Application of TaPKMT-5A gene and related biological materials thereof in regulating and controlling protein content of plant seeds

Abstract

The invention discloses an application of a TaPKMT-5A gene and a related biological material thereof in regulating and controlling the protein content of plant seeds. The gene TaPKMT-5A is cloned from wheat, the gene TaPKMT-5A is overexpressed in wheat Fielder, a TaPKMT-5A gene overexpressed plant is obtained, and a detection result shows that the protein content in wheat seeds can be remarkably increased by 13.3%-16.9% or above through TaPKMT-5A gene overexpression, so that the TaPKMT-5A gene has the effect of regulating and controlling the protein content in the plant seeds, and the protein content in the wheat seeds can be remarkably increased by 13.3%-16.9% or above. The method can be applied to plant breeding or quality improvement.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to the application of TaPKMT-5A gene and related biological materials in regulating the protein content of plant seeds. Background Art

[0002] wheat( Triticum aestivum L.) is one of the most important food crops in the world. Its quality and yield are directly related to human food security. Among many quality indicators, protein content is a key factor in measuring the nutritional value of wheat. Cultivating wheat varieties with high protein content is of great significance to improving the quality of human diet.

[0003] Protein is one of the most important substances that make up an organism. As the world's third largest food crop, wheat provides about 25% of human protein intake. The quality and quantity of wheat grain protein are important factors that determine nutrition and processing quality, and are also the most important indicator for international commodity grain pricing. Increasing wheat protein content and improving wheat quality are the main goals of wheat breeders in various countries. Therefore, further strengthening basic research on wheat quality improvement and revealing the molecular genetic mechanism that affects grain protein content are of great significance to improving China's wheat quality and cultivating high-quality special varieties. Summary of the invention

[0004] The purpose of the present invention is to provide the application of TaPKMT-5A gene and related biological materials in regulating the protein content of plant seeds.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of TaPKMT-5A gene and related biological materials in regulating the protein content of plant seeds.

[0006] The TaPKMT-5A gene of the present invention is derived from wheat ( Triticum aestivum ), which is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO: 2.

[0007] Further, the CDS sequence of the TaPKMT-5A gene is as follows: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence expresses a protein with the same function; iii) a nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization in a 0.1×SSPE solution containing 0.1% SDS or a 0.1×SSC solution containing 0.1% SDS at 65°C and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses a protein with the same function.

[0008] Furthermore, the regulation is positive regulation.

[0009] The plant includes wheat. Further, the biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0010] In a second aspect, the present invention provides a method for increasing the protein content of plant seeds, the method comprising: introducing the TaPKMT-5A gene into the plant via a plasmid or integrating it into the plant chromosome via genetic engineering.

[0011] In a third aspect, the present invention provides a method for increasing the protein content of wheat seeds, the method comprising: enhancing the expression of TaPKMT-5A gene in wheat.

[0012] Furthermore, the enhanced approach may be selected from the following 1) to 6), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on wheat chromosomes; 3) by changing the promoter sequence of the gene on the wheat chromosome; 4) by operably linking a strong promoter to the gene; 5) by introducing enhancers; 6) Enhanced by using genes or alleles encoding high activity of the corresponding enzyme or protein.

[0013] In a fourth aspect, the present invention provides the use of the transgenic plant obtained according to the method in plant breeding.

[0014] Further, breeding methods include but are not limited to transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides the application of TaPKMT-5A gene and related biological materials in regulating or increasing the protein content of plant seeds. The gene TaPKMT-5A is cloned from wheat and overexpressed in wheat Fielder to obtain transgenic plants overexpressing the TaPKMT-5A gene. The test results show that overexpression of the TaPKMT-5A gene can significantly increase the protein content in wheat seeds by 1.68% to 2.13%. Therefore, the TaPKMT-5A gene has the function of regulating the protein content of plant seeds and can be applied to plant breeding or quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a map of the TaOE (i.e., TaOE-PPT) carrier in a preferred embodiment of the present invention.

[0017] Figure 2 This is the map of the TaOE-TaPKMT-5A (i.e., TaPKMT-TaOE) carrier in a preferred embodiment of the present invention.

[0018] Figure 3 The RT-PCR detection results of the relative expression levels of the TaPKMT-5A gene in the overexpression strain and the wild-type plant in the preferred embodiment of the present invention.

[0019] Figure 4 The results are as follows in a preferred embodiment of the present invention, in which a near infrared spectrometer was used to detect the total protein content in the mature seeds of the overexpression strain and the wild-type plant. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0021] Example 1 Cloning and Functional Verification of Wheat TaPKMT-5A Gene 1. Experimental Materials and Reagents 1.1 Experimental Materials Wheat material “Fielder”: preserved in this laboratory.

[0022] 1.2 Reagents Escherichia coli Trans1-T1 Phage Resistant competent cells: Quanshijin Company, catalog number: CD501-03; RNA Reverse Transcription Kit EasyScript ® One-Step RT-PCR SuperMix: Quanshijin Company, catalog number: AE411-02; pEASY®-Uni Seamless Cloning and Assembly Kit: Quanshijin Company, catalog number: CU101-01; The wheat overexpression vector TaOE (the full sequence of the TaOE vector is composed of the sequences shown in SEQ ID NO:3-4) was kindly donated by Professor Liu Zhiyong from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences (Li, M., Zhang, H., Xiao, H. et al. A membrane associated tandem kinase from wild emmer wheat confers broad-spectrum resistance to powdery mildew. Nat Commun 15, 3124 (2024). https: / / doi.org / 10.1038 / s41467-024-47497-w); Agrobacterium EHA109 competent cells: Huayueyang Company, product number: GT707.

[0023] 2. Experimental Methods 2.1 In the early stage, a genome-wide association study (GWAS) was conducted using 350 germplasm resources, and a candidate site HWP3 related to the regulation of wheat grain protein content was obtained. Through positioning, it was found that the candidate site belongs to the TaPKMT-5A genome. PKMT (Protein-lysine N-methyltransferase) is a class of enzymes that can catalyze the methylation of lysine residues on histone tails and non-histone targets. This type of enzyme modifies proteins by monomethylation, dimethylation or trimethylation by transferring methyl groups from S-adenosylmethionine to the ε-amino group of lysine. These modifications have an important effect on the structure and compactness of chromatin, thereby regulating gene expression. Therefore, we cloned the TaPKMT-5A gene in wheat, and constructed related biological materials and phenotypic analysis.

[0024] 2.2 Total RNA was extracted from the leaves of the wheat material "Chinese spring", reversed into cDNA and used as a template. The forward primer (5'-cctaggcctactagg ATGGCGAGCGGCGCGG-3') and reverse amplification primer (5'-cgaaagctctgagct CTAAATGGGGGCGCCATCGC-3') were used for cloning and sequencing to obtain the wheat TaPKMT-5A gene, whose CDS sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by TaPKMT-5A is shown in SEQ ID NO:2.

[0025] 2.3 The PCR reaction program was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 68°C for 2 min, 40 cycles; extension at 68°C for 5 min.

[0026] 2.4 Construction of wheat TaPKMT-5A overexpression vector The TaPKMT-5A overexpression vector was constructed into TaOE vector by seamless cloning (vector map as shown in Figure 1 As shown in Figure 1, TaOE-PPT). The specific method is as follows: First, the TaOE vector was digested with restriction endonucleases BamHⅠ and Sac I at 37°C for 3 hours, and then recovered and purified. Then, the PCR product (3 uL), the linearized TaOE fragment (2 uL) and 2 × Assembly Mis (5 uL) were mixed and ligated at 50°C for 30 minutes. Finally, the ligation product was transformed into Escherichia coli Trans1-T1 competent cells and cultured at 37°C for 12 hours to obtain recombinant Escherichia coli, and positive monoclonal colonies were screened and sequenced.

[0027] 3.5 The correctly sequenced monoclonal colonies were amplified by shaking in LB liquid medium to extract the recombinant plasmid TaOE-TaPKMT-5A ( Figure 2 , namely TaPKMT-TaOE). The TaOE-TaPKMT-5A plasmid (10 uL) was transformed into Agrobacterium EHA105 competent cells and cultured on LB solid medium (containing kanamycin 50 mg / L and rifampicin 25 mg / L) at 28°C for 2 days. Positive clones were selected and PCR identified using forward primer F: 5'-GATGATGGCATATGCAGCAGCT-3' and reverse primer R: 5'-TGTCGAAACCGATGATACGAACGA-3'. The positive bacterial solution obtained by PCR identification was stored at -80°C for later use.

[0028] 3.6 Obtaining transgenic plants Referring to the wheat transformation method of Yuji Ishida et al. (Methods in Molecular Biology Volume 1223, 2015, pp189-198 doi: 101007 / 978-1-4939-1695-5 15. Wheat (Triticum aestivum L.) Transformation Using Immature Embryos), the Agrobacterium in the above step was transformed into the wheat Fielder material to obtain TaOE-TaPKMT-5A overexpressing transgenic wheat, and the detection primers of the Bar gene (forward primer: 5'-atgagcccagaacgacgcccg-3' and reverse primer: 5'-aactcgagtcaaatctcggt-3', product size 560 bp) were used to identify the transgenic plants, and more than 15 independent T0 generation TaOE-TaPKMT-5A overexpressing transgenic lines were obtained. The T0 generation transgenic overexpression lines were propagated and planted to obtain T1 generation plants, which were then identified by PCR using the Bar gene detection primers. Finally, the T1 generation positive individual seeds were harvested and further planted and tested to obtain T2 generation positive transgenic wheat homozygous lines.

[0029] The expression level of TaPKMT-5A gene in each line of TaOE-TaPKMT-5A transgenic T2 generation overexpressing wheat was detected, and three biological replicates were set up in the experiment. Each replicate experiment was set up as follows: total RNA from leaves of three transgenic overexpressing lines and wild-type (WT) wheat Fielder was extracted and reverse transcribed into cDNA. Real-time RT-PCR method was used to detect the relative expression level of TaPKMT-5A gene with wheat-actin (forward primer: 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer: 5'-CCAGACAACTCGCAACTTAGA-3') as the internal standard gene. The primer sequences of TaPKMT-5A are as follows: forward primer: 5'- GAGGAGCAGCGACGGCAGGA-3' and reverse primer: 5'-GGTTGTAGTCGTAGAAGGCGAAGTC -3'.

[0030] Test results such as Figure 3As shown, the expression level of TaPKMT-5A gene in the overexpression transgenic wheat lines TaOE-TaPKMT-5A-OE-1, TaOE-TaPKMT-5A-OE-2 and TaOE-TaPKMT-5A-OE-3 was significantly higher than that in the wild-type plant (WT) Fielder (P<0.001). The results indicated that the overexpression transgenic wheat TaOE-TaPKMT-5A-OE line was successfully constructed.

[0031] The total protein content in the mature seeds of the three TaOE-TaPKMT-5A transgenic overexpression lines and the wild-type (WT) wheat Fielder was measured using a near-infrared spectrometer (Perton DA7000). Figure 4 As shown, the total protein content in the seeds of wild-type plants (WT) was about 12.56%, while the total protein content in the seeds of transgenic lines TaOE-TaPKMT-5A-OE-1, TaOE-TaPKMT-5A-OE-2 and TaOE-TaPKMT-5A-OE-3 were 14.69%, 14.32% and 14.24%, respectively, which were significantly (P < 0.001) higher than the wild-type plants (WT) wheat Fielder, increased by 2.13%, 1.76% and 1.68%, respectively, indicating that overexpression in wheat TaPKMT-5A The gene can significantly increase the total protein content in the grain. It can be seen that overexpression of wheat TaPKMT-5A will lead to a significant increase in the protein content in wheat seeds, and the encoded protein TaPKMT-5A can regulate the protein content of wheat seeds and can be used in wheat breeding and variety improvement.

[0032] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Application of TaPKMT-5A gene and its related biological materials in regulating the protein content of plant seeds; The TaPKMT-5A gene is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent functions, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The regulation is positive regulation.

3. The use according to claim 1, characterized in that: The plant is wheat.

4. The use according to any one of claims 1 to 3, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

5. A method for increasing the protein content of plant seeds, characterized in that: The method comprises: introducing the TaPKMT-5A gene into the plant via a plasmid or integrating it into the plant chromosome via genetic engineering; The TaPKMT-5A gene is the same as that described in claim 1.

6. The method according to claim 5, characterized in that The plant is a grass plant, preferably wheat.

7. A method for increasing the protein content of wheat seeds, characterized in that: The method comprises: enhancing the expression of TaPKMT-5A gene in wheat; The TaPKMT-5A gene is the same as that described in claim 1.

8. The method according to claim 7, characterized in that The enhanced approach is selected from the following 1) to 6), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on wheat chromosomes; 3) by changing the promoter sequence of the gene on the wheat chromosome; 4) by operably linking a strong promoter to the gene; 5) by introducing enhancers; 6) Enhanced by using genes or alleles encoding high activity of the corresponding enzyme or protein.

9. Use of the transgenic plant obtained by the method according to any one of claims 5 to 8 in plant breeding.

10. The use according to claim 9, characterized in that: Breeding methods include transgenic, hybridization, backcrossing, selfing or asexual reproduction.

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