Application of ZmAAAP64 gene in regulating and controlling protein accumulation and nitrogen utilization of whole corn plant

Abstract

The invention discloses application of a ZmAAAP64 gene in regulation and control of protein accumulation and nitrogen utilization of a whole corn plant, and belongs to the technical field of biology. The nucleotide sequence of the ZmAAAP64 gene is as shown in SEQ ID NO. 11. A BC2S3 population is constructed based on wild corn Ames21814 and a common corn inbred line B73 for QTL positioning, a key gene ZmAAAP64 for regulating and controlling the protein content and nitrogen utilization efficiency of the whole corn plant is excavated, structural variation analysis finds that in different corn population materials, insertion and deletion variations exist in a plurality of conservative intervals of a ZmAAAP64 gene promoter region, and the ZmAAAP64 gene promoter region has a high expression in the whole corn plant protein content and the nitrogen utilization efficiency of the whole corn plant protein content and the nitrogen utilization efficiency of the whole corn plant protein content and the nitrogen utilization efficiency of the whole corn plant protein content. The accumulation amount of Gln and Asn in the stem of the ZmAAAP64 gene overexpression material is increased by about 30-60%, and the protein content of the stem, the protein content of the leaf and the nitrogen accumulation amount of the whole plant are remarkably increased. And a technical means is provided for cultivating corn with high protein and high nitrogen utilization efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the ZmAAAP64 gene in regulating protein accumulation and nitrogen utilization in maize. Background Technology

[0002] Protein content is a crucial indicator determining the nutritional quality and feed value of maize. With global population growth and rapid livestock development, increasing the protein levels of staple crops like maize has become a core objective for ensuring food security and promoting sustainable agricultural development. Modern cultivated maize (Zea mays L.), a globally important food and feed crop, is produced on a large scale, but it generally suffers from low whole-plant protein content and insufficient nitrogen use efficiency, especially in silage varieties. This characteristic necessitates the supplementation of feed with expensive exogenous protein sources, increasing breeding costs and hindering the green development of modern livestock farming.

[0003] The formation and accumulation of proteins in plants depend on nitrogen absorption, amino acid synthesis, and transport and redistribution among different tissues. The vascular tissue of the stem is a key channel for the transport of amino acids from roots to leaves and grains, playing a pivotal role in nitrogen distribution and protein accumulation throughout the plant. However, compared to the in-depth research on the transport mechanisms of photosynthetic products (carbohydrates), the molecular basis of transtissue transport of amino acids in maize remains unclear. Some studies have focused on the regulation of amino acid synthesis and storage, but a systematic analysis of key transport proteins, tissue-specific expression patterns, and genetic variation patterns in long-distance transport is lacking.

[0004] In plant amino acid transmembrane transport studies, the amino acid / auxin osmotic transporter (AAAP) family is considered to play a central role in amino acid uptake, intercellular transport, and long-distance transport. While some AAAP members have been shown to be associated with nitrogen metabolism regulation in model plants such as rice and Arabidopsis, systematic research on the functional differentiation, tissue specificity, and natural variation effects of its members in maize remains lacking. Furthermore, traditional transcriptomics and metabolomics studies are limited by the average signal across the entire tissue, making it difficult to elucidate spatial differences and metabolic flow patterns among different cell types, thus hindering a deeper understanding of the spatial dynamics and regulatory mechanisms of amino acid transport.

[0005] Although some progress has been made in the study of amino acid synthesis and metabolism, there are still gaps in the understanding of the core link in the formation of high protein in maize—the regulation of nitrogen and amino acid flow in the source-sink system.

[0006] 1. Lack of systematic understanding of source-sink-flow dynamics. Existing research focuses primarily on leaf (source) synthesis or grain (sink) accumulation, while studies on the transport "flow" process of amino acids in the stem vascular system are significantly insufficient. Since the stem is the central channel connecting root absorption and grain utilization, its regulation of "flow rate and direction" in whole-plant protein formation remains unclear, resulting in a lack of explanation for key aspects of the high-protein formation mechanism.

[0007] 2. Lack of functional gene resources that can promote efficient amino acid transport. Although the AAAP family and others are thought to be related to amino acid transport, no key transport genes that can directly enhance long-distance amino acid transport and promote nitrogen redistribution and protein accumulation have been identified in maize, and there is also a lack of molecular breeding applications based on these genes.

[0008] 3. Traditional techniques struggle to elucidate tissue-specific transport regulation mechanisms. Because transcriptomic and metabolomic analyses operate on a whole-tissue basis, they suffer from severe signal averaging effects, making it difficult to identify cell type-specific amino acid transport channels and their regulatory networks within stems, thus limiting the accuracy of high-protein molecule modification.

[0009] These issues have led to slow progress in improving nitrogen reuse efficiency, optimizing protein flow, and applying molecular breeding techniques in maize, resulting in a lack of effective gene targets and technological pathways. Therefore, there is an urgent need for a new gene that regulates the efficient trans-tissue transport of amino acids, enhancing source-sink nitrogen flow efficiency in the vascular system of the stem and achieving an overall improvement in protein content throughout the plant. Summary of the Invention

[0010] The purpose of this invention is to provide the application of the ZmAAAP64 gene in regulating whole-plant protein accumulation and nitrogen utilization in maize, in order to solve the problems existing in the prior art. This gene can promote the efficient transmembrane transport and long-distance redistribution of amino acids in the vascular tissue of maize stalks, thereby significantly improving the nitrogen utilization efficiency and whole-plant protein accumulation in maize, and solving the technical bottleneck problem of lacking effective molecular targets in the "source-sink-flow" regulation link in the prior art.

[0011] The purpose of this invention is to address the problems of low protein accumulation capacity, insufficient nitrogen use efficiency, and lack of clear molecular regulatory targets in existing maize varieties. It aims to provide a key gene capable of regulating long-distance amino acid transport and promoting whole-plant protein formation at the molecular level, along with its application method. To achieve the above objective, this invention provides the following solution:

[0012] In the first aspect, the present invention provides the application of the ZmAAAP64 gene in regulating protein accumulation and nitrogen utilization in maize whole plant, the nucleotide sequence of which is shown in SEQ ID NO.11.

[0013] Secondly, the present invention also provides the application of the protein encoded by the ZmAAAP64 gene in regulating protein accumulation and nitrogen utilization in the whole maize plant, the amino acid sequence of which is shown in SEQ ID NO.10.

[0014] Thirdly, the present invention also provides a gene expression cassette, wherein the gene expression cassette includes the ZmAAAP64 gene.

[0015] Fourthly, the present invention also provides a recombinant vector comprising the gene expression cassette described above.

[0016] Fifthly, the present invention also provides a recombinant bacterium, wherein the recombinant bacterium includes the recombinant vector described above.

[0017] In a sixth aspect, the present invention also provides the application of the ZmAAAP64 gene, the gene expression cassette, the recombinant vector, or the recombinant bacteria in regulating protein accumulation and nitrogen utilization in the whole maize plant.

[0018] In a seventh aspect, the present invention also provides a method for improving protein accumulation and nitrogen utilization in maize plants, the method comprising the steps of introducing the ZmAAAP64 gene, the gene expression cassette, the recombinant vector, or the recombinant bacteria into the plant.

[0019] Eighthly, the present invention also provides the application of molecular markers in identifying maize varieties with high whole-plant protein accumulation and nitrogen utilization. The molecular markers are located in the promoter region of the maize gene ZmAAAP64 and contain deletion variants of lengths of 46bp and 42bp, as well as insertion variants of length 58bp. The corresponding promoter insertion and deletion variant sequences are shown in SEQ ID NO.1-3.

[0020] Preferably, the presence of deletion variants of 46 bp and 42 bp and insertion variants of 58 bp in the promoter region of the ZmAAAP64 gene in the maize variety is detected by PCR amplification.

[0021] Preferably, the primer sequences used for the PCR amplification include the upstream primer sequence shown in SEQ ID NO.4 and the downstream primer sequence shown in SEQ ID NO.5.

[0022] The present invention discloses the following technical effects:

[0023] This invention identifies and functionally verifies the amino acid transporter gene ZmAAAP64 and its superior natural variants, and constructs a molecular regulatory system that can significantly improve nitrogen use efficiency and protein accumulation in maize, fundamentally solving the technical bottleneck of lacking clear molecular targets for regulating long-distance amino acid transport in existing technologies.

[0024] From a theoretical perspective, the technical concept and effects of this invention are reflected in the following aspects:

[0025] 1. Clear Spatial Molecular Recognition Mechanism: This invention utilizes a combined spatial transcriptomics and QTL localization method to precisely locate the key gene ZmAAAP64, associated with transtissue transport of amino acids, within the 8-16 Mb region of maize chromosome 10. This gene exhibits high specificity expression in the vascular bundles of the stem (especially xylem vessels), indicating its central role in the long-distance upward transport of amino acids, thus revealing the spatial regulatory mechanism of maize protein formation at the tissue level.

[0026] 2. Natural Variation Enhances Regulatory Activity: The promoter region of the Ames21814 ZmAAAP64 allele contains multiple insertion / deletion variants (Indels), resulting in significantly higher transcriptional activity than the B73 allele. Dual-luciferase assays confirmed that this natural variation enhances promoter driving capacity by 3-5 times, thereby significantly increasing gene expression levels without altering the coding sequence. This discovery reveals the molecular mechanism by which superior promoter variations promote high protein formation under natural selection, providing a usable variation resource for targeted design breeding.

[0027] 3. Confirmation of transmembrane transport function: Through subcellular localization and yeast complementation experiments, this invention confirms that the ZmAAAP64 encoded product is located in the plasma membrane and has a broad spectrum of amino acid uptake and transport capabilities. It can effectively mediate the uptake and translocation of major nitrogen source amino acids such as glutamic acid, aspartic acid, and asparagine, thereby achieving rapid cycling and redistribution of amino acids at the cellular and tissue levels.

[0028] 4. Significantly increased whole-plant protein content: In transgenic validation, ZmAAAP64 overexpressing plants showed significant advantages under both low-nitrogen and normal-nitrogen conditions: increased free amino acid content in roots, stems, and leaves; increased stem protein content; significantly improved whole-plant biomass and nitrogen use efficiency; and in near-isogenic lines, the grain protein content of materials carrying the Ames21814 promoter haplotype was higher than that of the B73 type.

[0029] 5. Achieving applicability in molecular breeding: The ZmAAAP64 gene and its variant promoter provided by this invention can be used as molecular markers and functional genes for breeding high-protein and high-nitrogen efficiency maize and other gramineous crops, and have stable heritability and significant breeding value.

[0030] In summary, this invention utilizes a systematic approach combining spatial joint genetic population localization, functional verification, and promoter natural variation to enhance expression. By leveraging the principle of enhanced transmembrane transport of amino acids and nitrogen redistribution in vascular tissues, it significantly improves the overall protein content and nitrogen use efficiency of the entire plant. This breakthrough overcomes the limitations of the traditional "synthesis-accumulation" model, proposing a novel "transport-redistribution" mechanism for high-protein formation, which has significant scientific value and industrial application potential. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The purpose of this study was to locate ZmAAAP64 and identify candidate genes. Among them, a) is the phenotypic determination of biomass, stem protein content and their product (as a proxy indicator of whole plant protein) of the population; b) is the analysis of spatial transcriptome signals in the same chromosomal region; c) is the further screening of candidate genes in the QTL peak region; and d) is the UMAP clustering results.

[0033] Figure 2 For the molecular function verification of ZmAAAP64; where a is the comparative analysis of the ZmAAAP64 promoter (bottom left, representative luminescence thermogram; bottom right, quantitative results; two-tailed test, ***P<0.001, bar chart represents mean ± standard error), b is the subcellular localization analysis results, c is the yeast complementation experiment results, and d is the yeast growth curve;

[0034] Figure 3 This study validated the phenotype and function of ZmAAAP64 overexpressing plants. Specifically, a) seedling performance under nitrogen treatment (scale bar = 5cm), b) fresh weight of roots and shoots of WT and OE lines under low and normal nitrogen conditions, c) comparison of free amino acid (Gln, Asn, Glu, Asp) content in roots and shoots of WT and OE seedlings, d) field stage traits, and e) seed protein content of near-isogenic lines. All quantitative results are expressed as mean ± standard error. Significance was indicated by *P<0.05, **P<0.01, and ***P<0.001 (two-tailed test). Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0040] This invention provides an amino acid transporter gene ZmAAAP64 that regulates protein accumulation in the whole maize plant and its applications. The technical solution includes the following:

[0041] I. Identification and Structural Characteristics of Genes

[0042] The ZmAAAP64 gene (gene number Zm00001eb408350) identified in this invention is located in the AAAP family tandem gene cluster on chromosome 10 of maize (approximately 8-16 Mb interval), and its encoded product belongs to the amino acid / auxin permease (AAAP) family.

[0043] The ZmAAAP64 gene coding sequence is approximately 1500 bp long, encoding 480 amino acids (SEQ ID NO.10). ProtParam calculations show that its theoretical molecular weight is approximately 51.5 kDa, theoretical isoelectric point pI=8.27, instability index is 35.02 (stable), aliphatic index is 96.54, and GRAVY=0.529 (significant overall hydrophobicity).

[0044] Predicted by TMHMM v2.0 and Phobius, this protein is a typical multi-transmembrane protein containing nine transmembrane helical domains, with coordinates of amino acid positions 45-67, 71-93, 123-145, 195-217, 289-311, 326-348, 386-405, 409-431, and 443-465. No signal peptide sequence was detected, indicating its location on the plasma membrane. This structural characteristic is consistent with known members of the AAAP family (such as AtAAP1 and OsAAP6), indicating that ZmAAAP64 possesses typical amino acid transmembrane transport functions.

[0045] Sequence alignment analysis between B73 and Ames21814 revealed multiple insertion / deletion variations (Indels) in the promoter region of the ZmAAAP64 allele of Ames21814, mainly including 42bp, 46bp and 58bp structural differences, which led to a significant enhancement of the transcriptional activity of the gene.

[0046] II. Gene Expression and Functional Characteristics

[0047] Spatial transcriptomics analysis showed that ZmAAAP64 was highly expressed in maize stalk vascular tissue, especially in xylem vessels (Xyl) cells.

[0048] Subcellular localization experiments showed that ZmAAAP64 is mainly located in the plant cell membrane and is a transmembrane amino acid transporter.

[0049] Yeast complementation experiments verified that ZmAAAP64 can mediate the absorption and utilization of various amino acids, including glutamic acid, aspartic acid, asparagine, alanine, phenylalanine, proline, and serine, demonstrating broad-spectrum amino acid transport activity.

[0050] III. Functional Verification and Application Results

[0051] Promoter activity was verified using a Nicotiana benthamiana dual-luciferase transient expression system. The results showed that the luciferase signal intensity driven by the Ames21814 promoter was approximately 3-5 times that of the B73 promoter.

[0052] Heterologous expression of ZmAAAP64 in an amino acid uptake-deficient yeast strain (22Δ10α) restored its growth ability under single nitrogen source (Asn, Asp, Glu, etc.) conditions, confirming that the gene has amino acid transmembrane transport function.

[0053] A ZmAAAP64 overexpression vector was constructed and transformed into maize to obtain transgenic plants. Greenhouse and field experiments showed that, under low nitrogen and normal nitrogen conditions, the biomass, nitrogen uptake efficiency, and stem protein content of the overexpression lines were significantly higher than those of the control; the accumulation of free amino acids (especially Gln and Asn) in the stems was also increased.

[0054] In the BC2S5 near-isogenic line, the seed protein content of the material carrying the Ames21814 type ZmAAAP64 promoter haplotype was significantly higher than that of the B73 type (difference P<0.01), further confirming that this natural variation can be directly used for molecular breeding.

[0055] IV. Application Methods

[0056] Genetic improvement vectors based on ZmAAAP64 or its promoter were constructed to improve nitrogen utilization and protein accumulation in maize through genetic transformation or gene editing.

[0057] We developed molecular markers based on ZmAAAP64 promoter variations for marker-assisted selection (MAS) of high-protein and high-nitrogen-efficiency maize.

[0058] Heterologous expression in other gramineous crops (such as rice, wheat, sorghum, etc.) can enhance amino acid transport capacity and increase protein content.

[0059] The amino acid sequence of the ZmAAAP64 protein described in this invention is shown in SEQ ID NO.10, and the nucleotide sequence of the gene encoding the ZmAAAP64 protein is shown in SEQ ID NO.11.

[0060] SEQ ID NO.10:

[0061] MAVSHNVGSKHGVAPLEVSVEAGNGGAAEWLDDDGRPRRTGTFWTASAHIVTAVIGSGVLSLAWAIAQLGWVAGPAAMLLFAFVTYYTATLLAECYRTGDPETGKRNYTYMDAVRSNLGGAKVAFCGVIQYANLVGVAIGYTIAASISMKAVRRAGCFHAHGHADPCNSSSTPYMILFGVVQILFSQIPDFDQIWWLSIVAAVMSFTYSSIGLSLGIAQTISNGGFMGSLTGISIGAGVTSTQKIWHTLQAFGDIAFAYSFSNILIEIQDTIKAPPPSESKVMQKATRLSVATTTIFYMLCGCMGYAAFGDKAPDNLLTGFGFFEPFWLIDIANVAIVVHLVGAYQVFCQPIFAFVERRAAAAWPDSAFVSQELRVGPFAVSVFRLTWRSSFVCVTTVVAMLLPFFGNVVGFLGAVSFWPLTVYFPVEMYIKQRRVPRGSTKWICLQTLSVSCLLVSVAAAAGSIADVIDALKVYRPFSG;

[0062] SEQ ID NO.11:

[0063]

[0064] Example 1: Localization of ZmAAAP64 and Screening of Candidate Genes

[0065] (1) Group building and QTL positioning

[0066] Using common maize inbred line B73 as the recurrent parent and wild maize Ames21814 (Zea mays ssp. parviglumis) as the donor parent, a BC2S3 population of 380 individual plants was constructed. Phenotypic analysis was performed in the field to determine stem protein content, total biomass, and their product (representing the overall protein accumulation capacity of the plant). Genome-wide association analysis (interval mapping) ultimately detected a major-effect QTL peak (significant LOD value) in the 8-16 Mb region of chromosome 10. Figure 1 (a)

[0067] (2) Spatial transcriptome combined analysis

[0068] To analyze the tissue-specific expression of genes within this region, 10× Genomics Visium spatial transcriptomics technology was used to sequence cross sections of B73 and Ames21814 stems. The results showed ( Figure 1 In the vascular bundles (xylem Xyl, phloem Phl, and sclerenchyma Scl), Ames21814 significantly upregulated a cluster of amino acid transporter genes, with ZmAAAP64 showing the most significant expression.

[0069] (3) Candidate gene identification

[0070] Further integration of gene structure, functional annotation, and expression enrichment information identified ZmAAAP64 as the primary candidate gene. Its UMAP clustering results showed ( Figure 1 In Ames 21814, ZmAAAP64 was mainly enriched in xylem cell groups (d).

[0071] Example 2: Identification of structural variations and verification of molecular function of ZmAAAP64

[0072] (1) Promoter variation analysis and activity verification

[0073] DNA was extracted from the genomes of maize inbred line B73 and wild maize Ames21814, and the promoter sequence (approximately 2kb) of the ZmAAAP64 gene was compared and analyzed using the Biostrings software of R / Bioconductor. The results showed (Table 1) significant insertion and deletion variations (Indels) in several conserved regions.

[0074] Table 1. ZmAAAP64 promoter mutation information

[0075] Mutation type (wild type compared to B73) Length (bp) Sequence (5'→3') Serial Number Missing 46bp CATCCCATTAGGATAAGTCATATATTGTATGCAGAACCAAGCACAC SEQ ID NO.1 insert 58bp TCTTTTTTCAAATTTAACAGTGTTTTATTAAAAGATTTATAACTTTTATTGGTGTCGA SEQ ID NO.2 Missing 42bp CGCACGGAGCGTGGCGGCGTGGCATCCGCAGTGCCTCGGTGG SEQ ID NO.3

[0076] To verify the activity differences among different promoter types, specific primers were designed to amplify approximately 2.5 kb promoter fragments using genomic DNA from maize inbred line B73 and wild maize Ames21814 as templates. These fragments were then cloned upstream of the LUC gene in the pGreenII0800-LUC dual-luciferase reporter vector, constructing pGreenII-B73pro::LUC and pGreenII-Ames21814pro::LUC, respectively. The specific amplification primer sequences are shown in Table 2. The nucleotide sequence of the ZmAAAP64 promoter region of maize inbred line B73 is shown in SEQ ID NO.14, and the nucleotide sequence of the ZmAAAP64 promoter region of wild maize Ames21814 is shown in SEQ ID NO.15.

[0077] Table 2 Primer sequences for ZmAAAP64 promoter amplification

[0078] Primer name Sequence (5'→3') Serial Number ProZmAAAP64-F CACATGAGCCATCCTAGAGCC SEQ ID NO.4 ProZmAAAP64-R GGTTGGCGTACTGTATGACGC SEQ ID NO.5

[0079] Two constructed vectors were transformed into Agrobacterium GV3101, and then injected into Nicotiana benthamiana leaves. After 48 hours of treatment, the LUC / REN ratio was determined using the Dual Luciferase Reporter Assay Kit. The experimental results showed ( Figure 2 (a) The expression level of luciferase driven by the Ames21814 promoter was about 3-5 times that of the B73 promoter (P<0.001), confirming that this Indel and other structural variations significantly enhanced the promoter activity of ZmAAAP64.

[0080] (2) Subcellular localization analysis

[0081] Primers were designed based on the ZmAAAP64 coding sequence to amplify the full-length CDS region (excluding the stop codon). Primer sequence information is shown in Table 3.

[0082] Table 3 CDS amplification primer sequences

[0083] Primer name Sequence (5'→3') Serial Number ZmAAAP64-CDS-F ATGGCCGTGTCACACAACGTT SEQ ID NO.6 ZmAAAP64-CDS-R TTAACCGCTGAATGGCCGGTAC SEQ ID NO.7

[0084] The full-length CDS of ZmAAAP64 was amplified using the primers described above, and the amplified product was cloned into the pCAMBIA3100 vector and fused with the N-terminus of the GFP gene to construct the 35S::ZmAAAP64-GFP fusion expression vector. Simultaneously, the plasma membrane marker genes PM and RFP were fused together to construct PM-RFP as a reference for plasma membrane localization. 35S::ZmAAAP64-GFP and PM-RFP (or GFP and PM-RFP as controls) were co-transformed into *Nicotiana benthamiana* leaves using *Agrobacterium* GV3101. Forty-eight hours after infection, fluorescence signals were observed using an Olympus FV3000 laser confocal microscope. The experimental results showed that ZmAAAP64-GFP and PM-RFP were completely co-localized, indicating that the ZmAAAP64 protein is mainly located in the plasma membrane and is a transmembrane amino acid transporter (…). Figure 2 (b)

[0085] (3) Yeast complementarity experiment

[0086] Amino acid uptake-deficient yeast strain 22Δ10α (negative control) and prototrophic strain 23344c (positive control) were used. The full-length CDS of ZmAAAP64 was amplified and cloned into the yeast expression vector pYES2 (GAL1 promoter, URA3 select marker), and transformed into strain 22Δ10α via the LiAc / PEG method. The empty vector pYES2 was simultaneously transformed into both control strains. Transformants were screened on nitrogen-free medium (YNB). Information on the amplification primers used is shown in Table 4.

[0087] Table 4. Primer sequences for homologous recombination

[0088] Primer name Sequence (5'→3') Serial Number ZmAAAP64-pYES2-F TTGGTACCGAGCTCGGATCCATGGCCGTGTCACACAACGT SEQ ID NO.8 ZmAAAP64-pYES2-R ATGCATGCTCGAGCGGCCGCTTAACCGCTGAATGGCCGGT SEQ ID NO.9

[0089] The bacterial solution was diluted to OD after washing. 600 =0.6, press 10 0 10 -1 10 -2 10 -3 Serial dilutions were performed, with 2.5 μL inoculated at each point onto solid YNB medium (3 mM total nitrogen equivalent) supplemented with different single amino acids (Ala, Asn, Asp, Glu, Phe, Pro, Ser) as the sole nitrogen source, and incubated upside down at 30°C for 48 h. The 22Δ10α strain expressing ZmAAAP64 was able to recover growth under various amino acid conditions, while the empty vector showed no growth.

[0090] In liquid culture assays, the same strain was inoculated into a solution containing a single amino acid (3 mmol / L). -1 In YNB liquid medium containing either (NH4)2SO4 or (NH4)2SO4 as a control, the culture was carried out for 60 h, and the OD was measured every 12 h. 600 (Each group of n=3 was cultured independently). The results showed ( Figure 2 The growth rate of strains carrying ZmAAAP64 was significantly increased under Asn, Asp, and Glu conditions, confirming that ZmAAAP64 has broad-spectrum amino acid transmembrane transport activity.

[0091] Example 3: Functional Verification and Application of ZmAAAP64 in Maize

[0092] (1) Obtaining ZmAAAP64 overexpression materials

[0093] 1) Replace the fragment between the Spe I and Bamh I restriction sites in the vector UBI-FLAG (the nucleotide sequence is obtained by sequentially linking SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18) with the DNA sequence shown in SEQ ID NO.11 to obtain the overexpression vector UBI-ZmAAAP64-FLAG, and perform sequencing verification.

[0094] 2) The prepared overexpression vector UBI-ZmAAAP64-FLAG was introduced into Agrobacterium EHA105 to obtain recombinant bacteria.

[0095] 3) The recipient maize B73 inbred line was transformed using recombinant bacteria to obtain T0 generation plants.

[0096] 4) Using primer pairs consisting of primers ZmAAAP64-OE-F (SEQ ID NO.12) and primers ZmAAAP64-OE-R (SEQ ID NO.13) (see Table 5), the obtained T0 generation plants were identified by PCR. The T0 generation plants that were identified as positive were selected for self-pollination to obtain T1 generation plants.

[0097] 5) Identify the T1 generation plants obtained in step 4) according to the method in step 4), select the T1 generation plants that are identified as positive and self-pollinate to obtain T2 generation plants.

[0098] 6) Identify the T2 generation plants obtained in step 5) according to the method in step 4), select the positive T2 generation plants for self-pollination to obtain T3 generation plants, and identify them according to the method in step 4), screen out the positive transgenic T3 generation plants, and name them OE1-ZmAAAP64 and OE2-ZmAAAP64.

[0099] Table 5 Detection Primer Sequences

[0100] Primer name Sequence (5'→3') Serial Number ZmAAAP64-OE-F TTCATGGGCAGTCTCACTGGCA SEQ ID NO.12 ZmAAAP64-OE-R AATCGATGTCGTGATCCTTATAGTCTCC SEQ ID NO.13

[0101] (2) Greenhouse nitrogen treatment and phenotypic determination

[0102] Plants re-transmitted with the empty vector served as the control group (WT). WT and OE plants were cultured under two nitrogen source conditions: low nitrogen (LN, 0.04 mM KNO3) and normal nitrogen (NN, 4 mM KNO3). After 11 days of culture, plant height, root and aboveground fresh weight were measured. Plant samples were dried at 65℃ to constant weight, ground, and the total nitrogen content was determined using the Dumas combustion method and converted to protein content. The contents of free amino acids (Gln, Asn, Glu, Asp) were also measured.

[0103] The results show that ( Figure 3 Under low nitrogen conditions, the aboveground biomass of OE1 and OE2 was significantly higher than that of WT (P<0.01). Under normal nitrogen conditions, both the aboveground and belowground biomass of OE plants were significantly higher than that of WT. Regarding amino acid accumulation, especially under normal nitrogen conditions, the accumulation of glutamine, glutamate, asparagine, and aspartic acid in the aboveground parts of OE plants was significantly higher than that of WT. This indicates that overexpression of ZmAAAP64 significantly promotes nitrogen uptake and amino acid transport.

[0104] (3) Field functional verification

[0105] WT and OE plants were cultivated in a field environment. Total aboveground biomass and stem and leaf tissue protein content were measured at maturity. Results showed that ( Figure 3 In the middle (d), the stem protein content, leaf protein content and total biomass of OE plants were significantly higher than those of WT plants.

[0106] (4) Validation of near-isogenic lines

[0107] BC2S5 near-isogenic lines (NILs) were constructed based on the B73×Ames21814 hybrid population. Materials with background purity >98% were screened by whole-genome resequencing, and haplotypes were distinguished based on the diagnostic Indel marker of the ZmAAAP64 promoter. Results showed that ( Figure 3 (e) The seed protein content of NILs carrying the Ames21814 promoter haplotype was significantly higher than that of the B73 haplotype (P<0.01), verifying that this natural variant can be directly used for high-protein breeding.

[0108] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of the ZmAAAP64 gene in modulating whole-plant protein accumulation and nitrogen utilization in maize, characterized in that, The nucleotide sequence of the ZmAAAP64 gene is shown as SEQ ID NO.

11.

2. The use of a protein encoded by the ZmAAAP64 gene of claim 1 in modulating whole plant protein accumulation and nitrogen utilization in maize, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO.

10.

3. A gene expression cassette, characterized in that, The gene expression cassette comprises the ZmAAAP64 gene as claimed in claim 1.

4. A recombinant vector, characterized in that, The recombinant vector comprises the gene expression cassette as claimed in claim 3.

5. A recombinant bacterium, characterized in that, The recombinant bacteria comprises the recombinant vector as claimed in claim 4.

6. Use of the ZmAAAP64 gene as claimed in claim 1, the gene expression cassette as claimed in claim 3, the recombinant vector as claimed in claim 4 or the recombinant bacteria as claimed in claim 5 in regulating maize whole plant protein accumulation and nitrogen utilization.

7. A method of increasing whole plant protein accumulation and nitrogen use in maize, characterized in that, The method comprises the step of introducing the ZmAAAP64 gene as claimed in claim 1, the gene expression cassette as claimed in claim 3, the recombinant vector as claimed in claim 4 or the recombinant bacteria as claimed in claim 5 into a plant.

8. Use of a molecular marker in identifying corn varieties with high corn whole-plant protein accumulation and nitrogen use, characterized in that, The molecular marker is located in the promoter region of the maize gene ZmAAAP64, and there are deletion variations of 46 bp and 42 bp and an insertion variation of 58 bp, and the corresponding promoter insertion and deletion variation sequences are shown as SEQ ID NO. 1-3.

9. Use according to claim 8, characterized in that, PCR amplification is used to detect whether the ZmAAAP64 gene promoter region in the corn variety has deletion variations of 46 bp and 42 bp and an insertion variation of 58 bp.

10. Use according to claim 9, characterized in that, The primer sequences used in the PCR amplification comprise an upstream primer sequence shown as SEQ ID NO. 4 and a downstream primer sequence shown as SEQ ID NO. 5.

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