Key genes controlling corn protein content and nitrogen efficiency

Abstract

The present invention discloses the use of the wild maize asparagine synthetase 4 gene Thp9 in improving the protein content of maize grains, the total nitrogen content of the plant and / or the nitrogen efficiency. By increasing the activity of asparagine synthetase 4 or enhancing the expression of asparagine synthetase 4, the protein content of maize and the utilization rate of nitrogen fertilizer can be increased. This has great economic significance for the creation of new high-protein maize germplasm resources, agricultural production and environmental protection.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural genetic engineering and relates to the application of wild corn asparagine synthetase 4 gene Thp9 in improving corn grain protein content, plant total nitrogen content and / or nitrogen high efficiency. Background Art

[0002] The quality of corn (Zea mays L.) directly impacts the yield and quality of meat and milk, and is a key determinant of the development of the livestock industry. Furthermore, with improving living standards, consumers are increasingly concerned about corn quality, with protein nutritional quality (total protein) being a key indicator of widespread concern. However, the protein content of corn typically ranges from 7% to 9%, requiring additional protein supplementation with soybean meal and other ingredients as feed, significantly increasing feed costs. Therefore, increasing corn kernel protein content and reducing or even eliminating the use of soybean meal in feed are important approaches to promoting the healthy development of my country's feed and livestock industries. Furthermore, increasing the total nitrogen content and free amino acid content of silage corn straw will also have significant implications for the livestock industry. Therefore, cloning genes that control total protein content in corn, elucidating the mechanisms of high-protein production, and creating new high-protein germplasm resources are crucial strategies for ensuring food security. A rice quantitative trait locus (QTL), qPC1, has been reported to be associated with rice protein content. The amino acid transporter OsAAP6, encoded by qPC1, is associated with high grain protein content (Peng et al., 2014). Furthermore, by measuring the total protein and storage protein content of over 400 rice germplasm accessions, map-based cloning and functional studies have revealed that qGPC-10 (OsGluA2) encodes a glutenin precursor, a storage protein in rice, significantly affecting rice protein content and ultimately nutritional quality (Yang et al., 2019). Near-infrared analysis of 961 accessions from 2009 and 2010 revealed a 7.32%–15.20% variation in maize grain protein content (Karn et al., 2017). However, the locus controlling this protein content has yet to be identified.

[0003] The maize kernel's endosperm is the primary storage organ for nutrients, with starch and protein being the two most important storage substances. Common maize inbred lines contain approximately 10% total protein, with starch accounting for approximately 70% (Flint-Garcia et al., 2009). Proteins are classified based on solubility into zeins, albumins, globulins, and glutelins (Wu and Messing, 2017). When used as feed or grain, the abundance and amino acid composition of different protein types vary greatly, determining their nutritional quality. Zeins are the primary storage protein in maize, accounting for over 60% of total protein. Zeins are classified based on amino acid homology into four subfamilies: α (19 and 22 kD), β (15 kD), γ (50, 27, and 16 kD), and δ (18 and 10 kD). α-zein is the most abundant zein, accounting for over 50% of the total zein content (Esen, 1987; Thompson and Larkins, 1994). However, almost all zeins lack the essential amino acids lysine and tryptophan, resulting in a significant deficiency of these two amino acids in total maize endosperm protein (Mertz et al., 1964). Opaque2 (O2) is a key transcription factor in the maize endosperm. In the O2 mutant, expression of the alcohol-soluble protein zein is reduced by over 60%. However, protein homeostasis leads to a compensatory upregulation of non-alcohol-soluble protein expression, resulting in only a slight decrease in total protein content. Non-alcohol-soluble proteins are rich in lysine, resulting in approximately twice the lysine content in the O2 mutant compared to conventional maize. Mice fed O2 maize grow significantly faster than controls (fed conventional maize) (Mertz et al., 1965). However, the O2 endosperm is powdery, making the kernels susceptible to shattering and mold disease, and the total protein content is low (approximately 8-9%), resulting in low yields, making it unsuitable for direct industrialization and cultivation. The creation of new high-quality, high-protein corn will have significant implications for food production and food security.

[0004] Several genes influencing efficient nitrogen use efficiency in rice have been cloned. Natural variation in the nitrate transporter gene NRT1.1B is a key factor mediating the differences in nitrogen use efficiency between indica and japonica rice varieties, with the indica-type NRT1.1B allele exhibiting high nitrogen use efficiency (Hu et al., 2015). Furthermore, genome-wide association studies (GWAS) revealed that OsTCP19, a member of the rice TCP transcription factor family, is a key factor in rice's adaptation to varying soil nitrogen levels (Liu et al., 2021). At the same time, studies have revealed that the GA signaling pathway coordinately regulates rice growth and nitrogen metabolism. The rice transcription factor GROWTH-REGULATING FACTOR 4 (GRF4) (Li et al., 2018) and the APETALA2 domain-containing transcription factor NITROGEN-MEDIATED TILLER GROWTHRESPONSE 5 (NGR5) are key factors mediating nitrogen regulation of tiller formation (Wu et al., 2020). Superior allelic variation in these genes increases and stabilizes yields under low-nitrogen growth conditions, providing a critical resource for efficient nitrogen utilization in rice. However, improving nitrogen fertilizer utilization or enhancing efficient nitrogen perception, absorption, assimilation, and transport in maize plants, as well as identifying and identifying molecular modules for efficient nitrogen utilization in maize, remain major scientific challenges that urgently need to be addressed in agricultural production. Summary of the Invention

[0005] Improving corn protein content requires a high-protein donor. We measured and analyzed the protein content of over 30 different wild corn accessions and found that wild corn contains approximately 30% protein. Therefore, wild corn is an excellent gene donor resource for creating new high-protein corn germplasm. Introducing wild corn into cultivated corn is a method for increasing corn protein content. To further characterize the major QTL loci controlling protein content in wild corn, we have been using the wild corn teosinte (Zea mays ssp. Parviglumis, Ames21814, hereinafter referred to as Ames21814 or Teosinte (Teo)) as the introgression donor for our population construction since 2012. Ames21814 boasts a protein content of 30%, with significant increases in both α-zein and the non-zein fraction, which is rich in the nutrient lysine, making it a representative natural donor for increasing corn protein content. After 10 years of hard work, analysis of a large number of genetic populations, the creation of 10 consecutive generations of near-isogenic line populations, and the determination of protein content in tens of thousands of samples, we cloned the first major QTL gene locus that controls total protein content in corn, and through third-generation sequencing, analyzed and assembled the high-quality wild corn Ames21814 genome sequence. The present invention discovered Thp9, a key gene in wild corn that controls the formation of high-protein corn. This gene can not only significantly increase corn protein content and biomass, but also increase the nitrogen use efficiency of corn and reduce the use of nitrogen fertilizer. In addition, we have also developed a molecular marker for this gene, introduced the wild corn high-protein gene Thp9 into cultivated corn, and cultivated and created a new high-protein corn germplasm resource. Accordingly, the present invention includes the technical solutions described below.

[0006] The first aspect of the present invention is to provide the use of wild corn asparagine synthetase 4 gene, such as Thp9, in increasing corn kernel protein content, plant total nitrogen content and / or nitrogen high efficiency.

[0007] Specifically, the application of the wild corn asparagine synthetase 4 gene, such as Thp9, can be selected from the following groups: introducing the wild corn asparagine synthetase 4 encoding gene, such as Thp9, into the common corn chromosome; causing corn to overexpress the wild corn asparagine synthetase 4 gene, such as Thp9; causing corn to overexpress the common corn original asparagine synthetase 4 gene ZmASN4; introducing the regulatory region that controls the expression amount of the wild corn asparagine synthetase 4 gene into corn to increase the expression amount of the gene, thereby increasing the activity of asparagine synthetase 4 or enhancing the expression of asparagine synthetase 4, and then increasing the asparagine content in corn.

[0008] The wild corn asparagine synthetase 4 encoding gene is a mutant of the original asparagine synthetase 4 of common corn.

[0009] The above-mentioned original asparagine synthetase 4 gene ZmASN4 of common corn refers to Zm00001d047736 for common cultivated corn such as inbred line corn B73.

[0010] In one embodiment, the nucleotide sequence of the wild corn asparagine synthetase 4 gene, such as Thp9, is selected from the group consisting of:

[0011] (A) The polynucleotide shown in SEQ ID NO: 1 is derived from wild Zea mays ssp. Parviglumis (Ames 21814), named Thp9 (Teosinte high protein locus in 9th chromosome), gene accession number Teo09G002926, NCBI Genome submission: SUB11272093;

[0012] (B) A polynucleotide having a homology of ≥80%, ≥85%, ≥90%, preferably ≥95%, more preferably ≥98% to the nucleotide sequence of SEQ ID NO: 1.

[0013] The wild corn asparagine synthetase 4 is a polypeptide selected from the group consisting of:

[0014] (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2;

[0015] (b) a polypeptide derived from (a) formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 and having the function of the polypeptide of (a);

[0016] (c) a polypeptide derived from (a) that has 95% or more homology, preferably 98% or more homology, and more preferably 99% or more homology with the polypeptide sequence defined in (a), and has the function of the polypeptide of (a); or

[0017] (d) A derivative polypeptide having a sequence containing the polypeptide sequence described in (a) or (b) or (c).

[0018] Among them, SEQ ID NO: 2 has the following amino acid sequence:

[0019] MCGILAVLGCSDCSQARRARILACSRRLKHRGPDWSGLYQHEGNFLAQQRLAIVSP LSGDQPLFNEDRTVVVVANGEIYNHKNVRKQFTGAHSFSTGSDCEVIIPLYEKYGENFVDMLDGVFAFVLYDTRDRTYVAARDAIGVNPLYIGWGSDGSVWMSSEMKALNEDCVRFEI FPPGHLYSSAAGGFRRWYTPHWFQEQVPRTPYQPLVLREAFEKAVIKRLMTDVPFGVLLSGGLDSSLVASVTKRHLVKTDAAEKFGTELHSFVVGLEGSPDLKAAREVADYLGTTHHE FHFTVQDGIDAIEEVIYHDETYDVTTIRASTPMFLMARKIKSLGVKMVLSGEGSDELLGGYLYFHFAPNREELHRETCRKVKALHQYDCLRANKATSAWGLEVRVPFLDKEFVDVAMG MDPEWKMYDKNLGRIEKWVLRKAFDDEEHPYLPEHILYRQKEQFSDGVGYNWIDGLKSFTEQQVTDEMMNNAAQMFPYNTPVNKEAYYYRMIFERLFPQDSARETVPWGPSIACS TPAAIEWVEQWKASNDPSGRFISSHDSAATDRTGDKLAVVNGDGHGAANGTVNGNDVAVAIAV(SEQ IDNO:2).

[0020] The amino acid sequence of asparagine synthetase 4 expressed in inbred maize line B73 is as follows:

[0021] MCGILAVLGCSDCSQARRARILACSRRLKHRGPDWSGLYQHEGNFLAQQRLAIVSP LSGDQPLFNEDRTVVVVANGEIYNHKNVRKQFTGAHSFSTGSDCEVIIPLYEKYGENFVDMLDGVFAFVLYDTRDRTYVAARDAIGVNPLYIGWGSDGSVWMSSEMKALNEDCVRFEI FPPGHLYSSAAGGFRRWYTPHWFQEQVPRTPYQPLVLREAFEKAVIKRLMTDVPFGVLL SGGLDSSLVASVTKRHLVKTDAAGKFGTELHSFVVGLEGSPDLKAAREVADYLGTTHHEFHFTVQDGIDAIEEVIYHDETYDVTTIRASTPMFLMARKIKSLGVKMVLSGEGSDELLGG YLYFHFAPNREELHRETCRKVKALHQYDCLRANKATSAWGLEVRVPFLDKEFVDVAMGMDPEWKMYDKNLGRIEKWVLRKAFDDEEHPYLPEHILYRQKEQFSDGVGYNWIDGLK AFTEQQVDGRRRS.

[0022] As one method of the above application, a method for introducing a wild corn asparagine synthetase 4 encoding gene, such as Thp9, into a corn chromosome comprises the following steps:

[0023] (1) cloning the wild corn asparagine synthetase 4 encoding gene, such as Thp9, into a plant expression vector suitable for expression in Agrobacterium to obtain an expression vector for the gene;

[0024] (2) After the vector is sequenced and verified, the expression vector of the gene is transformed into maize embryos using Agrobacterium-mediated method to obtain transgenic maize overexpressing the gene.

[0025] Preferably, step (2) can be to transform the expression vector of the gene into Agrobacterium competent cells after sequencing verification; use the transformant to transform corn embryos; and obtain positive plants through genome level and transcription level identification after corn culture and growth.

[0026] For example, the pCAMBIA vector can be a pCAMBIA3300 vector driven by a maize Ubiquitin promoter. The ZmASN4 gene can be loaded downstream of the Ubiquitin promoter.

[0027] The second aspect of the present invention is to provide a kit for implementing the above application, comprising: a Thp9 gene fragment of SEQ ID NO: 1 or its CDS sequence SEQ ID NO: 3, and PCR primers required for cloning the gene fragment or its CDS sequence into a plant expression vector;

[0028] Or it may comprise: the above-mentioned gene expression vector, and reagents for transferring the gene expression vector into Agrobacterium;

[0029] Or it may include: Agrobacterium transformed with the above gene expression vector, and reagents for transforming Agrobacterium into plants.

[0030] The third aspect of the present invention provides a method for detecting the above-mentioned gene in the corn genome, comprising the following steps:

[0031] When detecting the Thp9 gene with a nucleotide sequence of SEQ ID NO: 1, the forward primer thp9-F is CTCTGTGCCATGCATCCTCC, the reverse primer thp9-R is CGTCAGCGCTGGTTAGC, and the PCR product is 198 bp of SEQ ID NO: 4, which is a molecular marker for the Thp9 high protein site:

[0032] CTCTGTGCCATGCATCCTCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCA GTGACGGAGTGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGATCGCATCTCCTAGACATCGTACCGTACGTC GTCCAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 4);

[0033] Alternatively, the PCR product is 176 bp of SEQ ID NO: 5, which is also a molecular marker for the Thp9 high protein site:

[0034] CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGT TCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATTTATGGCCACGCATCTCCTAGACATCGTCGTCGTCGAAGTTAGC TAACCAGCGCTGACG (SEQ ID NO: 5);

[0035] The PCR product is 151 bp of SEQ ID NO: 6, which is the molecular marker of the maize B73 gene Zm00001d047736:

[0036] CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGT TCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATGGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 6);

[0037] When detecting whether the Thp9 gene with the nucleotide sequence of SEQ ID NO: 1 is inserted into the maize genome, the forward primer asn4-is-F: CCGTTCCTCGACAAGGAGTT, the reverse primer asn4-is-R: ATCAGAGCTGAAAGTGGGGC, the PCR product is SEQ ID NO: 7 of 455 bp, which is the molecular marker for the wild maize Ames21814 genotype insertion:

[0038] CCGTTCCTCGACAAGGAGTTCGTCGACGTCGCGATGGGCATGGACCCCGAGT GGAAAATGGTACTGACGCGGGCCTTTTTCGACACGGCCCGGCCCTGCCGCCGCA CGTCGGGGTCTCGGTTCTACGTATGATGATGACGCCTTCTTCTCTTCTTTGCGCAGTACGACAAGAACCTGGGTCGCATCGAGAAGTGGGTCCTGAGGAAGGCGTTCGAC GACGAGGAGCACCCTTACCTGCCCGAGGTAAGAACATCTTCAGAGAAGGCTGGTCGTTTACCTCTGTGTCTGTGTGATTTCAAGCCTGAACTGACGCCTCTGTGCCATGC ATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGAT(SEQ ID NO:7).

[0039] When detecting whether the Thp9 gene with the nucleotide sequence of SEQ ID NO: 1 is inserted into the corn genome, PCR detection can be performed using a common PCR MIX and program. If a band can be amplified, it means that the wild corn Ames21814 genotype has been inserted. If no band can be amplified, it means that the wild corn Ames21814 genotype is not carried.

[0040] The fourth aspect of the present invention is to provide a kit for implementing the above method, which comprises primers corresponding to SEQ ID NOs: 4-7, or DNA / RNA probes, or a microarray chip of DNA / RNA probes.

[0041] The present invention applies the wild corn asparagine synthetase 4 gene Thp9 to cultivated corn, which not only increases the protein content of corn, but also promotes the high nitrogen efficiency of corn, thereby improving the utilization rate of nitrogen fertilizer. This has great economic significance for the creation of new high-protein corn germplasm resources, agricultural production and environmental protection, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This study demonstrates the strong selection for protein content during the domestication of wild maize into cultivated maize. (a) Schematic diagram of the transformation of tillering wild maize into cultivated maize inbred lines over approximately 9,000 years of domestication. (b) Analysis of the grain protein content of over 30 accessions of two wild maize species, Parviglumis and Mexiana, showed approximately 28.6% ± 1.0%, while the protein content of 405 cultivated maize inbred lines ranged from 6.5% to 16%, with an average of 11.52%. (c) Analysis of alcohol-soluble and non-alcohol-soluble proteins in representative wild maize lines (accession numbers Ames and PI series) revealed significantly higher levels of both alcohol-soluble and non-alcohol-soluble proteins in wild maize than in the control B73 inbred line. (d) The free amino acid asparagine content in the roots, stems, and leaves of wild maize Ames21814 was significantly higher than that in the control B73 inbred line.

[0043] Figure 2 Results from a prolamin analysis of 500 natural inbred lines and a GWAS analysis of α-zein population variation are shown. (a) Prolamin analysis of 500 natural inbred lines; (b) α-zein content, which showed the greatest variation in zein content, was stratified into high and low α-zein levels. The differences in 19- and 22-kD α-zein content were then categorized into three levels (19-kD content higher, equal to, and lower than 22-kD α-zein content) and subsequently subjected to genome-wide association studies (GWAS). GWAS results indicate that the primary locus for the differences in 19- and 22-kD α-zein content is located on the short arm of chromosome 4.

[0044] Figure 3The results of the third-generation sequencing assembly of the wild maize genome and gliadin copy number analysis are shown. (a) Wild maize Z. mays ssp. Parviglumis Ames21814, the F1 line of B73 x Ames21814, and the cultivated maize B73; (b) Wild maize genome assembly flow chart; (c) High-quality wild maize genome, showing, from outermost circle to innermost circle, gene density, repeat density, TIR density, number of indels, number of SNPs, copia density, gypsy density, knob density, and GC content; (d) Tandem repeat copy number analysis of gliadin, showing the copy number of different gene clusters in the α-zein inbred line B73 with different tandem repeat sequences, the wild maize Teosinte, and the inbred line W22.

[0045] Figure 4 The results of protein detection during the population construction process and the analysis of the genetic basis of high protein are shown. Among them, a, seeds of inbred line B73, wild corn Teo, and B73 x Teo and B73 x Teo F2, seed protein determination data are marked above, n is the number of determinations; b, SDS-PAGE gel analysis of alcohol-soluble proteins in inbred line B73, wild corn Teo, and B73 x Teo; c, B73 x Teo Seed alcohol-soluble protein analysis of F2, all F2 are high protein, and B73 is used as the control; d, Protein analysis of different ears of F1BC2 population. The figure shows the alcohol-soluble protein analysis of 12 individual ears, and B73 is used as the control; e, 12 grains from the same high-protein ear of F1BC2 population were individually analyzed for protein content, and B73 was used as the control; F, Protein determination of 30 ears of F1BC3 population, the total protein content of the grains was separated into 10% and 15%; g, 8 high-protein ears of F1BC3 population, 7 grains from each were taken for protein determination, the protein content of each grain in the high-protein ear was ~15%, and B73 was used as the control; h, Protein determination of 30 ears of F1BC4 population, the total protein content of the grains was separated into 10% and ~15%; i, 8 high-protein ears of F1BC4 population, 7 grains from each were taken for protein determination, the protein content of each grain in the high-protein ear was ~15%, and B73 was used as the control.

[0046] Figure 5The results of the localization and expression analysis of the Thp9 gene are shown. a, BSA mapping G'value analysis of the F1BC4 population; b, analysis of introgressed genes by three BSA sequencing runs of F1BC4, F1BC6, and F1BC8; c, Thp9 positional cloning. Thp9 is located in a 147 kb interval between markers 143.7 and 143.8, containing only one gene with significantly altered expression, named Thp9 (Teosinte high protein locus in 9th chromosome, gene number Teo09G002926, NCBIGenome submission: SUB11272093), corresponding to ZmASN4, Zm00001d047736, in B73; d, schematic diagram of ASN4 transcripts in B73 and wild maize; e, statistical analysis of ASN4 transcripts in roots and leaves of B73 and wild maize using RNA-Seq sequencing; f, comparison of the near-isogenic line NILTHP9 and the control NILB73. Transcriptome analysis of roots and leaves showed that ZmAsn4 was significantly overexpressed in roots and leaves of NILTHP9; g, ZmASN4 protein analysis in roots and leaves of the near-isogenic line NILTHP9 and the control NILB73.

[0047] Figure 6 The results of linkage marker development and phenotypic analysis of the high-protein variant locus of Thp9 in wild maize are shown. (a) In the F2BC7 population, Asn4-B73 represents Thp9 with the B73 genotype, Asn4-H represents Thp9 with a heterozygous genotype, and Asn4-Teo represents Thp9 with a homozygous wild maize genotype. (b) Determination of grain protein content in the F2BC7 population for different Thp9 genotypes. Asn4-B73 represents Thp9 with the B73 genotype, Asn4-H represents Thp9 with a heterozygous genotype, and Asn4-Teo represents Thp9 with a homozygous wild maize genotype. (c) Determination of the free amino acid asparagine content in the roots of the F2BC7 population for different Thp9 genotypes. Asn4-B73 represents Thp9 with the B73 genotype, Asn4-H represents Thp9 with a heterozygous genotype, and Asn4-Teo represents Thp9 with a homozygous wild maize genotype.

[0048] Figure 7Figure 1 shows the results of phenotypic analysis of the near-isogenic line NILTHP9. (a) Protein content in grains of the near-isogenic line NILTHP9 and the control line NILB73 in different ecological zones: Shanghai, Sanya, and Northeast China; (b) Total nitrogen content in roots, stems, and leaves of the near-isogenic line NILTHP9 and the control line NILB73; (c) Free amino acid asparagine content in roots of the near-isogenic line NILTHP9 and the control line NILB73; (d) Plant height of the near-isogenic line NILTHP9 and the control line NILB73; (e) Plant height of the near-isogenic line NILTHP9 and the control line NILB73, planted in Sanya in 2021; (f) Fresh weight of leaves, stems, and whole plants of the near-isogenic line NILTHP9 and the control line NILB73.

[0049] Figure 8 The results of genetic verification of Thp9 are shown. Among them, a, relative expression of Thp9 in roots of two independent transgenic events OE-1 (Overexpression-1) and OE-2 (Overexpression-2) overexpressing Thp9; b, relative expression of Thp9 in leaves of two independent transgenic events OE-1 and OE-2 overexpressing Thp9; c, immunoblotting of THP9 in roots of two independent transgenic events OE-1 and OE-2 overexpressing Thp9; d, determination of grain protein content of two independent transgenic events OE-1 and OE-2 overexpressing Thp9; e, GWAS analysis of grain protein content of 405 and 438 inbred lines in 2019 and 2020, respectively, showed a significant signal at ASN4 on chromosome 9; f, 3 of ASN4 in natural populations Schematic diagram of the gene structure of the three haplotypes, where HAP1 is the wild maize Thp9 haplotype, HAP3 is the B73 haplotype with a 47bp deletion compared to HAP1, and HAP2 is a 22bp deletion compared to HAP1; g, Analysis of the protein content of the three haplotypes of ASN4 in natural populations.

[0050] Figure 9This figure shows the results of nitrogen efficiency trials conducted on the near-isogenic line NILTHP9 at the Shanghai Experimental Base in 2020. (a) Plants of NILB73 and NILTHP9 under normal and no nitrogen fertilization; (b) Roots of NILB73 and NILTHP9 under normal and no nitrogen fertilization; (c) ASN4 gene expression was induced by nitrogen fertilization, and the expression level of ASN4 in the near-isogenic line NILTHP9 under no nitrogen fertilization reached that of ASN4 in NILB73 under normal nitrogen fertilization; (d) Above-ground plant biomass of NILB73 and NILTHP9 under normal and no nitrogen fertilization; (e) Below-ground root biomass of NILB73 and NILTHP9 under normal and no nitrogen fertilization; (f) Total biomass of NILB73 and NILTHP9 under normal and no nitrogen fertilization; and (g) Grain protein content.

[0051] Figure 10 The results of the nitrogen high-efficiency test of the near-isogenic line NILTHP9 at the Sanya Experimental Base in 2020 are shown. Among them, a, 0%, 25%, 50% and 100% (the 100% level means that fertilizer was applied once at the seedling stage and once at the jointing stage, for a total of two applications, each time with 0-4-8-16g / plant, nitrogen content of 17%, and the other levels decreased successively, with a planting density of 0.6mx 0.25m) 4 gradient nitrogen application field tests, NILB73 on the left and NILTHP9 on the right; b, determination of plant height of NILB73 and NILTHP9 under 4 different nitrogen application levels; c, determination of aboveground biomass of NILB73 and NILTHP9 under 4 different nitrogen application levels; d, determination of total nitrogen content in roots of NILB73 and NILTHP9 under 4 different nitrogen application levels; e, determination of total nitrogen content in leaves of NILB73 and NILTHP9 under 4 different nitrogen application levels; f, determination of total nitrogen content in leaves of NILB73 and NILTHP9 under 4 different nitrogen application levels g, Determination of total nitrogen content in the stems of NILB73 and NILTHP9; g, Determination of protein content in the grains of NILB73 and NILTHP9 under four different nitrogen application levels.

[0052] Figure 11Shows the results of testing Thp9 hybrids and improving new varieties to create high-protein corn. Among them, a, phenotype of F2 hybrid ears created by using NILB73 and NILTHP9 with Mo17; b, comparison of 100-grain weight and protein content determination of hybrids carrying Thp9; c, high-protein Zhengdan 958THP9 plants created by Thp9 improvement of Zhengdan 958; d, hybrid ears of high-protein Zhengdan 958THP9 created by Thp9 improvement of Zhengdan 958; e, aboveground fresh weight determination of high-protein Zhengdan 958THP9 created by Thp9 improvement of Zhengdan 958 and control Zhengdan 958; f, plant height determination of high-protein Zhengdan 958THP9 created by Thp9 improvement of Zhengdan 958 and control Zhengdan 958; e, aboveground fresh weight determination of high-protein Zhengdan 958THP9 created by Thp9 improvement of Zhengdan 958 and control Zhengdan 958; g, high-protein Zhengdan 958THP9 created by Thp9 improvement of Zhengdan 958 h, Determination of the protein content in the grains of Zhengdan 958 (creating high-protein Zhengdan 958THP9) improved by Thp9 and the control Zhengdan 958; i, Determination of the total nitrogen in the stems of Zhengdan 958 (creating high-protein Zhengdan 958THP9) improved by Thp9 and the control Zhengdan 958; e, Determination of the total nitrogen in the leaves of Zhengdan 958 (creating high-protein Zhengdan 958THP9) improved by Thp9 and the control Zhengdan 958. DETAILED DESCRIPTION

[0053] High-protein maize is a key resource for modern hybrid maize breeding and a crucial agronomic trait. Because high protein content in maize is controlled by multiple genes with minor effects and complex genetic mechanisms, the quantitative trait loci (QTLs) controlling its formation in natural populations are difficult to clone and have yet to be reported. Furthermore, the mechanisms underlying the formation of high-protein maize remain unclear. Consequently, genetic improvement of high-protein maize and its expansion into new germplasm have been slow and difficult. This is not only time-consuming and labor-intensive, but also inefficient and directionally uncertain. Furthermore, each generation requires the arduous and tedious task of measuring protein content. After 10 years of persistent effort, we have finally cloned the key QTL for high-protein maize, Thp9, from wild maize for the first time. This QTL has been applied to the development of maize hybrids. The positive results indicate that the development and utilization of this key gene will significantly advance the genetic improvement and germplasm innovation of high-protein maize, with broad application prospects and economic value.

[0054] The inventors first cloned the high-protein gene Thp9 from wild maize (Ames21814). The allele of this gene in the B73 genome is ZmASN4, with the gene ID Zm00001d047736. Studies have shown that wild maize Thp9 can significantly increase the protein content of maize kernels and plants, increase maize biomass, and improve nitrogen fertilizer use efficiency.

[0055] The asparagine synthetase 4 gene ASN4 studied in this article includes both asparagine synthetase 4 genes from common cultivated corn, such as Zm00001d047736 from the inbred corn B73, and Thp9 from wild corn, with a nucleotide sequence of SEQ ID NO: 1.

[0056] For simplicity, the terms "asparagine synthetase 4" and its encoding gene, ASN4 (or "Asn4"), are sometimes used interchangeably herein. Those skilled in the art will understand that these refer to different substances in different contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of asparagine synthetase, "ASN4" refers to the protein; when describing a gene, "ASN4" refers to the gene encoding the enzyme.

[0057] In order to apply the Thp9 gene to corn germplasm improvement, the present invention also developed molecular markers for identifying the Thp9 high protein site, including 198 bp SEQ ID NO: 4 and 176 bp SEQ ID NO: 5, to facilitate the use of this high protein site in the genetic improvement of common corn.

[0058] On the other hand, in order to detect whether the Thp9 gene is inserted into the maize genome, the present invention further developed a molecular marker SEQ ID NO: 7 for identifying Thp9 high protein sites in natural populations, which is used to screen for superior allele variations in natural maize populations.

[0059] These molecular markers are easy to use and can be performed by a person skilled in the art through simple molecular biology experiments. For example, genomic DNA from corn leaves can be extracted and PCR reactions performed using the mutation site-specific primers we developed, followed by sequencing using the provided primers to detect the mutation site.

[0060] The ASN4 gene application and detection objects of the present invention are applicable to all natural corn populations, including but not limited to existing wild species, inbred lines, farm species and hybrids.

[0061] Considering the convenience of operation, as a preferred method, the application method and target gene detection method of the present invention can be carried out using a kit, which can concentrate the required materials in a kit. In a preferred embodiment, in addition to containing: ASN4 gene fragments, ASN4 gene amplification PCR primers, plant binary expression vectors, restriction endonucleases, Agrobacterium and necessary reagents, the above-mentioned kit can also include at least one of the following items: a carrying tool whose space is divided into a limited space that can accommodate one or more containers, 96-well plates or strips, such as test kits, medicine bottles, test tubes, and the like, each container containing a separate component for the method of the present invention; instructions, which can be written on the bottle, test tube, and the like, or on a separate piece of paper, or on the outside or inside of the container, such as a paper with a window for downloading an operation demonstration video APP, such as a QR code. The instructions can also be in multimedia form, such as a CD, USB flash drive, network disk, IC card, etc.

[0062] It is easy for those skilled in the art to understand that there are many ways to promote the overexpression of ASN4 genes (including Zm00001d047736 and Thp9) in corn, for example, through natural variation and artificial mutagenesis (all mutagen mutagenesis and genetic engineering methods) leading to mutations in the promoter region of the ASN4 gene of wild corn or common corn, and up-regulation of gene expression in the distal regulatory region of the gene; up-regulation of ASN4 gene expression by screening corn ASN4 upstream regulatory factors and their mutations, etc.

[0063] Based on the ASN4 gene (including Zm00001d047736 and Thp9) in the prior art, further improving the activity of the asparagine synthetase 4 gene is also expected in this technical field, for example, through natural variation and artificial mutagenesis (all mutagen mutagenesis and genetic engineering methods) to cause the ASN4 gene function to be acquired or changed; by screening the interacting proteins of corn ASN4 to change the function of ASN4; by screening for variation to cause the expression level of corn ASN4 to change, or the gene function to change.

[0064] The key gene Thp9 that controls high protein and nitrogen efficiency in maize, which we cloned from the wild maize Ames21814, has at least the following advantages:

[0065] 1. Thp9 is highly functional and genetically stable. Crossing wild corn Thp9 with different inbred corn lines can increase kernel protein content. This is theoretically effective for most inbred lines, significantly expanding the potential for high-protein corn germplasm resources. Furthermore, Thp9 is genetically very stable, with consistent phenotypes in Northeast China, Shanghai, and Sanya.

[0066] 2. Using Thp9 to improve high-protein corn takes less time. Because the genes and mechanisms controlling the formation of high-protein corn are unclear, conventional genetic improvement of high-protein corn requires years of large-scale field research at multiple locations to obtain stable material. However, when improving high-protein corn, simply crossbreed with other inbred lines and identify plants carrying the superior Thp9 locus using our developed molecular markers, SEQ ID NOs: 4-5, 7. The ears produced from these plants, whether self-pollinated or hybridized with other corn pollen, will all produce high-protein corn seeds. Furthermore, during the introduction process, at least one ear needs to be retained for crossbreeding and self-pollination to obtain stable hard-grained material, significantly reducing workload.

[0067] 3. Using Thp9 to improve high-protein corn is simple and suitable for large-scale operations. Hybridization and selfing techniques for corn are relatively simple and can be mastered by ordinary workers. We have developed Thp9 molecular marker primers (SEQ ID NOs: 4-5, 7), which can easily identify superior Thp9 genotypes. The corn leaf DNA extraction, PCR reaction, and sequencing involved are all routine molecular experiments that can be performed by ordinary laboratories and sequencing companies.

[0068] 4. Using Thp9 to improve high-protein corn is low-cost. Compared with traditional corn genetic improvement, using Thp9 to improve high-protein corn is more effective, more stable, and reduces time and workload, thereby significantly saving costs. In addition, molecular experiments for Thp9 genotype identification are also very routine and low-cost. Traditional identification of grain protein content requires using a nitrogen analyzer to measure the protein content of each ear. The nitrogen analyzer is expensive and has low throughput. Purchasing a Rapid N nitrogen analyzer also costs more than 500,000 yuan, and the maintenance is complex and the consumables are also expensive.

[0069] 5. Thp9 can be used to create high-protein hybrid corn. By introducing the Thp9 genotype into the parents of a major corn variety, we can quickly produce high-protein corn hybrids while maintaining the original hybrid vigor. This can create significant economic and social benefits if promoted. We have already introduced Thp9 into Zheng 58 and Chang 7-2, two parents of Zhengdan 958, to create high-protein corn hybrids.

[0070] 6. Thp9 is a naturally occurring major locus that increases protein content. Introducing the wild maize Thp9 locus into maize inbred lines and hybrid parents significantly increases protein content and biomass. Our research found that the nitrogen content in the grains, stems, and roots of the near-isogenic line NILThp9, which contains the high-protein locus Thp9, was significantly higher than that in the near-isogenic line NILB73, which does not contain the high-protein locus. Furthermore, overexpressing Thp9 in B73 significantly increased total nitrogen content in the grains, stems, and roots.

[0071] 7. Thp9 is a nitrogen-efficient gene that can significantly improve the nitrogen utilization efficiency of corn and reduce the use of nitrogen fertilizer. It is an important gene for starting a new green revolution in corn and other crops.

[0072] The present invention will be further described below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the present invention. In addition, it should be understood that after reading the concept of the present invention, various changes or adjustments made by those skilled in the art should fall within the scope of protection of the present invention, and these equivalent forms also fall within the scope defined by the claims appended to this patent.

[0073] Example

[0074] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages mentioned are by mass unless otherwise specified.

[0075] Materials and methods

[0076] Corn self-pollination, hybridization, genetic modification, and field breeding are carried out according to conventional breeding methods.

[0077] The primer synthesis and gene sequencing in the examples were all completed by Shanghai Boshang Biotechnology Co., Ltd.

[0078] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were performed primarily with reference to Molecular Cloning: A Laboratory Manual (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. Specific experimental conditions can be determined by simple experiments when necessary.

[0079] PCR amplification experiments should be performed according to the reaction conditions provided by the reagent supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0080] Example 1: Measuring corn protein and amino acid content to identify high-protein wild corn donor materials

[0081] 1.1 Analysis of corn kernel protein content using nitrogen analyzer

[0082] The corn kernels for which the total protein content was to be determined were dried in an oven at 60°C and then ground into powder using a crushing instrument. 50-70 mg of the dry corn powder was weighed to prepare a test sample, and the total protein content was determined using a Dumas rapid nitrogen analyzer (rapid N exceed) from Elementar, Germany.

[0083] 1.2 SDS-PAGE gel analysis of alcohol-soluble and non-alcohol-soluble proteins

[0084] (1) Dry the endosperm in a 37°C oven and grind it into powder using a grinder at 60 Hz for 60 seconds. Weigh 100 mg of the ground powder into a 2 mL tube, add 1 mL of alcohol-soluble protein extract, mix thoroughly, and let it sit at room temperature for more than 2 hours or overnight. (2) Place steel balls in the 2 mL tube, shake it in a grinder for 1 minute, place it on the table for 20 minutes, and centrifuge it at 15871g for 15 minutes. (3) Take 100 mL of the supernatant and place it in a new 1.5 mL tube. Add 10 μL of 10% (g / mL) SDS and vacuum-extract it at 45°C (select rotation and ethanol solution extraction) for 70 minutes. Add 100 μL of ddH2O and place it in a refrigerator at 4°C overnight. The extraction of alcohol-soluble protein Zein is complete. (4) Pour out the remaining liquid in (2) and add 1 mL of alcohol-soluble protein extract. Shake it and let it sit for more than 2 hours. Centrifuge it at 15871g for 15 minutes and remove the supernatant. Repeat this process 3 times, i.e., extract alcohol-soluble proteins with alcohol-soluble protein extract. (5) After repeating 3 times, remove the upper liquid and place the precipitate in a vacuum at 45℃ to evacuate (select rotation and ethanol solution extraction) for 40 minutes. (6) Add 1mL of non-alcohol-soluble extract, shake and vortex, let it stand for 2 hours, centrifuge at 15871g for 15 minutes, take 100mL of the upper liquid and put it in a new tube, and the non-alcohol-soluble protein extraction is completed. (7) Prepare a 15% SDS-PAGE gel for protein analysis. The amount of alcohol-soluble protein to be loaded is 3μL sample plus 8μL 2x loading buffer, and then denature at 95℃ for 5 minutes before loading; the amount of non-alcohol-soluble protein to be loaded is 4μL sample plus 8μL x loading buffer, and then denature at 95℃ for 5 minutes before loading. (8) Electrophoresis at 180V for 70 minutes, and the sample is ready just when the blue parallel lines run out. The cells were stained with Coomassie Brilliant Blue for 2 h and then destained with decolorizing solution three times, changing the decolorizing solution every 45 min.

[0085] Prolamin Extraction Solution

[0086]

[0087] Non-alcohol-soluble protein extraction solution

[0088]

[0089]

[0090] 1.3 Determination of free amino acid content in different tissues of corn

[0091] (1) Sample pretreatment: Dry the sample at 65°C, grind it, and pass it through a 100-mesh sieve. Weigh an appropriate amount of sample, add distilled water and shake it for 1 min. Soak it at 4°C for 8 hours, then add steel balls and homogenize it. The homogenate was centrifuged at 4500 g for 5 min, and the supernatant was taken for use; (2) Derivatization process: a standard mixture (concentrations of the mixture are shown in S1-S5 in the original data, amino acid kit: Beijing Mass Spectrometry Medical Research Co., Ltd., MSLAB50AA, batch number: MSLAB50AA211201#; asparagine Asn standard curve: y = 0.00147x + 0.00104 (r = 0.9984); y indicator name Asn, x analytical value, r correlation coefficient; concentrations of the mixture S1-S5 are 1.25 μmol / L, 6.25 μmol / L, 12.5 μmol / L, 50 μmol / L and 100 μmol / L respectively), 50 μl of the sample to be tested was added with 50 μl of protein precipitation agent (containing NVL), mixed and centrifuged at 13200 rpm for 4 minutes. 10 μl of the supernatant was taken, 50 μl of labeling buffer was added, mixed and centrifuged instantly. Add 20 μl of derivatization solution, mix thoroughly, centrifuge briefly, and then derivatize at 55°C for 15 minutes. After derivatization, cool the sample in the refrigerator, mix thoroughly, centrifuge briefly, and a 50 μl sample is analyzed. Instrument model: HPLC-MS / MS; LC: Dionex Ultimate 3000; MS: API 3200QTRAP; Amino Acid Kit: MSLAB50AA, Beijing Mass Spectrometry Medical Research Co., Ltd., batch number: MSLAB50AA170601#; Methanol, acetonitrile, etc. were purchased from Fisher.

[0092] Since 2012, we have been searching for, analyzing, and constructing a population of high-protein corn donor materials. To find high-protein donor materials, we measured the protein content of more than 30 wild corn seeds and common cultivated corn inbred lines. We found that the protein content of wild corn is 28.6% ± 1.0%, while the protein content of common corn inbred lines (B73 as a representative) is 10% ( Figure 1 We further analyzed the alcohol-soluble proteins and non-alcohol-soluble proteins of wild corn and inbred line B73 by SDS-PAGE gel and found that the alcohol-soluble proteins and non-alcohol-soluble proteins of wild corn were significantly increased compared with those of B73 ( Figure 1c). By measuring the free amino acid content of roots, stems and leaves of wild corn and B73 plants, it was found that the asparagine content in the free amino acid components decreased significantly (roots: 16611 μg / g of wild corn decreased to 4120 μg / g of cultivated corn; stems: 13668 μg / g of wild corn decreased to 2529 μg / g of cultivated corn; leaves: 14689 μg / g of wild corn decreased to 2946 μg / g of cultivated corn) ( Figure 1 These results indicate that wild maize is an excellent high-protein donor material that exists naturally.

[0093] Example 2: Protein analysis and determination of 500 inbred lines from a natural population

[0094] 2.1 SDS-PAGE gel electrophoresis analysis of alcohol-soluble protein content in 500 maize inbred lines

[0095] We planted 500 inbred lines in Harbin in 2014. After harvesting and drying at maturity, we collected kernels from the middle of three ears of each inbred line, removed the embryos, and mixed and ground the kernels. Prolamin extraction and SDS-PAGE analysis were performed as described in step 1.2 above for prolamin extraction.

[0096] 2.2 GWAS association analysis

[0097] 500 maize inbred seeds and corresponding genotype data were provided by Professor Lai Jinsheng's laboratory at China Agricultural University. The prolamin content of these 500 maize inbred lines was analyzed by SDS-PAGE gel electrophoresis. Zein levels, which showed the greatest variability, were categorized into high and low α-zein content. The differences between 19- and 22-kD α-zein content were then divided into three levels (19-kD content was higher, equal to, and lower than 22-kD α-zein). A genome-wide association study (GWAS) was then performed. The methods used in this study were similar to those published by our laboratory (Liu et al., PNAS, 2015).

[0098] By analyzing the protein content of inbred lines in natural populations, we found that there are also major factors for high protein in natural populations. The main storage protein of corn is alcohol-soluble protein, which accounts for more than 60% of the total protein. We further analyzed the alcohol-soluble protein of 500 natural populations by SAD-PAGE gel ( Figure 2 (a) found significant differences between inbred lines. Zein levels, which showed the greatest variation, were stratified by α-zein content, and a genome-wide association study (GWAS) was performed. The main locus for α-zein content variation was located on the short arm of chromosome 4, where 19- and 22-kD α-zein gene copies are clustered. This suggests that variation in zein content among inbred lines in natural populations is related to zein copy number. Figure 2(b) Is the high protein content in wild corn determined by the number of tandem repeats of prolamin?

[0099] Example 3: High-quality genome sequencing and assembly of wild maize Ames21814

[0100] To analyze the copy number of complex alcohol-soluble protein tandem repeats and provide reference sequences for downstream gene cloning, we completed the assembly and annotation of the high-quality genome of wild maize Ames21814 by third-generation sequencing ( Figure 3 Our sequencing material was the F1 hybrid of wild maize (Ames21814) and B73. Using the latest HiFi sequencing technology, we obtained 6,752,166 reads, totaling approximately 104 Gb of high-quality CCS sequences with an N50 read length of 15.4 Mb. This represents approximately 47x the data size of the 2.2 Gb B73 genome. Our assembly was based on the Trio-binning method, extracting wild maize CCS sequences using second-generation parental sequences. The sequences were then assembled into contigs using HiFisam and Yak programs. Using 375.56 Gb of Hi-C data, the genome was aligned to the PacBio genome assembly using juicer and bwa mem with default parameters, using R1 and R2. Chromosomes were clustered, sorted, and subjected to two rounds of error correction based on the interaction information provided by the HiC data. Finally, the HiC interaction matrix was imported into juicebox for visualization and manual inspection. After verification of anomalies, it was exported. 500 Ns were added between each contig. The final chromosome assembly efficiency was 91.30%, the genome size was 2460 Mb, the contig N50 was 62.29 Mb, and the scaffold N50 reached the gold standard of 243.71 Mb for the wild maize genome. BUSCO assessment determined the wild maize genome to be 96.8% complete. The wild maize genome contains 80.80% repetitive sequences, with LTR transposons being the most prevalent transposable element, accounting for approximately 61.48% of the genome. The wild maize genome was annotated with 58,092 genes encoding 108,712 transcripts. The assembled wild maize genome sequence has been uploaded to NCBI (Genomesubmission:SUB11272093). Based on the high-quality genome obtained, we continued to analyze and annotate the copies of all alcohol-soluble proteins in wild maize through sequence alignment. We found that the copy number of all alcohol-soluble proteins in wild maize Ames21814 did not change significantly compared with B73, indicating that the high protein content of wild maize is not caused by the number of alcohol-soluble protein copies ( Figure 3 (d)

[0101] Example 4: Analysis of the genetic basis of high protein and population construction

[0102] How is the high-protein trait of wild corn inherited? To solve this problem, we constructed a genetic population of wild corn Ames21814 and B73 for analysis. First, using B73 as the mother plant, the protein content of the F1 grains obtained was 11.6±0.8%, which was similar to the protein content of the mother plant B73 of 10.8±1%. We continued to self-pollinate the F1 and re-pollinate its pollen on B73. The protein content of the F2 seeds obtained by self-pollination of the F1 was 19.9±1.2%, and the protein content between different F2 seeds did not change, indicating that the factor controlling the formation of high protein is determined by the genotype of the mother plant ( Figure 4 We then planted the harvested F1BC1 and then used B73 as the male parent for backcrossing. The harvested F1BC2 (Fig. 4d and e), F1BC3 ( Figure 4 f and g) and F1BC4( Figure 4 Protein analysis was performed on both populations h and i, and it was found that the protein content was separated by ear. In the backcross population, the protein content between different ears varied by 10% to 15%, while the protein content of different kernels in the same ear was the same ( Figure 4 The above genetic population construction and protein analysis further demonstrate that the factors controlling protein content are determined by the genotype of the maternal plant, and that introgressing wild corn into cultivated maize inbred lines can increase protein content, becoming a key strategy for creating high-protein corn. Based on this genetic foundation, we determined the genetic population of each backcross generation, selected high-protein ears for further planting, and repeatedly backcrossed with B73 to create advanced near-isogenic lines for subsequent mapping of high-protein genes.

[0103] Example 5: BSA sequencing and map-based cloning of Thp9, a key gene controlling the formation of high-protein corn

[0104] 5.1 Leaf DNA extraction

[0105] (1) Place corn leaves in a 2 mL tube, add a steel column, treat with liquid nitrogen, and grind (60 Hz, 60 s). (2) After grinding, add 0.6 mL of CTAB extraction buffer and mix well. (3) Place in a 65°C oven for 60 min, mixing every 10-15 min. (4) Remove and place at room temperature for 5-10 min. Add an equal volume of chloroform:isoamyl alcohol (24:1) to the centrifuge tube, seal, and shake for 5 min. (5) Centrifuge at 15871 g for 15 min at room temperature. Pipette the supernatant into a new 1.5 mL centrifuge tube. (6) Add an equal volume of isopropanol, mix well by inverting, and place at -20°C for 20 min. (7) Centrifuge at 15871 g for 1 min at room temperature. Discard the supernatant. Wash the DNA precipitate with 1 mL of 75% ethanol 1 to 2 times, centrifuging at 15871 g for 1 min each time, and discard the ethanol. (8) Briefly centrifuge, aspirate excess liquid, and air-dry the DNA pellet at room temperature. (9) Add 0.3 mL of H2O to dissolve the DNA pellet.

[0106] CTAB extraction buffer

[0107]

[0108]

[0109] Chloroform:isoamyl alcohol (24:1): Add 20.8 mL of isoamyl alcohol to 500 mL of chloroform and mix well.

[0110] 5.2 BSA sequencing and analysis

[0111] Protein content was determined using a nitrogen analyzer (using the same method as in step 1.1 above) for the F1BC4, F1BC6, and F1BC8 populations. DNA was extracted from an equal number of individuals from each population with two extreme phenotypes, and equal amounts were pooled for BSA sequencing. Within the F1BC4 population, 100 samples (pool 1) of the high-protein phenotype (approximately 15%) and 100 samples (pool 2) of the B73 phenotype (approximately 10%) selected from the segregating population were subjected to high-throughput sequencing (X-Ten 100X, total data volume 500G). The recurrent parent, B73, was resequenced (30X, data volume 75G). The filtered, high-quality sequencing data were aligned to the B73 reference genome, and SNP detection was performed using GATK software. SNP index differences between the two pools and between the parental phenotypes were calculated to identify regions closely linked to the high-protein trait (this method can be found in the inventors' previously published paper, Huang et al., Plant Cell, 2019). For the F1BC6 population, 150 samples (Pool 1) of the high-protein genotype (approximately 15%) and 150 samples (Pool 2) of the B73 genotype (approximately 10%) selected from the segregant population were sequenced using high-throughput sequencing (X-Ten 100X, total data volume 500G). The analysis method was the same as above. For the F1BC8 population, 50 samples (Pool 1) of the high-protein genotype (approximately 15%) and 50 samples (Pool 2) of the B73 genotype (approximately 10%) selected from the segregant population were sequenced using high-throughput sequencing (X-Ten 50X, total data volume 250G). The analysis method was the same as above.

[0112] 5.3 Plotting and Data Analysis of F1BC4, F1BC6, and F1BC8

[0113] (1) Alignment with the reference genome: Align the B73 sample, the F1BC4 high-protein HP sample, and the low-protein LP sample data (the same for F1BC6 and F1BC8) to the combined B73 and Teo genome. (2) Statistical coverage depth of the Teo gene: Calculate the coverage depth of each window region using a sliding window of 50 kb and a step size of 25 kb for each sample's aligned bam file. (3) Data normalization: To make the coverage depths of different samples comparable, we normalized the coverage depth of each sample (formula: coverage depth of each sample / 50 kb - coverage depth of B73 sample / 50 kb). (4) Obtaining plot data: Subtract the normalized coverage depth of the low-protein LP sample (the same for BC4, BC6, and BC8) from the normalized coverage depth of the high-protein HP sample to obtain the delta value for plotting. (5) Plotting: Plotting was performed using the ggplot2 package in R.

[0114] 5.4 Molecular marker PCR detection method

[0115] Genome-wide polymorphic markers were designed and developed based on the wild corn genome and the B73 reference genome, and then the developed molecular markers were selected in wild corn, B73 and F1 in the high-protein population for verification. The available molecular markers of chromosome 9 140Mb-152Mb were screened, and 2000 F1BC9 populations were subjected to genotyping by PCR and agarose gel running using the above-screened molecular markers. At the same time, the protein content of the 2000 populations was determined, and the genotype genetic exchange information was analyzed with the grain protein content corresponding to the exchanged individual plants. PCR was amplified using 2×HieffTM PCR Master Mix (Shanghai Yishen Biotechnology Co., Ltd., 10102ES03) and standard procedures, and identified using 3% agarose gel.

[0116] Primer information for positional cloning molecular markers:

[0117]

[0118] 5.5 RNA extraction and RNA-seq analysis

[0119] (1) Take tissue samples from the experimental and control groups at the same time, such as roots and leaves, freeze them in liquid nitrogen, and quickly transfer them to a -80℃ ultra-low temperature freezer for storage. (2) Use a grinding machine to grind them thoroughly (60Hz, 70s) under liquid nitrogen freezing conditions and then store them in liquid nitrogen. (3) Add 1mL of Trizol extract to the thoroughly ground tissue powder, shake it thoroughly, and let it stand for 5 minutes; then add 200μL of chloroform to the mixture, shake it thoroughly, let it stand on ice for 5 minutes, centrifuge it at 13523g for 10 minutes at 4℃, and aspirate 500μL of the supernatant (do not aspirate the middle layer and the organic phase below) into a new centrifuge tube. (4) Add 500μL of isopropanol to the supernatant, shake it thoroughly, let it stand on ice for 10 minutes, centrifuge it at 13523g for 10 minutes at 4℃, and discard the supernatant. (5) Add 1 mL of 70% ethanol solution, flick the precipitate to float, place on ice for 1 minute, centrifuge at 13523g for 5 minutes at 4℃, and discard the supernatant. (6) Centrifuge briefly and remove excess liquid with a small pipette. Dry the RNA precipitate at room temperature for about 2 minutes, and add 100 μL of ddH2O to dissolve the precipitate. (7) Use Qiagen's RNeasy Plus Mini Kit (Qiagen, catalog number: 74,106) to purify the above-mentioned RNA by column according to the standard method of the kit, which involves the use of DNaseI (Qiagen, catalog number: 79,254) to remove DNA, and finally dissolve it with 30 μL of RNase-free H2O. (8) Use Pomega's reverse transcription kit (ImProm-IITM RNA was reverse transcribed using the RNA Reverse Transcription System according to the standard method in the kit.

[0120] 5.6 Quantitative PCR detection

[0121] Specific primers were designed and analyzed by NCBI. Primers Asn4-rt-1F / R were used for RT-qPCR, using Actin as an internal reference gene. Quantitative analysis was performed using the Takara SYBR Green kit standard protocol. Three technical replicates were performed for each sample. The reverse-transcribed cDNA samples were diluted 8-fold and quantitatively analyzed in a 20 μL reaction. A 20 μL reaction volume was prepared using SYBR Green Mix, with 10 μL of the mix, 1 μL of each primer, 2 μL of diluted cDNA, and 6 μL of ddH2O. Gene expression was determined using a two-step PCR amplification method using a BIO-RAD CFX fluorescence quantitative analyzer. The reaction conditions were: initial denaturation at 95°C for 30 s; amplification at 95°C for 5 s; amplification at 60°C for 35 s; and 40 cycles of termination at 95°C for 15 s; 60°C for 60 s; and 95°C for 15 s. The quantitative data were analyzed using EXCELL 2010 and the ΔΔCT method. The ZmAsn4 quantitative primers are as follows.

[0122]

[0123] 5.7 Western Blotting

[0124] (1) Total protein extraction: Weigh 100 mg of fresh root and leaf powder samples into a 2 mL centrifuge tube. Add 1 mL of non-alcohol-soluble protein extraction buffer and incubate at room temperature for 2 h. Centrifuge at 15871 g for 15 min. Transfer the supernatant to a new 1.5 mL centrifuge tube and store at 4°C until use.

[0125] (2) Western immunoblotting: Pipette 15 μL of extracted protein and 5 μL of 4x Protein Loading Buffer into a 200 μL centrifuge tube and vortex to mix. Incubate at 95°C for 5 minutes for denaturation using a PCR instrument. Prepare a 12% separation gel and a 4% stacking gel for PGAE gel. Use 1x SDS-PAGE Buffer for electrophoresis at 120V for 60 minutes. Cut a PVDF membrane of the same size as the gel and activate it with methanol for 5 seconds. After electrophoresis, use a BIO-RAD semi-dry transfer instrument for transfer. Place the transferred membrane in a clean hybridization box and block it with 5% imported skim milk powder dissolved in 1x TBST at 4°C overnight. The next day, dilute the primary antibody diluent with 1x TBST at a ratio of 1:1000 and incubate for hybridization at room temperature for 1 hour. Wash the membrane with 1x TBST, changing the TBST every 15 minutes, and repeat the washing process 4 times. Dilute the secondary antibody solution 1:5000 in 1x TBST and incubate at room temperature for 1 hour. Wash the membrane with 1x TBST, changing the TBST every 15 minutes, and repeat this wash four times. Add chemiluminescent solution for color development and imaging.

[0126] 5.8 Gene Cloning

[0127] To identify the genetic factors controlling the main effect of high-protein maize, we performed three BSA pool sequencing on F1BC4 (n=500), F1BC6 (n=1650), and F1BC8 (n=2000), and through deep resequencing of five high-protein and five low-protein stable F3BC6 materials, we found that the factor controlling high protein is located in the chromosome 9 interval ( Figure 5 (a and b). Further mapping of the gene was performed by map-based cloning. By measuring protein levels in 2,000 F1BC9 populations and developing and screening polymorphic markers, we narrowed down the Thp9 gene to markers 143.7 and 143.8. Based on wild maize haplotype sequences, this 147 kb interval contained only one gene, teo09G002926, that showed significant expression changes. Figure 5 (c) The corresponding B73 version of the gene is ZmAsn4, gene number Zm00001d047736, encoding asparagine synthetase 4, ASN4. By analyzing the three generations of wild maize genome sequences, we found that the wild maize Asn4 has a 47bp insertion in exon 10 compared to B73. This insertion causes the wild maize Asn4 gene transcript to be different from that of B73. By analyzing the Asn4 transcripts in the roots and leaves of wild maize and B73, we found that the transcript used by wild maize Asn4 is different from that in B73, and the wild maize transcript is significantly higher ( Figure 5Further analysis of RNA-Seq of roots and leaves of the near-isogenic line NILTHP9 carrying this high-protein locus and the control NILB73 revealed that Asn4 was significantly overexpressed in both roots and leaves of the near-isogenic line NILTHP9, while it was almost not expressed in NILB73 ( Figure 5 Immunoblotting analysis also demonstrated that ASN4 was highly accumulated in roots and leaves of near-isogenic lines ( Figure 5 (f) Through a series of mapping sequencing, large-scale population protein content measurement, and molecular marker screening, we ultimately located the major high-protein gene as Asn4-Thp9.

[0128] Example 6: Verification of the key gene Thp9 and linked markers for the formation of high-protein corn

[0129] The leaf DNA extraction and PCR identification methods are the same as those in step 5.4 above. The identification primers are:

[0130]

[0131]

[0132] Based on this 47 bp insertion, we developed a molecular marker SEQ ID NO: 4 and analyzed 200 individuals in the F3BC7 population. We found that the protein content of ZmAsn4-B73 (ZmAsn4 is the genotype of B73) in the population was significantly lower than that of the heterozygous ZmAsn4-H (ZmAsn4 is the heterozygous genotype) and the wild maize genotype Asn4-Teo (ZmAsn4 is the genotype of wild maize) ( Figure 6 In a and b), the content of free amino acid asparagine in the roots was also significantly higher in ZmAsn4-H and Asn4-Teo types than in ZmAsn4-B73 type ( Figure 6 (c) indicates that THP9 protein levels are linked to this marker. This result further demonstrates that high ZmAsn4 expression is a key factor in increasing maize protein content. The molecular marker we developed, SEQ ID NO: 4, can be used to identify introduced high-protein loci in wild maize and to identify population-level variation in these loci.

[0133] Example 7: Performance of Thp9 high-protein corn under different ecological conditions

[0134] The methods for protein content determination and amino acid content analysis were the same as those in steps 1.1 and 1.3 above. We tested the stability of protein content traits at different locations, and selected Shanghai, Sanya, and Harbin in Northeast China for testing in different years and locations. We found that the protein content of the near-isogenic line NILTHP9 seeds in Shanghai was approximately 13.1±0.38%, while the control NILB73 was approximately 9.69±0.43% in Shanghai; the protein content of NILTHP9 seeds in Sanya was approximately 15.39±0.95%, while the control NILB73 was approximately 11.17±0.95%; and the protein content of NILTHP9 seeds in Northeast China was also approximately 11.96±0.65%, while the control NILB73 was approximately 9.16±0.52% in Northeast China, which increased by 35.19%, 47.78%, and 30.57% in Shanghai, Sanya, and Northeast China, respectively. Figure 7 In addition, we found that NILTHP9 increased the total nitrogen content in the stem and roots in addition to increasing the protein content in the grain ( Figure 7 b). The free amino acid content determination showed that asparagine in NILTHP9 was significantly higher than that in NILB73 ( Figure 7 In addition, the plant height and aboveground biomass of NILTHP9 increased significantly, and the plant height also increased by 10% ( Figure 7 d and e), aboveground biomass increased by 20% ( Figure 7 (f) indicates that excessive asparagine accumulation by THP9 is beneficial to plant growth. THP9 has great potential in increasing corn kernel protein content, total nitrogen in silage corn stalks, and plant biomass and height.

[0135] Example 8: Genetic verification shows that Thp9 is a key gene controlling the formation of high-protein corn

[0136] 8.1 Construction of Thp9 overexpression vector and primers

[0137] The construction of the Thp9 overexpression vector was carried out by amplifying the cDNA of wild corn Thp9 as a template. The amplification primers were: forward primer ASN4-3300-FlgF:

[0138] aggtcgactctagaggatccATGgactacaaggaccatgacggtgactacaaggaccatgacattgactacaaggatgacgatg acaagggaggaggatgtggcatcttagccgtg;

[0139] Reverse primer ASN4-3300-R3: ggggaaattcgagctcTTACACCGCGATGGCGACAGC.

[0140] PCR conditions: Toyobo KOD-FX-NEO enzyme was used for PCR amplification. The system was mixed according to the KOD enzyme standard system, with pre-denaturation at 94°C for 2 min, denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 68°C for 2 min, for 35 cycles.

[0141] The PCR amplified fragment was cloned into the pCAMBIA3300 vector using homologous recombination (ClonExpress II One Step Cloning Kit, C112-02, Novozymes) and placed downstream of the maize Ubiquitin (UBI) promoter to construct a Thp9 overexpression vector.

[0142] 8.2 Genetic transformation

[0143] Agrobacterium EHA105 competent cells were prepared according to the Molecular Cloning Experimental Guide (3rd edition). The overexpression Thp9 vector was transformed into maize B73 immature embryos using Agrobacterium-mediated transformation to obtain overexpression transgenic maize. Genetic transformation was performed at Weimi Biotechnology (Jiangsu) Co., Ltd.

[0144] 8.3 Quantitative PCR Analysis and Western Blotting of Transgenic Maize

[0145] The specific analysis method is the same as 5.5 and 5.6, and the quantitative primers are:

[0146]

[0147] 8.4 Nitrogen Analyzer Analysis of 500 Corn Inbred Lines' Kernel Protein Content

[0148] One of 500 inbred lines was planted at the Damao base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan Province (2019 and 2020). Three ears of each material were inbred. After ripening and drying, six kernels from the middle of three ears of each inbred line were collected, mixed, and ground into a sample. Protein content was determined as described in step 1.1 above.

[0149] Furthermore, we overexpressed Thp9 from wild maize haplotype in the B73 background via the UBI promoter. The expression of Thp9 gene in roots and leaves of the overexpressing transgenic event was significantly increased ( Figure 8 a and b); Immunoblotting analysis showed that Thp9 was significantly accumulated in the roots of the overexpressing transgenic events ( Figure 8 c), the grain protein content increased from 12.08±0.88% in the control to 15.18±1.03% in Asn4-OE-1 and 15.81±1.13% in Asn4-OE-2 ( Figure 8d). Through the verification of transgenic inheritance, we proved that overexpression of Thp9 also has great potential in increasing the protein content of corn grains, increasing the total nitrogen of silage corn stalks, and increasing plant height, which once again proves the important value of Thp9. In addition, we measured the protein content of 405 and 438 inbred lines planted in the Damao base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan in 2019 and 2020, respectively. We found that the variation in protein content of the inbred lines in 2019 was 6.5%-16%, with an average of 11.52%; while the variation in protein content in 2020 was 7.7%-16.8%, with an average of 12.3%. The protein content of the inbred lines uniformly planted in the Damao base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan was subjected to GWAS analysis of the protein content of the natural population, and it was found that the main effect site controlling the protein content of the natural population is still the Thp9 gene site located on chromosome 9 ( Figure 8 In natural populations, there are three haplotypes of ASN4 gene at the insertion / deletion positions previously identified in wild maize and B73 ( Figure 8 f), analysis showed that the ASN4 gene (Zm00001d047736) of B73 belongs to a haplotype HAP3, which is 47bp missing compared to the wild maize haplotype HAP1, but has no function and is almost not expressed, with a protein content of about 10%; the wild maize haplotype HAP1 and the natural population haplotype HAP2 with a 22bp missing haplotype both significantly increased the protein content in the natural population ( Figure 8 (g) From wild maize to cultivated maize, ASN4 was selected during domestication, leading to divergence in natural populations. Population-level variation again demonstrates that ASN4 variation is significantly correlated with protein content.

[0150] Example 9: Thp9 nitrogen high efficiency test and field test

[0151] Protein content was determined using the same method as in step 1.1 above. By growing the Thp9-carrying near-isogenic line NILTHP9 and the non-THP9 control NILB73 in soils with varying nitrogen levels, we found that ZmAsn4 gene expression significantly increased with increasing nitrogen levels. Furthermore, under low nitrogen conditions (no artificial nitrogen fertilizer application), NILTHP9 achieved plant height, above- and below-ground biomass, and grain protein content comparable to those of the control NILB73 at normal nitrogen application (applied twice, 10 g each time, at the seedling and jointing stages). Figure 9 These results indicate that high expression of the wild maize ZmASN4 gene has the potential to be highly efficient in nitrogen utilization.

[0152] Furthermore, we conducted field experiments and set up four gradient nitrogen application tests: 0%, 25%, 50% and 100% (100% level: once at the seedling stage and once at the jointing stage, for a total of two applications, each with 0-4-8-16g / plant, nitrogen content 17%, and the other levels decreased in sequence; planting density 0.6mx 0.25m). Each group planted 300 seeds (Figure 10a). We found that NILTHP9 had a significant difference in plant height ( Figure 10 b), aboveground biomass ( Figure 10 c) and the total N content of roots, leaves and stems ( Figure 10 The NILTHP9 strain (ZmAsn4) showed increased seed protein content compared to the control (NILB73) (Figure 10g). Furthermore, at the 25% nitrogen level, NILTHP9 achieved plant height, biomass, and nitrogen content comparable to the 50% and 100% nitrogen levels of the control. Field experiments further demonstrated the high nitrogen efficiency of wild maize ZmAsn4. The introduction of the Thp9 gene has significant implications for reducing nitrogen fertilizer application and promoting environmental sustainability.

[0153] Example 10: Application potential and value of Thp9

[0154] The PCR identification method for the Thp9 locus was the same as in step 5.3, with primers thp9-F / R and asn4-is-F / R. The amplification information is shown in Example 6. To test the application potential of Thp9, hybrids were first created with the Thp9 near-isogenic line (B73 background) and the control NILB73 and Mo17. It was found that the protein content of the hybrid carrying the Thp9 locus was significantly increased ( Figure 11 (a and b). Furthermore, we backcrossed Thp9 into Zheng 58 and Chang 7-2, two parents of the main corn hybrid Zhengdan 958. Through three generations of backcrossing and self-pollination, we obtained Thp9-modified versions of Zheng 58 and Chang 7-2, respectively. The improved versions of the two parents were hybridized to obtain the improved version of Zhengdan 958. Planting tests in Sanya showed that the high-protein gene-modified Zhengdan 958 material had increased plant numbers and plant biomass ( Figure 11 The protein content of the improved version of Zhengdan 958-THP9 seeds was 11.14±1.13%, while that of the control Zhengdan 958 was 9.88±0.58%, an increase of 12.75% ( Figure 11 In addition, the total nitrogen content in roots, stems, and leaves of the improved version Zhengdan 958-THP9 increased ( Figure 11 my country's annual corn production is about 270 million tons. A 1 percentage point increase in protein content is equivalent to an additional 2.7 million tons of protein, which will generate huge potential and value in agricultural production.

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[0171] Huang,Y.,H.Wang,X.Huang,Q.Wang,J.Wang et al.,2019 Maize VKS1Regulates Mitosis and Cytokinesis During Early Endosperm Development.PlantCell 31:1238-1256. Sequence Listing <110> Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences <120> Key genes controlling corn protein content and nitrogen efficiency <130> SHPI2210108 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 3207 <212> DNA <213> Zea mays ssp. Parviglumis, Ames21814 <400> 1 atgtgtggca ttttagccgt gctcggatgc tccgactgct cccaggccag gagggctcgc 60 atcctcgcct gctccagaag gcaagcatcc gtccattgca ctgggaccga gctcttctca 120 tataggaacc ataaagaatc gaacggtaga gagactagga tgggccgatc tgaaaaggag 180 catgggaatc ttactcgttc aagcaaccct gtgtgtgtgt gcgcgcgctt ctgtgtcact 240 ggttacagta ttgtcgggtg ggcgcgatta gttgttggtt agtgtttaag ttttgacgac 300 tggcggacct gatgatggtc ggtcactccc acgtggtgtg caggctgaag cacaggggcc 360 ccgactggtc gggcctctac cagcacgagg gcaacttcct ggcgcagcag cggctcgcca 420 tcgtctcccc gctgtccggc gaccagccgc tgttcaacga ggaccgcacc gtcgtggtgg 480 tggtaagcta ataagatcca aatatgcacg cgcgcagcat gcatgctcaa gcgtcgtcta gctagttttg acggggcccta tctatgctgt agttcctcag catgcatgcg ctttgtttgc 600 tttttttact tcacaggcca atggagagat ctacaaccac aagaacgtcc ggaagcagtt caccggcgcg cacagcttca gcaccggcag tgactgcgag gtcatcatcc ccctggtgag 720 ccttacactg atcgtttcag ttctgaaaac caaacttgtt cttcgcttac acactaaaga acaaaaactt ctcctctact gatgctgcct ttgttattgc tgctgccgtc gtcggcgtca 840 tcgatctcca gtacgagaag tacggcgaga acttcgtgga catgctggac ggagtcttcg cgttcgtgct ctacgacacg cgagacagga cctacgtggc ggcacgcgac gccatcggcg 960 tcaacccgct ctacatcggc tggggcagcg acggtcagac tcagacacag cgtggcgtgg 1020 cattttcgca gtgcggtcgc gccaagcaga gcaccccagc taggtgggtc aagctgaagc 1080 tgaagctgac cgatcgattt tctcgcctcg ccttccctcc actactgcag gttccgtctg 1140 gatgtcgtcc gagatgaagg cgctgaacga ggactgcgtg cgcttcgaga tcttcccgcc 1200 ggggcacctc tactccagcg ccgccggcgg gttccgccgg tggtacaccc cgcactggtt 1260 ccaggagcag gtgccccgga cgccgtacca gccgctcgtc cttagagagg ccttcgagaa 1320 ggtgagtgac cttgcacttg ttgggtcgtc ggtggtattt aagcaataaa gatggccgtt 1380 actgacactg acctctggcc atgggccatg ggcccgtgcg ctgcaggcgg ttatcaagag 1440 gctcatgacc gacgtcccgt tcggggtcct cctctccggc ggcctcgact cctccctcgt 1500 cgcctccgtc accaagcgcc acctcgtcaa gaccgacgcc gccgaaaagt tcggcacaga 1560 gctccactcc ttcgtcgtcg gcctcgaggt tttgtttcgt tttttttggc attggtggtg 1620 cgcgtgtctt atttgtctcg gcgatagaat cgcgcgtggg acgggacgct gacgtttttt 1680 ttacgtctct ctcgatcgcc gaccggccgg cacgtacgct tcagggctcc cctgacctga 1740 aggccgcacg agaggtcgct gactacctcg gaaccaccca tcacgagttc catttcaccg 1800 tacaggcaag taaatcattc gcgcgcgcgc tcgcttttgg cgagaccgtg acgtaggctg 1860 acgagtggca aaaaaattac aaaaatggac catcatccat aggacggcat cgacgcgatc 1920 1980 atgttcctga tggctcgcaa gatcaagtcg ctgggcgtga agatggtgct gtccggggag 2040 ggctccgacg agctcctggg cggctacctc tacttccact tcgcccccaa cagggaggag 2100 ctccacagg agacctgccg caaggtgaag gccctgcacc agtacgactg cctgcgcgcc 2160 aacaaggcga cgtcggctg gggcctggag gtccgcgtgc cgttcctcga caaggagttc 2220 gtcgacgtcg cgatgggcat ggaccccgag tggaaaatgg tactgacgcg ggcctttttc 2280 gacacggccc ggccctgccg ccgcacgtcg gggtctcggt tctacgtatg atgatgacgc 2340 cttcttctct tctttgcgca gtacgacaag aacctgggtc gcatcgagaa gtgggtcctg aggaaggcgt tcgacgacga ggagcaccct tacctgcccg aggtaagaac atcttcagag 2520. aaggctggtc gtttacctct gtgtctgtgt gatttcaagc ctgaactgac gcctctgtgc catgcatcct ccgcagcata ttctgtacag gcagaaagaa cagttcagtg acggagtggg 2580 ctacaactgg atcgatggac tcaaatcctt caccgaacag caggttgatt tacggcccca ctttcagctc tgatcgcatc tcctagacat cgtaccgtac gtcgtccaag ttagctaacc agcgctgacg ttccccccca atgttcaggt gacggatgag atgatgaaca acgccgccca gatgttcccg tacaaccgc ccgtcaacaa ggaggcctac tactaccgga tgatattcga gaggctcttc cctcaggtga ttgattcagc tttcagccag cctccaacga tgcgcgtgtt gcactgcaca cgtggtagcc aattcaatac gcgcggcgtg ctgctgactg ttgggtcgtg aactcggtga tgcctgcctg catgcaggac tcggcgaggg agacggtgcc gtggggcccg 3000. agcatcgcct gcagcacgcc cgcggccatc gagtgggtgg agcagtgga ggcctccaac 3060 gacccctccg gccgcttcat ctcctcccac gactccgccg ccaccgaccg caccggagac 3120 aagctggcgg tggtcaacgg cgacgggcac ggcgcggcga acggcacggt caacggcaac 3180 gacgtcgctg tcgcgatcgc ggtgtaa 3207 <210> 2 <211> 588 <212> PRT <213> Zea mays ssp. Parviglumis, Ames21814 <400> 2 Met Cys Gly Ile Leu Ala Val Leu Gly Cys Ser Asp Cys Ser Gln Ala 1 5 10 15 Arg Arg Ala Arg Ile Leu Ala Cys Ser Arg Arg Leu Lys His Arg Gly 20 25 30 Pro Asp Trp Ser Gly Leu Tyr Gln His Glu Gly Asn Phe Leu Ala Gln 35 40 45 Gln Arg Leu Ala Ile Val Ser Pro Leu Ser Gly Asp Gln Pro Leu Phe 50 55 60 Asn Glu Asp Arg Thr Val Val Val Val Ala Asn Gly Glu Ile Tyr Asn 65 70 75 80 His Lys Asn Val Arg Lys Gln Phe Thr Gly Ala His Ser Phe Ser Thr 85 90 95 Gly Ser Asp Cys Glu Val Ile Ile Pro Leu Tyr Glu Lys Tyr Gly Glu 100 105 110 Asn Phe Val Asp Met Leu Asp Gly Val Phe Ala Phe Val Leu Tyr Asp 115 120 125 Thr Arg Asp Arg Thr Tyr Val Ala Ala Arg Asp Ala Ile Gly Val Asn 130 135 140 Pro Leu Tyr Ile Gly Trp Gly Ser Asp Gly Ser Val Trp Met Ser Ser 145 150 155 160 Glu Met Lys Ala Leu Asn Glu Asp Cys Val Arg Phe Glu Ile Phe Pro 165 170 175 Pro Gly His Leu Tyr Ser Ser Ala Ala Gly Gly Phe Arg Arg Trp Tyr 180 185 190 Thr Pro His Trp Phe Gln Glu Gln Val Pro Arg Thr Pro Tyr Gln Pro 195 200 205 Leu Val Leu Arg Glu Ala Phe Glu Lys Ala Val Ile Lys Arg Leu Met 210 215 220 Thr Asp Val Pro Phe Gly Val Leu Leu Ser Gly Gly Leu Asp Ser Ser 225 230 235 240 Leu Val Ala Ser Val Thr Lys Arg His Leu Val Lys Thr Asp Ala Ala 245 250 255 Glu Lys Phe Gly Thr Glu Leu His Ser Phe Val Val Gly Leu Glu Gly 260 265 270 Ser Pro Asp Leu Lys Ala Ala Arg Glu Val Ala Asp Tyr Leu Gly Thr 275 280 285 Thr His His Glu Phe His Phe Thr Val Gln Asp Gly Ile Asp Ala Ile 290 295 300 Glu Glu Val Ile Tyr His Asp Glu Thr Tyr Asp Val Thr Thr Ile Arg 305 310 315 320 Ala Ser Thr Pro Met Phe Leu Met Ala Arg Lys Ile Lys Ser Leu Gly 325 330 335 Val Lys Met Val Leu Ser Gly Glu Gly Ser Asp Glu Leu Leu Gly Gly 340 345 350 Tyr Leu Tyr Phe His Phe Ala Pro Asn Arg Glu Glu Leu His Arg Glu 355 360 365 Thr Cys Arg Lys Val Lys Ala Leu His Gln Tyr Asp Cys Leu Arg Ala 370 375 380 Asn Lys Ala Thr Ser Ala Trp Gly Leu Glu Val Arg Val Pro Phe Leu 385 390 395 400 Asp Lys Glu Phe Val Asp Val Ala Met Gly Met Asp Pro Glu Trp Lys 405 410 415 Met Tyr Asp Lys Asn Leu Gly Arg Ile Glu Lys Trp Val Leu Arg Lys 420 425 430 Ala Phe Asp Asp Glu Glu His Pro Tyr Leu Pro Glu His Ile Leu Tyr 435 440 445 Arg Gln Lys Glu Gln Phe Ser Asp Gly Val Gly Tyr Asn Trp Ile Asp 450 455 460 Gly Leu Lys Ser Phe Thr Glu Gln Gln Val Thr Asp Glu Met Met Asn 465 470 475 480 Asn Ala Ala Gln Met Phe Pro Tyr Asn Thr Pro Val Asn Lys Glu Ala 485 490 495 Tyr Tyr Tyr Arg Met Ile Phe Glu Arg Leu Phe Pro Gln Asp Ser Ala 500 505 510 Arg Glu Thr Val Pro Trp Gly Pro Ser Ile Ala Cys Ser Thr Pro Ala 515 520 525 Ala Ile Glu Trp Val Glu Gln Trp Lys Ala Ser Asn Asp Pro Ser Gly 530 535 540 Arg Phe Ile Ser Ser His Asp Ser Ala Ala Thr Asp Arg Thr Gly Asp 545 550 555 560 Lys Leu Ala Val Val Asn Gly Asp Gly His Gly Ala Ala Asn Gly Thr 565,570,575 Val Asn Gly Asn Asp Val Ala Val Ala Ile Ala Val 580 585 <210> 3 <211> 1767 <212> DNA <213> Zea mays ssp. Parviglumis, Ames21814 <400> 3 atgtgtggca tttagccgt gctcggatgc tccgactgct cccaggccag gagggctcgc 60 atcctgcct gctccagaag gctgaagcac aggggccccg actggtcggg cctctaccag 120 cacgagggca acttcctggc gcagcagcgg ctcgccatcg tctccccgct gtcggcgac 180 cagccgctgt tcaacgagga ccgcaccgtc gtggtggtgg ccaatggaga gatctacaac 240 cacaagaacg tccggaagca gttcaccggc gcgcacagct tcagcaccgg cagtgactgc 300 gaggtcatca tccccctgta cgagaagtac ggcgagaact tcgtggacat gctggacgga 360 gtcttcgcgt tcgtgctcta cgacacgcga gacaggacct acgtggcggc acgcgacgcc 420 atcggcgtca acccgctcta catcggctgg ggcagcgacg gttccgtctg gatgtcgtcc 480 gagatgaagg cgctgaacga ggactgcgtg cgcttcgaga tcttcccgcc ggggcacctc 540 tactccagcg ccgccggcgg gttccgccgg tggtacaccc cgcactggtt ccaggagcag 600 gtgccccgga cgccgtacca gccgctcgtc cttagagagg ccttcgagaa ggcggttatc 660 aagaggctca tgaccgacgt cccgttcggg gtcctcctct ccggcggcct cgactcctcc 720 ctcgtcgcct ccgtcaccaa gcgccacctc gtcaagaccg acgccgccga aaagttcggc 780 acagagctcc actcttcgt cgtcggcctc gagggctccc ctgacctgaa ggccgcacga 840 gaggtcgctg actacctcgg aaccacccat cacgagttcc atttcaccgt aggacggc 900 960 gccagcacgc ccatgttcct gatggctcgc aagatcaagt cgctgggcgt gaagatggtg 1020 ctgtccgggg agggctccga cgagctcctg ggcggctacc tctacttcca cttcgccccc 1080 aacagggagg agctccacag ggagacctgc cgcaaggtga aggccctgca ccagtacgac 1140 tgcctgcgcg ccaacaaggc gacgtcggcg tggggcctgg aggtccgcgt gccgttcctc 1200 gacaaggagt tcgtcgacgt cgcgatgggc atggaccccg agtggaaaat gtacgacaag 1260 aacctgggtc gcatcgaga gtgggtcctg aggaaggcgt tcgacgacga ggagcaccct tacctgcccg agcatattct gtacaggcag aaagaacagt tcagtgacgg agtgggctac aactggatcg atggactcaa atccttcacc gaacagcagg tgacggatga gatgatgaac aacgccgccc agatgttccc gtacaacacg cccgtcaaca aggaggccta ctactaccgg atgatattcg agaggctctt ccctcaggac tcggcgaggg agacggtgcc gtggggcccg 1560. agcatcgcct gcagcacgcc cgcggccatc gagtgggtgg agcagtggaa ggcctccaac 1620 gacccctccg gccgcttcat ctcctcccac gactccgccg ccaccgaccg caccggagac 1680 aagctggcgg tggtcaacgg cgacgggcac ggcgcggcga acggcacggt caacggcaac 1740 gacgtcgctg tcgcgatcgc ggtgtaa 1767 <210> 4 <211> 198 <212> DNA <213> Zea mays ssp. Parviglumis, Ames2 <400> 4 60. ctctgtgcca tgcatcctcc gcagcatatt ctgtacaggc agaaagaaca gttcagtgac ggagtgggct acaactggat cgatggactc aaatccttca ccgaacagca ggttgattta cggccccact ttcagctctg atcgcatctc ctagacatcg taccgtacgt cgtccaagtt 180 agctaaccag cgctgacg 198 <210> 5 <211> 176 <212> DNA <213> Artificial Sequence <400> 5 ctctgtgcca tgcatcctcc gcagcatatt ctgtacaggc agaaagaaca gttcagtgac 60 ggagtgggct acaactggat cgatggactc aaagccttca ccgaacagca ggttgattta 120 tggccacgca tctcctagac atcgtcgtcg tcgaagttag ctaaccagcg ctgacg 176 <210> 6 <211> 151 <212> DNA <213> Artificial Sequence <400> 6 ctctgtgcca tgcatcctcc gcagcatatt ctgtacaggc agaaagaaca gttcagtgac 60 ggagtgggct acaactggat cgatggactc aaagccttca ccgaacagca ggttgatggt 120 cgtcgtcgaa gttagctaac cagcgctgac g 151 <210> 7 <211> 455 <212> DNA <213> Zea mays ssp. Parviglumis, Ames21814 <400> 7 ccgttcctcg acaaggagtt cgtcgacgtc gcgatgggca tggaccccga gtggaaaatg 60 gtactgacgc gggccttttt cgacacggcc cggccctgcc gccgcacgtc ggggtctcgg 120 ttctacgtat gatgatgacg ccttcttctc ttctttgcgc agtacgacaa gaacctgggt 180 cgcatcgaga agtgggtcct gaggaaggcg ttcgacgacg aggagcaccc ttacctgccc 240 gaggtaagaa catcttcaga gaaggctggt cgtttacctc tgtgtctgtg tgatttcaag 300 cctgaactga cgcctctgtg ccatgcatcc tccgcagcat attctgtaca ggcagaaaga 360 acagttcagt gacggagtgg gctacaactg gatcgatgga ctcaaatcct tcaccgaaca 420 gcaggttgat ttacggcccc actttcagct ctgat 455

Claims

1. Application of a wild maize asparagine synthetase 4 gene in increasing maize grain protein content, plant total nitrogen content, or nitrogen efficiency, wherein the amino acid sequence of the wild maize asparagine synthetase 4 is SEQ ID NO:

2.

2. The use according to claim 1, characterized in that The CDS sequence of the wild corn asparagine synthetase 4 gene is SEQ ID NO:

3.

3. The use according to claim 1, characterized in that The nucleotide sequence of the wild corn asparagine synthetase 4 gene is SEQ ID NO:

1.

4. The use according to claim 1, wherein The application is selected from the following methods: introducing the wild corn asparagine synthetase 4 encoding gene into the common corn chromosome; causing corn to overexpress the wild corn asparagine synthetase 4 gene.

5. The use according to claim 4, characterized in that The method for introducing a wild corn asparagine synthetase 4 encoding gene into a corn chromosome comprises the following steps: (1) cloning the wild corn asparagine synthetase 4 encoding gene into a plant expression vector suitable for expression in Agrobacterium to obtain an expression vector for the gene; (2) After the vector is verified by sequencing, the expression vector of the gene is transformed into corn embryos using the Agrobacterium-mediated method to obtain transgenic corn that overexpresses the gene.

6. The use according to claim 5, characterized in that The expression vector is a pCAMBIA3300 vector driven by the maize Ubiquitin promoter.

7. A kit for implementing the use according to claim 2 or 3, comprising: the maize asparagine synthetase 4 gene as shown in SEQ ID NO: 1 or its CDS sequence SEQ ID NO: 3, and PCR primers required for cloning the gene or its CDS sequence into a plant expression vector; Or it comprises: the gene expression vector as claimed in claim 5 or 6, and a reagent for transferring the gene expression vector into Agrobacterium; Or it comprises: Agrobacterium transformed with the gene expression vector as claimed in claim 5 or 6, and a reagent for transforming plants with Agrobacterium.

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