type JM20. The TaCKS1 gene of wheat is identified for the first time, the function of the TaCKS1 gene for negatively regulating the grain length, grain width, thousand grain weight and single plant yield of wheat is proved through EMS mutation experience, and a new gene resource is provided for wheat breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of wheat TaCKS1 protein, encoding gene and mutant sequence thereof in regulating wheat grain development. Background Art

[0002] Wheat (Triticum aestivum L.) belongs to the genus Triticum of the family Gramineae. It originated in western Asia and is distributed all over the world, adapting to different latitudes and climatic conditions. Wheat grains are rich in carbohydrates, and their protein content is significantly higher than that of other cereal crops. The simultaneous improvement of its yield and quality is of great significance to ensuring national food security and public health. With the trend of global climate change, growing population, changing eating habits and increasing consumption of biofuels, there is still a large gap between the total output of wheat and the demand for wheat. Therefore, the cultivation and breeding of new high-yield and high-quality wheat varieties is of great significance to ensuring the safe production of wheat and the national and even world food security.

[0003] Wheat yield is mainly determined by the number of ears per unit area, thousand-grain weight and number of grains per ear. High-yield breeding is to gradually improve the levels of the three factors, coordinate the relationship between the three, and achieve the optimal combination effect under a specific ecological environment. Among them, thousand-grain weight is the most stable and heritable characteristic, which mainly depends on morphological construction (volume) and grain filling (fullness). Large thousand-grain weight indicates that the wheat grains are fuller, with more 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 vitality and growth of wheat seedlings, and indirectly affects the growth of plants and the final yield of wheat. Grain weight and grain size are the main components of wheat yield. Improving grain size and grain weight is an effective measure to increase yield at a certain stage of breeding. Therefore, in-depth analysis of grain-related traits is of great significance for variety improvement to improve wheat yield and quality.

[0004] The cell cycle plays a vital role in the process of cell development. The cell cycle process is mainly regulated by cyclin dependent kinase (CDK), cyclins and endogenous CDK inhibitors (CKI). Among them, CDK is the main cell cycle regulator, which can bind to cyclins to form cyclin CDK complexes, thereby phosphorylating hundreds of substrates and regulating interphase and mitotic processes. Cks1 (cyclin-dependent kinase subunit 1) is a structural subunit of the cyclin-dependent kinase complex CDK. It is highly conserved in sequence, with a protein size of about 18kDa, and belongs to the Cks / Suc1 family. Cks1 plays an important role in cell cycle progression, transcriptional regulation, protein ubiquitination and degradation by binding to CDK and other phosphorylated proteins, and is a key gene in the cell cycle regulation process. The Saccharomyces cerevisiae CDK complex contains the catalytic subunit CDK1, the cycle-specifically expressed regulatory subunit cyclin and the structural subunit Cks1. Saccharomyces cerevisiae is Cks1, and Schizosaccharomyces pombe is Suc1. The two are homologous proteins and belong to the Cks / Suc1 family of cell cycle regulatory proteins. At present, a large number of studies have shown that Cks1 is highly expressed in the pathological tissues of malignant tumors such as gastric cancer and liver cancer, and is related to a variety of human malignant tumor diseases; in Arabidopsis, it was found that Cks1 can bind to CDK to regulate the cell cycle. Overexpression of CKS1At will reduce leaf size and root growth rate, and also greatly reduce the size of meristems. As for whether Cks1 is related to wheat grain-related traits, the existing technology has not reported. Summary of the invention

[0005] The purpose of the present invention is to provide the application of wheat TaCKS1 protein, encoding gene and mutant sequence thereof in regulating wheat grain development to solve the problems existing in the above-mentioned 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 object, the present invention provides the following solutions:

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

[0008] The present invention also provides the use of a biological material related to the TaCKS1 protein in regulating wheat grain development, wherein the biological material comprises any one of the following:

[0009] A TaCKS1 gene encoding the TaCKS1 protein;

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

[0011] A recombinant vector containing the TaCKS1 gene or tacks1;

[0012] A recombinant microorganism containing the recombinant vector;

[0013] A mutant containing the tacks1;

[0014] The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NOs. 4-6;

[0015] The nucleotide sequence of tacks1 is shown in any one of SEQ ID NOs. 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 biological material in promoting the lengthening of wheat grains and / or the width of wheat grains and / or the increase of 1,000-grain weight.

[0019] The present invention also provides a method for promoting the lengthening of wheat grains and / or the width of wheat grains and / or the increase of 1000-grain weight, comprising the steps of reducing the transcription level of 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 in cultivating transgenic wheat with longer wheat grains and / or wider wheat grains and / or increased thousand-grain weight.

[0022] The present invention also provides a method for cultivating transgenic wheat with longer grain length and / or wider grain width and / or increased thousand-grain weight, comprising the steps of reducing the transcription level of 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] The present invention cloned the homologous gene TaCKS1 from Arabidopsis thaliana into wheat, screened the mutant of tacks1 in the EMS mutagenesis population of Jimai 20, and analyzed the grain phenotype of the tacks1 mutant and the wild type JM20, and found that the mutant had increased grain length, grain width, 1000-grain weight and single-plant yield compared with the wild type. The present invention identified the TaCKS1 gene of wheat for the first time, and verified the function of the TaCKS1 gene in negatively regulating wheat grain length, grain width, 1000-grain weight and single-plant yield through the EMS mutant, providing new gene resources for wheat breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is the TaCKS1 amino acid sequence alignment;

[0028] Figure 2 To analyze the subcellular localization of TaCKS1 protein;

[0029] Figure 3 Screening and identification of tacks1 mutations; A: tacks1 mutation position; B: tacks1 mutant and wild-type DNA alignment; C: tacks1-a1 mutant and wild-type CDS alignment.

[0030] Figure 4 For the expression analysis of tacks1 mutants;

[0031] Figure 5 The grain phenotype and single-plant yield of the tacks1 EMS mutant; A: Comparison of grain length between the tacks1 mutant and wild-type wheat; B: Comparison of grain width between the tacks1 mutant and wild-type wheat; C: Statistical graph of grain length between the tacks1 mutant and wild-type wheat; D: Statistical graph of grain width between the tacks1 mutant and wild-type wheat; E: Statistical graph of thousand-grain weight between the tacks1 mutant and wild-type wheat; F: Statistical graph of single-plant yield between the tacks1 mutant and wild-type wheat. DETAILED DESCRIPTION

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

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

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

[0038] Example 1 Gene cloning

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

[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 sequence of proteins encoded by TaCKS1-4A, TaCKS1-4B, TaCKS1-4D):

[0053] MGQIQYSEKYFDDTFEYRHVVLPPEVAKLLPKNRLLAENEWRALGVQQSRGWVHYA VHRPEPHIMLFRRPLNYQQQQDAAAAAAAQMMPK*.

[0054] Total RNA from the seeds of Kenong 199 at different stages after flowering was extracted according to the instructions of RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Catalog No.: DP441) of Tiangen Biochemical Technology (Beijing) Co., Ltd. To prevent RNA degradation, the entire extraction process must be carried out on ice.

[0055] The first strand cDNA was synthesized by reverse transcription using the extracted total RNA as a template according to the operating steps provided by the reverse transcription kit PrimeScriper 1st strand cDNA Synthesis Kit (Code No. 6110A) of TaKaRa.

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

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

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

[0059] The cDNA was used as a template to amplify the target gene by PCR reaction, and the target gene was connected to the cloning vector, and then sequenced and analyzed to obtain the TaCKS1 gene sequence.

[0060] Example 2 Bioinformatics analysis of TaCKS1

[0061] The CKS1At gene and protein sequences of Arabidopsis were used to search for wheat TaCKS1 in the Plant Genome Database (http: / / plants.ensembl.org / index.html).

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

[0063] Example 3 Analysis of subcellular localization of TaCKS1 protein

[0064] Genes related to grain development are usually located in the nucleus and perform their transcriptional regulation functions in the nucleus. Therefore, the subcellular localization of wheat TaCKS1 protein in tobacco leaves was analyzed using the fusion protein expression vector method.

[0065] 1. Vector construction

[0066] The primers used are as follows:

[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 homology arms was recombined into the GFP vector through seamless cloning technology to form a fusion protein with GFP, which was expressed by the 35S promoter.

[0070] 2. Injection of tobacco

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

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

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

[0074] (4) Treatment of bacteria: Centrifuge at room temperature and 4000 rpm for 10 min to collect the bacteria, and suspend the Agrobacterium bacteria in an inoculum solution (containing 10 mM MgCl2, 10 mM MES, 200 μM As, pH = 5.6) until the OD 600 =1.0. Leave at room temperature for 2-3 hours.

[0075] Mix equal volumes of the above Agrobacterium with the Agrobacterium containing the endoplasmic reticulum marker protein (KDEL-mCherry) expression vector, use a 1mL needle to gently make a small hole on the back of the tobacco leaf (be careful not to puncture it), then use a needle without the needle to draw up the bacterial solution and inject it into the leaf from the wound. Use a marker to mark the water-stained area of ​​the tobacco leaf.

[0076] 48 hours after the injection of tobacco, the area where the tobacco was injected with Agrobacterium was observed for fluorescence, and fluorescence imaging was performed using a laser confocal microscope.

[0077] 3. Observation of GFP expression

[0078] GFP expression was observed under a laser confocal microscope. 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 the TaCKS1 protein mainly functions in the nucleus and cytoplasm.

[0079] Example 4 Screening of TaCKS1 mutants

[0080] In order to analyze the biological function of TaCKS1, mutants of tacks1 were screened from a total of 629 JM20EMS mutagenesis populations previously grown in Zhao County, Hebei Province in our laboratory.

[0081] According to the TaCKS1 genome sequence, specific primers for the A, B, and D genomes were designed using SnapGene 6.0.2 software and synthesized by Sangon Biotechnology (Shanghai) Technology Services 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, and then sequencing analysis and sequence alignment were performed to screen out the tacks1 mutant.

[0085] The specific steps are as follows:

[0086] 1. Extraction of DNA from wheat grains

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

[0088] Table 2 Wheat grain DNA extraction solution formula

[0089]

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

[0091] (3) The supernatant (about 500 μL) obtained by the above centrifugation was transferred to a 1.5 mL tube, and the same volume of chloroform:isoamyl alcohol (24:1) was added. The tube was shaken on a shaker for 10-20 min and centrifuged at 12,000 rpm for 10 min.

[0092] (4) The supernatant obtained from the above centrifugation was transferred to a new 1.5 mL tube, and 0.7 times the volume of pre-cooled isopropanol was added. The mixture was mixed and placed at -20°C 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 and place it in a ventilated place for 5-10 minutes to dry the alcohol. Finally, use an appropriate amount of sterile water to dissolve the DNA.

[0096] The above extracted DNA was used as a template and PCR amplification was performed using the primers in Table 1. The PCR reaction system was 25 μL, including 1 μL template DNA, 13 μL KOD One, 1.5 μL upstream and downstream primers, and 25 μL was added with sterile water. PCR reaction program: 98°C pre-denaturation for 2 min; 98°C denaturation for 10 s, 58°C annealing for 5 s, 68°C extension for 5 s (5 kb / s), 35 cycles; 68°C extension for 7 min, and storage at 4°C.

[0097] 2. Mutant Screening

[0098] The PCR products were separated by agarose gel electrophoresis, cut from the gel and sent for testing, and sequencing was completed by Sangon Biotechnology (Shanghai) Technology Service Co., Ltd. SnapGene was used for sequence alignment, and the results are shown below.

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

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

[0101] The TaCKS1-4D gene has a base substitution in the third exon, from GTG to TTG, and is temporarily 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 shaded marks indicate the positions where base substitutions occurred.

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

[0106] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGATTGGCACGTCGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGTGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCGGCCGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

[0107] Note: The shaded marks indicate the 4 inserted bases.

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

[0109] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGTTCGTGCCCAAACCCTAGCCCCTCCCGATCTCATCCCCTCGGTGCGCCGCGGATGGCGAAACCCTAAGTAACCAACTAACACGGCCGATGTCGCCGCGCGCAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAATCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGTCCGCATTCCCGTAAGATCGCGTTTTCTCGCCGGATCGGCTCTCTGATGTTGCGAAATTGGGGTTTTTTTCCCCTTGCAGAACGAGTGGCGCGCGCTGGGCGTGCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGCTCAACTACCAGCAGCAGCAGGACGCGGCTGCTGCCGCCGCGGCGCAGATGATGCCCAAGTGA;

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

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

[0112] ATGGGCCAGATCCAGTACTCCGAGAAGTACTTCGACGACACCTTCGAGTACAGGTTCGTGCCCAAACCCTAGCCCCTCCCGATCTCATCCCTCGGTGCGCCGCGGATGGCGAAACCCTAAGTAACCAACTAACACGGCCGATGTCGCCGCGCAGGCACGTGGTCCTCCCGCCGGAAGTCGCCAAGCTCCTCCCCAAGAACCGCCTCCTCGCCGAGGTAACCGTCCAGATTTCCGT CCGCATTCCCGTAAGATCGCGTTTTCTCGCCGGATCGGCTCTCTGATGTTGCGAAATTGGGGTTTTTTTCCCCTTGCAGAACGAGTGGCGCGCTGGGCTTGCAGCAGAGCCGCGGGTGGGTGCACTACGCCGTCCACCGGCCGGAGCCGCACATCATGCTGTTCCGCCGCCCGTCCAACTACCAGCAGCAGCAGGACGCGGCTGCTGCCGCCGCGGCGCAGATGATGCCCAAGTGA.

[0113] Note: The shaded areas are where base substitutions occurred.

[0114] 3. The transcription level of the mutant is lower than that of the wild type

[0115] In order to detect whether the transcription level of the mutant has changed, the total RNA of the leaves of the tacks1 mutant was extracted according to the instructions of RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Catalog No.: DP441) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0116] The extracted total RNA was used as a template for reverse transcription into cDNA using the operating steps provided by TaKaRa's reverse transcription kit 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 the wild type, the transcription level of tacks1-a1 was reduced, and the transcription 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 selected 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, data such as plant height, ear length, number of grains per ear, number of spikelets, and number of tillers were measured 15 days after flowering.

[0121] Through the investigation of the agronomic traits of the tacks1 mutant and the wild type JM20 in the field, it was found that compared with the wild type, the plant height, ear length, spikelet number and tiller number of the mutant did not change significantly, and the number of grains per ear was lower than that of the wild type, but the difference was not significant (Table 3).

[0122] Table 3 Agronomic characteristics of tacks1 mutants

[0123]

[0124] After harvesting and threshing, the grain phenotypes of the tacks1 mutant and the wild type JM20 were analyzed, and it was found that compared with the wild type, the mutant had different degrees of increase in grain length and width ( Figure 5 ).

[0125] The Wansen SC-C automatic seed analysis and thousand-grain weight system were used to calculate the grain length, grain width, and thousand-grain weight. The results showed that compared with the wild type, the grain length, grain width, thousand-grain weight, and single-plant yield of the tacks1 mutant were significantly increased ( Figure 5 ).

[0126] The present invention utilizes EMS mutant technology to verify the function of TaCKS1 gene in negatively regulating wheat grain length, grain width and 1000-grain weight, providing new gene resources for wheat breeding.

[0127] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of TaCKS1 protein in regulating wheat grain development, characterized in that: The amino acid sequence of the TaCKS1 protein is shown in SEQ ID NO.

7.

2. Use of a biological material related to the TaCKS1 protein in claim 1 in regulating wheat grain development, characterized in that: The biological material includes any one of the following: A TaCKS1 gene encoding the TaCKS1 protein; The mutant sequence of the TaCKS1 gene is tacks1; A recombinant vector containing the TaCKS1 gene or tacks1; A recombinant microorganism containing the recombinant vector; A mutant containing the tacks1; The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NOs. 4-6; The nucleotide sequence of tacks1 is shown in any one of SEQ ID NOs. 19-21.

3. The use according to claim 1 or 2, characterized in that: The regulation is negative regulation.

4. The use according to claim 1 or 2, characterized in that: The wheat grain development includes grain length, grain width and thousand-grain weight.

5. Use of the TaCKS1 protein as described in claim 1 or the biological material as described in claim 2 in promoting the lengthening of wheat grains and / or the width of wheat grains and / or the increase of 1000-grain weight.

6. A method for promoting the lengthening of wheat grains and / or the width of wheat grains and / or the increase of 1000-grain weight, characterized in that: including the steps of reducing the transcription level of TaCKS1 gene in wheat; The nucleotide sequence of the TaCKS1 gene is shown in any one of SEQ ID NO.4-6.

7. Use of the TaCKS1 protein as claimed in claim 1 or the biological material as claimed in claim 2 in cultivating transgenic wheat with longer grain length and / or wider grain width and / or increased thousand-grain weight.

8. A method for cultivating transgenic wheat with longer grain length and / or wider grain width and / or increased thousand-grain weight, characterized in that: The method comprises the steps of reducing the transcription level of 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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