Wheat grain protein content related protein TaSKP1 and application of coding gene thereof

Abstract

The invention relates to the technical field of plant molecular breeding, in particular to a wheat grain protein content related protein TaSKP1 and application of a coding gene thereof. The amino acid sequence of the protein TaSKP1 related to the protein content of the wheat grains is shown as SEQ ID No.2 or 3; or the amino acid sequence is an amino acid sequence which is obtained by substitution, deletion or insertion of one or more amino acid residues of the amino acid sequence shown as SEO ID No.2 or 3 and has protein with the same function, and the amino acid sequence is obtained by substitution, deletion or insertion of one or more amino acid residues of the amino acid sequence shown as SEO ID No.2 or 3. By reducing the expression quantity of the protein TaSKP1 related to the wheat grain protein content or knocking out the coding gene of the protein TaSKP1, a wheat variety with high grain protein content can be obtained, a theoretical clue is provided for molecular genetic research of the wheat grain protein content, and an important gene resource is also provided for wheat quality molecular breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant breeding, and in particular to wheat grain protein content-related protein TaSKP1 and application of its encoding gene. Background Art

[0002] Wheat provides about 19% of human protein nutrition and is one of the main sources of protein for humans. The grain protein content and quality not only affect the commercial value of wheat, but also are an important material basis for processing and producing high-quality flour products, directly affecting the quality of processed foods (He et al., 2003). Improving wheat grain protein content (Grain protein content, GPC) is an important goal of wheat quality genetic breeding in my country.

[0003] However, so far, there are few reports on the cloning of genes related to wheat grain protein content. Triticum turgidum ssp. dicoccoides) on 6BS NAM-B1 It is the main gene controlling the protein content of grains. The cloning of this gene provides a material basis for the genetic improvement of wheat grain protein content. 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 their excellent allelic variations and analyzing 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] In order to solve the defects of the prior art, the present invention provides a wheat grain protein content-related protein TaSKP1 and the application of its encoding gene, and the specific scheme is as follows.

[0006] In a first aspect, the present invention provides the use of a protein having 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 the amino acid sequence shown in SEO ID No.2, which is obtained by replacing, deleting or inserting one or more amino acid residues and having the same functional protein.

[0007] In a second aspect, the present invention provides the use of a protein having 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 the amino acid sequence shown in SEO ID No.4, which is obtained by replacing, deleting or inserting one or more amino acid residues and having the same functional protein.

[0008] In a third aspect, the present invention provides the use of a coding gene with a nucleotide sequence as shown in SEQ ID No. 1 in regulating the protein content of wheat grains; Or the sequence of the coding gene is a nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID No.1.

[0009] In a fourth aspect, the present invention provides the use of a coding gene having a nucleotide sequence as shown in SEQ ID No. 3 in regulating the protein content of wheat grains; Or the sequence of the coding gene is a nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID No.3.

[0010] In a fifth aspect, the present invention provides the use of a protein having an amino acid sequence as shown in SEQ ID No. 2 or 4 or a coding gene having a nucleotide sequence as shown in SEQ ID No. 1 or 3 in any of the following: (1) Preparation of transgenic wheat; (2) Wheat breeding; (3) Wheat variety improvement.

[0011] In a sixth aspect, the use of a protein having 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 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 the present invention, the expression level of the protein with the amino acid sequence shown in SEQ ID No. 2 or 4 in the wheat plant is reduced, so that the protein content of the wheat grain is increased.

[0013] In the application provided by the present invention, the coding gene shown in SEQ ID No. 1 or 3 in wheat is knocked out, so that the protein content of wheat grains is increased.

[0014] In a seventh aspect, the present invention provides an sgRNA for knocking out a gene encoding a nucleotide sequence as shown in SEQ ID No.1, and the sequence of the sgRNA is shown in SEQ ID No.7.

[0015] In an eighth aspect, the present invention provides an sgRNA for knocking out a gene encoding a nucleotide sequence as shown in SEQ ID No.3, and the sequence of the sgRNA is shown in SEQ ID No.8.

[0016] The beneficial effects of the present invention are: The present invention proposes for the first time 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 the protein content of wheat grains and negatively regulate the protein content of wheat grains. The present invention provides theoretical clues for the molecular genetic research of the protein content of wheat grains and also provides important gene resources for molecular breeding of wheat quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are 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.

[0018] Picture 1 The present invention TaSKP1 Gene PCR. In the figure, M is marker. 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, respectively.

[0019] Picture 2 It is the editing type of knockout transgenic wheat of the present invention.

[0020] Picture 3 The invention relates to the determination of the grain protein content of the knockout transgenic wheat. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1 Cloning of the gene encoding the wheat grain protein content-related protein TaSKP1

[0023] The present invention takes the flag leaf of Fielder 25 days after flowering as the experimental sample, uses the Trizol method to extract the total RNA of the leaf, and uses the PrimeScript RT reagent Kit with gDNA Eraser reverse transcription kit (RR047A; TaKaRa) of TaKaRa company to perform reverse transcription to obtain wheat leaf cDNA.

[0024] The cDNA of wheat leaves was used as a template, and primers synthesized by Beijing Qingke Biotechnology Co., Ltd. were used for PCR amplification. The specific reaction procedure was: 94℃ pre-denaturation for 5 min; 98℃ 10 s, 60℃ 15 s, 68℃ 1.5 min, 35 cycles; 68℃ extension for 5 min. After agarose gel electrophoresis, a PCR amplification product of about 1500 bp was obtained ( Picture 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 amino acid sequence encoded by the gene is named TaSKP1-5B protein, and the amino acid sequence is shown in SEQ ID No.2.

[0025] By using the wheat genome for multiple sequence alignment, the allele was obtained, and the nucleotide sequence included the DNA sequence shown in SEQ ID NO:3, which was named TaSKP1-5A The amino acid sequence encoded by the gene is named 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 example provides a method for constructing TaSKP1 knockout transgenic wheat, the steps are as follows: (1) The CDS sequence of the TaSKP1-5B gene was placed in the E-crispr (http: / / www.e-crisp.org / E-CRISP / designcrispr.html) website to design gRNA targets. The specificity of the targets was then detected using the Ensemble plant (http: / / plants.ensembl.org / index.html) website to obtain two targets, sgRNA1 (SEQ ID No.7: 5'-CCCGAAGGAGCAAGCCCCTA-3') and sgRNA2 (SEQ ID No.8: 5'-GATGGGTGAGCAACCCCTTA-3'), which were conserved in TaSKP1-5B and TaSKP1-5A. The sgRNAs were constructed into the pCBC-MT1T2 vector by PCR reaction. The PCR products were purified and used for restriction digestion and ligation reactions.

[0028] (2) Perform restriction enzyme digestion and ligation of PCR products. The total reaction system is 15 µL: 2 µL of PCR product in 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 on ice for cooling.

[0029] (3) According to the E. coli transformation instructions of Beijing Bomade Gene Technology Co., Ltd., the enzyme-digested ligation product was transformed into DH5α, evenly spread on an LB plate containing 50 mg / ml Kana, and inverted and cultured at 37°C overnight; colony PCR detection was performed using detection primers 414-seq-F (SEQ ID No.9): 5'-TTTCCCAGTCACGACGTTGT-3' and 414-seq-R (SEQ ID No.10): 5'-ATCTCTAGAGAGGGGCACGA-3', and the positive product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing; the cloned plasmid with correct sequencing was extracted and transformed into EHA105 Agrobacterium 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 to obtain T0 generation plants. The plant DNA was extracted using the CTAB method and the target sites were sequenced and identified using primers (Table 1) to obtain the positive strains of transgenic knockout KO-1 and KO-2 ; By adding generation, we can obtain TaSKP1Double mutant homozygous transgenic knockout strains, whose editing types are as follows Picture 2 shown.

[0031] Table 1 Primers for detecting target sites of knockout transgenic wheat

[0032] (5) TaSKP1 Determination of protein content in grains of transgenic knockout wheat lines

[0033] 1) Harvesting TaSKP1 transgenic knockout lines KO-1 , KO-2 And the mature kernels of wild-type Fielder.

[0034] 2) Use the Botong DA7200 near-infrared grain analyzer to measure different TaSKP1 The protein content of wheat grains of transgenic knockout lines was repeated three times and the mean value was finally taken for data analysis. Picture 3 .

[0035] (6) Results analysis

[0036] By wild-type Fielder, TaSKP1 Transgenic knockout strains KO-1 and KO-2 The comparison of grain protein content showed that compared with wild-type Fielder, KO-1 and KO-2 The protein content of wheat grains in the two lines increased significantly, which indicated that TaSKP1 Genes can negatively regulate wheat grain protein content.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of a protein having 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 the amino acid sequence shown in SEO ID No.2, which is obtained by replacing, deleting or inserting one or more amino acid residues and having the same functional protein.

2. Application of the protein having the amino acid sequence as shown in SEQ ID No. 4 in regulating the protein content of wheat grains; Or the amino acid sequence is the amino acid sequence shown in SEO ID No.4, which is obtained by replacing, deleting or inserting one or more amino acid residues and having the same functional protein.

3. Application of the coding gene with the nucleotide sequence as shown in SEQ ID No. 1 in regulating the protein content of wheat grains; Or the sequence of the coding gene is a nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID No.

1.

4. Application of the coding gene with the nucleotide sequence as shown in SEQ ID No. 3 in regulating the protein content of wheat grains; Or the sequence of the coding gene is a nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID No.

3.

5. Use of a protein having 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 transgenic wheat; (2) Wheat breeding; (3) Wheat variety improvement.

6. Use of a protein having 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 increasing the protein content of wheat grains, characterized in that: The coding gene negatively regulates the protein content of wheat grains.

7. The use according to claim 6, characterized in that: The expression level of the protein with the amino acid sequence shown in SEQ ID No. 2 or 4 in the wheat plant is reduced, so that the protein content of the wheat grain is increased.

8. The use according to claim 6, characterized in that: Knocking out the coding gene shown in SEQ ID No. 1 or 3 in wheat increases the protein content of wheat grains.

9. A sgRNA for knocking out a gene encoding a nucleotide sequence as shown in SEQ ID No. 1, characterized in that: The sequence of the sgRNA is shown in SEQ ID No.

7.

10. An sgRNA for knocking out a gene encoding a nucleotide sequence as shown in SEQ ID No. 3, characterized in that: The sequence of the sgRNA is shown in SEQ ID No.8.

Images