CN114920810A - Application of nitrate absorption related protein in regulation and control of nitrate absorption of corn - Google Patents

Abstract

The invention discloses application of nitrate absorption related protein or substances for regulating the content or activity of the nitrate absorption related protein in regulating and controlling nitrate absorption of plants or in preparing products for regulating and controlling nitrate absorption of plants. Transgenic experiments for introducing genes for coding nitrate absorption related proteins into corn prove that the overexpression related genes remarkably promote the absorption and utilization of the nitrogen in the corn under the low-nitrogen environment, show that the overexpression related genes are related to the absorption and utilization of the nitrogen, can be used for promoting plants to absorb and utilize the nitrogen, and improve the yield of the plants.

Description

Application of nitrate uptake-related proteins in the regulation of nitrate uptake in maize

technical field

The invention relates to the application of a nitrate absorption-related protein in the field of biotechnology in regulating the nitrate absorption of corn.

Background technique

Nitrogen is one of the essential and most demanded mineral elements in plant growth and development, accounting for about 1%-5% of plant dry weight (Li et al., 2009). Of all the essential nutrients, nitrogen is the primary factor limiting plant growth and yield formation. Nitrogen plays a central role in the building up of plant cells, tissues, organs and yields, so it is called the element of life. Nitrogen absorbed by plants is metabolized by assimilation in the body to produce various forms of nitrogen-containing compounds, such as amino acids, proteins, nucleic acids, hormones, etc., which are involved in important physiological processes such as photosynthesis and respiration (Williams et al., 2001) .

Corn is one of the major crops in the world and is widely grown in many countries. As one of the key crops, corn has long been the basic food source for human survival and the main raw material for animal feed; in addition, corn is indispensable for the processing of many daily necessities; in recent years, the use of corn to produce ethanol fuel has become a new trend. A major trend in energy development. These urgently need to increase corn planting area and unit yield; and the increase of corn yield requires a large amount of nitrogen fertilizer input, so it is necessary to improve the nitrogen efficiency of corn. Maize is a dryland crop that mainly absorbs and utilizes soil nitrate nitrogen. The absorption of nitrate from the soil by plants is the first step in their assimilation and utilization of nitrate nitrogen, and the research on this process is the key to improving the nitrogen absorption and utilization capacity of crops, which will help to find the limiting factor of efficient nitrogen utilization (Garnett et al. al., 2013; Plett et al., 2010). Studying the process and law of nitrate absorption and utilization in maize can provide theoretical basis and genetic resources for cultivating new maize varieties with high nitrogen efficiency by molecular means.

Nitrate has important effects on plant metabolism and growth as both a nutrient element and a signal. The NO ₃ ^{-concentration} levels in soil varied greatly, and the difference was up to 100-fold (Lark et al., 2004). Plant roots require different nitrate uptake systems for efficient nitrogen uptake in response to changes in soil NO ₃ ^{-concentration} levels. In the model plant Arabidopsis thaliana, the AtNRT1.1 gene encodes a dual-affinity nitrate transporter, which can switch between high-affinity and low-affinity nitrate transport activities by sensing the concentration level of nitrate in the environment . Changes in protein nitroaffinity are regulated by phosphorylation of the 101st threonine (Thr) in the amino acid sequence (Martin et al., 2008). When the external nitrate concentration is low, Thr101 is phosphorylated, which promotes AtNRT1.1 to have high-affinity nitrate transport activity; when the external nitrate concentration is high, Thr101 is dephosphorylated, enabling AtNRT1.1 to have low-affinity nitrate transport. activity.

The research group of Chu Chengcai, Institute of Genetics, Chinese Academy of Sciences found that OsNRT1.1B can improve the nitrogen use efficiency of japonica rice (Hu et al., 2015). The high nitrogen use efficiency gene OsNRT1.1B was cloned from indica rice by map-based cloning strategy. OsNRT1.1B has only one amino acid difference (T327M) between indica and japonica rice, resulting in higher nitrate uptake and transport activity in indica rice than in japonica. . The indica rice type OsNRT1.1B was introduced into the japonica rice varieties. The results of the field test showed that the japonica rice varieties containing the indica rice type OsNRT1.1B increased the yield by 30-33% and the nitrogen use efficiency by 30% compared with the control under half fertilization conditions; Under normal nitrogen application conditions, the yield is increased by 7-13%, and the nitrogen utilization efficiency is increased by about 10%.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve the absorption and utilization of nitrogen by plants in a low nitrogen environment.

In order to solve the above technical problems, the present invention provides an application of a nitrate absorption-related protein or a substance that regulates the content or activity of the nitrate absorption-related protein in regulating the absorption of nitrate by a plant or in the preparation of a product for regulating the absorption of nitrate by a plant, The gene encodes a nitrate uptake-related protein, and the nitrate uptake-related protein is a protein of the following A1, A2 or A3:

A1. The amino acid sequence is a protein of the amino acid sequence shown in either Sequence 3 or Sequence 4 in the sequence listing;

A2. The amino acid sequence shown in either Sequence 3 or Sequence 4 in the Sequence Listing is 80% identical to the protein shown in A1) obtained by substitution and/or deletion and/or addition of one or more amino acid residues A protein that is identical to the above and is associated with plant powdery mildew resistance and/or sheath blight resistance;

A3. A fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of A1) or A2).

In the above application, Sequence 3 in the Sequence Listing consists of 608 amino acid residues, and Sequence 4 in the Sequence Listing consists of 595 amino acid residues.

In the above applications, identity refers to the identity of amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as the Matrix, and set the Gap existence cost, Per residue gap cost and Lambda ratio to be respectively 11, 1 and 0.85 (default value) and search for the identity of a pair of amino acid sequences to calculate the identity value (%).

In the above applications, the identity of more than 80% may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identical.

In the above application, the nitrate absorption-related protein can be derived from corn.

In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant; further, the monocotyledonous plant is a gramineous plant, and the dicotyledonous plant is a cruciferous plant; further, the gramineous plant is corn , the cruciferous plant is Arabidopsis thaliana.

The second object of the present invention is the application of a substance for regulating gene expression in regulating the absorption of nitrate by a plant or in the preparation of a product for regulating the absorption of nitrate by a plant, the gene encoding the nitrate absorption-related protein.

In the above application, the material for regulating gene expression may be a material for at least one of the following 6 kinds of regulation: B1) regulation at the level of gene transcription; B2) regulation after the gene is transcribed (ie regulation of splicing or processing of the primary transcript of the gene); B3) regulation of RNA transport of the gene (ie regulation of the mRNA transport of the gene from the nucleus to the cytoplasm) B4) regulation of the translation of the gene; B5) regulation of the mRNA degradation of the gene; B6) post-translation regulation of the gene (that is, the activity of the protein translated by the gene is carried out control).

In the above application, the material for regulating gene expression is a biological material related to the nitrate absorption-related protein; the biological material is any one of the following C1-C3:

C1, a nucleic acid molecule encoding the nitrate absorption-related protein;

C2. Nucleic acid molecules that improve the expression of the nitrate absorption-related protein;

C3. An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule described in C1 or C2.

In the above application, the nucleic acid molecule described in C1 or C2 can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

In the above application, the nucleic acid molecule described in C1 may specifically be the gene shown in D1 or D2 as follows:

D1. The coding sequence (ORF) of the coding strand is the DNA molecule shown in Sequence Listing Sequence 1 or Sequence Listing Sequence 2;

D2. The nucleotide sequence is the DNA molecule shown in Sequence Listing Sequence 1 or Sequence Listing Sequence 2.

In the above application, the regulation of gene expression may be to increase the gene expression.

In order to solve the above-mentioned technical problems, the present invention provides a plant reagent for promoting the absorption of nitrate by plants, containing the above-mentioned nitrate absorption-related protein or the above-mentioned substance for regulating gene expression, and the plant reagent is a plant that promotes the absorption and utilization of nitrogen by plants. reagents.

The active components of the above reagents may also contain other biological components or/and non-biological components, which can be determined by those skilled in the art according to the nitrate absorption effect of plants.

In order to solve the above-mentioned technical problems, the present invention also provides a method for promoting the absorption of nitrate by plants, comprising the steps of: increasing the expression of the nitrate absorption-related protein in the recipient plant, and increasing the abundance of the nitrate absorption-related protein in the recipient plant. degree to obtain a target plant with a higher nitrate absorption rate than the recipient plant in a low nitrogen environment.

In the above-mentioned method, the nucleic acid encoding the above-mentioned protein is introduced into the recipient plant to obtain the target plant whose nitrate absorption rate is higher than that of the recipient plant.

In the above method, the nitrate absorption may be nitrate absorption in a low nitrogen environment. The nitrate concentration in the low nitrogen environment is 0-0.2 mM/L.

In the above method, the plant is a monocotyledonous plant or a dicotyledonous plant; further, the monocotyledonous plant is a gramineous plant, and the dicotyledonous plant is a cruciferous plant; further, the gramineous plant is corn , the cruciferous plant is Arabidopsis thaliana.

The present invention provides the application of a substance regulating gene expression in improving the absorption of nitrate by plants or in the preparation of a product for improving the absorption of nitrate by maize. 1B gene. Transgenic experiments in which ZmNRT1.1A and ZmNRT1.1B genes were introduced into Arabidopsis thaliana and maize, respectively, showed that transgenic plants overexpressing ZmNRT1.1A or ZmNRT1.1B genes significantly promoted the low nitrogen environment compared with recipient plants. The absorption and utilization of nitrate nitrogen by maize indicates that ZmNRT1.1A gene and ZmNRT1.1B gene are genes related to nitrogen absorption and utilization. ZmNRT1.1A gene and ZmNRT1.1B gene can be used to promote the absorption and utilization of nitrogen by plants and improve Plant yield.

Description of drawings

FIG. 1 is the colinear map analysis and phylogenetic tree analysis diagram of ZmNRT1.1A/B and other homologous genes of Gramineae origin in Example 1. FIG. Among them, A in FIG. 1 is a collinear map analysis, and B in FIG. 1 is an analysis diagram of a phylogenetic tree.

Figure 2 shows the expression characteristics of ZmNRT1.1A/B gene in Example 1 after nitrogen deficiency in maize B73 was restored to nitrate for different periods of time and the expression level analysis of long-term culture under different nitrate concentrations. Among them, A in Figure 2 is the expression characteristics of ZmNRT1.1A/B gene after nitrogen deficiency in maize B73 to restore nitrate supply for different periods of time; B in Figure 2 is ZmNRT1.1A/B in maize B73 cultured for a long time under different nitrate concentrations Analysis of the expression level of B gene; using real-time quantitative PCR (qPCR) to analyze the transcriptional level expression of ZmNRT1.1A/B gene in maize B73.

Figure 3 is the amplification result of the open reading frame of the complete maize ZmNRT1.1A/B obtained by amplification in Example 2. In the figure, the right lane M is the DNA molecular weight standard, and the left lane is the amplification results of ZmNRT1.1A and ZmNRT1.1B open reading frame sequences.

4 is the RT-PCR molecular verification results of the transgenic Arabidopsis plants overexpressing the ZmNRT1.1A gene or the ZmNRT1.1B gene in Example 3 and the results of ¹⁵ NO ₃ ^-transient absorption under high and low nitrogen. Panel A of Figure 4 shows the gene expression levels of the transgenic lines; Panel B of Figure 4 shows the nitrate uptake rates of the transgenic lines under different nitrogen concentrations (0.2 mM KNO ₃ , 5 mM KNO ₃ ). Among them, chl1-5-p35S::ZmNRT1.1A#4, chl1-5-p35S::ZmNRT1.1A#11, chl1-5-p35S::ZmNRT1.1A#13 belong to the chl1-5 background to ZmNRT1.1A Genes of Arabidopsis lines chl1-5-p35S::ZmNRT1.1A; chl1-5-p35S::ZmNRT1.1B#5, chl1-5-p35S::ZmNRT1.1B#13, chl1-5-p35S: :ZmNRT1.1B#14 belongs to the Arabidopsis thaliana line chl1-5-p35S::ZmNRT1.1B with the ZmNRT1.1B gene in the chl1-5 background. Data are presented as mean ± standard deviation, the number of replicates is 4, and different lowercase letters indicate significant differences.

Figure 5 shows the gene expression levels and physiological phenotypes of maize plants overexpressing ZmNRT1.1A gene or overexpressing ZmNRT1.1B gene. Figure A is the gene expression level of the transgenic ZmNRT1.1A maize plant; Figure B is the gene expression level of the transgenic ZmNRT1.1B maize plant; Figure C is the statistical result of the aboveground biomass (g/plant) of the transgenic ZmNRT1.1A maize line; Figure D is the statistical result of the above-ground biomass (g/plant) of the transformed ZmNRT1.1B maize line; Figure E is the statistical result of the aerial nitrogen uptake (mg/plant) of the transformed ZmNRT1.1A maize line; Figure F is the transformed ZmNRT1 .1B Statistical results of nitrogen uptake in the shoots of maize lines (mg/plant). Among them, A1-OE, A2-OE and A3-OE are all transformed ZmNRT1.1A maize lines, and B1-OE, B2-OE and B3-OE are all transformed ZmNRT1.1B maize lines. WT is a wild-type line; data are expressed as mean ± standard deviation, the number of replicates is 12, and * indicates that the result of significance analysis is P<0.05.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can serve as a guide for those of ordinary skill in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, are conventional biochemical reagents, which can be obtained from commercial sources.

In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3'-terminal nucleus of the corresponding DNA/RNA. Glycosides.

1. Carrier

In the following examples, the cloning vector pGEM T-Easy is a product of Promega Corporation.

The plant expression vectors pPTKan and pCAMBIA1301 in the following examples are described in the non-patent document "Sutter et al., Selective Mobility and Sensitivity to SNAREs Is Exhibited by the Arabidopsis KAT1K1 Channel at the Plasma Membrane, 2006, Plant Cell 18: 935-954". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The vector pPT-Hyg in the following examples is described in the non-patent document "Gu et al., Characterization of AMT-mediated High-affinity Ammonium Uptake in Roots of Maize, 2013, Plant CellPhysiology: 1515-1524". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The vector p1301-UBI-PPT in the following examples is described in the non-patent document "Zhao et al., OverExpression of the Maize ZmAMT1; 1a Gene Enhances Root Ammonium UptakeEfficiency Under Low Ammonium Nutrition, 2018, PlantBiotechnology Reports 12:47-56". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

2. Strain

Escherichia coli DH5α in the following examples belongs to Escherichia coli and was purchased from Tiangen Biochemical Technology Co., Ltd.

The Agrobacterium tumefaciens strains GV3101 and EHA105 in the following examples both belong to Agrobacterium tumefaciens and were purchased from Tiangen Biochemical Technology Co., Ltd.

3. Plant strains

The Arabidopsis chl1-5 mutant in the following examples is described in the non-patent document "Ho et al., CHL1 Function as a Nitrate Sensor in Plants, 2009, Cell: 1184-1194". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The Arabidopsis Col-0 wild type in the following examples is described in the non-patent document "Ho et al., CHL1 Function as a Nitrate Sensor in Plants, 2009, Cell: 1184-1194". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The maize inbred line B73 in the following examples is described in the non-patent document "Schnable et al., The B73 Maize Genome: Complexity, Diversity, and Dynamics, 2009, Science: 1112-1114.". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The corn hybrid HiII in the following examples is described in the non-patent document "An et al., Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7gene (ZmALDH7B6) in Response to Abiotic Stresses, 2014, Journal of Intergrative Agriculture: 1900-1908.". The public can obtain it from China Agricultural University to replicate the experiments in this application and cannot be used for other purposes.

The corn variety PH6WC in the following examples is an inbred line of DuPont Pioneer Company, which is recorded in the non-patent document "Liu Jiguo et al., Xianyu 335 female parent line PH6WC, 2010, New Agriculture: 30". The public can obtain it from the gene bank or China Agricultural University to repeat the experiments of this application and cannot be used for other purposes.

4. Reagents

In the following examples, various restriction endonucleases are products of Promega company; cDNA library construction kits are products of Invitrogen company; various Taq enzymes and Trizol RNA mini-extraction kits are products of Takara company; dNTP mixture is Shanghai Raw products; T4 DNA ligase is the product of Promega Company; Ampicillin (Amp), Kanamycin (Kan), Spectinomycin (Spe), Rifampicin (Rif) are all products of Xinjingke Company.

In the following example, the Hoagland nutrient solution for Arabidopsis hydroponics is K ₂ SO ₄ , MgSO ₄ .7H ₂ O, KH ₂ PO ₄ , H ₃ BO ₃ , MnSO ₄ .H ₂ O, ZnSO ₄ .7H ₂ O, CuSO ₄ .5H ₂ O, Na ₂ MoO ₄ .2H ₂ O, NaFe-EDTA, NH ₄ NO ₃ (nitrogen source), CaCl ₂ .2H ₂ O were dissolved in sterile water to obtain a pH of 5.8-6.0 The solution. In the Hoagland nutrient solution for Arabidopsis hydroponics, the concentration of K ₂ SO ₄ is 0.25 mM, the concentration of MgSO ₄ .7H ₂ O is 1 mM, the concentration of KH ₂ PO ₄ is 1 mM, the concentration of H ₃ BO ₃ is 30 μM, The concentration of MnSO ₄ .H ₂ O was 5 μM, the concentration of ZnSO ₄ .7H ₂ O was 1 μM, the concentration of CuSO ₄ .5H ₂ O was 1 μM, the concentration of Na ₂ MoO ₄ .2H ₂ O was 1 μM, and the concentration of NaFe-EDTA was 1 μM. _The concentration was 0.1 mM, the concentration of _NH4NO3 was ₂ mM, and the concentration of _CaCl2.2H2O was 0.25 mM.

In the following example, the Hoagland nutrient solution for corn hydroponics is K ₂ SO ₄ , MgSO ₄ .7H ₂ O, KH ₂ PO ₄ , H ₃ BO ₃ , MnSO ₄ .H ₂ O, ZnSO ₄ .7H ₂ O , CuSO ₄ .5H ₂ O, Na ₂ MoO ₄ .2H ₂ O, NaFe-EDTA, Ca(NO ₃ ) ₂ (nitrogen source), CaCl ₂ .2H ₂ O (used to balance calcium ions) dissolved in sterile water The obtained solution was pH 5.8-6.0. In the Hoagland nutrient solution for corn hydroponics, the concentration of K ₂ SO ₄ is 1 mM, the concentration of MgSO ₄ .7H ₂ O is 0.6 mM, the concentration of KH ₂ PO ₄ is 0.1 mM, the concentration of H ₃ BO ₃ is 30 μM, and the concentration of MnSO _4. The concentration of H ₂ O is 0.5 μM, the concentration of ZnSO ₄ .7H ₂ O is 0.5 μM, the concentration of CuSO ₄ .5H ₂ O is 0.2 μM, the concentration of Na ₂ MoO ₄ .2H ₂ O is 0.07 μM, and the concentration of NaFe The concentration of -EDTA is 0.1mM, the concentration of Ca(NO ₃ ) ₂ is set according to the final concentration of NO ₃ ^- is 0.04mmol/L, 0.4mmol/L, 4mmol/L, CaCl ₂ .2H ₂ O is used to balance calcium ions .

5. PCR amplification primers

P-A-1-F: 5'-ATGGTCGGACTCCTCCCC-3';

P-A-1-R: 5'-TCAGTGGAGCGTGGGC-3';

P-B-1-F: 5'-ATGGCCTCCGTCCTGCC-3';

P-B-1-R: 5'-TCAGTGGCCGACGGC-3';

PA-2-F: 5'-C TCTAGA ATGGTCGGACTCCTCCCC-3' (the sequence indicated by the underline is the Xba I enzyme recognition site sequence);

PA-2-R: 5'-CCG CTCGAG TCAGTGGACGCGTGGGCT-3' (the sequence indicated by the underline is the Xho I enzyme recognition site sequence);

PB-2-F: 5'-C TCTAGA ATGGCCTCCGTCCTGCCG-3' (the underlined sequence is the Xba I enzyme recognition site sequence);

PB-2-R: 5'-CCG CTCGAG TCAGTGGCCGACGGCAATAG-3' (the underlined sequence is the Xho I enzyme recognition site sequence);

PA-3-F: 5'-TC GAGCTC ATGGTCGGACTCCTCCCC-3' (the underlined sequence is the Sac I enzyme recognition site sequence);

PA-3-R: 5'-TC GAGCTC TCAGTGGACGTGGGCT-3' (the sequence indicated by the underline is the Sac I enzyme recognition site sequence);

PB-3-F: 5'-CG GGATCC ATGGCCTCCGTCCTGCCG-3' (the sequence indicated by the underline is the BamH I enzyme recognition site sequence);

PB-3-R: 5'-CG GGATCC TCAGTGGCCGACGGCAATAG-3' (the underlined sequence is the BamH I enzyme recognition site sequence);

P-A-4-F: 5'-TCCTCCAGCAAGAAGAGCAAGC-3';

P-A-4-R: 5'-TTCACCTCCTCCACGTCCGT-3';

P-B-4-F: 5'-TGCTGCTGCCAGTGCCACAA-3';

P-B-4-R: 5'-ACACGTTAATTAGCTCGACCTGCG-3';

PT-F: 5'-GCTATCCTGTGATCTGCCCTGA-3';

PT-R: 5'-CGCCAAACTTAATAACCCAGTA-3'.

The above primers were synthesized by Beijing Qingke Company.

In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA, and the last position is the 3'-terminal nucleotide of the corresponding DNA.

Quantitative tests in the following examples, unless otherwise specified, were set up to repeat the experiment three times, and the results were averaged, and * indicates that the significance analysis result is P<0.05.

Example 1. Expression analysis of maize nitrate transporter ZmNRT1.1A/ZmNRT1.1B

1. Evolutionary analysis of ZmNRT1;1s gene

So far, there are 4 NRT1.1s genes homologous to the model plant AtNRT1.1 gene in maize (ZmNRT1.1A/B/C/D) and 3 in rice (OsNRT1.1A/B/C) ), three in sorghum (SbNRT1.1A/B/C), and four in Brachypodium (BdNRT1.1A/B/C/D) (Plett et al., 2010, The PLoS One). Gene information search was performed in Phytozome/MaizeGDB database, and the nucleotide sequence, amino acid sequence and corresponding gene expression data of ZmNRT1.1A/B/C/D were obtained. Transcriptome data showed that the gene ZmNRT1.1A/B was mainly expressed in the root system, and the gene ZmNRT1.1C/D was rarely expressed, which may be redundant genes.

Figure 1 shows the collinear map analysis (A in Figure 1) and phylogenetic tree analysis (B in Figure 1) of ZmNRT1.1s. According to the collinear map, ZmNRT1.1B and ZmNRT1.1D were produced due to the doubling of the whole genome during the evolution, while ZmNRT1.1B and ZmNRT1.1C were produced by the tandem recombination of the genome. From the phylogenetic tree analysis, it can be seen that ZmNRT1.1B and OsNRT1.1B have higher homology; ZmNRT1.1A has higher homology with OsNRT1.1A.

2. Real-time quantitative PCR (Real-Time PCR) analysis of the expression pattern of ZmNRT1.1s gene in maize roots

The following two sets of experimental treatments were carried out:

(1) The maize inbred line B73 plants were first cultured in 2mM NH ₄ NO ₃ aqueous solution for 8 days, followed by nitrogen deficiency for 4 days, and then restored to 4 mM KNO ₃ for 1 hour/3 hours/6 hours/18 hours/72 hours respectively , harvesting corn roots with liquid nitrogen.

(2) The corn inbred B73 plants were planted in 0 mM/0.04 mM/4 mM/10 mM KNO ₃ aqueous solution for 12 days, and the corn roots were harvested with liquid nitrogen.

Total RNA of each treated maize root was extracted by Trizol method. The obtained total RNA was reverse transcribed into cDNA after the DNA in the total RNA was removed by a reverse transcription kit. Real-time PCR was used to amplify maize ZmNRT1 with primers P-A-4-F and P-A-4-R; 1A gene was used to amplify maize ZmNRT1 with primers P-B-4-F and P-B-4-R; 1B gene, Using PT-F and PT-R amplified maize ZmTUB1 gene as internal reference control, the expression of ZmNRT1.1A/ZmNRT1.1B gene in the same sample was divided by the expression of ZmTUB1 gene as ZmNRT1.1A/ZmNRT1.1B gene expression. The relative expression levels were used to study the expression characteristics of ZmNRT1.1A/ZmNRT1.1B genes in different tissue parts of maize.

The above-mentioned Real-Time PCR operation steps are as follows:

1. Extract total RNA from different samples with Trizol kit (TaKaRa company, catalog number: 9109).

2. Take 1.5 μg of total RNA and remove genomic DNA with reverse transcription kit (TaKaRa company, catalog number: RR047A). The method is as follows:

Reaction system 1 (10 μl):

5x gDNA Eraser Buffer 2μl

gDNA Eraser 1μl

RNase Free dH ₂ O to a final volume of 10 μl;

The reaction was carried out at 42°C for 5 minutes.

3. Reverse transcription to synthesize the first strand of cDNA by conventional methods, the method is as follows:

Reaction system 2 (20 μl):

42°C for 60 minutes; 95°C for 5 minutes.

4. Real-time PCR was used to detect gene abundance. The reagents were SYBR Green RealtimePCR Master Mix (catalog number: RR820A) from TAKARA, and the quantitative PCR instrument model was IQ5. The inversion product was diluted 10 times as the Real-time PCR template.

reaction system:

PCR reaction program: 50°C for 2 minutes, 95°C for 2 minutes, 40 cycles (95°C for 15 seconds, 61°C for 15 seconds, 72°C for 30 seconds);

Melting curve steps: 95°C for 15 seconds, a cycle of 10 seconds, each cycle increases the rate of 0.5°C from 60°C to 95°C for 70 cycles;

Using ZmTUB1 as an internal reference, the relative expression of ZmNRT1.1s gene in different tissue parts of maize was calculated by relative quantitative algorithm. The results are shown in Figure 2, the maize ZmNRT1;1B gene was induced by nitrate, and the ZmNRT1;1A gene was not induced by nitrate. The expression levels of ZmNRT1;1A gene and ZmNRT1;1B gene in maize both increased with the increase of nitrogen concentration.

Example 2. Cloning of ZmNRT1;1s gene and construction of Arabidopsis expression vector

1. Amplification of CDS of ZmNRT1.1A gene and ZmNRT1.1B gene

According to the sequence of ZmNRT1.1A/ZmNRT1.1B obtained by bioinformatics, primers were designed: the primer pair for amplifying ZmNRT1.1A consisted of upstream primer P-A-1-F and downstream primer P-A-1-R. The primer pair for amplifying ZmNRT1.1B consisted of the upstream primer P-B-1-F and the downstream primer P-B-1-R. Both P-A-1-F and P-B-1-F contain exactly the translation initiation codon ATG, and both P-A-1-R and P-B-1-R contain sequences that are reverse complementary to the translation termination codon TGA.

Using the reverse transcribed cDNA of corn root total RNA (see step (2) of Example 1) as a template, use the high-fidelity enzyme Primer STAR (TaKaRa company, catalog number: R010Q) to amplify, respectively obtain the complete open reading frame containing the cDNA. ZmNRT1; 1A gene fragment and ZmNRT1; 1B gene fragment.

The amplification system is as follows:

Make up to 50 μl with H ₂ O.

PCR reaction conditions: pre-denaturation at 98°C for 2 minutes; then 30 cycles of 98°C for 10 seconds, 65°C for 10 seconds, and 72°C for 3 minutes and 30 seconds; then add 1 μl of Taq enzyme, and extend and tail at 72°C for 20 minutes.

8 μl PCR products were taken and detected by electrophoresis on a 1.0% agarose gel. The detection results are shown in Figure 2. The mRNA sequence of ZmNRT1.1A consists of 2492 bases, and its open reading frame (ORF) is from the 5' end. Bases 326-2153, the fragment size is 1827bp; the mRNA sequence of ZmNRT1.1A consists of 2286 bases, and its open reading frame (ORF) is the bases 187-1975 from the 5' end, and the fragment size is 1788bp . The electrophoresis results are shown in Figure 3. The right lane M is the DNA molecular weight standard, and the left lane is the amplification results of the ZmNRT1.1A open reading frame (ORF) sequence and the ZmNRT1.1B open reading frame (ORF) sequence.

The target band was recovered, and the recovered product was ligated into the pGEM T-Easy vector, transformed into E. coli DH5α competent cells, and the plasmid was extracted and verified by sequencing. The sequencing results showed that the open reading frames of 1827bp (ZmNRT1.1A) and 1788bp (ZmNRT1.1B) were obtained respectively, and the comparison found that this sequence could completely match the sequence obtained from the online database. The nucleotide sequence of ZmNRT1;1A is shown in sequence 1 in the sequence listing; the amino acid sequence encoded by it is shown in sequence 3 in the sequence listing. The nucleotide sequence of ZmNRT1; 1B is shown in Sequence 2 in the sequence listing; the amino acid sequence encoded by it is shown in Sequence 4 in the sequence listing.

2. Construction of plant expression vector

1. Construction of Arabidopsis ZmNRT1; 1A gene overexpression vector (pPT-ZmNRT1; 1A) and Arabidopsis ZmNRT1; 1B gene overexpression vector (pPT-ZmNRT1; 1B)

Select the correct target gene of sequencing in step 1, re-amplify the target gene with the upstream primer containing the Xba I enzyme recognition site sequence and the downstream primer containing the Xho I enzyme recognition site sequence, specifically: with P-A-2-F (containing P-A-2-F). Xba I enzyme recognition site sequence) and P-A-2-R (containing Xho I enzyme recognition site sequence) to amplify the target gene ZmNRT1; 1A; P-B-2-F (containing Xba I enzyme recognition site sequence) and P-B -2-R (containing the Xho I enzyme recognition site sequence) amplifies the target gene ZmNRT1; 1B. The amplified products were concentrated to 10 μl, digested with Xba I enzyme, recovered by electrophoresis, and concentrated to 10 μl to obtain ZmNRT1; 1A gene target fragment and ZmNRT1; 1B gene target fragment respectively.

At the same time, Xba I digestion of the vector pPT-Hyg was carried out, and the linear pPT-Hyg plasmid DNA was recovered, and then the vector was dephosphorylated. The specific operation of dephosphorylation is to add the following reagents to a new centrifuge tube:

Linear pPT-Hyg plasmid DNA 5μg

10x CIPA Reaction Buffer 10μl

Diluted CIAP (0.01U/μl) 5μl

Add water to make up to 100 μl.

The following procedures are performed in the PCR machine:

Take the dephosphorylated product obtained in the previous step, add 100 μl of Tris saturated phenol and 100 μl of chloroform, mix and shake well; centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to another tube, add the same volume of chloroform, mix and shake well; centrifuge at 12,000 rpm for 10 min; transfer the supernatant to another tube, add 1/10 volume of 3M ammonium acetate, 3 times the volume of no Water-cooled ethanol, mixed well, precipitated at -80°C for 1 hour, 4°C, 12,000 rpm, centrifuged for 10 minutes, discarded the supernatant, added 700 μl of 70% ethanol to wash once, and dissolved in 10 μl of water to obtain the dephosphorylated vector fragment.

Take the target fragment after enzyme digestion and the dephosphorylated vector fragment for ligation reaction. The reaction system (10 μl) is as follows:

After ligation at 16°C for 16-20 hours, transfer into 50 μl DH5α competent cells, spread on LB plates containing kanamycin, cultivate at 37°C for 12 hours, collect all cells, extract plasmid DNA and verify by enzyme digestion , and sequenced for the sequencing company to confirm the correct sequence of the vector, which carries the selection marker gene for hygromycin resistance.

Among them, the vector with the correct sequence obtained by ligating the ZmNRT1; 1A gene target fragment and the vector fragment after transfection is pPT-ZmNRT1; 1A, and the ZmNRT1; 1B gene target fragment and the vector fragment after ligation. pPT-ZmNRT1; 1B.

2. Construction of the overexpression vector (p1301-Bar-ZmNRT1.1A) of transgenic maize ZmNRT1; 1A gene and the overexpression vector (p1301-Bar-ZmNRT1.1B) of transgenic maize ZmNRT1; 1B gene

Select the correctly sequenced plasmid pPT-ZmNRT1; 1A, re-amplify the target gene ZmNRT1 with primers P-A-3-F and P-A-3-R containing the Sac I enzyme recognition site sequence; 1A, recover the target fragment ZmNRT1; 1A. The target fragment ZmNRT1; 1A was digested with SacI enzyme and recovered by electrophoresis. The vector p1301-UBI-PPT was digested with SacI enzyme and recovered, followed by dephosphorylation of the vector.

Select the correctly sequenced plasmid pPT-ZmNRT1; 1B, re-amplify the target gene ZmNRT1; 1B with primers P-B-3-F and P-B-3-R containing the BamH I enzyme recognition site sequence; recover the target fragment ZmNRT1; 1B. The target fragment ZmNRT1; 1B was digested with BamH I enzyme and recovered by electrophoresis. The vector p1301-UBI-PPT was digested with BamHI and recovered, followed by dephosphorylation of the vector.

The dephosphorylation of the carrier is performed by adding the following reagents to a new centrifuge tube:

Linear p1301-UBI-PPT plasmid DNA 5μg

10x CIPA Reaction Buffer 10μl

Diluted CIAP (0.01U/μl) 5μl

Add water to make up to 100 μl.

The following procedures are performed in the PCR machine:

Take the dephosphorylated product obtained in the previous step, add 100 μl of Tris saturated phenol and 100 μl of chloroform, mix and shake well; centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to another tube, add the same volume of chloroform, mix and shake well; centrifuge at 12,000 rpm for 10 min; transfer the supernatant to another tube, add 1/10 volume of 3M ammonium acetate, 3 times the volume of no Water-cooled ethanol, mixed well, precipitated at -80°C for 1 hour, 4°C, 12,000 rpm, centrifuged for 10 min, discarded the supernatant, added 700 μl of 70% ethanol to wash once, and dissolved in 10 μl of water to obtain the dephosphorylated vector fragment.

Take the target fragment after enzyme digestion and the dephosphorylated vector fragment for ligation reaction. The reaction system (10 μl) is as follows:

After ligation at 16°C for 16-20 hours, transfer into 50 μl DH5α competent cells, spread on LB plates containing kanamycin, cultivate at 37°C for 12 hours, collect all cells, extract plasmid DNA and verify by enzyme digestion , and sequenced for the sequencing company to confirm the correct sequence of the vector, which carries the selection marker gene for hygromycin resistance.

Among them, the correct sequence of the vector obtained by transfection after connecting the target fragment ZmNRT1;1A and the vector fragment is to insert the ZmNRT1;1A gene (as shown in sequence 1 in the sequence table) into the restriction nucleic acid of the p1301-UBI-PPT vector The fragment of the cleavage site of Dicer SacI, keeping the other sequences of the p1301-UBI-PPT vector unchanged, obtained the overexpression vector of the transgenic maize ZmNRT1; 1A gene, named p1301-Bar-ZmNRT1.1A.

The correct sequence of the vector obtained by transfection after the target fragment ZmNRT1; 1B is connected with the vector fragment is the restriction endonuclease for inserting the ZmNRT1; 1B gene (as shown in sequence 2 in the sequence table) into the p1301-UBI-PPT vector The fragment of the BamHI restriction site, keeping other sequences of the p1301-UBI-PPT vector unchanged, obtained the overexpression vector of the transgenic maize ZmNRT1;1B gene, named p1301-Bar-ZmNRT1.1B.

Example 3. Phenotypic identification of transgenic plants overexpressing ZmNRT1.1A and ZmNRT1.1B

1. Overexpression ZmNRT1; 1A transgenic line Arabidopsis thaliana material and overexpressing ZmNRT1; 1B transgenic line Arabidopsis material ¹⁵ N transient uptake phenotype experiments

In order to study whether overexpression of ZmNRT1.1A gene or overexpression of ZmNRT1.1B gene can improve plant nitrogen uptake efficiency, transgenic Arabidopsis was constructed with the overexpression vector in Example 2, as follows:

Using the Arabidopsis chl1-5 mutant lacking the AtNRT1.1 gene as the transformation receptor, the chl1-5 mutant was transformed with p1301-Bar-ZmNRT1.1A by Agrobacterium-mediated genetic transformation, and the hygromycin resistance was screened The T ₀ generation transgenic plants were obtained. The obtained T ₀ generation transgenic plants were identified by PCR, and the positive ZmNRT1.1A plants were amplified to obtain a 1827bp band. The obtained positive transgenic ZmNRT1.1A plants were selfed, screened and identified to obtain 3 T ₃ generation transgenic ZmNRT1.1A lines (chl1-5-p35S::ZmNRT1.1A): chl1-5-p35S::ZmNRT1 .1A#4, chl1-5-p35S::ZmNRT1.1A#11, chl1-5-p35S::ZmNRT1.1A#13. The gene expression level of ZmNRT1.1A was verified by RT-PCR (the control was chl1-5), and the primers were PA-4-F and PA-4-R. The relative expression of ZmNRT1.1A gene was significantly higher than the control chl1-5.

Using the Arabidopsis chl1-5 mutant as the transformation receptor, the chl1-5 mutant was transformed with p1301-Bar-ZmNRT1.1B by Agrobacterium-mediated genetic transformation, and the T ₀ generation transgene was obtained by screening for hygromycin resistance. plant. The obtained T ₀ generation transgenic plants were identified by PCR, and the positive ZmNRT1.1B plants were amplified to obtain a 1788bp band. The positive transgenic ZmNRT1.1B plants were selfed, screened and identified to obtain 3 T ₃ generation transgenic ZmNRT1.1B lines (chl1-5-p35S::ZmNRT1.1B): chl1-5-p35S::ZmNRT1. 1B#5, chl1-5-p35S::ZmNRT1.1B#13, chl1-5-p35S::ZmNRT1.1B#14. The gene expression level of ZmNRT1.1B was verified by RT-PCR (the control was chl1-5), and the primers were PB-4-F and PB-4-R. The relative expression of ZmNRT1.1B gene was significantly higher than the control chl1-5.

Arabidopsis thaliana T ₃ homozygotes (chl1-5-p35S::ZmNRT1.1A#4, chl1-5-p35S::ZmNRT1.1A#11, chl1-5-p35S: : ZmNRT1.1A#13), under the condition of ¹⁵ NO ₃ -concentration ^of 0.2mmol/L and 5mmol/L NO ₃ ^-to measure the nitrate absorption rate of root system. The chl1-5 mutant and the Col-0 wild type were used as controls.

Arabidopsis thaliana T ₃ homozygotes (chl1-5-p35S::ZmNRT1.1B#5, chl1-5-p35S::ZmNRT1.1B#13, chl1-5-p35S: : ZmNRT1.1B#14), under the condition of ¹⁵ NO ₃ -concentration ^of 0.2mmol/L and 5mmol/L NO ₃ ^-to measure the nitrate absorption rate of root system. The chl1-5 mutant and the Col-0 wild type were used as controls.

Arabidopsis hydroponics method: firstly, Arabidopsis thaliana was cultured with Hoagland nutrient solution for 6 weeks, and the nutrient solution was changed every 3 days. Afterwards, the cells were cultured in Hoagland nutrient solution containing 0.2mmol/L and 5mmol/L KNO ₃ (except for the change of N source, other components remained unchanged) for 1 week. Incubate for 10 minutes in Hoagland nutrient solution containing 0.2mmol/L and 5mmol/L K ¹⁵ NO ₃ (except for the change of N source, other components remain unchanged), soak in saturated CaSO ₄ for 1 minute, collect Arabidopsis root samples, and analyze them by mass spectrometry. The ¹⁵ N abundance was analyzed and the instantaneous nitrate uptake rate of Arabidopsis roots under different treatment conditions was calculated.

The results are shown in Panel B of Figure 4. Under the condition that 5 mM K ¹⁵ NO ₃ was the nitrogen source, the transgenic line chl1-5-p35S::ZmNRT1.1A (the left side of the B panel in Figure 4) and the transgenic line chl1-5- The instantaneous ¹⁵ N uptake rates of p35S::ZmNRT1.1A (the right side of panel B in Figure 4) were significantly higher than those of chl1-5; under the condition that 0.2 mM K ¹⁵ NO ₃ was the nitrogen source, the transgenic line chl1-5-p35S: :ZmNRT1.1A (the left side of panel B in Figure 4) and the transgenic line chl1-5-p35S::ZmNRT1.1A (the right side of panel B in Figure 4) were significantly higher than those of ^chl1-5 . These results indicate that ZmNRT1; 1A gene or ZmNRT1; 1B gene transfer into Arabidopsis chl1-5 mutant can improve the nitrate uptake capacity of Arabidopsis thaliana.

It was proved that expressing the exogenous maize genes ZmNRT1; 1A and ZmNRT1; 1B in Arabidopsis can enhance the absorption of nitrate in Arabidopsis. Therefore, it has the potential application value of improving the nitrate nitrogen absorption and utilization efficiency of crops.

2. Phenotypic experiments of different nitrogen concentrations in maize materials overexpressing ZmNRT1; 1A transgenic lines and maize materials overexpressing ZmNRT1; 1B transgenic lines

In order to study whether overexpression of ZmNRT1.1A gene or overexpression of ZmNRT1.1B gene can improve the nitrogen absorption efficiency of maize plants, the overexpression vector in Example 2 was used to construct transgenic Arabidopsis, as follows:

The maize hybrid HiII (inbred line A188×B73) was used as the transformation receptor, and p1301-Bar-ZmNRT1.1A was used to transform the maize hybrid _HiII by Agrobacterium-mediated genetic transformation. Generation of transgenic plants. The obtained T ₀ generation transgenic plants were identified by PCR, and the positive ZmNRT1.1A plants were amplified to obtain a 1827bp band. After backcrossing (the backcross parent is PH6WC), selfing, screening and identification, three BC ₃ F ₃ -generation transgenic ZmNRT1.1A lines were obtained: A1-OE, A2-OE, A3-OE. The relative expression level of ZmNRT1.1A was verified by RT-PCR (the control was maize that did not contain the p1301-Bar-ZmNRT1.1A fragment in the progeny of BC ₃ F ₃ self-segregation, that is, the wild-type control BC ₃ F ₃ , the parent of HiII without Transgenic treatment), the primers were PA-4-F and PA-4-R, the results are shown in Figure A of Figure 5, indicating that the relative expression of ZmNRT1.1A gene in the three lines was significantly higher than that of the control.

The maize hybrid HiII (inbred line A188×B73) was used as the transformation receptor, and p1301-Bar-ZmNRT1.1B was used to transform the maize hybrid _HiII by Agrobacterium-mediated genetic transformation. Generation of transgenic plants. The obtained T ₀ generation transgenic plants were identified by PCR, and the positive ZmNRT1.1B plants were amplified to obtain a 1788bp band. After backcrossing (the backcross parent is PH6WC), selfing, screening and identification, three BC ₃ F ₃ -generation ZmNRT1.1B lines were obtained: B1-OE, B2-OE, B3-OE. The relative expression of ZmNRT1.1B was verified by RT-PCR (the control was maize without the p1301-Bar-ZmNRT1.1B fragment in the progeny of BC ₃ F ₃ self-segregation, that is, the wild-type control BC ₃ F ₃ , the parent of HiII without Transgenic treatment), the primers were PB-4-F and PB-4-R, the results are shown in Figure B of Figure 5, indicating that the relative expression of ZmNRT1.1B gene in the three lines was significantly higher than that of the control.

Maize BC ₃ F ₃ generation homozygotes (A1-OE, A2-OE, A3-OE) transfected with ZmNRT1.1A gene in hydroponics were cultured under 0.04mmol/L, 0.4mmol ^/ L, 4mmol/L NO ₃ -conditions After 14 days, the biomass and nitrogen concentration of the plants were measured. The maize that did not contain the p1301-Bar-ZmNRT1.1A fragment in the self-segregated progeny was used as a control.

Maize BC ₃ F ₃ generation homozygotes (B1-OE, B2-OE, B3-OE) transformed into ZmNRT1.1B gene by hydroponics, under the conditions of 0.04mmol/L, 0.4mmol/L, 4mmol/L NO ₃ ^- After culturing for 14 days, the biomass and nitrogen concentration of the plants were measured. The maize that did not contain the p1301-Bar-ZmNRT1.1B fragment in the progeny of self-segregation was used as a control.

Maize hydroponics method: cultured with Hoagland nutrient solution with different NO ₃ ^{-concentrations} (0.04mmol/L, 0.4mmol/L, 4mmol/L) for 14 days, and the nutrient solution was changed every 2 days.

The statistical results of above-ground biomass and nitrogen uptake are shown in Figure 5. Under the condition that 0.2mM or 2mM Ca(NO ₃ ) ₂ was the only nitrogen source, the above-ground biomass and nitrogen content of transgenic lines were not significantly different from wild-type controls. Under the condition of 0.02mM Ca(NO ₃ ) ₂ , the aboveground biomass and nitrogen content of transgenic lines were significantly higher than those of wild-type controls.

These results indicate that increasing the expression of ZmNRT1; 1A gene or ZmNRT1; 1B gene can improve the ability of transgenic maize to absorb and utilize nitrate under low nitrogen environment, thereby improving the nitrogen efficiency of plants in low nitrogen environment. Under normal nitrogen environment, it has no effect on the absorption and utilization of nitrate.

Studies in rice have found that OsNRT1.1A can significantly improve the nitrogen efficiency of rice under both high nitrogen and low nitrogen conditions, and can also promote early maturity under high nitrogen; while OsNRT1.1B has excellent allelic variation, this Allelic variation can also improve nitrogen efficiency in rice under different nitrogen environments. From the evolutionary relationship, ZmNRT1.1A/ZmNRT1.1B gene was found to be the homologous gene of OsNRT1.1A/OsNRT1.1B gene in maize. However, unlike rice, OsNRT1.1a/OsNRT1.1b could improve nitrogen efficiency of rice under different nitrogen environments, while ZmNRT1.1A/ZmNRT1.1B only improved nitrogen efficiency of maize under low nitrogen environment. This reflects the similarity of homologous genes and the specificity between species. The present invention proves that overexpression of ZmNRT1.1A or ZmNRT1.1B gene in maize can increase the biomass and nitrogen content of maize under low nitrogen conditions. Therefore, it has the application value of improving nitrogen utilization efficiency under low nitrogen, and has potential breeding value.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experimentation, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. While the invention has been given particular embodiments, it should be understood that the invention can be further modified. In conclusion, in accordance with the principles of the present invention, this application is intended to cover any alterations, uses or improvements of the invention, including changes made using conventional techniques known in the art, departing from the scope disclosed in this application. The application of some of the essential features can be made within the scope of the following appended claims.

sequence listing

<110> China Agricultural University

<120> Application of nitrate uptake-related proteins in the regulation of nitrate uptake in maize

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gtcgcgcgcc gcgtcagcgg caacccgcac ggcctcaccc cgctgcagcg gatcgccgtc 1320

ggcctcgcgc tctccgtcgt cgccatggcg ggcgccgcgc tcacggaggt ccgccgcctc 1380

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cccgactgcg tgccggcgaa cgggacgcag ctcggggtgc tatacctggg tctgtacctg 480

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ggccgccgct ggggctacgg catctgcgcc gtcggcatcc tgtgcgggct gggcgtcttc 720

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gccgccgtga cggccgccgc ctggagcaag cgcgcgctgc cgctgccgtc cgacccggac 840

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ctgccccaca gcaaggaatg caggttcctg gaccacgcgg ccatcgtcgt cgtcgacggc 960

ggcggcgagt cgtcaccggc ggcgagcaag tgggcgctgt gcacgcggac ggacgtggag 1020

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ggcggcggct cgggcttcct catccccgcg ggctccctca ccgtcttcct catcggctcc 1200

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ggcaacccgc acggcctcac cccgctgcag cgcgtcttcg tcggcctcct cctctccgtc 1320

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Met Val Gly Leu Leu Pro Glu Thr Asn Ala Ala Ala Glu Thr Asp Val

1 5 10 15

Leu Leu Asp Ala Trp Asp Phe Lys Gly Arg Pro Ala Pro Arg Ala Thr

20 25 30

Thr Gly Arg Trp Gly Ala Ala Ala Met Ile Leu Val Ala Glu Leu Asn

35 40 45

Glu Arg Leu Thr Thr Leu Gly Ile Ala Val Asn Leu Val Thr Tyr Leu

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Thr Gly Thr Met His Leu Gly Asn Ala Glu Ser Ala Asn Val Val Thr

65 70 75 80

Asn Phe Met Gly Thr Ser Phe Met Leu Cys Leu Leu Gly Gly Phe Val

85 90 95

Ala Asp Ser Phe Leu Gly Arg Tyr Leu Thr Ile Ala Ile Phe Thr Ala

100 105 110

Val Gln Ala Ser Gly Val Thr Ile Leu Thr Ile Ser Thr Ala Ala Pro

115 120 125

Gly Leu Arg Pro Ala Ser Cys Ser Ala Thr Gly Gly Gly Val Val Gly

130 135 140

Glu Cys Ala Arg Ala Ser Gly Ala Gln Leu Gly Val Leu Tyr Leu Ala

145 150 155 160

Leu Tyr Leu Thr Ala Leu Gly Thr Gly Gly Leu Lys Ser Ser Val Ser

165 170 175

Gly Phe Gly Ser Asp Gln Phe Asp Glu Ser Asp Gly Gly Glu Lys Arg

180 185 190

Gln Met Met Arg Phe Phe Asn Trp Phe Phe Phe Phe Ile Ser Leu Gly

195 200 205

Ser Leu Leu Ala Val Thr Val Leu Val Tyr Val Gln Asp Asn Leu Gly

210 215 220

Arg Arg Trp Gly Tyr Gly Ala Cys Ala Cys Ala Ile Ala Ala Gly Leu

225 230 235 240

Leu Val Phe Leu Ala Gly Thr Arg Arg Tyr Arg Phe Lys Lys Leu Ala

245 250 255

Gly Ser Pro Leu Thr Gln Ile Ala Ala Val Val Val Ala Ala Trp Arg

260 265 270

Lys Arg Arg Leu Pro Leu Pro Ala Asp Pro Ala Met Leu Tyr Asp Val

275 280 285

Asp Val Gly Lys Ala Ala Ala Val Glu Asp Gly Ser Ser Ser Lys Lys

290 295 300

Ser Lys Arg Lys Glu Arg Leu Pro His Thr Asp Gln Phe Arg Phe Leu

305 310 315 320

Asp His Ala Ala Ile Asn Glu Asp Pro Ala Ala Gly Ala Ser Ser Ser

325 330 335

Ser Lys Trp Arg Leu Ala Thr Leu Thr Asp Val Glu Glu Val Lys Thr

340 345 350

Val Ala Arg Met Leu Pro Ile Trp Ala Thr Thr Ile Met Phe Trp Thr

355 360 365

Val Tyr Ala Gln Met Thr Thr Phe Ser Val Ser Gln Ala Thr Thr Met

370 375 380

Asp Arg Arg Val Gly Gly Ser Phe Gln Ile Pro Ala Gly Ser Leu Thr

385 390 395 400

Val Phe Phe Val Gly Ser Ile Leu Leu Thr Val Pro Val Tyr Asp Arg

405 410 415

Leu Val Val Pro Val Ala Arg Arg Val Ser Gly Asn Pro His Gly Leu

420 425 430

Thr Pro Leu Gln Arg Ile Ala Val Gly Leu Ala Leu Ser Val Val Ala

435 440 445

Met Ala Gly Ala Ala Leu Thr Glu Val Arg Arg Leu Arg Val Ala Arg

450 455 460

Asp Ser Ser Glu Ser Ala Ser Gly Gly Val Val Pro Met Ser Val Phe

465 470 475 480

Trp Leu Ile Pro Gln Phe Phe Leu Val Gly Ala Gly Glu Ala Phe Thr

485 490 495

Tyr Ile Gly Gln Leu Asp Phe Phe Leu Arg Glu Cys Pro Lys Gly Met

500 505 510

Lys Thr Met Ser Thr Gly Leu Phe Leu Ser Thr Leu Ser Leu Gly Phe

515 520 525

Phe Val Ser Ser Ala Leu Val Ala Ala Val His Arg Val Thr Gly Asp

530 535 540

Arg His Pro Trp Ile Ala Asn Asp Leu Asn Lys Gly Arg Leu Asp Asn

545 550 555 560

Phe Tyr Trp Leu Leu Ala Ala Val Cys Leu Ala Asn Leu Leu Val Tyr

565 570 575

Leu Val Ala Ala Arg Trp Tyr Lys Tyr Lys Ala Gly Arg Pro Gly Ala

580 585 590

Asp Gly Ser Val Asn Gly Val Glu Met Ala Asp Glu Pro Thr Leu His

595 600 605

<210> 4

<211> 595

<212> PRT

<213> Corn (Zea mays)

<400> 4

Met Ala Ser Val Leu Pro Asp Thr Ala Ser Asp Gly Lys Ala Leu Thr

1 5 10 15

Asp Ala Trp Asp Tyr Lys Gly Arg Pro Ala Ser Arg Ala Thr Thr Gly

20 25 30

Gly Trp Ala Cys Ala Ala Met Ile Leu Gly Ala Glu Leu Phe Glu Arg

35 40 45

Met Thr Thr Leu Gly Ile Ala Val Asn Leu Val Pro Tyr Met Thr Gly

50 55 60

Thr Met His Leu Gly Asn Ala Ser Ala Ala Asn Thr Val Thr Asn Phe

65 70 75 80

Ile Gly Ala Ser Phe Met Leu Cys Leu Leu Gly Gly Phe Val Ala Asp

85 90 95

Thr Tyr Leu Gly Arg Tyr Leu Thr Ile Ala Ile Phe Thr Ala Val Gln

100 105 110

Ala Thr Gly Val Met Ile Leu Thr Ile Ser Thr Ala Ala Pro Gly Leu

115 120 125

Arg Pro Pro Ala Cys Ala Asp Ala Lys Gly Ala Ser Pro Asp Cys Val

130 135 140

Pro Ala Asn Gly Thr Gln Leu Gly Val Leu Tyr Leu Gly Leu Tyr Leu

145 150 155 160

Thr Ala Leu Gly Thr Gly Gly Leu Lys Ser Ser Val Ser Gly Phe Gly

165 170 175

Ser Asp Gln Phe Asp Glu Ala His Gly Gly Glu Arg Lys Arg Met Leu

180 185 190

Arg Phe Phe Asn Trp Phe Tyr Phe Phe Val Ser Ile Gly Ala Leu Leu

195 200 205

Ala Val Thr Val Leu Val Tyr Val Gln Asp Asn Val Gly Arg Arg Trp

210 215 220

Gly Tyr Gly Ile Cys Ala Val Gly Ile Leu Cys Gly Leu Gly Val Phe

225 230 235 240

Leu Leu Gly Thr Arg Arg Tyr Arg Phe Arg Lys Leu Val Gly Ser Pro

245 250 255

Leu Thr Gln Val Ala Ala Val Thr Ala Ala Ala Trp Ser Lys Arg Ala

260 265 270

Leu Pro Leu Pro Ser Asp Pro Asp Met Leu Tyr Asp Val Asp Asp Ala

275 280 285

Ala Ala Ala Gly Ala Asp Val Lys Gly Lys Glu Lys Leu Pro His Ser

290 295 300

Lys Glu Cys Arg Phe Leu Asp His Ala Ala Ile Val Val Val Asp Gly

305 310 315 320

Gly Gly Glu Ser Ser Pro Ala Ala Ser Lys Trp Ala Leu Cys Thr Arg

325 330 335

Thr Asp Val Glu Glu Val Lys Gln Val Val Arg Met Leu Pro Ile Trp

340 345 350

Ala Thr Thr Ile Met Phe Trp Thr Ile His Ala Gln Met Thr Thr Phe

355 360 365

Ser Val Ala Gln Ala Glu Val Met Asp Arg Ala Leu Gly Gly Gly Ser

370 375 380

Gly Phe Leu Ile Pro Ala Gly Ser Leu Thr Val Phe Leu Ile Gly Ser

385 390 395 400

Ile Leu Leu Thr Val Pro Val Tyr Asp Arg Leu Leu Ala Pro Leu Ala

405 410 415

Arg Arg Leu Thr Gly Asn Pro His Gly Leu Thr Pro Leu Gln Arg Val

420 425 430

Phe Val Gly Leu Leu Leu Ser Val Ala Gly Met Ala Val Ala Ala Leu

435 440 445

Val Glu Arg His Arg Gln Val Ala Ser Gly His Gly Ala Thr Leu Thr

450 455 460

Val Phe Leu Leu Met Pro Gln Phe Val Leu Val Gly Ala Gly Glu Ala

465 470 475 480

Phe Thr Tyr Met Gly Gln Leu Ala Phe Phe Leu Arg Glu Cys Pro Lys

485 490 495

Gly Met Lys Thr Met Ser Thr Gly Leu Phe Leu Ser Thr Cys Ala Leu

500 505 510

Gly Phe Phe Phe Ser Thr Leu Leu Val Thr Ile Val His Lys Val Thr

515 520 525

Ala His Ala Gly Arg Asp Gly Trp Leu Ala Asp Asn Leu Asp Asp Gly

530 535 540

Arg Leu Asp Tyr Phe Tyr Trp Leu Leu Ala Val Ile Ser Ala Ile Asn

545 550 555 560

Leu Val Leu Phe Thr Phe Ala Ala Arg Gly Tyr Val Tyr Lys Glu Lys

565 570 575

Arg Leu Ala Asp Ala Gly Ile Glu Leu Ala Asp Glu Glu Ser Ile Ala

580 585 590

Val Gly His

595

Claims (10)

1. the application of nitrate absorption-related protein or regulating and controlling the nitrate absorption-related protein content or active substance in regulating plant to nitrate absorption or in the preparation of regulating plant to nitrate absorption product, and described gene encodes nitrate absorption-related protein, the nitrate absorption-related protein is the following A1, A2 or A3 protein: A1. The amino acid sequence is a protein of the amino acid sequence shown in either Sequence 3 or Sequence 4 in the sequence listing; A2. The amino acid sequence shown in either Sequence 3 or Sequence 4 in the Sequence Listing is 80% identical to the protein shown in A1) obtained by substitution and/or deletion and/or addition of one or more amino acid residues A protein that is identical to the above and is associated with plant powdery mildew resistance and/or sheath blight resistance; A3. A fusion protein obtained by linking a protein tag to the N-terminus or/and C-terminus of A1) or A2). 2. The application according to claim 1, wherein the plant is a monocotyledonous plant or a dicotyledonous plant. 3. The application according to claim 2, wherein the monocotyledonous plant is a gramineous plant, and the dicotyledonous plant is a cruciferous plant. 4. The application according to claim 3, wherein the grasses are maize, and the crucifers are Arabidopsis. 5. The application of a substance that regulates gene expression in regulating the absorption of nitrate by a plant or in the preparation of a product for regulating the absorption of nitrate by a plant, wherein the gene encodes the nitrate absorption-related protein of claim 1. 6. The application according to claim 5, wherein the material for regulating gene expression is a biological material related to the nitrate absorption-related protein; the biological material is any of the following C1-C3 A sort of: C1, a nucleic acid molecule encoding the nitrate absorption-related protein of claim 1; C2, the nucleic acid molecule that improves the expression of the nitrate absorption-related protein of claim 1; C3. An expression cassette, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ comprising the nucleic acid molecule described in C1 or C2. 7. application according to claim 6 is characterized in that, the nucleic acid molecule described in C1 is the gene shown in following D1 or D2: D1. The coding sequence of the coding strand is the DNA molecule shown in Sequence Listing Sequence 1 or Sequence Listing Sequence 2; D2. The nucleotide sequence is the DNA molecule shown in Sequence Listing Sequence 1 or Sequence Listing Sequence 2. 8. A plant agent, characterized in that it contains the protein of claim 1 or the substance that regulates gene expression according to any one of claims 5-7, and the plant agent is a plant agent that promotes the absorption and utilization of nitrogen by plants. 9. A method for promoting the absorption of nitrate by a plant, comprising the steps of: improving the expression of the nitrate absorption-related protein of claim 1 in the recipient plant, improving the nitrate absorption-related protein of claim 1 The abundance of the target plants was obtained with a higher nitrate uptake rate than the recipient plants in a low nitrogen environment. 10 . The method according to claim 9 , comprising introducing the nucleic acid encoding the protein of claim 1 into a recipient plant to obtain a target plant with a higher nitrate absorption rate than the recipient plant. 11 .

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