CN114920810B - 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 the gene for coding the nitrate absorption related protein into the corn prove that the overexpression related gene remarkably promotes the absorption and utilization of the nitrogen by the corn in a low-nitrogen environment, so that the overexpression related gene is related to the absorption and utilization of the nitrogen, can be used for promoting the absorption and utilization of the nitrogen by plants and improving the yield of the plants.

Description

Application of nitrate absorption related protein in regulation and control of nitrate absorption of corn

Technical Field

The invention relates to application of nitrate absorption related protein in regulating and controlling the absorption of nitrate by corn in the field of biotechnology.

Background

Nitrogen is one of the most essential and required mineral elements in the plant growth and development process, and accounts for about 1% -5% of the dry weight of the plant (Li et al, 2009). Among all the essential nutrients, nitrogen is the primary factor limiting plant growth and yield development. Nitrogen plays a central role in plant cell, tissue, organ and yield establishment and is therefore called a vital element. Nitrogen absorbed by plants is metabolized in the body by assimilation 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 world's major crops and is widely cultivated in many countries. As one of the key crops, corn has long been a basic food source and a major animal feed ingredient for human survival; in addition, the processing of a plurality of living goods is less, and corn is not used as a raw material; in recent years, the production of ethanol fuel from corn is becoming a major trend in the development of new energy. These urgent needs are to improve corn planting area and specific yield; the improvement of the corn yield needs a large amount of investment of nitrogen fertilizer, so the improvement of the nitrogen efficiency of the corn is necessary. Corn is a dry-land crop and mainly absorbs and utilizes soil nitrate nitrogen. The absorption of nitrate from soil by plants is the first step in the assimilation and utilization of nitrate nitrogen, and the research on the process is the key to improving the absorption and utilization capacity of nitrogen in crops, which is helpful for finding out a limiting factor for the efficient utilization of nitrogen fertilizer (Garnett et al, 2013, plettet al, 2010. The research on the process and rule of the absorption and utilization of the nitrate by the corn can provide theoretical basis and gene resources for the cultivation of new corn varieties with high nitrogen efficiency by molecular means.

Nitrate nitrogen is used as a nutrient element and also as a signal, and has important influence on the metabolism and growth of plants. NO in soil ₃ ^- The concentration levels varied widely, with a degree of difference of up to 100-fold (Lark et al, 2004). The plant root system needs different nitrate absorption systems to deal with the soil NO ₃ ^- The concentration level is varied to efficiently absorb nitrogen. In the model plant Arabidopsis thaliana, the AtNRT1.1 gene encodes a double-affinity nitrate transporter that switches between high-affinity and low-affinity nitrate transport activities by sensing the nitrate concentration level in the environment. The alteration of protein nitroaffinity is regulated by phosphorylation of threonine 101 (Thr) on the amino acid sequence (Martin et al, 2008). When the concentration of external nitrate is low, thr101 is phosphorylated, so that AtNRT1.1 is promoted to have the activity of high-affinity nitrate transport; when the external nitrate concentration is high, thr101 dephosphorylates, so that AtNRT1.1 has low-affinity nitrate transport activity.

OsNRT1.1B is found to improve the utilization efficiency of nitrogen fertilizer of japonica rice by the problem group of the Chinese academy of sciences. The high nitrogen utilization efficiency gene OsNRT1.1B is cloned from indica rice through a map-based cloning strategy, and only one amino acid difference (T327M) exists between indica rice and japonica rice in OsNRT1.1B, so that indica rice has higher nitrate absorption and transport activity than japonica rice. The indica type OsNRT1.1B is introduced into japonica rice varieties, and the results of field experiments show that the japonica rice varieties containing the indica type OsNRT1.1B increase the yield by 30-33% and improve the utilization efficiency of nitrogen fertilizers by 30% compared with a control under the condition of half fertilization; under the normal nitrogen applying condition, the yield is increased by 7-13%, and the utilization efficiency of the nitrogen fertilizer is improved by about 10%.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the absorption and utilization of nitrogen of 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 nitrate absorption of plants or in preparing a product for regulating the nitrate absorption of plants, wherein the gene encodes the nitrate absorption-related protein, and the nitrate absorption-related protein is a protein of A1, A2 or A3 as follows:

a1, protein of which the amino acid sequence is shown in any one of a sequence 3 or a sequence 4 in a sequence table;

a2, protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in any one of the sequence 3 or the sequence 4 in the sequence table, has the identity of more than 80 percent with the protein shown in A1) and is related to the resistance of powdery mildew and/or the resistance of sheath blight of plants;

a3, and a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

In the application, the sequence 3 in the sequence table consists of 608 amino acid residues, and the sequence 4 in the sequence table 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 web page of the NCBI home web site. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, per residual Gap cost, and Lambda ratio to 11,1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

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

In the above application, the nitrate uptake-associated protein may be derived from corn.

In the above application, the plant is a monocotyledon or dicotyledon; further, the monocotyledon is a gramineae plant, and the dicotyledon is a cruciferae plant; further, the gramineous plant is corn and the cruciferous plant is arabidopsis thaliana.

The second purpose of the invention is the application of the substance for regulating and controlling the expression of the gene in regulating and controlling the absorption of the nitrate by the plant or in preparing the product for regulating and controlling the absorption of the nitrate by the plant, and the gene codes the protein related to the absorption of the nitrate.

In the above application, the substance for regulating gene expression may be a substance for regulating at least one of the following 6 kinds of regulation: b1 Regulation at the level of transcription of said gene; b2 Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); b3 Regulation of the RNA transport of the gene (i.e., regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); b4 Control of translation of the gene; b5 Regulation of mRNA degradation of the gene; b6 Post-translational regulation of the gene (i.e., regulation of the activity of a protein translated from the gene).

In the above application, the substance 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, nucleic acid molecules encoding the nitrate uptake-associated protein;

c2, nucleic acid molecules for improving the expression of the nitrate uptake-associated protein;

c3, an expression cassette containing the nucleic acid molecule described in C1 or C2, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ.

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

In the above application, the nucleic acid molecule of C1 may specifically be a gene represented by D1 or D2:

d1, the coding sequence (ORF) of the coding chain is a DNA molecule shown in sequence 1 of the sequence table or sequence 2 of the sequence table;

d2, the nucleotide sequence is a DNA molecule shown in sequence 1 of the sequence table or sequence 2 of the sequence table.

In the above application, the regulating gene expression may be increasing the gene expression.

In order to solve the above-mentioned problems, the present invention provides a plant agent for promoting the uptake of nitrate into plants, comprising the above-mentioned nitrate uptake-associated protein or the above-mentioned substance that regulates gene expression, wherein the plant agent is a plant agent for promoting the uptake and utilization of nitrogen into plants.

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

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

The method specifically comprises introducing a nucleic acid encoding the protein into a recipient plant to obtain a target plant having a higher nitrate uptake rate than the recipient plant.

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

In the above method, the plant is a monocotyledon or a dicotyledon; further, the monocotyledon is a gramineae plant, and the dicotyledon is a cruciferae plant; further, the graminaceous plant is corn and the cruciferous plant is arabidopsis thaliana.

The invention provides an application of a substance for regulating gene expression in improving the absorption of plants to nitrate or an application in preparing a product for improving the absorption of corn to nitrate, wherein the gene codes nitrate absorption related protein and is ZmNRT1.1A gene and ZmNRT1.1B gene. Transgenic experiments for respectively introducing the ZmNRT1.1A gene and the ZmNRT1.1B gene into arabidopsis thaliana and corn prove that transgenic plants over expressing the ZmNRT1.1A gene or the ZmNRT1.1B gene remarkably promote the absorption and utilization of nitrate nitrogen in corn under a low-nitrogen environment compared with receptor plants, and therefore the ZmNRT1.1A gene and the ZmNRT1.1B gene are genes related to the absorption and utilization of nitrogen, and the ZmNRT1.1A gene and the ZmNRT1.1B gene can be used for promoting the absorption and utilization of nitrogen in plants and improving the yield of the plants.

Drawings

FIG. 1 is a graph of colinearity map analysis and phylogenetic tree analysis of ZmNRT1.1A/B and other homologous genes derived from Gramineae in example 1. Wherein, A in FIG. 1 is colinear map analysis, and B in FIG. 1 is a phylogenetic tree analysis diagram.

FIG. 2 is the expression profile of the ZmNRTT 1.1A/B gene in example 1 restored to supply nitrate for various periods of time after nitrogen deficiency in maize B73 and the expression level analysis of long-term culture at various nitrate concentrations. Wherein, A in figure 2 is the expression characteristic of the ZmNRT1.1A/B gene for recovering the nitrate supply for different time lengths after the nitrogen deficiency of the maize B73; FIG. 2B is a graph showing an analysis of the expression level of ZmNRT1.1A/B gene in maize B73 cultured for a long period at different nitrate concentrations; the transcriptional level expression of the ZmNRT1.1A/B gene in maize B73 was analyzed by fluorescent real-time quantitative PCR (qPCR).

FIG. 3 is the result of amplification of the open reading frame to obtain intact maize ZmNRT1.1A/B in example 2. In the figure, the right lane M is a DNA molecular weight standard, and the left lane is the amplification results of ZmNRT1.1A and ZmNRT1.1B open reading frame sequences in this order.

FIG. 4 shows the RT-PCR molecular validation results and the contents of high and low nitrogen in the transgenic Arabidopsis plants of example 3 over-expressing ZmNRTT 1.1A gene or over-expressing ZmNRTT 1.1B gene ¹⁵ NO ₃ ^- And (5) instantly absorbing the result. FIG. 4A is a diagram showing the gene expression level of a transgenic line; FIG. 4B is a graph showing the transgenic lines at different nitrogen concentrations (0.2 mM KNO) ₃ 、5mM KNO ₃ ) The nitrate uptake rate of (a). Wherein chl1-5-p35S, zmNRT1.1A #4, chl1-5-p35S, zmNRT1.1A #11, chl1-5-p35S, zmNRT1.1A #13 belongs to Chl1-5 background and the Arabidopsis thaliana of ZmNRT1.1A gene is transferredThe strain chl1-5-p35S is 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 strain Chl1-5-p35S of ZmNRT1.1B gene under the Chl1-5 background, zmNRT1.1B. Data are presented as mean ± sd, number of repeats is 4, and different lower case letters indicate significant differences.

FIG. 5 shows the gene expression level and physiological phenotype of maize plants over-expressing ZmNRT1.1A gene or over-expressing ZmNRT1.1B gene. FIG. A shows the gene expression level of ZmNRT1.1A transformed maize plants; panel B shows gene expression levels of ZmNRT1.1B transgenic maize plants; FIG. C is the statistics of aboveground biomass (g/strain) of ZmNRTT 1.1A transformed maize line; FIG. D shows the statistics of aboveground biomass (g/strain) of ZmNRTT 1.1B maize line; FIG. E shows the statistics of nitrogen uptake (mg/strain) in the aerial part of ZmNRTT 1.1A maize line; FIG. F shows the statistics of the nitrogen uptake (mg/strain) in the aerial part of ZmNRTT 1.1B maize line. Wherein, A1-OE, A2-OE and A3-OE are ZmNRT1.1A transgenic corn strains, and B1-OE, B2-OE and B3-OE are ZmNRT1.1B transgenic corn strains. WT is a wild-type strain; data are expressed as mean ± sd, with 12 repeats, indicating a significance analysis of P < 0.05.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are all conventional biochemical reagents and are commercially available unless otherwise specified.

In the following examples, unless otherwise specified, the 1 st 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 nucleotide of the corresponding DNA/RNA.

1. Carrier

The cloning vector pGEM T-Easy in the examples below is a product of Promega.

The following examples plant expression vectors pPTKan and pCAMBIA1301 are described in non-patent literature "Sun et al," Selective Mobility and Sensitivity to SNAREs Is inhibited by the Arabidopsis KAT1K1 Channel at the plasmid Membrane,2006, plant Cell 18. The public is available from the university of agriculture in China to repeat the experiments of the present application and is not available for other uses.

The vector pPT-Hyg in the following examples is described in non-patent document "Gu et al," Characterisation of AMT-mediated High-affinity Ammonium upstake in Roots of Maize,2013, plant Cell physiology 1515-1524". The public is available from the university of agriculture in China to repeat the experiments of the present application and not for other uses.

The vectors p1301-UBI-PPT in the following examples are described in non-patent literature "ZHao et al, over Expression of the same Maize ZmAMT1;1a Gene enzymes Root Ammonium Uptake Efficiency Under Low Ammonium Nutrition,2018, plantaBiotechnology Reports 12. The public is available from the university of agriculture in China to repeat the experiments of the present application and is not available for other uses.

2. Strain of bacillus

Escherichia coli DH 5. Alpha. In the following examples belongs to Escherichia coli (Escherichia coli) and is purchased from Tiangen Biochemical technology Ltd.

The Agrobacterium tumefaciens strains GV3101 and EHA105 in the examples described below both belong to Agrobacterium tumefaciens (Agrobacterium tumefaciens) and are both available from Tiangen Biochemical technology Ltd.

3. Plant lines

The Arabidopsis thaliana CHL1-5 mutants in the following examples are described in non-patent literature "Ho et al, CHL1 Function as a Nitrate Sensor in Plants,2009, cell:1184-1194". The public is available from the university of agriculture in China to repeat the experiments of the present application and not for other uses.

Arabidopsis thaliana Col-0 wild-type in the following examples is described in non-patent literature "Ho et al, CHL1 Function as a Nitrate Sensor in Plants,2009, cell:1184-1194". The public is available from the university of agriculture in China to repeat the experiments of the present application and is not available for other uses.

Maize inbred line B73 in The following examples is described in non-patent literature "Schnable et al, the B73 Maize Genome: complexity, diversity, and Dynamics,2009, science 1112-1114.". The public is available from the university of agriculture in China to repeat the experiments of the present application and not for other uses.

The Maize hybrid HiII in the following examples is described in non-patent document "An et al," transactional Regulation of Expression of Maize Aldehyde Dehydrogene 7gene (ZmALDH 7B 6) in stress to Absolute Stresses,2014, journal of interstitial agricultural residues 1900-1908 ". The public is available from the university of agriculture in China to repeat the experiments of the present application and is not available for other uses.

The maize variety PH6WC in the following examples is an inbred line from du pont pioneer company, described in non-patent document "liu jun et al, pioneer yu 335 parent line PH6WC,2010, new agriculture: 30". The public is available from germplasm repository or from the university of Chinese agriculture to repeat the experiments of the present application and not for other uses.

4. Reagent

In the following examples, each restriction enzyme is a product of Promega corporation; the cDNA library construction kit is a product of invitrogen company; various Taq enzyme and Trizol RNA small-amount extraction kits are products of Takara company; the dNTP mixture is a Shanghai biological product; t4 DNA ligase is a product of Promega corporation; ampicillin (Amp), kanamycin (Kan), spectinomycin (Spe), and rifampin (Rif) are all products of Xinjing Corp.

In the following embodiment, the Hoagland nutrient solution for the water culture of arabidopsis thaliana 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 is dissolved in sterile water to obtain a solution with pH of 5.8-6.0. In Hoagland nutrient solution for hydroponics of arabidopsis thaliana, K ₂ SO ₄ Has a concentration of 0.25mM, mgSO ₄ .7H ₂ The concentration of O is 1mM, KH ₂ PO ₄ At a concentration of 1mM, H ₃ BO ₃ Has a concentration of 30 μ M and MnSO ₄ .H ₂ The concentration of O is 5 μ M, znSO ₄ .7H ₂ The concentration of O is 1 mu M and CuSO ₄ .5H ₂ The concentration of O is 1 μ M and Na ₂ MoO ₄ .2H ₂ O concentration of 1. Mu.M, naFe-EDTA concentration of 0.1mM, NH ₄ NO ₃ In a concentration of 2mM, caCl ₂ .2H ₂ The concentration of O was 0.25mM.

In the following embodiment, the Hoagland nutrient solution for corn water culture 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 (for balancing calcium ions) in sterile water to obtain a solution with pH of 5.8-6.0. In Hoagland nutrient solution for corn water culture, K ₂ SO ₄ Is 1mM, mgSO ₄ .7H ₂ The concentration of O was 0.6mM, KH ₂ PO ₄ Is in a concentration of 0.1mM ₃ BO ₃ Has a concentration of 30. Mu.M, mnSO ₄ .H ₂ The concentration of O is 0.5 mu M, znSO ₄ .7H ₂ O concentration of 0.5. Mu.M, cuSO ₄ .5H ₂ The concentration of O is 0.2. Mu.M, na ₂ MoO ₄ .2H ₂ O concentration was 0.07. Mu.M, naFe-EDTA concentration was 0.1mM, ca (NO) ₃ ) ₂ In accordance with the concentration of NO ₃ ^- The final concentration of (C) is set to be 0.04mmol/L, 0.4mmol/L and 4mmol/L, caCl ₂ .2H ₂ O is used to balance calcium ions.

5. PCR amplification primer

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'；

P-A-2-F:5'-CTCTAGAATGGTCGGACTCCTCCCCC-3' (the underlined sequence is the Xba I enzyme recognition site sequence);

P-A-2-R:5'-CCGCTCGAGTCAGTGGAGCGTGGGCT-3' (the sequence underlined is the Xho I enzyme recognition site sequence);

P-B-2-F:5'-CTCTAGAATGGCCTCCCGTCCTGCGG-3' (the sequence underlined is the Xba I enzyme recognition site sequence);

P-B-2-R:5'-CCGCTCGAGTCAGTGGCCGACGGCAATAG-3' (the sequence indicated by underlining is the Xho I enzyme recognition site sequence);

P-A-3-F:5'-TCGAGCTCATGGTCGGACTCCTCCCCC-3' (the underlined sequence is the Sac I enzyme recognition site sequence);

P-A-3-R:5'-TCGAGCTCTCAGTGGAGCGTGGGCT-3' (the sequence indicated by underlining is the sequence of the SacI enzyme recognition site);

P-B-3-F:5'-CGGGATCCATGGCCTCCGTCTGCCG-3' (the sequence indicated by underlining is a BamH I enzyme recognition site sequence);

P-B-3-R:5'-CGGGATCCTCAGTGGCCGACGGCAATAG-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 Peking Ongkoku corporation.

In the following examples, unless otherwise specified, the 1 st 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.

The quantitative tests in the following examples, unless otherwise specified, were performed in triplicate and the results averaged to give a significance analysis result of P < 0.05.

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

1. ZmNRT1;1s Gene evolution analysis

Currently reported nrt1.1s genes homologous to the model plant atnrt1.1 gene are 4 in maize (zmnrt 1.1 a/B/C/D), 3 in rice (osnrt 1.1 a/B/C), 3 in sorghum (sbnrt 1.1 a/B/C), and 4 in brachypodium distachyon (bdnrt 1.1 a/B/C/D) (Plett et al, 2010, the PLoS One). Searching gene information in a Phytozome/MaizeGDB database to obtain the nucleotide sequence, the amino acid sequence and the corresponding gene expression data of ZmNRT1.1A/B/C/D. Transcriptome data show that the gene ZmNRT1.1A/B is mainly expressed in root systems, and the gene ZmNRT1.1C/D has extremely low expression level and is possibly a redundant gene.

FIG. 1 shows the co-linear mapping analysis (A in FIG. 1) and the evolutionary tree analysis (B in FIG. 1) of ZmNRTT 1.1 s. From the colinear maps, zmNRTT 1.1B and ZmNRTT 1.1D are generated by whole genome duplication in the course of evolution, while ZmNRTT 1.1B and ZmNRTT 1.1C are generated by genome tandem recombination. As can be seen from the phylogenetic tree analysis chart, zmNRTT 1.1B has higher homology with OsNRT1.1B; zmNRTT 1.1A has a high homology with OsNRT1.1A.

2. Fluorescent Real-Time quantitative PCR (Real-Time PCR) analysis of expression pattern of ZmNRT1.1s gene of corn root system

Two sets of experimental treatments were performed:

(1) Firstly, 2mM NH is carried out on the maize inbred line B73 plant ₄ NO ₃ Culturing in water solution for 8 days, then lack nitrogen for 4 days, and respectively recovering 4mM KNO ₃ 1 hour/3 hours/6 hours/18 hours/72 hours, and harvesting the corn root system with liquid nitrogen.

(2) The maize inbred line B73 plants were planted at 0mM/0.04mM/4mM/10mM KNO, respectively ₃ Culturing in water solution for 12 days, and collecting corn root system with liquid nitrogen.

The Trizol method is used for extracting the total RNA of the root of each treated corn. The total RNA is subjected to DNA removal by a reverse transcription kit and then is subjected to reverse transcription to form cDNA. Amplifying the corn ZmNRT1 by using primers P-A-4-F and P-A-4-R by using a Real-time PCR method; 1A gene, and amplifying corn ZmNRT1 by using primers P-B-4-F and P-B-4-R; 1B gene, the expression level of ZmNRT1.1A/ZmNRT1.1B gene in the same sample divided by the expression level of ZmTUB1 gene as the relative expression level of ZmNRT1.1A/ZmNRT1.1B gene, using PT-F and PT-R amplified maize ZmTUB1 gene as internal reference control, to study the expression characteristics of ZmNRT1.1A/ZmNRT1.1B gene in different tissue sites of maize.

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

1. total RNA was extracted from different samples using Trizol kit (TaKaRa, cat: 9109).

2. Mu.g of total RNA was collected and genomic DNA was removed using a reverse transcription kit (TaKaRa, cat: RR 047A) in the following manner:

reaction 1 (10 μ l):

5x gDNA Eraser Buffer 2μl

gDNA Eraser 1μl

RNase Free dH ₂ o to a final volume of 10. Mu.l;

the reaction was carried out at 42 ℃ for 5 minutes.

3. The first strand cDNA is synthesized by reverse transcription in the conventional manner as follows:

reaction 2 (20 μ l):

reacting at 42 ℃ for 60 minutes; the reaction was carried out at 95 ℃ for 5 minutes.

4. The Real-time PCR is used for detecting gene abundance, SYBR Green Real PCR Master Mix (catalog number: RR 820A) of TAKARA company and quantitative PCR instrument model IQ5 are selected as reagents, and the reverse product is diluted by 10 times and used as a Real-time PCR template

Reaction system:

PCR reaction procedure: 2 minutes at 50 ℃,2 minutes at 95 ℃,40 cycles (15 seconds at 95 ℃, 15 seconds at 61 ℃,30 seconds at 72 ℃);

a curve melting step: 95 ℃ for 15 seconds, circulating for 10 seconds, increasing the temperature from 60 ℃ to 95 ℃ at a speed of 0.5 ℃ per circulation, and performing 70 cycles;

and (3) calculating the relative expression quantity of the ZmNRT1.1s gene in different tissue parts of the corn by using ZmTUB1 as an internal reference and adopting a relative quantitative algorithm. Results are shown in figure 2, maize ZmNRT1;1B gene is induced by nitrate, zmNRT1; the 1A gene was not induced by nitrate. Corn ZmNRT1;1A gene and ZmNRT1; the expression level of 1B gene increases with the increase of nitrogen concentration.

Example 2, zmNRT1; cloning of 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, a primer is designed: the primer pair for amplifying ZmNRT1.1A consists of an upstream primer P-A-1-F and a downstream primer P-A-1-R. The primer pair for amplifying ZmNRT1.1B consists of an upstream primer P-B-1-F and a downstream primer P-B-1-R. Wherein each of P-A-1-F and P-B-1-F comprises exactly the translation initiation codon ATG, and each of P-A-1-R and P-B-1-R comprises a sequence complementary to the reverse of the translation termination codon TGA.

Using cDNA reverse-transcribed from total RNA of corn roots (see step (2) of example 1) as a template, amplifying using Hi-Fi enzyme Primer STAR (TaKaRa Co., cat.: R010Q) to obtain ZmNRT1 containing intact open reading frames, respectively; 1A gene fragment and ZmNRT1;1B gene fragment.

The amplification system was as follows:

supplement H ₂ O to 50. Mu.l.

And (3) PCR reaction conditions: pre-denaturation at 98 ℃ for 2 min; then 30 cycles of 10 seconds at 98 ℃,10 seconds at 65 ℃ and 3 minutes and 30 seconds at 72 ℃; then 1. Mu.l of Taq enzyme was added, extended at 72 ℃ and tailed for 20 minutes.

Taking 8 mul of PCR products, carrying out electrophoresis detection on 1.0% agarose gel, wherein the detection result is shown in figure 2, the mRNA sequence of ZmNRT1.1A consists of 2492 bases, the Open Reading Frame (ORF) is 326-2153 bases from the 5' end, and the fragment size is 1827bp; the mRNA sequence of ZmNRT1.1A consists of 2286 bases, the Open Reading Frame (ORF) thereof is 187-1975 bases from the 5' end, and the fragment size is 1788bp. The electrophoresis results are shown in FIG. 3, wherein the right lane M is a DNA molecular weight standard, and the left lane is the amplification results of ZmNRTT 1.1A Open Reading Frame (ORF) sequence and ZmNRTT 1.1B Open Reading Frame (ORF) sequence in this order.

Recovering the target band, connecting the recovered product on a pGEM T-Easy vector, transforming Escherichia coli DH5 alpha competent cells, extracting plasmids and sequencing for verification. Sequencing results show that 1827bp (ZmNRT1.1A) and 1788bp (ZmNRT1.1B) open reading frames are respectively obtained, and the sequence can be completely matched with a sequence obtained from a database on the Internet through comparison. ZmNRT1; the nucleotide sequence of 1A is shown as sequence 1 in the sequence table; the coded amino acid sequence is shown as a sequence 3 in a sequence table. ZmNRT1; the nucleotide sequence of 1B is shown as a sequence 2 in a sequence table; the coded amino acid sequence is shown as a sequence 4 in a sequence table.

2. Construction of plant expression vectors

1. Transgenic arabidopsis ZmNRT1;1A gene over-expression vector (pPT-ZmNRT 1; 1A) and transgenic Arabidopsis ZmNRT1; construction of 1B Gene overexpression vector (pPT-ZmNRT 1; 1B)

Selecting the target gene with correct sequencing in the first step, and re-amplifying the target gene by using an upstream primer containing an Xba I enzyme recognition site sequence and a downstream primer containing an Xho I enzyme recognition site sequence, wherein the specific steps are as follows: amplifying a target gene ZmNRT1 by using P-A-2-F (containing an Xba I enzyme recognition site sequence) and P-A-2-R (containing an Xho I enzyme recognition site sequence); 1A; amplifying a target gene ZmNRT1 by using P-B-2-F (containing an Xba I enzyme recognition site sequence) and P-B-2-R (containing an Xho I enzyme recognition site sequence); 1B. Respectively concentrating the amplification products to 10 mu l, carrying out enzyme digestion by Xba I enzyme, carrying out electrophoresis recovery, and concentrating to 10 mu l to respectively obtain ZmNRT1;1A gene target fragment and ZmNRT1;1B gene target fragment.

At the same time, xba I enzyme digestion of the vector pPT-Hyg is carried out, and linear pPT-Hyg plasmid DNA is obtained by recovery, and then dephosphorylation of the vector is carried out. The specific procedure for dephosphorylation was to add the following reagents to a new centrifuge tube:

linear pPT-Hyg plasmid DNA 5. Mu.g

10x CIPA Reaction Buffer 10μl

Diluted CIAP(0.01U/μl) 5μl

Add water to make up to 100 μ l.

The following procedure was performed in the PCR instrument:

taking the dephosphorylated product obtained in the previous step, adding 100 mu l of Tris saturated phenol and 100 mu l of trichloromethane, mixing and shaking up; centrifuging at 12000 rpm for 10min. Sucking the supernatant into another tube, adding chloroform with the same volume, mixing and shaking up; centrifuging at 12000 rpm for 10min; sucking supernatant into another tube, adding 1/10 volume of 3M ammonium acetate and 3 times volume of anhydrous cold ethanol, mixing uniformly, precipitating at-80 ℃ for 1 hour, precipitating at 4 ℃, rotating at 12000 r, centrifuging for 10min, discarding supernatant, washing with 700 μ l of 70% ethanol once, and dissolving with 10 μ l of water to obtain dephosphorylated vector fragment.

Taking the target fragment after enzyme digestion and the carrier fragment after dephosphorylation to carry out a ligation reaction, wherein the reaction system (10 mu l) is as follows:

connecting at 16 ℃ for 16-20 hours, transferring into 50 mul DH5 alpha competent cells, coating on LB plate containing kanamycin, culturing at 37 ℃ for 12 hours, collecting all thalli, extracting plasmid DNA, performing enzyme digestion verification, and determining a vector with correct sequence for sequencing of a sequencing company, wherein the vector carries a hygromycin resistance screening marker gene.

Wherein, zmNRT1; the vector with correct sequence obtained by transfection after the target fragment of the 1A gene is connected with the vector fragment is pPT-ZmNRT1;1A, with ZmNRT1; the vector with correct sequence obtained by transfection after the target fragment and the vector fragment of the 1B gene are connected is pPT-ZmNRT1;1B.

2. Corn ZmNRT1 is transformed; 1A gene overexpression vector (p 1301-Bar-ZmNRT1.1A) and maize transgenic ZmNRTT 1; construction of 1B Gene overexpression vector (p 1301-Bar-ZmNRT1.1B)

Selecting a plasmid pPT-ZmNRT1 with correct sequencing; 1A, re-amplifying a target gene ZmNRT1 by using primers P-A-3-F and P-A-3-R containing a sequence of a Sac I enzyme recognition site; 1A, recovering a target fragment ZmNRT1;1A. Digesting the target fragment ZmNRT1 by using SacI enzyme; 1A, running electrophoresis recovery. The vector p1301-UBI-PPT was digested with SacI enzyme and recovered, followed by dephosphorylation of the vector.

Selecting a plasmid pPT-ZmNRT1 with correct sequencing; 1B, re-amplifying a target gene ZmNRT1 by using primers P-B-3-F and P-B-3-R containing a BamH I enzyme recognition site sequence; 1B; recovering the target fragment ZmNRT1;1B. Cutting a target fragment ZmNRT1 by using BamH I enzyme; and 1B, running electrophoresis recovery. The vector p1301-UBI-PPT was digested with BamH I and recovered, followed by dephosphorylation of the vector.

Dephosphorizing the carrier, specifically adding the following reagents into a new centrifuge tube:

linear p1301-UBI-PPT plasmid DNA 5. Mu.g

10x CIPA Reaction Buffer 10μl

Diluted CIAP(0.01U/μl) 5μl

Add water to make up to 100 μ l.

The following procedure was performed in the PCR instrument:

taking the dephosphorylated product obtained in the previous step, adding 100 mu l of Tris saturated phenol and 100 mu l of trichloromethane, mixing and shaking up; centrifuging at 12000 rpm for 10min. Sucking the supernatant into another tube, adding chloroform with the same volume, mixing and shaking up; centrifuging for 10min at 12000 rpm; sucking supernatant into another tube, adding 1/10 volume of 3M ammonium acetate and 3 times volume of anhydrous cold ethanol, mixing uniformly, precipitating at-80 ℃ for 1 hour, precipitating at 4 ℃, rotating at 12000 r, centrifuging for 10min, discarding supernatant, washing with 700 μ l of 70% ethanol once, and dissolving with 10 μ l of water to obtain dephosphorylated vector fragment.

Taking the target fragment after enzyme digestion and the carrier fragment after dephosphorylation to carry out a ligation reaction, wherein the reaction system (10 mu l) is as follows:

connecting at 16 ℃ for 16-20 hours, transferring into 50 mul DH5 alpha competent cells, coating on LB plate containing kanamycin, culturing at 37 ℃ for 12 hours, collecting all thalli, extracting plasmid DNA, performing enzyme digestion verification, and determining a vector with correct sequence for sequencing of a sequencing company, wherein the vector carries a hygromycin resistance screening marker gene.

Wherein, the target fragment ZmNRT1;1A and the carrier fragment are connected and transfected to obtain a carrier with correct sequence, namely ZmNRT1;1A gene (shown as a sequence 1 in a sequence table) is inserted into a fragment of a restriction endonuclease SacI enzyme cutting site of a p1301-UBI-PPT vector, and other sequences of the p1301-UBI-PPT vector are kept unchanged to obtain a maize ZmNRT1; the over-expression vector of the 1A gene is named as p1301-Bar-ZmNRT1.1A.

As a target fragment ZmNRT1;1B and the carrier fragment are connected and transfected to obtain a carrier with correct sequence, namely ZmNRT1;1B gene (shown as sequence 2 in a sequence table) is inserted into a fragment of a restriction endonuclease BamH I enzyme cutting site of a p1301-UBI-PPT vector, and other sequences of the p1301-UBI-PPT vector are kept unchanged to obtain a maize ZmNRT1; the over-expression vector of the 1B gene is named as p1301-Bar-ZmNRT1.1B.

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

1. Overexpresses ZmNRT1;1A transgenic arabidopsis material and overexpression ZmNRT1;1B transgenic arabidopsis material ¹⁵ Phenotypic experiment of N transient uptake

In order to study whether the nitrogen absorption efficiency of plants can be improved after over-expressing the ZmNRT1.1A gene or over-expressing the ZmNRT1.1B gene, the transgenic Arabidopsis thaliana is constructed by using the over-expression vector in the embodiment 2, which is specifically as follows:

using chl1-5 mutant of arabidopsis thaliana deletion AtNRT1.1 gene as transformation receptor, adopting p1301-Bar-ZmNRT1.1A to transform chl1-5 mutant by agrobacterium-mediated genetic transformation method, and screening hygromycin resistance to obtain T ₀ Transgenic plants are generated. For the obtained T ₀ PCR identification is carried out on the generation transgenic plant, and a 1827bp band which is a positive transformation ZmNRT1.1A plant is obtained through amplification. The obtained positive ZmNRT1.1A plants are subject to selfing generation-adding, screening and identification to obtain 3T ₃ Generation-to-ZmNRT1.1A line (chl 1-5-p35S:: zmNRT1.1A): chl1-5-p35S, zmNRT1.1A #4, chl1-5-p35S, zmNRT1.1A #11, chl1-5-p35S, zmNRT1.1A #13. RT-PCR is used for verifying the gene expression quantity of ZmNRTT 1.1A (the control is chl 1-5), the primers are P-A-4-F and P-A-4-R, and the result is shown in the left side of the A diagram of FIG. 4, which shows that the relative expression quantity of the ZmNRTT 1.1A gene in 3 strains is obviously higher than that of the control chl1-5.

The method comprises the steps of taking the arabidopsis thaliana chl1-5 mutant as a transformation receptor, adopting an agrobacterium-mediated genetic transformation method, transforming the chl1-5 mutant by utilizing p1301-Bar-ZmNRT1.1B, and screening hygromycin resistance to obtain T ₀ Transgenic plants are generated. For the obtained T ₀ PCR identification is carried out on the generation transgenic plant, and a positive ZmNRT1.1B transgenic plant with 1788bp bands is obtained through amplification. The positive ZmNRT1.1B plant is subjected to selfing generation-adding, screening and identification to obtain 3T ₃ Generation-to-ZmNRT1.1B strain (chl 1-5-p35S:: zmNRT1.1B): chl1-5-p35S, zmNRT1.1B #5, chl1-5-p35S, zmNRT1.1B #13, chl1-5-p35S, zmNRT1.1B #14. RT-PCR is used for verifying the gene expression quantity of ZmNRTT 1.1B (the control is chl 1-5), the primers are P-B-4-F and P-B-4-R, and the result is shown in the right side of the A picture of figure 4, which shows that the relative expression quantity of the ZmNRTT 1.1B gene in 3 strains is obviously higher than that of the control chl1-5.

Water culture transferred ZmNRT1.1A transgenic arabidopsis thaliana T ₃ The generation-pure complexes (chl 1-5-p35S:: zmNRT1.1A #4, chl1-5-p35S:: zmNRT1.1A #11, chl1-5-p35S:: zmNRT1.1A # 13) in ¹⁵ NO ₃ ^- The concentrations of 0.2mmol/L and 5mmol/L NO ₃ ^- And (3) measuring the nitrate absorption rate of the root system under the condition. The chl1-5 mutant and Col-0 wild type were used as controls.

Hydroponic cultureTransgenic Arabidopsis thaliana T transformed with ZmNRT1.1B ₃ The generation-pure complex (chl 1-5-p35S:: zmNRT1.1B #5, chl1-5-p35S:: zmNRT1.1B #13, chl1-5-p35S:: zmNRT1.1B # 14) in ¹⁵ NO ₃ ^- The concentrations of 0.2mmol/L and 5mmol/L NO ₃ ^- And (3) measuring the nitrate absorption rate of the root system under the condition. The chl1-5 mutant and Col-0 wild type were used as controls.

The water culture mode of arabidopsis thaliana: the Arabidopsis thaliana was first hydroponically cultured for 6 weeks in Hoagland nutrient solution, and the nutrient solution was changed every 3 days. Then, the mixture is added with KNO of 0.2mmol/L and 5mmol/L ₃ Hoagland nutrient solution (except for N source change, other components do not change) in culture for 1 week. Then the content of K is 0.2mmol/L and 5mmol/L ¹⁵ NO ₃ Culturing in Hoagland nutrient solution (except N source change, other components are unchanged) for 10min, and adding saturated CaSO ₄ Soaking for 1min, collecting root sample of Arabidopsis thaliana, and measuring by mass spectrometry ¹⁵ And (4) calculating the instantaneous nitrate absorption rate of the root system of the arabidopsis thaliana under different treatment conditions according to the N abundance.

The results are shown in Panel B of FIG. 4 at 5mM K ¹⁵ NO ₃ Of the transgenic line chl1-5-p 35S:ZmNRT1.1A (left side of B diagram in FIG. 4) and of the transgenic line chl1-5-p 35S:ZmNRT1.1A (right side of B diagram in FIG. 4) under nitrogen source conditions ¹⁵ The N instantaneous absorption rates are all obviously higher than chl1-5; at 0.2mM K ¹⁵ NO ₃ Under nitrogen source conditions, of the transgenic line chl1-5-p35S:: zmNRT1.1A (left side of B diagram in FIG. 4) and of the transgenic line chl1-5-p35S:: zmNRT1.1A (right side of B diagram in FIG. 4) ¹⁵ The N instantaneous absorption rate is obviously higher than chl1-5. These results demonstrate that ZmNRT1;1A gene or ZmNRT1; the nitrate absorption capacity of arabidopsis thaliana can be improved by transferring the 1B gene into the arabidopsis thaliana chl1-5 mutant.

Proves that the exogenous maize gene ZmNRT1 is expressed in arabidopsis; 1A and ZmNRT1;1B can enhance the nitrate uptake of Arabidopsis thaliana. Therefore, the method has the application value of potentially improving the absorption and utilization efficiency of the nitrate nitrogen of crops.

2. Overexpresses ZmNRT1;1A transgenic line maize material and overexpressing ZmNRT1; phenotype experiment of 1B transgenic line corn material with different nitrogen concentrations

In order to investigate whether the nitrogen absorption efficiency of maize plants can be improved after over-expressing the ZmNRT1.1A gene or over-expressing the ZmNRT1.1B gene, the transgenic Arabidopsis thaliana was constructed by the over-expression vector in example 2, specifically as follows:

transforming maize hybrid HiII (inbred line A188 xB 73) by using maize hybrid HiII as a transformation receptor and adopting an agrobacterium-mediated genetic transformation method, namely transforming the maize hybrid HiII by using p1301-Bar-ZmNRT1.1A, and screening glyphosate resistance to obtain T ₀ Transgenic plants are generated. For the obtained T ₀ PCR identification is carried out on the generation transgenic plant, and a 1827bp band which is a positive transformation ZmNRT1.1A plant is obtained through amplification. Obtaining 3 BC through backcross (the backcross parent is PH6 WC), selfing, screening and identification ₃ F ₃ Transformation into ZmNRT1.1A strain: A1-OE, A2-OE, A3-OE. RT-PCR was used to verify the relative expression level of ZmNRT1.1A (control is BC) ₃ F ₃ Maize that did not contain the p1301-Bar-ZmNRT1.1A fragment in the progeny isolated by selfing, i.e., wild-type control BC ₃ F ₃ The parent HiII is not treated by transgenosis), the primers are P-A-4-F and P-A-4-R, the result is shown in an A picture of figure 5, and the results show that the relative expression level of the ZmNRT1.1A genes in 3 strains is obviously higher than that of a control.

Transforming maize hybrid HiII (inbred line A188 xB 73) by using maize hybrid HiII as a transformation receptor and adopting an agrobacterium-mediated genetic transformation method, namely transforming the maize hybrid HiII by using p1301-Bar-ZmNRT1.1B, and screening glyphosate resistance to obtain T ₀ Transgenic plants are generated. For the obtained T ₀ PCR identification is carried out on the generation transgenic plant, and a positive ZmNRT1.1B transgenic plant with 1788bp bands is obtained through amplification. Obtaining 3 BC through backcross (the backcross parent is PH6 WC), selfing, screening and identification ₃ F ₃ Transformation into ZmNRT1.1B strain: B1-OE, B2-OE and B3-OE. RT-PCR was used to verify the relative expression level of ZmNRT1.1B (control BC) ₃ F ₃ Maize that did not contain the p1301-Bar-ZmNRT1.1B fragment in the progeny isolated by selfing, wild type control BC ₃ F ₃ The parent HiII is not treated by transgenosis), the primers are P-B-4-F and P-B-4-R, the result is shown in a B picture of figure 5, and the relative expression quantity of the ZmNRT1.1B gene in 3 lines is obviously higher than that of a control line.

Water culture ZmNRT1.1A gene-transferred corn BC ₃ F ₃ The purity of the substituted homozygote (A1-OE, A2-OE, A3-OE) was 0.04mmol/L or 0.4mmol/L/L、4mmol/L NO ₃ ^- The plants were cultured for 14 days under the conditions and the biomass and nitrogen concentration of the plants were measured. Maize that did not contain the p1301-Bar-ZmNRT1.1A fragment in the progeny isolated by selfing was used as a control.

Corn BC with ZmNRTT 1.1B gene transferred in hydroponic culture ₃ F ₃ The generation of pure complex (B1-OE, B2-OE, B3-OE) at 0.04mmol/L, 0.4mmol/L, 4mmol/L NO ₃ ^- The plants were cultured for 14 days under the conditions and the biomass and nitrogen concentration of the plants were measured. Maize that did not contain the p1301-Bar-ZmNRT1.1B fragment in the progeny isolated by selfing was used as a control.

The water culture mode of the corn: with different NO ₃ ^- Hoagland nutrient solution with concentration (0.04 mmol/L, 0.4mmol/L, 4 mmol/L) was cultured for 14 days, and the nutrient solution was changed every 2 days.

The above-ground biomass and nitrogen uptake were measured at 0.2mM or 2mM Ca (NO) as shown in FIG. 5 ₃ ) ₂ Under the condition of a unique nitrogen source, the biomass and the nitrogen content of the overground part of the transgenic line have no obvious difference with wild type contrast; and at 0.02mM Ca (NO) ₃ ) ₂ Under the condition, the biomass and nitrogen content of the overground part of the transgenic line are obviously higher than those of the wild type.

These results demonstrate an increase in ZmNRT1;1A gene or ZmNRT1; the expression of the 1B gene can improve the absorption and utilization capacity of the transgenic corn to nitrate in a low-nitrogen environment, so that the nitrogen efficiency of the plant in the low-nitrogen environment is improved, and the absorption and utilization capacity of the transgenic corn to nitrate in a normal nitrogen environment is not influenced.

Research in rice shows that OsNRT1.1A can remarkably improve the nitrogen efficiency of rice under high-nitrogen and low-nitrogen conditions, and can promote precocity under high nitrogen; osNRT1.1B has excellent allelic variation, and the allelic variation can improve the nitrogen efficiency of rice under different nitrogen environments. The evolutionary relationship shows that the gene ZmNRT1.1A/ZmNRT1.1B is the homologous gene of the gene OsNRT1.1A/OsNRT1.1B in maize. However, unlike rice, osNRT1.1a/OsNRT1.1b can improve nitrogen efficiency of rice under different nitrogen environments, and ZmNRT1.1A/ZmNRT1.1B can improve nitrogen efficiency of corn only under low nitrogen environments. This demonstrates the similarity of homologous genes and specificity between species. The invention proves that the biomass and nitrogen content of corn under the low-nitrogen condition can be improved by over-expressing ZmNRT1.1A or ZmNRT1.1B genes in the corn. Therefore, the method has the application value of improving the utilization efficiency of the nitrogen fertilizer under low nitrogen and has potential breeding value.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

Application of nitrate absorption related protein in regulation and control of nitrate absorption of corn

<130> GNCSY210027

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1827

<212> DNA

<213> corn (Zea mays)

<400> 1

atggtcggac tcctccccga gaccaatgcc gcggcggaga cggacgtcct cctcgacgcc 60

tgggacttca agggccggcc ggccccgcgc gccaccaccg gccgctgggg cgccgccgcc 120

atgatcctag tggcggagct gaacgagcgg ctgacgacgc tgggcatcgc cgtgaacctg 180

gtgacgtacc tgacgggcac catgcacctg ggcaacgccg agtccgccaa cgtcgtcacc 240

aacttcatgg gcacctcctt catgctctgc ctcctcggcg gcttcgtcgc cgactccttc 300

ctcggccgct acctcaccat cgccatcttc accgccgtcc aggcctcggg cgtgacgatc 360

ctgacgatct cgacggcggc gccggggcta cggccggcgt cctgctccgc gaccggcgga 420

ggcgtcgtcg gggagtgcgc gcgggcgtcg ggggcgcagc tgggggtgct gtacctggcg 480

ctgtacctga cggcgctggg cacgggtggg ctaaagtcga gcgtgtcggg gttcgggtcg 540

gaccagttcg acgagtcgga cggcggggag aagcggcaga tgatgcgctt cttcaactgg 600

ttcttcttct tcatctcgct ggggtcgctg ctggccgtca ccgtgctggt gtacgtccag 660

gacaacctgg gcaggcgctg gggctacggc gcctgcgcct gcgccatcgc cgcgggcctc 720

ctcgtcttcc tggccggcac acgcaggtac cgcttcaaga agctggccgg cagccccctc 780

acgcagatcg ccgccgtcgt cgtcgccgcc tggcgcaagc gccgcctccc tctccccgcc 840

gaccccgcca tgctctacga cgtcgacgtc ggcaaggccg ccgccgtcga ggatgggtcc 900

tccagcaaga agagcaagcg caaggagcgc ctcccccaca ccgaccagtt ccgcttcctg 960

gaccacgcgg cgatcaacga ggatccggcg gcgggggcga gcagcagcag caagtggcgg 1020

ctggcgacgc tgacggacgt ggaggaggtg aagacggtgg cgcggatgct gccgatctgg 1080

gcgaccacga tcatgttctg gacggtgtac gcgcagatga ccaccttctc ggtgtcgcag 1140

gccaccacca tggaccgccg cgtcgggggc tcgttccaga tccccgcggg ctccctcacc 1200

gtcttcttcg tcggctccat cctgctcacc gtgcccgtct acgaccgcct ggtggtgccc 1260

gtcgcgcgcc gcgtcagcgg caacccgcac ggcctcaccc cgctgcagcg gatcgccgtc 1320

ggcctcgcgc tctccgtcgt cgccatggcg ggcgccgcgc tcacggaggt ccgccgcctc 1380

cgcgtcgcgc gcgattcctc cgagtccgcc tccggaggcg tcgtgcccat gtccgtgttc 1440

tggctcatcc cgcagttctt cctcgtgggg gcgggcgagg cgttcacgta catcggccag 1500

ctcgacttct tcctgcgcga gtgccccaag gggatgaaga ccatgagcac ggggctgttc 1560

ctcagcaccc tgtcgctggg attcttcgtc agctccgcgc tcgtcgccgc cgtgcacagg 1620

gtcacgggcg accgccaccc ctggatcgcc aacgacctca acaagggccg cctcgacaac 1680

ttctactggc tgctcgccgc cgtctgcctc gccaacctac tagtctacct cgtcgccgcc 1740

cgctggtaca agtacaaggc gggccgcccc ggcgccgacg gcagcgtcaa cggcgtcgag 1800

atggccgacg agcccacgct ccactga 1827

<210> 2

<211> 1788

<212> DNA

<213> Zea mays

<400> 2

atggcctccg tcctgccgga tactgcgtcg gatggcaagg ccttgacgga cgcctgggac 60

tacaagggcc gccccgctag ccgcgccacc accggcggct gggcgtgcgc cgccatgata 120

ctaggcgcgg agctgttcga gcggatgacg acgctgggca tcgcggtgaa cctggtgccg 180

tacatgaccg gcaccatgca cctcggcaat gcctccgccg ccaacaccgt caccaacttc 240

atcggggctt ccttcatgct ctgcctcctc ggcgggttcg tcgccgacac ctacctcggc 300

cgctacctca ccatcgccat cttcaccgcc gtccaggcca cgggggtgat gatcctgacg 360

atctcaacgg ccgctcccgg gctgcgtccg ccggcgtgtg cggacgccaa gggggcgagc 420

cccgactgcg tgccggcgaa cgggacgcag ctcggggtgc tatacctggg tctgtacctg 480

acggcgctgg gcacgggcgg gctcaagtcc agcgtgtcgg gcttcggctc cgaccagttc 540

gacgaggcgc acggcggcga gcgcaagagg atgctgcgct tcttcaactg gttctacttc 600

ttcgtcagca tcggcgcgct gctggccgtc acggtgctgg tgtacgtgca ggacaacgtg 660

ggccgccgct ggggctacgg catctgcgcc gtcggcatcc tgtgcgggct gggcgtcttc 720

ctgctgggca cccggaggta ccggttcagg aagctggtgg ggagcccgct cacccaggtg 780

gccgccgtga cggccgccgc ctggagcaag cgcgcgctgc cgctgccgtc cgacccggac 840

atgctctacg acgtggacga cgcggccgcc gccggcgccg acgtcaaggg gaaggagaaa 900

ctgccccaca gcaaggaatg caggttcctg gaccacgcgg ccatcgtcgt cgtcgacggc 960

ggcggcgagt cgtcaccggc ggcgagcaag tgggcgctgt gcacgcggac ggacgtggag 1020

gaggtgaagc aggtggtgcg gatgctgccc atctgggcca ccaccatcat gttctggacc 1080

atccacgcgc agatgaccac cttctcggtg gcgcaggccg aggtcatgga ccgggccctc 1140

ggcggcggct cgggcttcct catccccgcg ggctccctca ccgtcttcct catcggctcc 1200

atcctgctca ccgtgcccgt ctacgaccgc ctcctggcgc ccctcgcccg ccgcctcacg 1260

ggcaacccgc acggcctcac cccgctgcag cgcgtcttcg tcggcctcct cctctccgtc 1320

gccggcatgg ccgtggccgc gctcgtcgag cgccaccgcc aggtggcctc cggccacggg 1380

gccacgctca cggtgttcct gctcatgccg cagttcgtgc tcgtcggcgc gggcgaggca 1440

ttcacgtaca tgggccagct cgccttcttc ctgcgcgagt gccccaaggg catgaagacc 1500

atgagcacgg gcctgttcct cagcacctgc gcgctcgggt tcttcttcag caccctgctc 1560

gtcaccatcg tgcacaaggt cacggcccac gccggccgtg acggttggct cgccgacaac 1620

ctcgacgacg ggaggctcga ctacttctac tggctgctcg ccgtcatcag cgccatcaac 1680

ctcgtcctct tcacgttcgc cgccaggggc tacgtctata aggagaagcg cctggccgac 1740

gccggcatcg agctcgcaga cgaggagtct attgccgtcg gccactga 1788

<210> 3

<211> 608

<212> PRT

<213> corn (Zea mays)

<400> 3

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

50 55 60

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 (2)

1. A method for promoting nitrate absorption by plants, comprising the steps of: improving the expression of the nitrate absorption related protein in the receptor corn, and improving the abundance of the nitrate absorption related protein to obtain the target corn with the nitrate absorption rate higher than that of the receptor corn in a low-nitrogen environment;

the nitrate absorption related protein is the following protein A1 or A2:

a1, protein of which the amino acid sequence is shown in any one of a sequence 3 or a sequence 4 in a sequence table;

a2, fusion protein obtained by connecting protein labels to the N terminal or/and the C terminal of A1);

NO in the low nitrogen environment ^3- Is 0.04mM.

2. The method of claim 1, comprising introducing a nucleic acid encoding the protein of claim 1 into recipient corn to obtain a corn of interest having a higher nitrate uptake than the recipient corn

The nucleic acid is a gene shown as the following D1 or D2:

d1, the coding sequence of the coding chain is a DNA molecule shown in a sequence 1 or a sequence 2 in a sequence table;

d2, the nucleotide sequence is a DNA molecule shown in sequence 1 of the sequence table or sequence 2 of the sequence table.

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