Wheat grain protein content related protein taskp1 and application of coding gene thereof
By cloning and utilizing wheat grain protein-related protein TaSKP1 and its encoding gene, the problem of difficulty in increasing grain protein content in existing technologies has been solved, resulting in a significant increase in grain protein content and providing breeding resources and theoretical support.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2024-10-15
- Publication Date
- 2026-07-21
AI Technical Summary
In existing technologies, few genes related to wheat grain protein content have been cloned and many have lost their function, making it difficult to increase grain protein content in breeding practices.
By cloning and utilizing the wheat grain protein-related protein TaSKP1 and its encoding gene, the protein content of grains can be increased by knocking out or regulating its expression level.
It significantly increased the protein content of wheat grains, providing important genetic resources and theoretical clues for molecular breeding of wheat quality.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant breeding technology, and in particular to the application of TaSKP1, a protein related to wheat grain protein content, and its encoding gene. Background Technology
[0002] Wheat provides approximately 19% of the protein nutrition needed by humans, making it one of the main sources of protein. The protein content and quality of wheat grains not only affect the commercial value of wheat but are also a crucial material basis for processing and producing high-quality wheat-based foods, directly influencing the quality of these processed foods (He et al., 2003). Improving wheat grain protein content (GPC) is an important goal of wheat quality genetic breeding in my country.
[0003] However, to date, there have been few reports of cloning genes related to wheat grain protein content. Previous studies have shown that genes located in wild emmer wheat ( Triticum turgidum ssp. dicoccoides) on 6BS NAM-B1 This is the major gene controlling grain protein content, and its cloning provides a material basis for the genetic improvement of wheat grain protein content. However, most common wheat... NAM-B1 Genes are often missing or mutated, resulting in loss of function, and are not fully utilized in breeding practices.
[0004] Therefore, discovering more genes related to wheat grain protein content, screening for their superior allelic variants, and elucidating their mechanisms of action can not only provide theoretical clues for molecular genetic research on wheat grain protein content, but also provide important genetic resources for molecular breeding of wheat quality. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention provides the application of TaSKP1, a protein related to wheat grain protein content, and its encoding gene, as detailed below.
[0006] In a first aspect, the present invention provides the application of a protein with an amino acid sequence as shown in SEQ ID No. 2 in regulating the protein content of wheat grains; Or the amino acid sequence is an amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEO ID No.2.
[0007] In a second aspect, the present invention provides the application of a protein with an amino acid sequence as shown in SEQ ID No. 4 in regulating the protein content of wheat grains; Or the amino acid sequence is an amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEO ID No.4.
[0008] Thirdly, the present invention provides the application of a gene encoding a nucleotide sequence as shown in SEQ ID No. 1 in regulating the protein content of wheat grains; Alternatively, the sequence encoding the gene may be a nucleotide sequence encoding a protein with the same function obtained by substituting, deleting, or inserting one or more nucleotides from the nucleotide sequence shown in SEQ ID No. 1.
[0009] Fourthly, the present invention provides the application of a gene encoding a nucleotide sequence as shown in SEQ ID No. 3 in regulating the protein content of wheat grains; Alternatively, the sequence encoding the gene may be a nucleotide sequence encoding a protein with the same function obtained by substituting, deleting, or inserting one or more nucleotides from the nucleotide sequence shown in SEQ ID No. 3.
[0010] Fifthly, the present invention provides the use of a protein encoding a protein with an amino acid sequence as shown in SEQ ID No. 2 or 4, or a gene encoding a nucleotide sequence as shown in SEQ ID No. 1 or 3, in any of the following: (1) Preparation of genetically modified wheat; (2) Wheat breeding; (3) Wheat variety improvement.
[0011] Sixthly, the application of a protein encoding gene with an amino acid sequence as shown in SEQ ID No. 2 or 4 or a nucleotide sequence as shown in SEQ ID No. 1 or 3 in increasing the protein content of wheat grains, characterized in that the encoding gene negatively regulates the protein content of wheat grains.
[0012] In the application provided by this invention, the expression level of proteins with amino acid sequences such as SEQ ID No. 2 or 4 in wheat plants is reduced, thereby increasing the protein content of wheat grains.
[0013] In the application provided by this invention, knocking out the coding gene shown in SEQ ID No. 1 or 3 in wheat increases the protein content of wheat grains.
[0014] In a seventh aspect, the present invention provides an sgRNA that knocks out the nucleotide sequence of the gene encoding the gene as shown in SEQ ID No. 1, wherein the sequence of the sgRNA is shown in SEQ ID No. 7.
[0015] In an eighth aspect, the present invention provides an sgRNA that knocks out the nucleotide sequence of the gene encoding the gene as shown in SEQ ID No. 3, the sequence of which is shown in SEQ ID No. 8.
[0016] The beneficial effects of this invention are as follows: This invention is the first to propose that the coding gene shown in SEQ ID No. 1 or 3, and the amino acid sequence shown in SEQ ID No. 2 or 4, are related to and negatively regulate wheat grain protein content. This invention provides theoretical clues for molecular genetic research on wheat grain protein content and also provides important genetic resources for molecular breeding of wheat quality. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.
[0018] Figure 1 This is the present invention. TaSKP1 Gene PCR. In the figure, M is the marker, and the band sizes from top to bottom are 5000 bp, 3000 bp, 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, and 100 bp.
[0019] Figure 2 This invention relates to the type of editing that knocks out genetically modified wheat.
[0020] Figure 3 This invention relates to the determination of grain protein content in knockout genetically modified wheat. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.
[0022] Example 1: Cloning of the gene encoding TaSKP1, a protein related to wheat grain protein content.
[0023] This invention uses flag leaves 25 days after flowering of *Fielder* as experimental samples, and extracts total RNA from the leaves using the Trizol method. Wheat leaf cDNA is obtained by reverse transcription using the TaKaRa PrimeScript RT reagent Kit with gDNA Eraser (RR047A; TaKaRa).
[0024] Using wheat leaf cDNA as a template, PCR amplification was performed using primers synthesized by Beijing Qingke Biotechnology Co., Ltd.: F (SEQ ID No. 5): 5'-GCCTAGAACAAACAGACAGC-3'; R (SEQ ID No. 6): 5'-TGTTCCCGTCCCTAGTACTC-3'. The specific reaction program was: 94℃ pre-denaturation for 5 min; 98℃ for 10 s, 60℃ for 15 s, 68℃ for 1.5 min, 35 cycles; extension at 68℃ for 5 min. The PCR amplification product of approximately 1500 bp was obtained by agarose gel electrophoresis. Figure 1 The PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The results showed that the nucleotide sequence of the PCR product included the DNA sequence shown in Sequence 1, and it was named... TaSKP1-5B The gene was named the amino acid sequence encoded by the gene as the TaSKP1-5B protein, and the amino acid sequence is shown in SEQ ID No. 2.
[0025] Alleles were obtained by multiple sequence alignment using the wheat genome. The nucleotide sequence included the DNA sequence shown in Sequence 3, and was named... TaSKP1-5A The gene was named the amino acid sequence encoded by the gene as TaSKP1-5A protein, and the amino acid sequence is shown in SEQ ID No. 4.
[0026] Example 2: Obtaining TaSKP1 knockout transgenic wheat
[0027] This embodiment provides a method for constructing TaSKP1 knockout transgenic wheat, the steps of which are as follows: (1) The CDS sequence of the TaskP1-5B gene was put into the E-crispr (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) website to design gRNA targets. Then, the specificity of the targets was detected by the Ensemble Plant (http: / / plants.ensembl.org / index.html) website. Two conserved targets in TaskP1-5B and TaskP1-5A were obtained: sgRNA1 (SEQ ID No.7: 5'-CCCGAAGGAGCAAGCCCCTA-3') and sgRNA2 (SEQ ID No.8: 5'-GATGGGTGAGCAACCCCTTA-3'). The sgRNA was constructed into the pCBC-MT1T2 vector by PCR reaction. The PCR product was purified and used for enzyme digestion and ligation reaction.
[0028] (2) Perform enzyme digestion and ligation of the PCR product. The total reaction volume is 15 µL: 2 µL of PCR product from step (1), 2 µL of pBUE414, 1.5 µL of 10×NEB T4 Buffer, 1.5 µL of CutSmart Buffer, 1 µL of Bsa I, 1 µL of T4 DNA Ligase, and 6 µL of ddH2O. Reaction conditions: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min. After the reaction, place the mixture on ice to cool.
[0029] (3) The enzyme digestion and ligation products were transformed into DH5α according to the E. coli transformation instructions of Beijing Bomaide Gene Technology Co., Ltd., and evenly spread on LB plates containing 50 mg / ml Kana. The plates were incubated overnight at 37°C with the plates inverted. Colony PCR was performed using the detection primers 414-seq-F (SEQ ID No. 9): 5'-TTTCCCAGTCACGACGTTGT-3' and 414-seq-R (SEQ ID No. 10): 5'-ATCTCTAGAGAGGGGCACGA-3'. The positive products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The plasmids with correct sequencing were extracted and transformed into Agrobacterium tumefaciens EHA105 to obtain the recombinant strain.
[0030] (4) The recombinant strain was sent to the transgenic platform of the Wheat Research Center of China Agricultural University to transform the recipient wheat Fielder, and T0 generation plants were obtained. The plant DNA was extracted by CTAB method and the target sites were sequenced and identified using primers (Table 1) to obtain transgene knockout positive lines. KO-1 and KO-2 ; through addition, obtain TaSKP1Double-mutant homozygous transgenic knockout lines, whose editing types are as follows: Figure 2 As shown.
[0031] Table 1 Primers for detecting the target site of knockout transgenic wheat
[0032] (5) TaSKP1 Determination of grain protein content in transgenic knockout wheat lines
[0033] 1) Harvest the TaskP1 transgenic knockout line KO-1 , KO-2 And the mature seeds of wild-type Fielder.
[0034] 2) Using a Wavecom DA7200 near-infrared grain analyzer to determine different... TaSKP1 The protein content of wheat grains from transgenic knockout lines was analyzed using three biological replicates, with the final mean value taken. (See attached data.) Figure 3 .
[0035] (6) Results Analysis
[0036] Through the study of wild-type Fielder, TaSKP1 Transgenic knockout lines KO-1 and KO-2 A comparison of seed protein content revealed that, compared to wild-type Fielder, KO-1 and KO-2 The protein content of wheat grains in both lines was significantly increased, a result indicating that... TaSKP1 Genes can negatively regulate the protein content of wheat grains.
[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. Application of a protein with the amino acid sequence shown in SEQ ID No. 2 in regulating the protein content of wheat grains; The application method involves reducing the expression level of proteins with amino acid sequences as shown in SEQ ID No. 2 in wheat plants, thereby increasing the protein content of wheat grains.
2. Application of the gene encoding the nucleotide sequence shown in SEQ ID No. 1 in regulating the protein content of wheat grains; The application method involves knocking out the coding gene shown in SEQ ID No. 1 in wheat, thereby increasing the protein content of wheat grains.