CN116987721A - Key gene for controlling corn protein content and nitrogen high efficiency - Google Patents

Abstract

The invention discloses application of a wild corn asparagine synthetase 4 gene Thp9 in improving corn kernel protein content, total nitrogen content of plants and/or nitrogen high efficiency, which can improve the protein content of corn and the utilization rate of nitrogen fertilizer by improving the activity of asparagine synthetase 4 or enhancing the expression of asparagine synthetase 4 and has great economic significance for creating new germplasm resources of high-protein corn, agricultural production and protecting environment.

Description

Key genes controlling maize protein content and nitrogen efficiency

Technical Field

The 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

The quality of corn (Zea mays L.) directly affects the yield and quality of meat and milk, and is an important determinant of the level of animal husbandry development. At the same time, with the improvement of people's living standards, consumers are paying more and more attention to the quality of corn, and protein nutritional quality (total protein) is an important indicator of widespread concern. However, the protein content of corn in production is generally between 7% and 9%. As a feed, it must be supplemented with protein by adding soybean meal, etc., which greatly increases the cost of feed. Therefore, increasing the protein content of corn grains and reducing or even zeroing the addition of soybean meal in feed are important ways to promote the healthy development of my country's feed industry and animal husbandry. At the same time, as an increase in the total nitrogen content of silage corn straw, the increase in the content of free amino acids will also have great significance for animal husbandry. Therefore, cloning the genes that control the total protein content of corn, analyzing the mechanism of high protein formation, and creating new germplasm resources with high protein content are important strategies to ensure food security. It has been reported that a rice quantitative trait locus (QTL) qPC1 has been cloned in rice. The amino acid transporter OsAAP6 encoded is related to rice protein content, and high expression of OsAAP6 is associated with high grain protein content (Peng et al., 2014). In addition, by measuring the total protein and storage protein content of more than 400 rice germplasm resources, and through map-based cloning and functional studies, it was determined that qGPC-10 (OsGluA2) is responsible for encoding the gluten precursor in rice storage protein, which can significantly affect the protein content of rice and ultimately affect the nutritional quality of rice (Yang et al., 2019). Scientists measured 961 population materials in 2009 and 2010 through near-infrared analysis and found that there was a variation of 7.32%-15.20% in the protein content of corn grains (Karn et al., 2017), but the gene locus that controls its protein content has not yet been cloned.

The endosperm of corn kernels is the main storage organ for nutrients, of which starch and protein are the two main storage substances. The total protein content of common corn inbred lines is about 10%, and starch is about 70% (Flint-Garcia et al., 2009). Proteins are divided into zein, albumin, globulin and glutelin according to solubility (Wu and Messing, 2017). When corn is used as feed and food, the abundance and amino acid composition of different types of protein vary greatly, which determines the nutritional quality of corn. The main storage protein of corn is alcohol-soluble protein, i.e. zein, which accounts for more than 60% of the total protein. Zein is divided into four subfamilies according to amino acid homology: α (19 and 22-kD), β (15-kD), γ (50, 27 and 16-kD) and δ (18 and 10-kD). α-zein has the highest abundance, accounting for more than 50% of the total zein content (Esen, 1987; Thompson and Larkins, 1994). However, almost all zeins do not contain the essential amino acids lysine and tryptophan, which leads to an extreme lack of these two amino acids in the total protein of corn endosperm (Mertz et al., 1964). Opaque2 (O2) is an important transcription factor in corn endosperm. In the O2 mutant, the expression of alcohol-soluble protein zein decreased by more than 60%. However, due to the protein balance mechanism, the expression of non-alcohol-soluble protein was compensatory up-regulated, and the final total protein content only decreased slightly. The lysine content in non-alcohol-soluble protein is rich, so the lysine content in the O2 mutant is about twice that of ordinary corn; mice fed with O2 corn grow significantly faster than the control (using ordinary corn) (Mertz et al., 1965). However, O2 is a powdery endosperm, the kernels are easy to break, and are susceptible to mold and disease, the total protein content is low (about 8-9%), and the yield is low, so it cannot be directly industrialized and planted. The creation of new high-quality, high-protein corn will have significant implications for food production and security.

It has been reported that several genes affecting rice nitrogen efficiency have been cloned in the study of rice nitrogen efficiency. The natural variation of the nitrate transporter gene NRT1.1B is a key factor mediating the different nitrogen utilization efficiencies of indica and japonica rice. The indica rice NRT1.1B allele has the characteristic of nitrogen efficiency (Hu et al., 2015). In addition, using genome-wide association study (GWAS), it was found that the rice TCP transcription factor family member gene OsTCP19 is a key factor controlling rice's adaptation to different soil nitrogen high and low environments (Liu et al., 2021). At the same time, the study revealed that the GA signaling pathway synergistically regulates rice growth and nitrogen metabolism. The rice transcription factor GROWTH-REGULATING FACTOR 4 (GRF4) (Li et al., 2018) and the APETALA2 domain transcription factor NITROGEN-MEDIATED TILLERGROWTH RESPONSE 5 (NGR5) are key factors mediating nitrogen regulation of tiller formation (Wu et al., 2020). The excellent allelic variation of these genes achieves increased and stable yields under low-nitrogen growth conditions, providing an important resource for efficient nitrogen utilization in rice. However, how to improve the utilization rate of nitrogen fertilizer or improve the efficient perception, absorption, assimilation and transport of nitrogen in corn plants, and to explore the mining and molecular modules of corn nitrogen efficient genes are major scientific problems that need to be solved in agricultural production at this stage.

Summary of the invention

In order to improve the protein content of corn, materials with high protein content need to be used as donors. We measured and analyzed the protein content of more than 30 different wild corns and found that the protein content of wild corn was about 30%. Therefore, wild corn is an excellent gene donor resource for creating new high-protein corn germplasm. Introducing wild corn as a donor into cultivated corn is a method to increase the protein content of corn. In order to deeply analyze the major QTL gene loci that control protein content in wild corn, we have selected wild corn teosinte (Zea mays ssp.Parviglumis, Ames21814, hereinafter referred to as Ames21814 or Teosinte (Teo)) as the introduction donor for our population construction since 2012. The protein content of wild corn Ames21814 reaches 30%, and both α-zein and non-zein parts rich in nutrient lysine content are significantly increased, which is one of the natural donors for increasing the protein content of corn. After 10 years of hard work, analysis of a large number of genetic populations, creation of 10 consecutive generations of near-isogenic line populations, and determination of tens of thousands of protein contents, we cloned the first major QTL gene locus that controls the total protein content of corn, and through three-generation sequencing, parsed and assembled the high-quality wild corn Ames21814 genome sequence. The present invention mines the key gene Thp9 that controls the formation of high-protein corn in wild corn. This gene can not only significantly increase the protein content and biomass of corn, but also increase the nitrogen utilization 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 germplasm resource of high-protein corn. Accordingly, the present invention includes the technical scheme described below.

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

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 chromosome of common corn; causing corn to overexpress the wild corn asparagine synthetase 4 gene, such as Thp9; causing corn to overexpress the original asparagine synthetase 4 gene ZmASN4 of common corn; 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.

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

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

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

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

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

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

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

(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);

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

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

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

MCGILAVLGCSDCSQARRARILACSRRLKHRGPDWSGLYQHEGNFLAQQRLAIVSP LSGDQPLFNEDRTVVVVANGEIYNHKNVRKQFTGAHSFSTGSDCEVIIPLYEKYGENFVD MLDGVFAFVLYDTRDRTYVAARDAIGVNPLYIGWGSDGSVWMSSEMKALNEDCVRFEI FPPGHLYSSAAGGFRRWYTPHWFQEQVPRTPYQPLV LREAFEKAVIKRLMTDVPFGVLL SGGLDSSLVASVTKRHLVKTDAAEKFGTELHSFVVGLEGSPDLKAAREVADYLGTTHHEFHFTVQDGIDAIEEVIYHDETYDVTTIRASTPMFLMARKIKSLGVKMVLSGEGSDELLGG YLYFHFAPNREELHRETCRKVKALHQYDCLRANKATSAWGLEVRVPFLDKEFVDVAMG MDPEWKMYDKNLGRIEKWVLRKAFDDEEHPYLPEHILYRQKEQFSDGVGYNWIDGLK SFTEQQVTDEMMNNAAQMFPYNTPVNKEAYYYRMIFERLFPQDSARETVPWGPSIACS TPAAIEWVEQWKASNDPSGRFISSHDSAATDRTG DKLAVVNGDGHGAANGTVNGNDVA VAIAV (SEQ ID NO: 2).

The amino acid sequence of asparagine synthetase 4 expressed in the inbred corn line B73 is:

MCGILAVLGCSDCSQARRARILACSRRLKHRGPDWSGLYQHEGNFLAQQRLAIVSP LSGDQPLFNEDRTVVVVANGEIYNHKNVRKQFTGAHSFSTGSDCEVIIPLYEKYGENFVD MLDGVFAFVLYDTRDRTYVAARDAIGVNPLYIGWGSDGSVWMSSEMKALNEDCVRFEI FPPGHLYSSAAGGFRRWYTPHWFQEQVPRTPYQPLV LREAFEKAVIKRLMTDVPFGVLL SGGLDSSLVASVTKRHLVKTDAAGKFGTELHSFVVGLEGSPDLKAAREVADYLGTTHHEFHFTVQDGIDAIEEVIYHDETYDVTTIRASTPMFLMARKIKSLGVKMVLSGEGSDELLGG YLYFHFAPNREELHRETCRKVKALHQYDCLRANKATSAWGLEVRVPFLDKEFVDVAMG MDPEWKMYDKNLGRIEKWVLRKAFDDEEHPYLPEHILYRQKEQFSDGVGYNWIDGLK AFTEQQVDGRRRS.

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

(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 of the gene;

(2) After the vector is verified by sequencing, the expression vector of the gene is transformed into maize embryos using the Agrobacterium-mediated method to obtain transgenic maize overexpressing the gene.

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 culture and grow the corn to obtain positive plants through genome level and transcription level identification.

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.

The second aspect of the present invention is to provide a kit for implementing the above application, which comprises: 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;

Or it may include: the above-mentioned gene expression vector, and reagents for transferring the gene expression vector into Agrobacterium;

Or it may include: Agrobacterium into which the above gene expression vector has been transferred, and reagents for transforming plants with Agrobacterium.

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

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

CTCTGTGCCATGCATCCTCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCA GTGACGGAGTGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGG TTGATTTACGGCCCCACTTTCAGCTCTGATCGCATCTCCTAGACATCGTACCGTACGTC GTCCAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 4);

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

CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGT TCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAAC AGCAGGTTGATTTATGGCCACGCATCTCCTAGACATCGTCGTCGTCGAAGTTAGC TAACCAGCGCTGACG (SEQ ID NO: 5);

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

CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGT TCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAAC AGCAGGTTGATGGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 6);

When detecting whether the Thp9 gene with the nucleotide sequence of SEQ ID NO: 1 is inserted into the corn 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 corn Ames21814 genotype insertion:

CCGTTCCTCGACAAGGAGTTCGTCGACGTCGCGATGGGCATGGACCCCGAGT GGAAAATGGTACTGACGCGGGCCTTTTTCGACACGGCCCGGCCCTGCCGCCGCA CGTCGGGGTCTCGGTTCTACGTATGATGATGACGCCTTCTTCTTCTTTGCGCAG TACGACAAGAACCTGGGTCGCATCGAGAAGTGGGTCCTGAGGAAGGCGTTCGACGACGAGGAGCACCCTTACCTGCCCGAGGTAAGAACA TCTTCAGAGAAGGCTGGT CGTTTACCTCTGTGTCTGTGTGATTTCAAGCCTGAACTGACGCCTCTGTGCCATGC ATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGG GCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGAT (SEQ ID NO: 7).

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 ordinary PCR MIX and programs. If a band can be amplified, it means that the wild corn Ames21814 genotype is inserted, and if it cannot be amplified, it does not carry the high protein site.

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.

The present invention applies the wild corn asparagine synthetase 4 gene Thp9 to cultivated corn, which can not only increase the protein content of corn, but also promote the nitrogen efficiency of corn and thus improve 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

Figure 1 shows the phenomenon of strong selection of protein content during the domestication of wild corn to cultivated corn. Among them, a, schematic diagram of the selection of wild corn with tillers to cultivated corn inbred lines after about 9,000 years of domestication; b, the analysis and determination of the grain protein content of more than 30 types of wild corn Parviglumis and Mexiana was about 28.6% ± 1.0%, while the protein content of 405 cultivated corn inbred lines was 6.5%-16%, with an average of 11.52%; c, representative wild corn (registration number Ames and PI series) was selected for alcohol-soluble and non-alcohol-soluble protein analysis, and it was found that the content of alcohol-soluble protein and non-alcohol-soluble protein in wild corn was significantly higher than that of the control B73 inbred line; d, the content of free amino acid asparagine in the roots, stems and leaves of wild corn Ames21814 was significantly higher than that of the control B73.

Figure 2 shows the results of the analysis of alcohol-soluble proteins in 500 inbred lines of natural populations and the GWAS analysis of α-zein population variation. Among them, a, the analysis of alcohol-soluble proteins in 500 inbred lines of natural populations; b, the α-zein content of the 500 inbred lines with the largest variation in corn alcohol-soluble protein content was graded, and the difference in 19- and 22-kD α-zein content was divided into three levels (19-kD content is higher, equal to, and lower than 22-kD α-zein) and then subjected to genome-wide association analysis GWAS. The GWAS results showed that the main effect locus of the difference in 19- and 22-kD α-zein content was on the short arm of chromosome 4.

Figure 3 shows the results of the third generation sequencing assembly of the wild corn genome and the analysis of the alcohol-soluble protein copy number. Among them, a, wild corn Z. mays ssp. Parviglumis Ames21814, F ₁ of B73 x Ames21814 and plants of common cultivated corn B73; b, wild corn genome assembly flow chart; c, wild corn high-quality genome, from the outer circle to the inner circle, respectively shows gene density, repeat sequence density, TIR density, Indel number, SNP number, Copia density, Gypsy density, Knob density and GC content; d, alcohol-soluble protein tandem repeat copy number analysis, respectively counting the copy numbers of different gene clusters of different tandem gene repeat sequences of α-zein inbred line B73, wild corn Teosinte and inbred line W22.

Figure 4 shows the analysis of the genetic basis of high protein and the results of protein detection during population construction. Among them, a, seeds of inbred line B73, wild corn Teo, and B73 x Teo and B73 x Teo F ₂ , the seed protein determination data is marked above, n is the number of determinations; b, SDS-PAGE gel analysis of alcohol-soluble proteins of inbred line B73, wild corn Teo, and B73 x Teo; c, alcohol-soluble protein analysis of seeds of B73 x Teo F ₂ , F ₂ are all high protein, B73 is the control; d, the protein analysis of different ears in F ₁ BC ₂ population is the analysis of alcohol-soluble proteins of 12 separate ears, B73 is the control; e, 12 grains from the same high-protein ear in F ₁ BC ₂ population were taken for separate protein content analysis, B73 is the control; F, 30 ears of protein in F ₁ BC ₃ population were determined, and the total protein content of the grains was separated into 10% and 15%; g, F ₁ BC 8 high-protein ears in ₃ populations, 7 grains were taken from each for protein determination, and the protein content of each grain in the high-protein ears was ~15%, and B73 was used as a control; h, 30 ears _{in F1BC4} population were protein determined, and the total protein content of the grains was separated into 10% and ~15%; i, 8 high-protein ears _{in F1BC4} population, 7 grains were taken from each for protein determination, and the protein content of each grain in the high-protein ears was ~15%, and B73 was used as a control.

Figure 5 shows the results of the localization and expression analysis of the Thp9 gene. Among them, a, BSA localization G'value analysis of the F ₁ BC ₄ population; b, analysis of the introgressed genes in three BSA sequencings of F ₁ BC ₄ , F ₁ BC ₆ , and F ₁ BC ₈ ; c, Thp9 map cloning, Thp9 is located in the 147kb interval between markers 143.7 and 143.8, and only one gene expression level has changed significantly, 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 root and leaf RNA-Seq sequencing in B73 and wild maize; f, Transcriptome analysis of roots and leaves of the near-isogenic line NILTHP9 and the control NILB73, ZmAsn4 was significantly overexpressed in the roots and leaves of NILTHP9; g, ZmASN4 protein analysis of roots and leaves of the near-isogenic line NILTHP9 and the control NILB73.

Figure 6 shows the results of linkage marker development and phenotypic analysis of the high-protein variation site of Thp9 wild corn. Among them, a, ears of F ₂ BC ₇ population, Asn4-B73 represents Thp9 with B73 genotype, Asn4-H represents Thp9 with heterozygous genotype, Asn4-Teo represents Thp9 with homozygous wild corn genotype; b, determination of grain protein content of different Thp9 genotypes in ears of F ₂ BC ₇ population, Asn4-B73 represents Thp9 with B73 genotype, Asn4-H represents Thp9 with heterozygous genotype, Asn4-Teo represents Thp9 with homozygous wild corn genotype; c, determination of free amino acid asparagine content in roots of different Thp9 genotypes in ears of F ₂ BC ₇ population, Asn4-B73 represents Thp9 with B73 genotype, Asn4-H represents Thp9 with heterozygous genotype, Asn4-Teo represents Thp9 with homozygous wild corn genotype.

Figure 7 shows the results of phenotypic analysis of the near-isogenic line NILTHP9. Among them, a, the protein content of the grains of the near-isogenic line NILTHP9 and the control NILB73 in different ecological zones, Shanghai, Sanya and Northeast China; b, the total nitrogen content in the roots, stems and leaves of the near-isogenic line NILTHP9 and the control NILB73; c, the determination of the free amino acid asparagine content in the roots of the near-isogenic line NILTHP9 and the control NILB73; d, the plants of the near-isogenic line NILTHP9 and the control NILB73; e, the plant height determination of the near-isogenic line NILTHP9 and the control NILB73, the plants were planted in Sanya in 2021; f, the fresh weight of the leaves, stems and whole plants of the near-isogenic line NILTHP9 and the control NILB73.

Figure 8 shows the genetic verification results of Thp9. Among them, a, relative expression level of Thp9 in the roots of two independent transgenic events OE-1 (Overexpression-1) and OE-2 (Overexpression-2) overexpressing Thp9; b, relative expression level of Thp9 in the leaves of two independent transgenic events OE-1 and OE-2 overexpressing; c, immunoblotting of THP9 in the roots of two independent transgenic events OE-1 and OE-2 overexpressing; d, determination of grain protein content of two independent transgenic events OE-1 and OE-2 overexpressing; 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, schematic diagram of the genetic structure of three haplotypes of ASN4 in natural populations, among which HAP1 is the wild corn Thp9 haplotype, and HAP3 is the B73 haplotype ratio. HAP1 lacks 47bp, and HAP2 lacks 22bp compared to HAP1; g, Analysis of protein contents of three haplotypes of ASN4 in natural populations.

Figure 9 shows the results of the nitrogen high-efficiency test of the near-isogenic line NILTHP9 at the Shanghai Experimental Base in 2020. Among them, a, plants of NILB73 and NILTHP9 under normal nitrogen application and without nitrogen application; b, roots of NILB73 and NILTHP9 plants under normal nitrogen application and without nitrogen application; c, the expression of the ASN4 gene is induced by nitrogen application, and the expression level of ASN4 in the near-isogenic line NILTHP9 under no nitrogen application reaches the expression of ASN4 in NILB73 under normal nitrogen application; d, biomass determination of the aboveground plants of NILB73 and NILTHP9 under normal nitrogen application and without nitrogen application; e, biomass determination of the underground roots of NILB73 and NILTHP9 under normal nitrogen application and without nitrogen application; f, total biomass determination of NILB73 and NILTHP9 under normal nitrogen application and without nitrogen application; g, determination of grain protein content.

Figure 10 shows the results of the nitrogen high-efficiency test of the near-isogenic line NILTHP9 at the Sanya Experimental Base in 2020. Among them, a, 0%, 25%, 50% and 100% (100% level is, once applied at the seedling stage and once at the jointing stage, a total of two fertilizations, each time 0-4-8-16g/plant, nitrogen content 17%, other levels decrease successively, planting density 0.6m x 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 of roots of NILB73 and NILTHP9 under 4 different nitrogen application levels; e, determination of total nitrogen content of leaves of NILB73 and NILTHP9 under 4 different nitrogen application levels; f, determination of total nitrogen content of NILB73 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.

FIG. 11 shows the test results of Thp9 hybrid test and the test results of improving new varieties to create high protein corn. Among them, a, phenotype of hybrid _F2 ears created by NILB73 and NILTHP9 with Mo17; b, comparison of 100-grain weight and protein content determination of hybrids carrying Thp9; c, Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 plants; d, Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 hybrid ears; e, determination of aboveground fresh weight of high-protein Zhengdan 958THP9 created by Thp9 and control Zhengdan 958; f, determination of plant height of high-protein Zhengdan 958THP9 created by Thp9 improved Zhengdan 958 and control Zhengdan 958; e, determination of aboveground fresh weight of high-protein Zhengdan 958THP9 created by Thp9 improved Zhengdan 958 and control Zhengdan 958; g, Thp9 improved Zhengdan 958 to create high-protein Zhengdan 958THP9 h, determination of total nitrogen in roots of Zhengdan 958 THP9 improved by Thp9 and the control Zhengdan 958; i, determination of total nitrogen in stems of Zhengdan 958 THP9 improved by Thp9 and the control Zhengdan 958; e, determination of total nitrogen in leaves of Zhengdan 958 THP9 improved by Thp9 and the control Zhengdan 958.

DETAILED DESCRIPTION

High-protein corn is an important germplasm resource for modern hybrid corn breeding, and it is also an important agronomic trait. Since high protein in corn is controlled by micro-effect polygenes and has a complex genetic mechanism, it is difficult to clone the quantitative trait locus QTL that controls its formation in natural populations, and no reports have been made so far. At the same time, the formation mechanism of high-protein corn is also unclear. Therefore, the genetic improvement of high-protein corn and the expansion of its genetic germplasm are very slow and difficult. It is not only time-consuming and labor-intensive, but also inefficient and has a very slow progress and unclear direction. Each generation also needs to measure the protein content, which is a huge and tedious task. After years of analysis of the genetic laws of high-protein corn, genome sequencing and assembly, difficult gene cloning, and rigorous genetic verification and field trials, after 10 years of persistence and hard work, we finally cloned the key QTL-Thp9 that controls the formation of high-protein corn from wild corn for the first time, and applied it to the creation of corn hybrids. The positive experimental results indicate that the development and utilization of this key gene will greatly promote the genetic improvement and germplasm resource innovation of high-protein corn, and has very broad application prospects and economic value.

The inventors cloned the high-protein gene Thp9 from wild corn (Ames21814) for the first time. The allele of this gene in the B73 genome is ZmASN4, and the gene number is Zm00001d047736. Studies have shown that wild corn Thp9 can significantly increase the protein content of corn kernels and plants, increase the biomass of corn, and improve the nitrogen fertilizer utilization efficiency of corn.

The asparagine synthetase 4 gene ASN4 studied in this article includes both the asparagine synthetase 4 gene 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.

In this article, for the sake of simplicity, a certain protein such as asparagine synthetase 4 is sometimes used interchangeably with its encoding gene ASN4 (or Asn4). Those skilled in the art should understand that they represent different substances in different description occasions. Those skilled in the art can easily understand their meanings based on the context. For example, for ASN4, when used to describe the function or category of asparagine synthetase, it refers to the protein; when described as a gene, it refers to the gene encoding the enzyme.

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, so as to facilitate the use of the high-protein site in the genetic improvement of common corn.

On the other hand, in order to detect whether the Thp9 gene is inserted into the corn 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 corn populations.

These molecular markers are very convenient to use and can be performed by ordinary technicians in this field through simple molecular biology experiments. For example, extracting corn leaf genomic DNA, using the above mutation site specific primers developed by us for PCR reaction, and then sequencing with the provided primers, the above mutation site can be detected.

The ASN4 gene application and detection object 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.

Considering the convenience of operation, as a preferred mode, the application mode and target gene detection mode of the present invention can be carried out using a kit, and the required materials are concentrated in a kit. In a preferred embodiment, in addition to the ASN4 gene fragment, ASN4 gene amplification PCR primer, plant binary expression vector, restriction endonuclease, 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 a kit, a medicine bottle, a test tube, and the like, each of which contains a separate component for the method of the present invention; instructions, which can be written on bottles, test tubes and the like, or on a separate piece of paper, or on the outside or inside of a container, such as a paper with an operation demonstration video APP download window such as a QR code, and the instructions can also be in the form of multimedia, such as a CD, a U disk, a network disk, an IC card, etc.

It is easy for those skilled in the art to understand that there are many ways to promote the overexpression of ASN4 gene (including Zm00001d047736 and Thp9) in corn, for example, through natural variation and artificial mutagenesis (all mutagen mutagenesis and genetic engineering methods) leading to mutation of the promoter region of wild corn or common corn ASN4 gene, 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.

On the basis of the ASN4 gene (including Zm00001d047736 and Thp9) in the prior art, further improving the activity of 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.

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

1. Thp9 has very strong functions and good genetic stability. Crossing wild corn Thp9 into different corn inbred lines can increase the protein content of its grains. In theory, it is effective for most inbred lines, which will greatly expand the high-protein corn germplasm resources. Moreover, Thp9 has very good genetic stability, and its phenotype is very stable in Northeast China, Shanghai and Sanya.

2. The time for improving high-protein corn with Thp9 is short. Since the genes and mechanisms that control the formation of high-protein corn are unclear, conventional genetic improvement of high-protein corn requires large-scale field surveys at multiple locations over many years to obtain stable materials. However, when improving high-protein corn now, it is only necessary to crossbreed with other inbred lines and identify plants carrying excellent Thp9 loci through the molecular markers SEQ ID NOs:4-5, 7 developed by us; the ears grown from them, whether self-pollinated or hybridized with other corn pollen, all produce seeds of high-protein corn. Moreover, during the introduction process, at least one ear needs to be retained for crossbreeding and self-pollination to obtain stable hard grain materials, which can greatly save workload.

3. The operation of using Thp9 to improve high-protein corn is simple and suitable for large-scale operations. The hybridization and self-pollination techniques of 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 the excellent variant Thp9 genotype. The corn leaf DNA extraction, PCR reaction and sequencing involved are all conventional molecular experiments that can be completed in ordinary laboratories and sequencing companies.

4. The cost of using Thp9 to improve high-protein corn is low. Compared with traditional corn genetic improvement, the use of Thp9 to improve high-protein corn is more effective, more stable, and reduces time and workload, thereby greatly saving costs. In addition, the molecular experiments for Thp9 genotype identification are also very routine and low-cost. The traditional identification of grain protein content requires the use of a nitrogen analyzer to measure the protein content of each ear. The cost of the nitrogen analyzer is expensive and the throughput is low. The purchase of a Rapid N nitrogen determination instrument also costs more than 500,000 yuan. The maintenance during the period is complicated and the consumables are also expensive.

5. Thp9 can be used to create high-protein hybrid corn. We introduced the Thp9 genotype into the parents of the main corn varieties, and quickly obtained high-protein corn hybrids while maintaining the original hybrid vigor. The promotion can create huge economic and social benefits. We have introduced Thp9 into the two parents of Zhengdan 958, Zheng 58 and Chang 7-2, and created high-protein corn hybrids.

6. Thp9 is a natural major locus for increasing protein content. Introducing the wild corn Thp9 locus into corn inbred lines and hybrid parents can significantly increase the protein content and biomass of inbred lines and hybrids. Our research found that the nitrogen content in the grains, stems and roots of the near-isogenic line NILThp9 containing the high-protein locus Thp9 was significantly higher than that in the near-isogenic NILB73 without the high-protein locus. At the same time, overexpression of Thp9 in B73 can significantly increase the total nitrogen content in grains, stems and roots.

7. Thp9 is highly nitrogen efficient and 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.

The present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only for illustrative purposes 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 belong to the scope defined by the claims attached to this patent.

Example

The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

Materials and methods

Maize self-pollination, hybridization, genetic modification, and field breeding are carried out according to conventional breeding methods.

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

The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

PCR amplification experiments were performed according to the reaction conditions provided by the reagent supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

Example 1: Measuring corn protein content and amino acid content to explore high-protein wild corn donor materials

1.1 Analysis of corn kernel protein content by nitrogen analyzer

The corn kernels whose total protein content needs to be determined are placed in an oven at 60°C for drying, then ground into powder using a crushing instrument, 50-70 mg of dry corn powder is weighed to prepare a test sample, and the total protein is determined using a Dumas rapid nitrogen analyzer (rapid N exceed) from German elementar.

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

(1) Dry the endosperm in a 37°C oven, grind it into powder at 60Hz for 60s, weigh 100mg of the ground and dried powder into a 2mL tube, add 1mL of alcohol-soluble protein extract, mix thoroughly, and leave at room temperature for more than 2h or overnight. (2) Put steel balls into the 2mL tube, shake the grinder for 1min, leave it on the table for 20min, and centrifuge at 15871g for 15min. (3) Take 100mL of the supernatant into a new 1.5mL tube, add 10μL of 10% (g/mL) SDS, and vacuum at 45℃ (select rotation and ethanol solution) for 70min. Add 100μL ddH ₂ O, and leave it in a refrigerator at 4℃ overnight. The extraction of alcohol-soluble protein Zein is complete. (4) Pour out the remaining liquid in (2), add 1mL of alcohol-soluble protein extract, shake, leave it for more than 2h, centrifuge at 15871g for 15min, and remove the supernatant. Repeat this process 3 times, and extract alcohol-soluble protein 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, vortex, place for 2 hours, centrifuge at 15871g for 15 minutes, take 100mL of the upper liquid into 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 180V, 70min, the sample is ready when the blue parallel lines just run out. The cells were stained with Coomassie Brilliant Blue for 2 h, and then destained with destaining solution for 3 times, changing the destaining solution every 45 min.

Prolamin Extraction Solution

Non-alcohol-soluble protein extraction solution

1.3 Determination of free amino acid content in different tissues of corn

(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: Take the standard mixed standard (the concentration of the mixed standard is 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 index name Asn, x analytical value, r correlation coefficient; the concentrations of the mixed standard S1-S5 are 1.25μmol/L, 6.25μmol/L, 12.5μmol/L, 50μmol/L and 100μmol/L respectively), 50μl of the sample to be tested, add 50μl of protein precipitant (including NVL), mix well, and centrifuge at 13200 rpm for 4 minutes. Take 10μl of the supernatant, add 50μl of labeling buffer, mix well, and centrifuge instantly. Add 20 μl of derivatization solution, mix well, centrifuge, and then place at 55°C for 15 minutes. After derivatization, cool the sample in a refrigerator, mix well, centrifuge, and take 50 μl for detection. Instrument model: HPLC-MS/MS; LC liquid phase: Dionex Ultimate3000; MS mass spectrometer: American AB company: API 3200QTRAP; Amino acid kit: Beijing Mass Spectrometry Medical Research Co., Ltd. MSLAB50AA, batch number: MSLAB50AA170601#; methanol, acetonitrile, etc. were purchased from Fisher.

Since 2012, we have started to search, analyze and construct a population of high-protein donor materials for corn. In order to find high-protein donor materials, we measured the protein content of more than 30 wild corn seeds and common cultivated corn inbred lines, and found that the protein content of wild corn was 28.6% ± 1.0%, while the protein content of common corn inbred lines was 10% (B73 as a representative) (a and b in Figure 1). We further analyzed the alcohol-soluble proteins and non-alcohol-soluble proteins of wild corn and inbred line B73 through SDS-PAGE gel, and found that the alcohol-soluble proteins and non-alcohol-soluble proteins of wild corn were significantly increased compared with B73 (c in Figure 1). 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 component decreased significantly (root: 16611μg/g of wild corn decreased to 4120μg/g of cultivated corn; stem: 13668μg/g of wild corn decreased to 2529μg/g of cultivated corn; leaf: 14689μg/g of wild corn decreased to 2946μg/g of cultivated corn) (Figure 1d). These evidences show that wild corn is an excellent high-protein donor material that exists naturally.

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

2.1 Analysis of alcohol-soluble protein content in 500 maize inbred lines by SDS-PAGE gel electrophoresis

We planted 500 inbred lines in Harbin in 2014. After harvesting and drying, we took the kernels from the middle of three ears of each inbred line, removed the embryos, mixed and ground the samples. The method for alcohol-soluble protein extraction and SDS-PAGE gel analysis was the same as the alcohol-soluble protein extraction in step 1.2 above.

2.2 GWAS association analysis

500 maize inbred line seeds and corresponding genotype data were provided by Professor Lai Jinsheng's laboratory at China Agricultural University. The alcohol-soluble protein content of 500 maize inbred lines was analyzed by SDS-PAGE gel electrophoresis. The α-zein content with the largest change in zein content was divided into high and low levels. The difference in 19- and 22-kD α-zein content was divided into three levels (19-kD content is higher, equal to, and lower than 22-kD α-zein), and then a genome-wide association analysis (GWAS) was performed. The method of genome-wide association analysis was based on the method published in this laboratory (Liu et al., PNAs, 2015).

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 through SAD-PAGE gel (a in Figure 2), and found that there were great differences between different inbred lines. We also graded the α-zein content, which has the largest change in corn alcohol-soluble protein content, and conducted a genome-wide association analysis GWAS. The main effect site of the difference in α-zein content is on the short arm of chromosome 4, that is, the region where 19- and 22-kD α-zein gene copies are clustered, suggesting that the change in alcohol-soluble protein content in inbred lines of natural populations is related to the number of alcohol-soluble protein copies (b in Figure 2). So is the high protein content of wild corn determined by the number of copies of alcohol-soluble protein tandem repeats?

Example 3: High-quality genome sequencing and assembly of wild corn Ames21814

In order 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 corn Ames21814 through 3rd generation sequencing (ac in Figure 3). Our sequencing material is the F ₁ after hybridization between wild corn (Ames21814) and B73. After sequencing using the latest HiFi mode, we obtained 6752166 sequences, a total of about 104Gb of high-quality CCS sequences, and the read length N50 reached 15.4Mb. According to the B73 genome size of 2.2Gb, the data volume is about 47X. Our assembly is based on the Trio-binning method. After extracting the wild corn CCS sequence by borrowing the second-generation parent sequence, the Hifisam and Yak programs were used to assemble it into contig level. At the same time, 375.56Gb of Hi-C data were used, and the PacBio assembled genome was aligned by juicer and bwa mem default parameters, R1 and R2, respectively. The chromosomes were clustered, sorted and two rounds of error correction were performed according to the interaction information provided by the HiC data. Finally, the HiC interaction matrix was imported into juicebox for visualization and manual inspection. After confirming that there were no abnormalities, it was exported. 500 Ns were added between each contig. The final chromosome mounting rate was 91.30%, the genome size was 2460Mb, the contig N50 was 62.29Mb, and the scaffoldN50 reached the gold level of 243.71Mb wild corn genome. BUSCO evaluated that the completeness of the wild corn genome was 96.8%. The wild corn genome contains 80.80% of repetitive sequences, of which LTR transposons are the most important transposable elements, accounting for about 61.48% of the genome. The wild corn genome was annotated with 58,092 genes, encoding 108,712 transcripts. The assembled sequence of the wild corn genome 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 corn through sequence alignment, and found that the copy number of all alcohol-soluble proteins in wild Ames21814 did not change significantly compared with B73, indicating that the high protein content of wild corn is not caused by the copy number of alcohol-soluble proteins (Figure 3d).

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

How is the trait of controlling high protein in wild corn inherited? In order 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 factors controlling the formation of high protein are determined by the genotype of the mother plant (ac in Figure 4). We then continued to plant the harvested F ₁ BC ₁ , and then backcrossed with B73 as the male parent. We performed protein analysis on the harvested F ₁ BC ₂ (Figure 4 d and e), F ₁ BC ₃ (Figure 4 f and g) and F ₁ BC ₄ (Figure 4 h and i) populations, and found that the protein content was separated by ear, and the protein content between different ears in the backcross population varied by 10% to 15%, while the protein content of different grains on the same ear was the same (Figure 4 di). The above genetic population construction and protein analysis further proved that the factors controlling protein content are determined by the genotype of the mother plant, and that the introduction of wild corn into cultivated corn inbred lines can increase protein content, becoming an important strategy for creating high-protein corn. Based on this genetic basis, we determined the genetic population of each generation of backcrossing, selected high-protein ears for continued planting, and continuously backcrossed with B73 to create high-generation near-isogenic lines for subsequent high-protein gene positioning.

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

5.1 Leaf DNA extraction

(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, and mix well every 10-15 min. (4) 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 room temperature at 15871 g for 15 min, and 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 room temperature at 15871 g for 1 min, and 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 H ₂ O to dissolve the DNA pellet.

CTAB extraction buffer

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

5.2 BSA sequencing and analysis

The protein content of F ₁ BC ₄ , F ₁ BC ₆ and F ₁ BC ₈ populations was determined using a nitrogen analyzer (the method is the same as step 1.1 above), and the same number of individuals of two extreme phenotypes in each population were selected for DNA extraction, and equal amounts were mixed for BSA sequencing. In the F ₁ BC ₄ population, 100 samples of high-protein type (content of about 15%) (mixed pool 1) and 100 samples of B73 type (content of about 10%) (mixed pool 2) selected from the segregated population were subjected to high-throughput sequencing (X-Ten 100X, total data volume 500G), and the recurrent parent B73 was resequenced (30X, data volume 75G). The filtered high-quality sequencing data was aligned to the B73 reference genome, and SNP detection was performed using GATK software. The SNP index difference between the two pools and between the parents was calculated to analyze the segments closely linked to the high-protein trait (this method can be referred to the inventor's previously published paper Huang et al., Plant Cell, 2019). The _{F 1} BC ₆ population performed high-throughput sequencing (X-Ten 100X, total data volume 500G) on 150 samples of high-protein type (content about 15%) selected from the segregated population (pool 1) and 150 samples of B73 type (content about 10%) (pool 2), and the analysis method was the same as above. The F ₁ BC ₈ population performed high-throughput sequencing (X-Ten 50X, total data volume 250G) on 50 samples of high-protein type (content about 15%) selected from the segregated population (pool 1) and 50 samples of B73 type (content about 10%) (pool 2). The analysis method is the same as above.

5.3 Plotting and data analysis of F ₁ BC ₄ , F ₁ BC ₆ and F ₁ BC ₈

(1) Alignment with the reference genome: Align the B73 sample, the high-protein HP sample of _{F1BC4 ,} and the _low -protein LP sample data (the same for _F1BC6 and _{F1BC8 )} to the genome after the merger of B73 and Teo; (2) Count the coverage depth of the Teo gene: Count the coverage depth of each window area of the bam file after each sample is aligned, with a window size of 50kb and a step size of 25kb; (3) Data normalization: In order to make the coverage depth of different samples comparable, we normalized the coverage depth of each sample (formula: coverage depth of each sample/50kb-coverage depth of B73 sample/50kb); (4) Obtain drawing data: Subtract the coverage depth of the low-protein LP sample (the same for BC4, BC6, and BC8) from the standardized coverage depth of the high-protein HP sample to obtain the difference (Delta) for drawing. (5) Drawing: Use the ggplot2 package of R language for drawing.

5.4 Molecular marker PCR detection method

Based on the wild corn genome and the B73 reference genome, genome-wide polymorphic markers were designed and developed, and then the developed molecular markers were selected in wild corn, B73 and F ₁ in the high-protein population for verification. The available molecular markers of chromosome 9 140Mb-152Mb were screened, and 2000 F ₁ BC ₉ populations were genotyped by PCR and agarose gel running with the above-screened molecular markers. At the same time, the protein content of the 2000 populations was determined, and the genotype genetic exchange information and the grain protein content corresponding to the exchanged individual plants were analyzed. PCR was amplified using 2×HieffTM PCR Master Mix (Shanghai Yisheng Biotechnology Co., Ltd., 10102ES03) and standard procedures, and 3% agarose gel was used for identification.

Primer information for positional cloning molecular markers:

5.5 RNA extraction and RNA-seq analysis

(1) Take tissue samples from the experimental group and the control group 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 fully grind (60Hz, 70s) under liquid nitrogen freezing conditions and then store them in liquid nitrogen. (3) Add 1mL of Trizol extraction solution to the fully ground tissue powder, shake it thoroughly, and let it stand for 5 minutes; then add 200μL of chloroform to the mixed solution, 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 min, centrifuge at 13523 g for 5 min at 4°C, 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 min, and add 100 μL of ddH ₂ O to dissolve the precipitate. (7) Use Qiagen's RNeasy Plus Mini Kit (Qiagen, catalog number: 74,106) to purify the above-mentioned RNA through a 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 H ₂ O. (8) Use Pomega's reverse transcription kit (ImProm-II ^TM Reverse Transcription System) to reverse transcribe RNA according to the standard method in the kit.

5.6 Quantitative PCR detection

Specific primers were designed by NCBI and specific analysis was performed. Primer Asn4-rt-1F/R was used for RT-qPCR, and Actin was used as the internal reference gene. Quantitative analysis was performed according to the standard method of Takara's SYBR Green kit. Three technical replicates were performed for each sample, and the cDNA samples after reverse transcription were uniformly diluted 8 times and quantitatively analyzed using a 20μL reaction system. SYBR Green Mix was used, according to a 20μL reaction system, with 10μL of mix, 1μL of each primer, 2μL of diluted cDNA, and 6μL of ddH ₂ O. Gene expression was detected by two-step PCR amplification using the BIO-RAD fluorescence quantitative analyzer CFX. The reaction conditions were: pre-denaturation 95℃, 30s; amplification: 95℃, 5s; 60℃, 35s; 40 cycles; termination: 95℃, 15s; 60℃, 60s; 95℃, 15s. The quantitative data were analyzed using EXCELL 2010 and the △△CT method. The quantitative primers for ZmAsn4 are as follows.

5.7 Western Blotting

(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, take the supernatant into a new 1.5 mL centrifuge tube, and store in a 4°C refrigerator for later use.

(2) Western immunoblotting: Pipette 15 μL of the 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 min for denaturation using a PCR instrument. Prepare 12% separation gel and 4% PGAE gel for stacking. Use 1x SDS-PAGE Buffer for electrophoresis at 120V for 60 min. Cut a PVDF membrane of the same size as the gel and activate it with methanol for 5 s. After electrophoresis, use a BIO-RAD semi-dry transfer instrument for transfer. Place the transferred membrane in a clean hybridization box and block it overnight at 4°C using 5% imported skimmed milk powder dissolved in 1x TBST. 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 h. Wash the membrane with 1x TBST, changing TBST every 15 min, and repeat the washing 4 times. Dilute the secondary antibody diluent with 1x TBST at a ratio of 1:5000 and incubate at room temperature for 1 hour. Wash the membrane with 1x TBST, changing TBST every 15 minutes, and repeat the washing 4 times. Add chemiluminescent solution for color development and imaging.

5.8 Gene cloning

In order to identify the genetic factors controlling the main effect of high-protein corn, we performed three BSA pool sequencing on F ₁ BC ₄ (n=500), F ₁ BC ₆ (n=1650) and F ₁ BC ₈ (n=2000), and through deep resequencing of 5 high-protein and 5 low-protein stable F ₃ BC ₆ materials, we found that the factor controlling high protein was located in the chromosome 9 interval (a and b in Figure 5). The gene was further located by map-based cloning. By measuring the protein content of 2000 _{F1 BC9} populations and developing and screening gene polymorphism markers, we narrowed down the Thp9 gene to between markers 143.7 and 143.8. According to the wild maize haplotype sequence, this 147 kb interval only contained one gene, teo09G002926, whose expression level changed significantly (Figure 5c). Its corresponding B73 version gene was ZmAsn4, gene number Zm00001d047736, encoding asparagine synthetase 4, ASN4. By analyzing the genome sequence of wild maize for three generations, we found that the wild maize Asn4 had a 47bp insertion in the 10th exon relative to B73, and this insertion caused 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 transcripts used by wild maize Asn4 were different from those in B73, and the wild maize transcripts were significantly highly expressed (Figure 5d). Further analysis of the RNA-Seq of the roots and leaves of the NILTHP9 near-isogenic line carrying the high-protein site and the control NILB73 revealed that Asn4 was significantly highly expressed in the roots and leaves of the near-isogenic line NILTHP9, while it was almost not expressed in NILB73 (Figure 5e). Immunoblotting analysis also demonstrated that ASN4 was highly accumulated in the roots and leaves of the near-isogenic lines (Figure 5f). Through a series of positioning sequencing, large-scale population protein content measurement, and molecular marker screening, we finally located the high-protein major gene as Asn4-Thp9.

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

The leaf DNA extraction and PCR identification methods are the same as step 5.4 above, and the identification primers are:

Based on the 47 bp insertion, we developed a molecular marker SEQ ID NO:4 and analyzed 200 individuals in the F ₃ BC ₇ 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 corn genotype Asn4-Teo (ZmAsn4 is the genotype of wild corn) (a and b in Figure 6). The free amino acid asparagine content in the roots was also significantly higher in ZmAsn4-H and Asn4-Teo than in ZmAsn4-B73 (c in Figure 6), indicating that the high or low THP9 protein content is linked to the marker. The results further proved that high expression of ZmAsn4 is an important factor in increasing the protein content of corn. The molecular marker SEQ ID NO:4 developed by us can be used to identify the introduced wild corn high protein site and the variation of this site at the population level.

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

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 about 13.1±0.38%, and the control NILB73 was about 9.69±0.43% in Shanghai; the protein content of NILTHP9 seeds in Sanya was about 15.39±0.95%, and the control NILB73 was about 11.17±0.95%; and the protein content of NILTHP9 seeds in Northeast China was also about 11.96±0.65%, and the control NILB73 was about 9.16±0.52% in Northeast China, which was 35.19%, 47.78%, and 30.57% higher in Shanghai, Sanya, and Northeast China, respectively (Figure 7a). In addition, we found that NILTHP9 increased the total nitrogen content in the stem and the total nitrogen content in the root of the plant in addition to increasing the protein content in the grain (Fig. 7b). The free amino acid content determination found that asparagine in NILTHP9 was significantly higher than that in NILB73 (Fig. 7c). In addition, the plant height and aboveground biomass of NILTHP9 increased significantly, and the plant height also increased by 10% (Fig. 7d and e), and the aboveground biomass increased by 20% (Fig. 7f), indicating that excessive accumulation of asparagine in THP9 is beneficial to plant growth. THP9 has great potential in increasing the protein content of corn grains, increasing the total nitrogen of silage corn stems, and increasing plant biomass and plant height.

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

8.1 Construction of Thp9 overexpression vector and primers

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

aggtcgactctagaggatccATGgactacaaggaccatgacggtgactacaaggaccatgacattgactacaaggatgacgatg acaagggaggaggatgtggcatcttagccgtg;

Reverse primer ASN4-3300-R3: ggggaaattcgagctcTTACACCGCGATGGCGACAGC.

PCR conditions: Toyobo's 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.

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

8.2 Genetic transformation

According to the Molecular Cloning Experiment Guide (3rd edition), Agrobacterium EHA105 competent cells were prepared. The overexpression Thp9 vector was transformed into maize B73 immature embryos by Agrobacterium-mediated method to obtain overexpression transgenic maize. The genetic transformation was carried out at Weimi Biotechnology (Jiangsu) Co., Ltd.

8.3 Quantitative PCR analysis and Western blotting of transgenic maize

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

8.4 Analysis of protein content in kernels of 500 maize inbred lines using nitrogen analyzer

500 inbred lines were planted at the Damao base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan (2019 and 2020). Three ears were inbred from each material. After maturity, harvesting and drying, 6 grains from the middle of three ears were taken from each inbred line and mixed and ground. The protein content determination method is shown in step 1.1 above.

Furthermore, we overexpressed Thp9 of wild maize haplotype in B73 background through UBI promoter. The expression of Thp9 gene in roots and leaves of overexpressed transgenic events was significantly increased (Figure 8a and b); immunoblot analysis showed that Thp9 was significantly accumulated in roots of overexpressed transgenic events (Figure 8c), and the grain protein content increased from 12.08±0.88% in 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 corn grain protein content, increasing total nitrogen in 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, and found that the variation of protein content in the inbred lines in 2019 was 6.5%-16%, with an average of 11.52%; while the variation of protein content in 2020 was 7.7%-16.8%, with an average of 12.3%. The protein content of the inbred lines planted uniformly in the Damao base of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences in Sanya, Hainan was analyzed by GWAS 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 locus located on chromosome 9 (e in Figure 8). There are three haplotypes of ASN4 gene in natural populations at the insertion and deletion positions of wild corn and B73 identified previously (Figure 8f). Analysis shows that the ASN4 gene of B73 (Zm00001d047736) belongs to a haplotype HAP3, which is 47bp missing compared to the wild corn haplotype HAP1, but has no function, almost no expression, and the protein content is about 10%; the wild corn haplotype HAP1 and the HAP2 haplotype in the natural population with a 22bp deletion significantly increase the protein content in the natural population (Figure 8g). From wild corn to cultivated corn, ASN4 was selected during the domestication process and differentiation occurred in natural populations. The population variation level once again proves that the variation of ASN4 is significantly correlated with protein content.

Example 9: Thp9 nitrogen high efficiency test and field test

The protein content determination method was the same as step 1.1 above. By planting the high-protein near-isogenic line NILTHP9 carrying Thp9 and the control NILB73 without Thp9 in soils with different nitrogen content levels, it was found that the expression of the ZmAsn4 gene increased significantly with the increase of nitrogen level; and the plant height, above-ground and underground biomass and grain protein content of NILTHP9 under low nitrogen (no artificial nitrogen fertilizer application) level reached the biomass and plant height of the control NILB73 under normal nitrogen application (applied twice, at the seedling stage and jointing stage, 10g each time) (Figure 9a-g). This shows that the high expression of the wild maize ZmASN4 gene has the potential for high nitrogen efficiency.

Furthermore, through field experiments, we set up four gradient nitrogen application experiments, namely 0%, 25%, 50% and 100% (the 100% level was fertilized once at the seedling stage and once at the jointing stage, for a total of two times, each time with 0-4-8-16 g/plant and a nitrogen content of 17%, and the other levels decreased successively; the planting density was 0.6m x 0.25m), and 300 seeds were planted in each group (Figure 10a). It was found that the plant height (Figure 10b), aboveground biomass (Figure 10c), total N content of roots, leaves and stems (Figures 10d-f), and grain protein content of NILTHP9 at each level were higher than those of the control NILB73 (Figure 10g), and the plant height, biomass and plant nitrogen content of NILTHP9 at the 25% level were the same as those at the 50% and 100% levels of the control. Field experiments further proved that wild corn ZmAsn4 has high nitrogen efficiency and the introduction of Thp9 gene is of great significance for reducing the application of nitrogen fertilizer and being environmentally friendly.

Example 10: Application potential and value of Thp9

The PCR identification method of Thp9 locus is the same as step 5.3, the primers are thp9-F/R and asn4-is-F/R, and the amplification information is shown in Example 6. In order to detect the application potential of Thp9, the Thp9 near-isogenic line (B73 background) and the control NILB73 were firstly hybridized with Mo17, and it was found that the protein content of the hybrid carrying the Thp9 locus was significantly increased (a and b in Figure 11). Further, we backcrossed Thp9 into the two parents of Zheng 58 and Chang 7-2 of the main corn hybrid Zhengdan 958, and obtained the improved versions of Thp9 Zheng 58 and Chang 7-2 through backcrossing for 3 generations and then selfing. The improved versions of the parents were hybridized to obtain the improved version of Zhengdan 958. Through testing in Sanya, it was found that the high-protein gene-modified version of Zhengdan 958 increased the number of plants and plant biomass (Figure 11 c-f), and the protein content was significantly higher than the control original hybrid. The protein content of the improved version of Zhengdan 958-THP9 seeds was 11.14±1.13%, while the control Zhengdan 958 was 9.88±0.58%, an increase of 12.75% (Figure 11 g). In addition, the total nitrogen content of the roots, stems, and leaves of the improved version of Zhengdan 958-THP9 increased (Figure 11 h-j). my country's annual corn production is about 270 million tons. Every 1 percentage point increase in protein is equivalent to an additional production of 2.7 million tons of protein, which will generate huge potential and value in agricultural production.

References:

Peng,B.,H.Kong,Y.Li,L.Wang,M.Zhong et al.,2014OsAAP6 functions as an important regulator of grain protein content and nutritional quality inrice.Nat Commun 5:4847.

Yang,Y.,M.Guo,S.Sun,Y.Zou,S.Yin et al.,2019Natural variation ofOsGluA2 is involved in grain protein content regulation in rice.Nat Commun10:1949.

Karn, A., J.D.Gillman and S.A.Flint-Garcia, 2017Genetic Analysis of Teosinte Alleles for Kernel Composition Traits in Maize.G3(Bethesda)7:1157-1164.

Flint-Garcia, S.A., A.L. Bodnar and M.P. Scott, 2009 Wide variability inkernel composition, seed characteristics, and zein profiles among diverse maizeinbreds, landraces, and teosinte. Theor Appl Genet 119:1129-1142.

Wu, Y., and Messing, J. (2017). Understanding and improving protein traits in maize. In Achieving sustainable cultivation of maize Vol 1: From improved varieties to local applications, D. Watson, ed (Cambridge, UK Burleigh DoddsScience Publishing.

Esen,A.(1987).A proposed nomenclature for the alcohol-solubleproteins(zeins)of maize (Zea mays L.).Journal of Cereal Science 5,117-128.

Thompson, G., and Larkins, B. (1994). Characterization of Zein Genes and Their Regulation in Maize Endosperm. In The Maize Handbook, M. Freeling and V. Walbot, eds (Springer New York), pp. 639-647.

Mertz,E.T.,Bates,L.S.,and Nelson,O.E.(1964).Mutant Gene That ChangesProtein Composition And Increases Lysine Content Of Maize Endosperm.Science145,279-280.

Mertz,E.T.,Veron,O.A.,Bates,L.S.,and Nelson,O.E.(1965).Growth of RatsFed on Opaque-2 Maize.Science 148,1741-1742.

Hu,B.,W.Wang,S.Ou,J.Tang,H.Li et al.,2015Variation in NRT1.1Bcontributes to nitrate-use divergence between rice subspecies.Nat Genet 47:834-838.

Liu,Y.,H.Wang,Z.Jiang,W.Wang,R.Xu et al.,2021Genomic basis ofgeographical adaptation to soil nitrogen in rice.Nature 590:600-605.

Li, S., Y. Tian, K. Wu, Y. Ye, J. Yu et al., 2018Modulating plant growth-metabolism coordination for sustainable agriculture. Nature 560:595-600.

Wu,K.,S.Wang,W.Song,J.Zhang,Y.Wang et al.,2020Enhanced sustainablegreen revolution yield via nitrogen-responsive chromatin modulation inrice.Science 367.

Gaufichon,L.,S.J.Rothstein and A.Suzuki,2016Asparagine MetabolicPathways in Arabidopsis.Plant Cell Physiol 57:675-689.

Liu,H.,J.Shi,C.Sun,H.Gong,X.Fan et al.,2016Gene duplication confersenhanced expression of 27-kDa gamma-zein for endosperm modification inquality protein maize.Proc Natl Acad Sci U S A 113:4964 -4969.

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 540

gctagttttg acgggcccta tctatgctgt agttcctcag catgcatgcg ctttgtttgc 600

tttttttact tcacaggcca atggagagat ctacaaccac aagaacgtcc ggaagcagtt 660

caccggcgcg cacagcttca gcaccggcag tgactgcgag gtcatcatcc ccctggtgag 720

ccttacactg atcgtttcag ttctgaaaac caaacttgtt cttcgcttac acactaaaga 780

acaaaaactt ctcctctact gatgctgcct ttgttatattgc tgctgccgtc gtcggcgtca 840

tcgatctcca gtacgagaag tacggcgaga acttcgtgga catgctggac ggagtcttcg 900

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 aacaaattac aaaaatggac catcatccat aggacggcat cgacgcgatc 1920

gaggaggtga tctaccacga cgagacgtac gacgtgacga cgatccgggc cagcacgccc 1980

atgttcctga tggctcgcaa gatcaagtcg ctgggcgtga agatggtgct gtccggggag 2040

ggctccgacg agctcctggg cggctacctc tacttccact tcgcccccaa cagggaggag 2100

ctccacaggg agacctgccg caaggtgaag gccctgcacc agtacgactg cctgcgcgcc 2160

aacaaggcga cgtcggcgtg 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 2400

aggaaggcgt tcgacgacga ggagcacct tacctgcccg aggtaagaac atcttcagag 2460

aaggctggtc gtttacctct gtgtctgtgt gatttcaagc ctgaactgac gcctctgtgc 2520

catgcatcct ccgcagcata ttctgtacag gcagaaagaa cagttcagtg acggagtggg 2580

ctacaactgg atcgatggac tcaaatcctt caccgaacag caggttgatt tacggcccca 2640

ctttcagctc tgatcgcatc tcctagacat cgtaccgtac gtcgtccaag ttagctaacc 2700

agcgctgacg ttccccccca atgttcaggt gacggatgag atgatgaaca acgccgccca 2760

gatgttcccg tacaacacgc ccgtcaacaa ggaggcctac tactaccgga tgatattcga 2820

gaggctcttc cctcaggtga ttgattcagc tttcagccag cctccaacga tgcgcgtgtt 2880

gcactgcaca cgtggtagcc aattcaatac gcgcggcgtg ctgctgactg ttgggtcgtg 2940

aactcggtga tgcctgcctg catgcaggac tcggcgaggg agacggtgcc gtggggcccg 3000

agcatcgcct gcagcacgcc cgcggccatc gagtgggtgg agcagtggaa 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 ttttagccgt gctcggatgc tccgactgct cccaggccag gagggctcgc 60

atcctcgcct gctccagaag gctgaagcac aggggccccg actggtcggg cctctaccag 120

cacgagggca acttcctggc gcagcagcgg ctcgccatcg tctccccgct gtccggcgac 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 actccttcgt cgtcggcctc gagggctccc ctgacctgaa ggccgcacga 840

gaggtcgctg actacctcgg aaccacccat cacgagttcc atttcaccgt acaggacggc 900

atcgacgcga tcgaggaggt gatctaccac gacgagacgt acgacgtgac gacgatccgg 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 gcatcgagaa gtgggtcctg aggaaggcgt tcgacgacga ggagcaccct 1320

tacctgcccg agcatattct gtacaggcag aaagaacagt tcagtgacgg agtgggctac 1380

aactggatcg atggactcaa atccttcacc gaacagcagg tgacggatga gatgatgaac 1440

aacgccgccc agatgttccc gtacaacacg cccgtcaaca aggaggccta ctactaccgg 1500

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, Ames21814

<400> 4

ctctgtgcca tgcatcctcc gcagcatatt ctgtacaggc agaaagaaca gttcagtgac 60

ggagtgggct acaactggat cgatggactc aaatccttca ccgaacagca ggttgattta 120

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 tggacccccga 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 (10)

1. Application of wild corn asparagine synthase 4 gene in improving corn grain protein content, plant total nitrogen content and/or nitrogen efficiency. 2. Application as claimed in claim 1, characterized in that the application is selected from the following group of methods: introducing the gene encoding wild corn asparagine synthase 4 into the common corn chromosome; making corn overexpress wild corn asparagine. Synthetase 4 gene; make corn overexpress the original asparagine synthase 4 gene ZmASN4 of common corn; introduce the regulatory region that controls the expression of wild corn asparagine synthase 4 gene into corn to increase the expression of this gene. 3. The application according to claim 2, characterized in that the original asparagine synthase 4 gene ZmASN4 of common corn refers to Zm00001d047736 for the inbred corn B73. 4. Application as claimed in claim 1, characterized in that the nucleotide sequence of the wild corn asparagine synthase 4 gene is selected from the following group: (A) The polynucleotide shown in SEQ ID NO: 1, named Thp9, gene number Teo09G002926, NCBIGenome submission: SUB11272093; (B) A polynucleotide having a homology of ≥80%, ≥85%, ≥90%, preferably ≥95%, and more preferably ≥98% with the nucleotide sequence shown in SEQ ID NO:1. 5. Application as claimed in claim 1, characterized in that the wild corn asparagine synthase 4 is a polypeptide selected from the following group: (a) A polypeptide having the amino acid sequence of SEQ ID NO:2; (b) A polypeptide derived from (a) that is formed by substituting, deleting or adding one or more amino acid residues to the SEQ ID NO:2 amino acid sequence and having the function of (a) polypeptide; (c) Having more than 95% homology, preferably more than 98% homology, and more preferably more than 99% homology with the polypeptide sequence defined in (a), and having the polypeptide function of (a). Derivatized peptides; or (d) A derived polypeptide whose sequence contains the polypeptide sequence described in (a) or (b) or (c). 6. Application as claimed in claim 2 or 4, characterized in that, the method for introducing the wild corn asparagine synthase 4 encoding gene into the corn chromosome includes the following steps: (1) Clone the wild corn asparagine synthase 4 encoding gene into a plant expression vector suitable for expression in Agrobacterium to obtain an expression vector of the gene; (2) After the vector is verified by sequencing, the expression vector of the gene is transformed into young corn embryos using Agrobacterium-mediated method to obtain transgenic corn that overexpresses the gene. 7. The application according to claim 5, characterized in that the pCAMBIA vector is the pCAMBIA3300 vector driven by the corn Ubiquitin promoter. 8. A kit for implementing the application as claimed in claim 4, which contains: the Thp9 gene fragment of SEQ ID NO: 1 or its CDS sequence SEQ ID NO: 3, used to combine the gene fragment or its CDS sequence PCR primers required for cloning into plant expression vectors; Or it includes: the gene expression vector as described in claim 5 or 6, a reagent for transforming the gene expression vector into Agrobacterium; Or it includes: Agrobacterium transformed with the gene expression vector as described in claim 5 or 6, and a reagent for transforming Agrobacterium into plants. 9. A method for detecting the gene of claim 3 or 4 in the corn genome, comprising the following steps: When detecting the Thp9 gene whose nucleotide sequence is SEQ ID NO: 1, the forward primer thp9-F: CTCTGTGCCATGCATCCTCC, the reverse primer thp9-R: CGTCAGCGCTGGTTAGC, the PCR product is 198 bp SEQ ID NO: 4, which is the high protein position of Thp9 Molecular markers of points: CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGATCGCATCTCCTAGACATCGTACCGTACGTCGTCCAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 4); Or the PCR product is 176bp SEQ ID NO:5, which is also a molecular marker of the Thp9 high protein site: CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATTTATGGCCACGCATCTCCTAGACATCGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 5); The PCR product is 151 bp SEQ ID NO: 6, which is the molecular marker of the maize B73 gene Zm00001d047736: CTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAAGCCTTCACCGAACAGCAGGTTGATGGTCGTCGTCGAAGTTAGCTAACCAGCGCTGACG (SEQ ID NO: 6); When detecting whether the Thp9 gene with the nucleotide sequence 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 455 bp SEQ ID NO :7, which is the molecular marker inserted into the wild maize Ames21814 genotype: CCGTTCCTCGACAAGGAGTTCGTCGACGTCGCGATGGGCATGGACCCCGAGTGGAAAATGGTACTGACGCGGGCCTTTTTCGACACGGCCCGGCCCTGCCGCCGCACGTCGGGGTCTCGGTTCTACGTATGATGATGACGCCTTCTTCTCTTCTTTGCAGTACGACAAGAACCTGGGTCGCATCGAGAAGTGGGTCCTGAGGAAGGCGTTCGACGACGAGGAGCACCCTTACCTGCCCGAGGTAAGAACAT CTTCAGAGAAGGCTGGTCGTTTACCTCTGTGTCTGTGTGATTTCAAGCCTGAACTGACGCCTCTGTGCCATGCATCCTCCGCAGCATATTCTGTACAGGCAGAAAGAACAGTTCAGTGACGGAGTGGGCTACAACTGGATCGATGGACTCAAATCCTTCACCGAACAGCAGGTTGATTTACGGCCCCACTTTCAGCTCTGAT (SEQ ID NO: 7). 10. A kit for implementing the method according to claim 9, characterized in that it includes a microarray for detecting corresponding primers of SEQ ID NOs: 4-7, or DNA/RNA probes, or DNA/RNA probes. array chip.