Application of TaSnRK2.8-5D gene and related biological materials thereof in regulation and control of protein content of plant seeds

Abstract

The invention discloses an application of a TaSnRK2.8-5D gene and a related biological material thereof in regulating and controlling the protein content of plant seeds. The gene TaSnRK2.8-5D is cloned from wheat, the gene TaSnRK2.8-5D is overexpressed in wheat Fielder, a TaSnRK2.8-5D gene overexpressed plant is obtained, and a detection result shows that the protein content in wheat seeds can be remarkably increased by more than 1.31% through TaSnRK2.8-5D gene overexpression, so that the TaSnRK2.8-5D gene has an effect of increasing the protein content in the plant seeds, and the protein content in the wheat seeds can be increased by more than 1.31%. The method 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 text of which is incorporated herein by reference. Technical Field

[0003] The invention relates to the technical field of plant genetic engineering, in particular to the application of TaSnRK2.8-5D gene and related biological materials in regulating the protein content of plant seeds. Background Art

[0004] wheat( Triticum aestivum L.) As one of the world's important food crops, its quality and yield are directly related to human food security. Protein content is a key factor in measuring the nutritional value of wheat. Increasing the protein content in wheat grains has many benefits. In terms of nutritional quality, it can provide the human body with high-quality protein and meet the needs of amino acids, promote growth and development, and maintain physiological functions; in terms of processing characteristics, the flour gluten network structure ground from wheat grains with high protein content is more perfect, suitable for making fermented pasta products, and can also expand processing uses; for agricultural production, it can enhance wheat stress resistance, promote sustainable development, reduce dependence on nitrogen fertilizers and reduce environmental pollution; in terms of economic value, it can increase income, improve industrial competitiveness, and promote the upgrading and development of the wheat industry. Therefore, cultivating wheat varieties with high protein content is of great significance to improving the quality of human diet.

[0005] As a key crop for high-quality agricultural development, wheat quality improvement is particularly important. Although some progress has been made in wheat quality trait research after years of genetic improvement, wheat quality traits are complex, among which wheat grain protein content is a key indicator for measuring wheat quality, which directly affects the processing quality and nutritional quality of wheat.

[0006] Studies have shown that the protein content of wheat seeds is a typical quantitative trait affected by multiple genes. Therefore, discovering the genes that affect the protein content of wheat seeds is of great significance for improving the quality of wheat protein. Summary of the invention

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

[0008] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides the use of TaSnRK2.8-5D gene and related biological materials in regulating the protein content of plant seeds.

[0009] The TaSnRK2.8-5D 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.

[0010] Furthermore, the CDS sequence of the TaSnRK2.8-5D 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.

[0011] Furthermore, the regulation is positive regulation.

[0012] The plants include wheat.

[0013] Furthermore, the biological material includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0014] In a second aspect, the present invention provides a method for increasing the protein content of plant seeds, the method comprising: introducing the TaSnRK2.8-5D gene into a plant via a plasmid or integrating it into a plant chromosome via genetic engineering.

[0015] 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 TaSnRK2.8-5D gene in wheat.

[0016] 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.

[0017] In a fourth aspect, the present invention provides the use of the transgenic plant obtained according to the method in plant breeding.

[0018] Further, breeding methods include but are not limited to transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0019] 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 TaSnRK2.8-5D gene and related biological materials in regulating or increasing the protein content of plant seeds. The gene TaSnRK2.8-5D is cloned from wheat and overexpressed in wheat Fielder to obtain transgenic plants overexpressing the TaSnRK2.8-5D gene. The test results show that overexpression of the TaSnRK2.8-5D gene can significantly increase the protein content in wheat seeds by 1.31% to 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a map of the TaOE (i.e., TaOE-PPT) carrier in a preferred embodiment of the present invention.

[0021] Figure 2 This is the carrier spectrum of TaOE-TaSnRK2.8-5D (i.e., TaSnRK2.8-TaOE) in a preferred embodiment of the present invention.

[0022] Figure 3 The RT-PCR test results of the relative expression levels of the TaSnRK2.8-5D gene in the overexpression strain and the wild-type plant in the preferred embodiment of the present invention.

[0023] Figure 4 The results of using a near-infrared spectrometer to detect the total protein content in mature seeds of overexpression strains and wild-type plants in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0024] 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.

[0025] Example 1 Cloning and Functional Verification of Wheat TaSnRK2.8-5D Gene 1. Experimental Materials and Reagents 1.1 Experimental Materials Wheat material “Fielder”: preserved in this laboratory.

[0026] 1.2 Reagents Escherichia coli Trans 1-T1 Phage Resistant competent cells: Quanshijin Company, catalog number: CD501-03; RNA Reverse Transcriptase 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.

[0027] 2. Experimental Methods 2.1 In the early stage, a genome-wide association study (GWAS) was conducted using 350 germplasm resources, and the candidate site HWP2 related to the regulation of wheat grain protein content was obtained. Through positioning, it was found that the candidate site belongs to the TaSnRK2.8-5D genome. The SnRK (Sucrose Non-Fermenting 1-Related Protein Kinase 2) family plays an important role in plant stress response and growth regulation, and participates in regulating plant metabolic processes and stress responses by phosphorylating multiple target proteins. Therefore, we cloned the TaSnRK2.8-5D gene in wheat, constructed related transgenic materials, and conducted phenotypic analysis and identification.

[0028] 2.2 Total RNA was extracted from the leaves of the wheat variety "Chinese spring" and reverse transcribed into cDNA using an RNA reverse transcriptase kit. Then, the reverse transcribed cDNA was used as a template, and the forward primer (5'-cctaggcctactaggATGGCAGGGGCGGCGCCG-3') and the reverse amplification primer (5'-cgaaagctctgagctTCACATCGCATACACGATCT-3') were used for cloning and sequencing to obtain the wheat TaSnRK2.8-5D gene, whose 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.

[0029] 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.

[0030] 2.4 The wheat TaSnRK2.8-5D DNA fragment was constructed into the TaOE vector using a seamless cloning kit (the vector map is shown in Figure 1 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 ligated product was transformed into Escherichia coli. Trans 1-T1 competent cells were transformed and cultured at 37°C for about 12 hours to obtain recombinant E. coli. Finally, positive monoclonal colonies were screened and sequenced.

[0031] 3.5 The correctly sequenced monoclonal colonies were amplified by shaking in LB liquid medium to extract the recombinant plasmid TaOE-TaSnRK2.8-5D ( Figure 2 , namely TaSnRK2.8-TaOE). The TaOE-TaSnRK2.8-5D 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.

[0032] 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-TaSnRK2.8-5D 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-TaSnRK2.8-5D 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.

[0033] The expression level of TaSnRK2.8-5D gene in each line of TaOE-TaSnRK2.8-5D 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 TaSnRK2.8-5D gene with wheat-actin (forward primer: 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer: 5'-CCAGACAACTCGCAACTTAGA-3') as the internal standard gene. The primer sequences of TaSnRK2.8-5D are as follows: forward primer: 5'-CACCGACCCATCTTGCTATTGTC-3' and reverse primer: 5'-TCTTCAAGCGAGGAGCAGGA-3'.

[0034] Test results such as Figure 3 As shown, the expression level of TaSnRK2.8-5D gene in the overexpressing transgenic wheat TaOE-TaSnRK2.8-5D-OE-1, TaOE-TaSnRK2.8-5D-OE-2 and TaOE-TaSnRK2.8-5D-OE-3 lines was significantly higher (P<0.001) than that in the wild-type wheat Fielder. The results indicated that the overexpressing transgenic wheat TaOE-TaSnRK2.8-5D-OE line was successfully constructed.

[0035] The total protein content in the mature seeds of the three TaOE-TaSnRK2.8-5D transgenic overexpression lines and the wild-type (WT) wheat Fielder was measured using a near-infrared spectrometer (Perton DA7000). Figure 4 As shown in the figure, the Fielder of wild-type plants (WT) wheat was about 12.03%, while the total protein contents 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 were 13.69%, 13.45% and 13.34%, respectively, which were significantly (P < 0.001) higher than the Fielder of wild-type plants (WT) wheat, increased by 1.66%, 1.42% and 1.31%, respectively, indicating that the total protein content of wheat overexpression was 1.66%, 1.42% and 1.31%, respectively, which was significantly higher than that of wild-type plants (WT) wheat Fielder. TaSnRK2.8-5D The gene can significantly increase the total protein content in the grain.

[0036] It can be seen that overexpression of wheat TaSnRK2.8-5D will lead to a significant increase in the protein content in wheat seeds, and the encoded protein TaSnRK2.8-5D can regulate the protein content in wheat seeds and can be used in wheat breeding and variety improvement.

[0037] 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 TaSnRK2.8-5D gene and its related biomaterials in regulating the protein content of plant seeds; The TaSnRK2.8-5D 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 TaSnRK2.8-5D gene into the plant via a plasmid or integrating it into the plant chromosome via genetic engineering; The TaSnRK2.8-5D 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 TaSnRK2.8-5D gene in wheat; The TaSnRK2.8-5D 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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