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

Abstract

The invention discloses an application of a TaNLP-3B gene and a related biological material thereof in regulating and controlling the protein content of plant seeds. The gene TaNLP-3B is cloned from wheat, the gene TaNLP-3B is overexpressed in wheat Fielder to obtain a TaNLP-3B transgenic overexpressed plant, and a detection result shows that the protein content in wheat seeds can be remarkably increased by more than 40% through TaNLP-3B gene overexpression, so that the TaNLP-3B gene has an effect of regulating and controlling the protein content in the plant seeds, and the protein content in the wheat seeds can be increased by more than 40%. 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 TaNLP-3B 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] The protein content of wheat seeds is one of the important factors affecting the nutritional quality and processing quality of wheat flour. The protein content of wheat seeds is a typical quantitative trait affected by multiple genes. Therefore, exploring the genes that affect the protein content of wheat seeds is of great significance for improving the protein quality of wheat. Increasing the protein content in wheat grains is of great significance for enhancing the nutritional value of food, improving food processing performance, improving economic benefits, coping with climate change, reducing dependence on animal protein, promoting sustainable agricultural development, and improving global food security. By increasing the protein content of wheat, it can provide more abundant nutrition for people who mainly eat wheat, while improving the elasticity and extensibility of dough, which is particularly important for pasta processing. In addition, high-protein wheat has a higher market value and can bring better economic returns to farmers. In the context of climate change, high-protein wheat varieties may show stronger stress resistance and help ensure food production. Reducing dependence on animal protein helps protect the environment. At the same time, by increasing the protein content of wheat, the use of fertilizers and pesticides can be reduced, promoting the sustainable development of agriculture. Globally, increasing the protein content of wheat helps increase food supply and has a positive effect on reducing hunger and malnutrition, thus having a profound impact on global food security. Summary of the invention

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

[0006] The TaNLP-3B 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] Furthermore, the CDS sequence of the TaNLP-3B 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 plants include wheat.

[0010] 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.

[0011] In a second aspect, 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.

[0012] 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 TaNLP-3B gene in wheat.

[0013] 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.

[0014] In a fourth aspect, the present invention provides the use of the transgenic plants obtained according to the method in plant breeding.

[0015] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, selfing or asexual reproduction.

[0016] 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 TaNLP-3B gene and its related biological materials in regulating or increasing the protein content of plant seeds. The gene TaNLP-3B is cloned from wheat and overexpressed in wheat Fielder to obtain transgenic plants overexpressing the TaNLP-3B gene. The test results show that overexpression of the TaNLP-3B gene can significantly increase 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a map of the TaOE (i.e., TaOE-PPT) carrier in a preferred embodiment of the present invention.

[0018] Figure 2 This is the map of the TaOE-TaNLP-3B carrier in a preferred embodiment of the present invention.

[0019] Figure 3 The RT-PCR test results of the relative expression levels of the TaNLP-3B gene in the overexpression strain and the wild-type plant in the preferred embodiment of the present invention.

[0020] 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

[0021] 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.

[0022] Example 1 Cloning and Functional Verification of Wheat TaNLP-3B Gene 1. Experimental Materials and Reagents 1.1 Experimental Materials Wheat material “Fielder”: preserved in this laboratory.

[0023] 1.2 Reagents Escherichia coli Trans1-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.

[0024] 2. Experimental Methods 2.1 In the early stage, a genome-wide association study (GWAS) was conducted using 350 wheat germplasm resources, and a candidate gene TaNLP-3B related to the regulation of wheat grain protein content was obtained. By consulting the literature, it was found that NLP (NIN-like protein) family proteins are involved in the regulation of nitrogen and phosphorus nutrient signaling pathways and play a role in plant growth and development and stress response. Therefore, we cloned the TaNLP-3B gene in wheat, constructed related biological materials and phenotypic analysis.

[0025] 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'-cctaggcctactaggATGGACATCGACCCTTCCTC-3') and reverse amplification primer (5'-cgaaagctctgagctTCAACCGGAGCTTCCACAAGAA-3') were used for cloning and sequencing to obtain the wheat TaNLP-3B gene, whose 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.

[0026] 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.

[0027] 2.4 Construction of wheat TaNLP-3B overexpression vector The TaNLP-3B overexpression vector was constructed into TaOE vector by seamless cloning (vector map as 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 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.

[0028] 3.5 The correctly sequenced monoclonal colonies were amplified by shaking in LB liquid medium to extract the recombinant plasmid TaOE-TaNLP-3B ( Figure 2 ). The TaOE-TaNLP-3B 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 use.

[0029] 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-TaNLP-3B 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-TaNLP-3B 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.

[0030] 3.7 The expression level of TaNLP-3B gene in each line of TaOE-TaNLP-3B transgenic T2 overexpression 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 overexpression lines and wild-type (WT) wheat Fielder was extracted and reverse transcribed into cDNA. The relative expression level of TaNLP-3B gene was detected by RT-PCR (Real-time PCR) method with wheat-actin (forward primer: 5'-CCTTAGTACCTTCCAACAGATGT-3' and reverse primer: 5'-CCAGACAACTCGCAACTTAGA-3') as the internal standard gene. The primer sequences of TaNLP-3B are as follows: forward primer: 5'-CAAGCAAAAAGTGCCTGAGTGGAC-3' and reverse primer: 5'-TTTACTGCCTCAAGAGCTTT-3'.

[0031] Test results such as Figure 3As shown, the expression level of TaNLP-3B gene in the overexpression transgenic wheat TaOE-TaNLP-3B-OE-1, TaOE-TaNLP-3B-OE-2 and TaOE-TaNLP-3B-OE-3 lines was significantly higher (P<0.001) than that in the wild-type wheat Fielder. The results indicated that the overexpression transgenic wheat TaOE-TaNLP-3B-OE line was successfully constructed.

[0032] The total protein content in the mature seeds of the three TaOE-TaNLP-3B transgenic overexpression lines and the wild-type (WT) wheat Fielder was measured using a near-infrared spectrometer (Perton DA7000). Figure 4 As shown, the Fielder of wild-type (WT) wheat 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 were 17.74%, 17.45% and 17.13%, respectively, which were significantly (P < 0.001) higher than the Fielder of wild-type (WT) wheat, increased by 3.38%, 3.09% and 2.77%, respectively, indicating that overexpressing the TaNLP-3B gene in wheat can significantly increase the total protein content in grains.

[0033] It can be seen that overexpression of wheat TaNLP-3B will lead to a significant increase in the protein content in wheat seeds, and the encoded protein TaNLP-3B can regulate the protein content in wheat seeds and can be used in wheat breeding and variety improvement.

[0034] 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 TaNLP-3B gene and its related biological materials in regulating the protein content of plant seeds; The TaNLP-3B 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 TaNLP-3B gene into the plant via a plasmid or integrating it into the plant chromosome via genetic engineering; The TaNLP-3B 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 TaNLP-3B gene in wheat; The TaNLP-3B 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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