Application of TaNLP-3B gene and related biological materials in regulating protein content in plant seeds

Abstract

The application discloses TaNLP-3B gene and application of related biological materials in regulating plant seed protein content. The gene TaNLP-3B is cloned from wheat, and the gene is overexpressed in wheat Fielder to obtain a trans-TaNLP-3B gene overexpression plant. The detection result shows that the overexpression of the TaNLP-3B gene can significantly increase the content of the protein in the wheat seed, and the increase is more than 40%. Therefore, the TaNLP-3B gene has the function of regulating the content of the protein in the plant seed, and can be applied to plant breeding or quality improvement.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of the TaNLP-3B gene and related biomaterials in regulating the protein content of plant seeds. Background Technology

[0002] wheat( Triticum aestivum Wheat (L.) is one of the world's most important food crops, and 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. Developing wheat varieties with high protein content is of great significance for improving the quality of human diets.

[0003] The protein content of wheat seeds is a crucial factor influencing the nutritional and processing quality of wheat flour. As a typical quantitative trait, wheat seed protein content is influenced by multiple genes; therefore, identifying these genes is essential for improving wheat protein quality. Increasing the protein content of wheat grains is significant for enhancing the nutritional value of food, improving food processing performance, increasing economic efficiency, addressing climate change, reducing dependence on animal protein, promoting sustainable agricultural development, and improving global food security. Increasing wheat protein content can provide richer nutrition for populations whose staple food is wheat, while also improving dough elasticity and extensibility, which is particularly important for pasta processing. Furthermore, high-protein wheat has a higher market value, bringing better economic returns to farmers. In the context of climate change, high-protein wheat varieties may exhibit greater resilience, contributing to food production. Reducing dependence on animal protein contributes to environmental protection; simultaneously, increasing wheat protein content can reduce the use of chemical fertilizers and pesticides, promoting sustainable agricultural development. Globally, increasing wheat protein content helps increase food supply, playing a positive role in reducing hunger and malnutrition, thus having a profound impact on global food security. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the TaNLP-3B gene and related biological materials in regulating the protein content of plant seeds.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides the application of the TaNLP-3B gene and related biological materials in regulating the protein content of plant seeds.

[0006] The TaNLP-3B gene of this invention is derived from wheat ( Triticum aestivum ), which is a gene encoding either protein (a) or (b) as follows:

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0008] (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.

[0009] Furthermore, the CDS sequence of the TaNLP-3B gene is as follows:

[0010] i) The nucleotide sequence shown in SEQ ID NO:1;

[0011] 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;

[0012] 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

[0013] iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0014] Furthermore, the regulation described is a positive regulation.

[0015] The plant mentioned includes wheat.

[0016] 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.

[0017] Secondly, the present invention provides a method for increasing the protein content of plant seeds, the method comprising: introducing the TaNLP-3B gene into the plant via a plasmid or integrating it into the plant chromosome via genetic engineering.

[0018] Thirdly, the present invention provides a method for increasing the protein content of wheat seeds, the method comprising: enhancing the expression of the TaNLP-3B gene in wheat.

[0019] Furthermore, the enhancement can be selected from the following 1) to 6), or an optional combination thereof:

[0020] 1) By importing a plasmid containing the gene;

[0021] 2) By increasing the copy number of the aforementioned genes on the wheat chromosome;

[0022] 3) By altering the promoter sequences of the aforementioned genes on the wheat chromosome;

[0023] 4) By operatively linking a strong promoter to the gene;

[0024] 5) By introducing enhancers;

[0025] 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0026] Fourthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0027] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0028] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0029] This invention provides the application of the TaNLP-3B gene and related biological materials in regulating or increasing the protein content of plant seeds. The TaNLP-3B gene was cloned from wheat and overexpressed in wheat Fielder to obtain TaNLP-3B overexpressing plants. The results showed that TaNLP-3B gene overexpression significantly increased the protein content in wheat seeds by more than 40%. Therefore, the TaNLP-3B 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

[0030] Figure 1 This is a TaOE (i.e., TaOE-PPT) carrier spectrum in a preferred embodiment of the present invention.

[0031] Figure 2 The image shows the TaOE-TaNLP-3B vector spectrum in a preferred embodiment of the present invention.

[0032] Figure 3 The results of RT-PCR detection of the relative expression levels of the TaNLP-3B gene in overexpression lines and wild-type plants in a preferred embodiment of the present invention are shown.

[0033] Figure 4 The 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

[0034] 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.

[0035] Example 1: Cloning and functional verification of the wheat TaNLP-3B gene

[0036] 1. Experimental materials and reagents

[0037] 1.1 Experimental Materials

[0038] Wheat material “Fielder”: preserved in this laboratory.

[0039] 1.2 Reagents

[0040] Escherichia coli Trans1-T1 Phage Resistant competent cells: TransGen Biotech, catalog number: CD501-03;

[0041] RNA Reversal Kit EasyScript ® One-Step RT-PCR SuperMix: TransGen Biotech, Product No.: AE411-02;

[0042] pEASY®-Uni Seamless Cloning and Assembly Kit: TransGen Corporation, Catalog No.: CU101-01;

[0043] 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).

[0044] Agrobacterium EHA109 competent cells: Huayueyang Company, catalog number: GT707.

[0045] 2. Experimental Methods

[0046] 2.1 In the previous stage, a genome-wide association study (GWAS) was performed on 350 wheat germplasm resources, which identified the candidate gene TaNLP-3B, which is related to the regulation of wheat grain protein content. Literature review revealed that NLP (NIN-like protein) family proteins are involved in the regulation of nitrogen and phosphorus signaling pathways and play a role in plant growth, development, and stress response. Therefore, we cloned the TaNLP-3B gene in wheat and constructed related biological materials and performed phenotypic analysis.

[0047] 2.2 Total RNA was extracted from the leaves of the wheat variety "Chinese spring", reversed to cDNA, and used as a template. The wheat TaNLP-3B gene was cloned and sequenced using forward primer (5'-cctaggcctactaggATGGACATCGACCCTTCCTC-3') and reverse amplification primer (5'-cgaaagctctgagctTCAACCGGAGCTTCCACAAGAA-3'). Its CDS sequence is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by TaNLP-3B is shown in SEQ ID NO:2.

[0048] 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.

[0049] 2.4 Construction of wheat TaNLP-3B overexpression vector: The expression vector was constructed into the TaOE vector using a seamless cloning method (vector map shown in Figure 1). 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 and cultured at 37℃ for 12 hours to obtain recombinant *E. coli*. Positive single colonies were screened and sequenced.

[0050] 3.5 Sequencingly sequenced single-clone colonies were amplified by shaking in LB liquid medium, and the recombinant plasmid TaOE-TaNLP-3B was extracted. Figure 2The TaOE-TaNLP-3B plasmid (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.

[0051] 3.6 Obtaining transgenic plants

[0052] 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 obtained in the above steps yielded TaOE-TaNLP-3B 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-TaNLP-3B 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.

[0053] 3.7 The expression level of the TaNLP-3B gene in individual lines of T2 generation overexpressing TaOE-TaNLP-3B transgenic wheat was detected. Three biological replicates were set up. The experimental setup for each replicate was as follows: Total RNA was extracted from leaves of the three transgenic overexpression lines and wild-type (WT) wheat Fielder plants, reverse transcribed into cDNA, and the relative expression level of the TaNLP-3B gene was detected using RT-PCR (Real-time PCR) with wheat-actin (forward primer: 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer: 5'-CCAGACAACTCGCAACTTAGA-3') as an internal control gene. The TaNLP-3B primer sequences are as follows: forward primer: 5'-CAAGCAAAAAGTGCCTGAGTGGAC-3' and reverse primer: 5'-TTTACTGCCTCAAGAGCTTT-3'.

[0054] Test results as follows Figure 3 As shown, in the transgenic wheat lines TaOE-TaNLP-3B-OE-1, TaOE-TaNLP-3B-OE-2, and TaOE-TaNLP-3B-OE-3, the expression level of the TaNLP-3B gene was significantly higher (P<0.001) than that of wild-type wheat Fielder, indicating that the transgenic wheat line TaOE-TaNLP-3B-OE was successfully constructed.

[0055] The total protein content in mature seeds of the three TaOE-TaNLP-3B transgenic overexpression lines and wild-type (WT) wheat Fielder was determined using a near-infrared spectroscopy (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 14.18%, while the total protein content in the seeds of transgenic lines TaOE-TaNLP-3B-OE-1, TaOE-TaNLP-3B-OE-2, and TaOE-TaNLP-3B-OE-3 was 17.74%, 17.45%, and 17.13%, respectively, all significantly higher (P < 0.001) than that of wild-type (WT) wheat Fielder, increasing by 3.38%, 3.09%, and 2.77%, respectively. This indicates that total overexpression of the TaNLP-3B gene in wheat can significantly increase the total protein content in the grains.

[0056] Therefore, it can be seen that overexpression of TaNLP-3B in wheat leads to a significant increase in protein content in wheat seeds, and the encoded protein TaNLP-3B can regulate the protein content of wheat seeds, which can be applied to wheat breeding and variety improvement.

[0057] 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. Application of TaNLP-3B gene and related biomaterials in regulating protein content in plant seeds; The TaNLP-3B gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO:2; The regulation is a positive regulation; The plant in question is wheat.

2. The application according to claim 1, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

3. A method for increasing the protein content of plant seeds, characterized in that, The method includes: introducing the TaNLP-3B gene into a plant via a plasmid or integrating it into the plant chromosome via genetic engineering. The TaNLP-3B gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO:2; The plant in question is wheat.

4. A method for increasing the protein content of wheat seeds, characterized in that, The method includes: enhancing the expression of the TaNLP-3B gene in wheat; The TaNLP-3B gene is a gene that encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

5. The method according to claim 4, characterized in that, The enhancement method is selected from the following 1) to 5), or an optional combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on the wheat chromosome; 3) By altering the promoter sequences of the aforementioned genes on the wheat chromosome; 4) By operatively linking a strong promoter to the gene; 5) By importing enhancers.

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