TaSnRK2.8-5D gene and related biological materials in regulating plant seed protein content

Abstract

The application discloses TaSnRK2.8-5D gene and application of related biological materials thereof in regulating protein content of plant seeds. The gene TaSnRK2.8-5D is cloned from wheat, and the gene is overexpressed in wheat Fielder to obtain a TaSnRK2.8-5D gene overexpression plant. The detection result shows that the overexpression of the TaSnRK2.8-5D gene can significantly increase the protein content in the wheat seeds by more than 1.31%, and therefore, the TaSnRK2.8-5D gene has the effect of increasing the protein content of the plant seeds and can be applied to plant breeding or quality improvement.

Description

[0001] Cross-reference to related applications

[0002] This invention claims priority to Chinese Patent Application No. CN202411863249.5, filed on December 17, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of the TaSnRK2.8-5D gene and related biomaterials in regulating the protein content of plant seeds. Background Technology

[0004] wheat( Triticum aestivum As one of the world's most important food crops, wheat's quality and yield are directly related to human food security. Protein content is a key factor in measuring the nutritional value of wheat, and increasing the protein content of wheat grains has numerous benefits. In terms of nutritional quality, it provides the human body with high-quality protein and meets amino acid requirements, promoting growth and development and maintaining physiological functions. In terms of processing characteristics, wheat grains with high protein content produce flour with a more complete gluten network structure, making them suitable for fermented flour products and expanding processing applications. For agricultural production, it enhances wheat's resistance to stress, promotes sustainable development, reduces dependence on nitrogen fertilizers, and lowers environmental pollution. In terms of economic value, it increases income, improves industrial competitiveness, and promotes the upgrading and development of the wheat industry. Therefore, cultivating high-protein wheat varieties is of great significance for improving the quality of human diets.

[0005] As a key crop for high-quality agricultural development, improving the quality of wheat is particularly important. Although research on wheat quality traits has made some progress after years of genetic improvement, wheat quality traits are complex. Among them, the protein content of wheat grains is a key indicator for measuring wheat quality, directly affecting its processing and nutritional quality.

[0006] Studies have shown that wheat seed protein content is a typical quantitative trait, influenced by multiple genes. Therefore, identifying the genes that affect wheat seed protein content is of great significance for improving wheat protein quality. Summary of the Invention

[0007] The purpose of this invention is to provide the application of the TaSnRK2.8-5D gene and related biological materials in regulating the protein content of plant seeds.

[0008] To achieve the objectives of this invention, in a first aspect, this invention provides the application of the TaSnRK2.8-5D gene and related biological materials in regulating the protein content of plant seeds.

[0009] The TaSnRK2.8-5D gene of this invention is derived from wheat ( Triticum aestivum ), which is a gene encoding either protein (a) or (b) as follows:

[0010] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0011] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0012] Furthermore, the CDS sequence of the TaSnRK2.8-5D gene is as follows:

[0013] i) The nucleotide sequence shown in SEQ ID NO:1;

[0014] ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0015] 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 at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or

[0016] iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0017] Furthermore, the regulation described is a positive regulation.

[0018] The plant mentioned includes wheat.

[0019] Furthermore, the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0020] Secondly, the present invention provides a method for increasing the protein content of plant seeds, the method comprising: introducing the TaSnRK2.8-5D gene into the plant via plasmid or integrating it into the plant chromosome via genetic engineering.

[0021] Thirdly, the present invention provides a method for increasing the protein content of wheat seeds, the method comprising: enhancing the expression of the TaSnRK2.8-5D gene in wheat.

[0022] Furthermore, the enhancement can be selected from the following 1) to 6), or an optional combination thereof:

[0023] 1) By importing a plasmid containing the gene;

[0024] 2) By increasing the copy number of the aforementioned genes on the wheat chromosome;

[0025] 3) By altering the promoter sequences of the aforementioned genes on the wheat chromosome;

[0026] 4) By operatively linking a strong promoter to the gene;

[0027] 5) By introducing enhancers;

[0028] 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0029] Fourthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0030] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] This invention provides the application of the TaSnRK2.8-5D gene and related biological materials in regulating or increasing the protein content of plant seeds. The TaSnRK2.8-5D gene was cloned from wheat and overexpressed in wheat Fielder to obtain transgenic plants overexpressing the TaSnRK2.8-5D gene. The results showed that overexpression of the TaSnRK2.8-5D gene significantly increased the protein content in wheat seeds by 1.31%–1.66%. Therefore, the TaSnRK2.8-5D gene has the function of regulating the protein content of plant seeds and can be applied to plant breeding or quality improvement. Attached Figure Description

[0033] Figure 1 This is a TaOE (i.e., TaOE-PPT) carrier spectrum in a preferred embodiment of the present invention.

[0034] Figure 2 The image shows the vector spectrum of TaOE-TaSnRK2.8-5D (i.e., TaSnRK2.8-TaOE) in a preferred embodiment of the present invention.

[0035] Figure 3 The results of RT-PCR detection of the relative expression levels of the TaSnRK2.8-5D gene in the overexpression lines and wild-type plants in the preferred embodiments of the present invention are shown.

[0036] Figure 4The results of detecting the total protein content in mature seeds of overexpression lines and wild-type plants using a near-infrared spectroscopy in a preferred embodiment of the present invention are shown. Detailed Implementation

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0038] Example 1: Cloning and functional verification of the wheat TaSnRK2.8-5D gene

[0039] 1. Experimental materials and reagents

[0040] 1.1 Experimental Materials

[0041] Wheat material “Fielder”: preserved in this laboratory.

[0042] 1.2 Reagents

[0043] E. coli Trans 1-T1 Phage Resistant competent cells: TransGen Biotech, catalog number: CD501-03;

[0044] RNA Reversal Kit EasyScript ® One-Step RT-PCR SuperMix: TransGen Biotech, Product No.: AE411-02;

[0045] pEASY®-Uni Seamless Cloning and Assembly Kit: TransGen Corporation, Catalog No.: CU101-01;

[0046] The wheat overexpression vector TaOE (the full sequence of the TaOE vector is formed by tandemly connecting the sequences shown in SEQ ID NO:3-4) was kindly provided by Professor Zhiyong Liu of 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 bread-spectrum resistance to powdery mildew. Nat Commun 15, 3124 (2024). https: / / doi.org / 10.1038 / s41467-024-47497-w).

[0047] Agrobacterium EHA109 competent cells: Huayueyang Company, catalog number: GT707.

[0048] 2. Experimental Methods

[0049] 2.1 In the early stages, genome-wide association analysis (GWAS) was performed on 350 germplasm resources, identifying HWP2 as a candidate locus related to the regulation of wheat grain protein content. Localization revealed that this candidate locus belongs to the TaSnRK2.8-5D genome. SnRK (Sucrose Non-Fermenting 1-Related Protein Kinase 2) family plays a crucial role in plant stress response and growth regulation, participating in the regulation of plant metabolic processes and stress responses by phosphorylating multiple target proteins. Therefore, we cloned the TaSnRK2.8-5D gene from wheat, constructed related transgenic materials, and conducted phenotypic analysis and identification.

[0050] 2.2 Total RNA was extracted from the leaves of the wheat variety "Chinese Spring" and reverse transcribed into cDNA using an RNA reverse transcription kit. Then, using the reverse-transcribed cDNA as a template, the wheat TaSnRK2.8-5D gene was cloned and sequenced using both forward primers (5'-cctaggcctactaggATGGCAGGGGCGGCGCCG-3') and reverse primers (5'-cgaaagctctgagctTCACATCGCATACACGATCT-3'). Its CDS sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by TaSnRK2.8-5D is shown in SEQ ID NO:2.

[0051] 2.3 The PCR reaction procedure is as follows: 98℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 58℃ annealing for 15 seconds, 68℃ extension for 2 minutes, 40 cycles; 68℃ extension for 5 minutes.

[0052] 2.4 The above wheat TaSnRK2.8-5D DNA fragment was constructed into the TaOE vector using a seamless cloning kit (vector map shown below). Figure 1 As shown, this is the TaOE-PPT vector. The specific method is as follows: First, the TaOE vector was digested with restriction endonucleases BamHI and SacI at 37℃ for 3 hours, and then purified. Next, the PCR product (3 μL), the linearized TaOE fragment (2 μL), and 2 × Assembly Mis (5 μL) were mixed and ligated at 50℃ for 30 minutes. Finally, the ligation product was transformed into *E. coli*. Trans1-T1 competent cells were transformed and cultured at 37°C for about 12 hours to obtain recombinant Escherichia coli. Finally, positive monoclonal colonies were screened and sequenced.

[0053] 3.5 Sequencing-correct single-clone colonies were amplified by shaking in LB liquid medium, and the recombinant plasmid TaOE-TaSnRK2.8-5D was extracted. Figure 2 The plasmid TaOE-TaSnRK2.8-5D (10 μL) was transformed into Agrobacterium EHA105 competent cells and cultured at 28°C for 2 days on LB solid medium (containing 50 mg / L kanamycin and 25 mg / L rifampin). Positive clones were selected and identified by PCR using forward primer F: 5'-gatgatggcatatgcagcagct-3' and reverse primer R: 5'-tgtcgaaaccgatgatacgaacga-3'. The positive bacterial cultures obtained by PCR identification were stored at -80°C for later use.

[0054] 3.6 Obtaining transgenic plants

[0055] Following the wheat transformation method described by 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-mediated transformation of wheat Fielder material was used to obtain TaOE-TaSnRK2.8-5D overexpressing transgenic wheat. The transgenic plants were identified using Bar gene detection primers (forward primer: 5'-ATGAGCCCAGAACGACGCCCG-3' and reverse primer: 5'-AACTCGAGTCAAATCTCGGT-3', product size 560 bp). More than 15 independent T0 generation TaOE-TaSnRK2.8-5D overexpressing transgenic lines were obtained. The T0 generation transgenic overexpression lines were propagated and planted to obtain T1 generation individual plants. The T1 generation individual plants were then identified by PCR using the same primers for detecting the Bar gene. Finally, the seeds of the positive T1 generation individual plants were harvested and planted for further testing to obtain T2 generation positive transgenic wheat homozygous lines.

[0056] The expression level of the TaSnRK2.8-5D gene in three lines of T2 generation transgenic wheat overexpressing TaOE-TaSnRK2.8-5D was detected. Three biological replicates were set up for each replicate. Total RNA was extracted from leaves of the three transgenic overexpression lines and wild-type (WT) wheat Fielder plants, reverse transcribed into cDNA, and analyzed using Real-time RT-PCR with wheat-actin (forward primer: 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer: 5'-CCAGACAACTCGCAACTTAGA-3') as an internal control gene to detect the relative expression level of the TaSnRK2.8-5D gene. The primer sequences for TaSnRK2.8-5D are as follows: forward primer: 5'-CACCGACCCATCTTGCTATTGTC-3' and reverse primer: 5'-TCTTCAAGCGAGGAGCAGGA-3'.

[0057] Test results as follows Figure 3 As shown, in the transgenic wheat lines TaOE-TaSnRK2.8-5D-OE-1, TaOE-TaSnRK2.8-5D-OE-2, and TaOE-TaSnRK2.8-5D-OE-3, the expression level of the TaSnRK2.8-5D gene was significantly higher (P<0.001) than that of wild-type wheat Fielder, indicating that the transgenic wheat line TaOE-TaSnRK2.8-5D-OE was successfully constructed.

[0058] The total protein content in mature seeds of the three TaOE-TaSnRK2.8-5D transgenic overexpression lines and wild-type (WT) wheat Fielder was determined using a near-infrared spectrometer (Borton DA7000). The results are as follows: Figure 4 As shown, the total protein content in the seeds of wild-type (WT) wheat Fielder was approximately 12.03%, while the total protein content in the seeds of transgenic lines TaOE-TaSnRK2.8-5D-OE-1, TaOE-TaSnRK2.8-5D-OE-2, and TaOE-TaSnRK2.8-5D-OE-3 was 13.69%, 13.45%, and 13.34%, respectively, all significantly higher (P < 0.001) than that in wild-type (WT) wheat Fielder, increasing by 1.66%, 1.42%, and 1.31%, respectively. This indicates that the total protein content in the seeds of transgenic lines TaOE-TaSnRK2.8-5D-OE-1, TaOE-TaSnRK2.8-5D-OE-2, and TaOE-TaSnRK2.8-5D-OE-3 was significantly higher (P < 0.001) than that in wild-type (WT) wheat Fielder, increasing by 1.66%, 1.42%, and 1.31%, respectively. TaSnRK2.8-5D Genes can significantly increase the total protein content in grains.

[0059] Therefore, overexpression of wheat TaSnRK2.8-5D leads to a significant increase in protein content in wheat seeds, and the encoded protein TaSnRK2.8-5D can regulate the protein content of wheat seeds, which can be applied to wheat breeding and variety improvement.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. TaSnRK2.8-5D gene and related biological materials in improving the protein content of wheat seeds; TaSnRK2.8-5D gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO: 2; The biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector or an engineered bacterium.

2. A method of increasing the protein content of wheat seed, characterized in that, The method comprises: introducing TaSnRK2.8-5D gene into plants through a plasmid or integrating it into the plant chromosome through genetic engineering means; TaSnRK2.8-5D gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO:

2.

3. A method of increasing the protein content of wheat seed, characterized in that, The method comprises: enhancing the expression of TaSnRK2.8-5D gene in wheat; TaSnRK2.8-5D gene is a gene encoding a protein with an amino acid sequence as shown in SEQ ID NO:

2.

4. The method of claim 3, wherein, The enhanced approach is selected from the following 1) ~ 5), or optional combination: 1) by introducing a plasmid with the gene; 2) by increasing the copy number of the gene on the wheat chromosome; 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 an enhancer.

5. The application of transgenic wheat obtained by the method according to any one of claims 2-4 in breeding to improve the protein content of wheat seeds.

6. Use according to claim 5, characterized in that, The breeding method comprises transgenesis, crossing, backcrossing, selfing or vegetative reproduction.

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