CN107779468B

CN107779468B - Application of rice NRT1.1A gene and coded protein thereof in breeding for improving plant yield

Info

Publication number: CN107779468B
Application number: CN201610680206.2A
Authority: CN
Inventors: 储成才; 王威; 胡斌; 李华; 张志华; 刘永强
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2016-08-17
Filing date: 2016-08-17
Publication date: 2020-04-28
Anticipated expiration: 2036-08-17
Also published as: WO2018033083A1; CN107779468A

Abstract

The invention discloses a rice NRT1.1A gene and application of a coded protein thereof in breeding for improving plant yield. The application provided by the invention is specifically the application of the protein or the coding gene thereof in regulating and controlling the growth and development of plants; the growth and development are embodied as single plant yield and/or plant height and/or spike grain number and/or flowering time and/or bolting time and/or rosette leaf size; the protein is any one of the following proteins: (1) the amino acid sequence is a protein shown as a sequence 1 in a sequence table; (2) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 in the sequence table, is related to the regulation of the growth and development of plants and is derived from the sequence 1. The invention has important significance for cultivating new varieties of high-yield rice.

Description

Application of rice NRT1.1A gene and coded protein thereof in breeding for improving plant yield

Technical Field

The invention belongs to the technical field of biology, and relates to a rice NRT1.1A gene and application of a protein coded by the gene in breeding for improving plant yield.

Background

Plants require a variety of mineral nutrients to sustain their normal growth, with nitrogen being the most mineral nutrient required by plants. Generally, nitrogen can comprise 1.5-2% of the plant body by dry weight. Nitrogen is a constituent of proteins, nucleic acids, phospholipids, and organic nitrogen compounds essential for plant growth and development, and these substances are structural or functional components on which living cells live, and thus nitrogen is also called a vital element. In pursuit of high yield of crops, a large amount of nitrogen fertilizer is often applied in agricultural production. Wherein the application amount of the nitrogen fertilizer of the rice is far more than that of any other crops, and the loss amount of the nitrogen fertilizer accounts for 70 percent of the total application amount of the fertilizer. A series of problems of excessive use of nitrogen fertilizer, low nitrogen utilization efficiency, environmental deterioration caused by nitrogen fertilizer loss and the like generally exist in China.

Improving the nitrogen utilization efficiency of crops and reducing nitrogen fertilizer application are key to solving this serious environmental problem. Nitrate is one of the most important nitrogen sources in soil, and the absorption and utilization of nitrate by plants largely determines the nitrogen utilization efficiency of crops. Nitrate transporters are the most direct functional performers of plants for uptake, transport and storage of nitrate.

Disclosure of Invention

The invention aims to provide a new application of rice NRT1.1A gene and its coded protein.

The new application provided by the invention is specifically the application of the protein or the coding gene thereof in regulating and controlling the growth and development of plants; the growth and development are embodied as single plant yield (including biomass) and/or plant height and/or spike grain number and/or flowering time and/or bolting time and/or rosette leaf size;

wherein the flowering time is the morning and evening of the time point of starting flowering; and the bolting time is the morning and evening of the bolting starting time point.

The protein is any one of the following proteins:

(1) the amino acid sequence is a protein (namely NRT1.1A protein) shown as a sequence 1 in a sequence table;

(2) and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 in the sequence table, is related to the regulation of the growth and development of plants and is derived from the sequence 1.

The protein or the coding gene thereof for regulating and controlling the growth and development of the plant is specifically embodied in that: the higher the expression level of the protein in the plant, the higher the yield (including biomass) of the plant per plant, the higher the plant height, the more the number of grains per ear, the earlier the flowering time, the earlier the bolting time and/or the larger the rosette leaves; the lower the expression level of the protein in the plant, the lower the yield (including biomass) per plant, the lower the plant height, the smaller the number of grains per panicle, the later the flowering time, the later the bolting time, and/or the smaller the rosette leaves of the plant.

The application of the protein or the coding gene thereof in breeding plant varieties with increased single plant yield (including biomass), increased plant height, increased spike grain number, advanced flowering time, advanced bolting time and/or increased rosette leaves also belongs to the protection scope of the invention;

the protein is any one of the following proteins:

In practical application, when the yield (including biomass) of a selected single plant is increased, and/or the plant height is increased, and/or the number of grains per ear is increased, and/or the flowering time is advanced, and/or the bolting time is advanced, and/or rosette leaves are increased, plants with higher protein expression amount are required to be used as parents for hybridization.

Still another object of the present invention is a method for breeding transgenic plants with increased yield per plant (including biomass) and/or increased plant height and/or increased panicle number and/or earlier flowering time and/or earlier bolting time and/or increased rosette leaves.

The method for cultivating the transgenic plant with increased single plant yield (including biomass) and/or increased plant height and/or increased spike grain number and/or advanced flowering time and/or advanced bolting time and/or increased rosette leaves, which is provided by the invention, can comprise the step of expressing or over-expressing protein in a target plant or comprises the step of improving the activity of the protein in the target plant;

the protein is any one of the following proteins:

(1) the amino acid sequence is a protein shown as a sequence 1 in a sequence table;

(2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the sequence 1 in the sequence table, is related to regulation of plant growth and development and is derived from the sequence 1;

further, the method may specifically comprise the steps of a) and b) as follows:

a) introducing a coding gene of the protein into the target plant to obtain a transgenic plant expressing the coding gene;

b) obtaining transgenic plants with increased single plant yield (including biomass) and/or increased plant height and/or increased panicle number and/or advanced flowering time and/or advanced bolting time and/or increased rosette leaves compared with the target plants from the transgenic plants obtained in the step a).

The application of the protein or the coding gene thereof in the following (A) or (B) also belongs to the protection scope of the invention:

(A) promoting nitrate absorption or transport;

(B) promoting the expression of nitrate metabolism related genes; the nitrate metabolism related gene is selected from any one of the following genes: NRT1.1b, NRT2.1, NRT2.3a, NAR1, and NAR 2;

the protein is any one of the following proteins:

(2) and (b) a protein which is formed by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 1 in the sequence table, is related to nitrate transport and is derived from the sequence 1.

In the present invention, the promotion of nitrate absorption or transport in (a) is specifically promotion of nitrate absorption or transport in xenopus laevis oocytes, or promotion of nitrate absorption or transport in rice; the expression of the gene related to promotion of nitrate metabolism in (B) is specifically the expression of the gene related to promotion of nitrate metabolism in rice; the nitrate metabolism related gene is selected from any one of the following genes: OsNRT1.1B, OsNRT2.1, OsNRT2.3a, OsNAR1 and OsNAR 2.

The application of the protein or the coding gene thereof in the following (C) or (D) also belongs to the protection scope of the invention:

(C) breeding plant varieties with improved nitrate absorption or transport capacity;

(D) breeding a plant variety with improved expression level of nitrate metabolism related genes; the nitrate metabolism related gene is selected from any one of the following genes: NRT1.1b, NRT2.1, NRT2.3a, NAR1, and NAR 2;

the protein is any one of the following proteins:

Another object of the present invention is to provide a method for breeding a transgenic plant having an improved nitrate uptake or transport capacity and/or an improved expression level of a gene involved in nitrate metabolism.

The cultivation of the transgenic plant with improved nitrate absorption or transport capacity and/or improved expression of the nitrate metabolism related gene provided by the invention can comprise a step of expressing or over-expressing the protein in a target plant or a step of improving the activity of the protein in the target plant;

the protein is any one of the following proteins:

further, the method may specifically comprise the steps of c) and d) as follows:

c) introducing a coding gene of the protein into the target plant to obtain a transgenic plant expressing the coding gene;

d) obtaining a transgenic plant with improved nitrate absorption or transport capacity and/or improved expression of a nitrate metabolism related gene compared with the target plant from the transgenic plant obtained in the step c);

the nitrate metabolism related gene is selected from any one of the following genes: NRT1.1b, NRT2.1, NRT2.3a, NAR1, and NAR 2.

In each of the above applications and methods, the encoding gene may be a DNA molecule as described in any one of (1) to (4):

(1) DNA molecule of sequence 2 in the sequence table;

(2) DNA molecule of sequence 3 in the sequence table;

(3) a DNA molecule which is hybridized with the DNA molecule limited in (1) or (2) under strict conditions and codes the protein with the amino acid sequence shown as the sequence 1 in the sequence table;

(4) and (3) DNA molecules which have more than 90% of homology with the DNA molecules limited in the step (1), the step (2) or the step (3) and code proteins with amino acid sequences shown in a sequence 1 in a sequence table.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

Wherein, the sequence 2 consists of 8143 nucleotides and is a genome sequence; the sequence 3 consists of 1812 nucleotides and is a cDNA sequence; the sequence 2 and the sequence 3 encode the protein shown in the sequence 1 in the sequence table, and the sequence 1 consists of 603 amino acid residues.

In both of the above-described methods, the coding gene may be introduced into the plant of interest via a recombinant expression vector containing the coding gene.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, can be added before the transcription initiation nucleotide, and can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a recombinant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

In the invention, the promoter for starting the transcription of the coding gene in the recombinant expression vector is specifically an Actin1 promoter or a self endogenous promoter (sequence 9) of a rice NRT1.1A gene. More specifically, the recombinant expression vector is a recombinant plasmid obtained by replacing a small fragment between XbaI and PstI of an enzyme cutting site of a pCAMBIA2300-Actin vector with a DNA fragment shown in a sequence 3 in a sequence table; or a DNA fragment shown in the 1 st-1814 th site of the sequence 9 in the sequence table is inserted between the enzyme cutting site KpnI and the EcoRI of the pCAMBIA2300 vector, and a DNA fragment shown in the sequence 3 in the sequence table is inserted between the enzyme cutting site EcoRI and the XmaI to obtain a recombinant plasmid; or a recombinant plasmid obtained by replacing a small fragment between the enzyme cutting sites Bam HI and Sal I of the pCAMBIA2300-35S-OCS vector with a DNA fragment shown in a sequence 3 in a sequence table.

In the above two methods, the introduction of the recombinant expression vector into the target plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In each of the above applications and methods, the plant may be either a monocot or a dicot. The monocotyledon can be rice; the dicotyledonous plant may specifically be arabidopsis thaliana. In one embodiment of the invention, the plant is in particular the rice variety Dongjin. In another embodiment of the invention, the plant is specifically Arabidopsis thaliana Columbia-0.

In each of the above applications and methods, when the plant is specifically rice, the nitrate metabolism-related gene is selected from any one of the following: OsNRT1.1B, OsNRT2.1, OsNRT2.3a, OsNAR1 and OsNAR 2.

In the invention, the nucleotide sequence of OsNRT1.1B is sequence 4 in a sequence table; the nucleotide sequence of OsNRT2.1 is a sequence 5 in a sequence table; the nucleotide sequence of OsNRT2.3a is sequence 6 in the sequence table; the nucleotide sequence of the OsNAR1 is a sequence 7 in a sequence table; the nucleotide sequence of the OsNAR2 is a sequence 8 in a sequence table.

The invention proves that NRT1.1A protein can transport nitrate by utilizing an in vitro transport system of Xenopus laevis oocytes. Transgenic experiments show that NRT1.1A can show obvious growth advantages by overexpression in a rice wild type, the grain number per ear and the plant height are obviously improved, the rice yield is further improved, NRT1.1A shows certain growth advantages in the seedling stage of overexpression in an arabidopsis wild type, and rosette leaves are obviously larger than wild type controls; after bolting, the overexpression lines bolting significantly earlier than the wild type controls. The above results suggest that NRT1.1A protein has great potential application in improving plant yield.

Drawings

FIG. 1 shows the expression analysis of NRT1.1A gene in various rice tissues.

FIG. 2 shows NRT1.1A proteinShows in Xenopus laevis oocytes¹⁵NO₃ ^-Transport activity.

FIG. 3 shows that the rice nrt1.1a mutant shows nitrate transport inhibition. Wherein A is the result after 3 hours of transferring into the new nutrient solution; and B is the result 24 hours after the nutrient solution is transferred into the nutrient solution. WT represents wild type control.

FIG. 4 shows that the expression levels of other nitrate transporter genes are suppressed in the context of the rice nrt1.1a mutant. WT represents wild type control.

FIG. 5 shows that NRT1.1A rice transgenic lines OX1-1 and OX2-6 with over-expression show obvious growth advantages. Wherein A is the seedling stage phenotype; b is a reproductive growth stage phenotype. WT represents wild type control.

FIG. 6 shows that the expression level of nitrate utilization related genes is remarkably up-regulated in the background of NRT1.1A overexpression rice transgenic lines OX1-1 and OX 2-6. WT represents wild type control.

FIG. 7 shows that NRT1.1A overexpression rice transgenic lines pNA-2 and pNA-4 show yield advantage under low/high nitrogen conditions in the field. Wherein A is a high nitrogen condition in the field; b is field low nitrogen condition.

FIG. 8 shows NRT1.1A overexpression Arabidopsis transgenic lines OX-3 and OX-17 showing significant growth advantages. Wherein A is the detection result of NRT1.1A gene expression level in WT, OX-3 and OX-17; b is seedling stage phenotype; c is bolting phase phenotype. WT means wild type control (Columbia-0).

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

pCAMBIA2300-Actin vector: described in "Wang, k.j., Tang, d., Wang, m., Lu, j.f., Yu, h.x, Liu, j.f., Qian, b.x., Gong, z.y., Wang, x, Chen, j.m., Gu, m.h.and Cheng, Z.K, (2009) MER3is required for normal medical cross over conversion, but not for regulatory alignment in rice.j Cell Sci,122, 2055-2063", publicly available from the applicant, only for use in recreating invention experiments.

Rice variety Dongjin: "Dongjin" as described in "Jakyung Yi, Gynheung an.Utilization of T-DNAsagging Lines in Rice.J.plant Biol. (2013)56: 85-90" is disclosed to be available from the applicant and can only be used in the experiments for replicating the invention.

Rice nrt1.1a mutant: from the Korean Rice mutant library (Crop Biotech Institute, KyungHee University, Republic of Korea, http:// www.postech.ac.kr/life/PFG/risd) at the ordering site of http:// signal.salk.edu/cgi-bin/Rice 5, the rice nrt1.1a mutant was correspondingly numbered PFG _1E-00433. L. The specific construction method is described in the text "Jakyung Yi, Gynheung an.Utilization of T-DNAsagging Lines in Rice.J.plant Biol. (2013)56: 85-90".

Xenopus ootheca (Xenopus oocyte) is described in "Suhong Xu, Feng Cheng, Juan Liang, et al, Maternal xForrin, a Canonical Wnt Signaling Agonist and TGF- β Antagonst, Controls Early neuro electrotechnical Specification in Xenopus, plos Biology, 2012", publicly available from the Applicant and only available for use in duplicate experiments.

pCS2⁺Vectors described in "Suhong Xu, Feng Cheng, Juan Liang, et al, MaternalxMorin, a Canonical Wnt Signaling Agonist and TGF- β Antagonst, Controls EarlyNeuroectoderm specificity in Xenopus, ploS Biology 2012," a material publicly available from the Applicant and only usable in duplicate experiments.

pCAMBIA2300-35S-OCS vector: the invention is described in the 'salty ocean, summer Yang, Zhang jin Wen, etc.. pCAMBIA2300-betA-BADH bivalent plant expression vector construction, Chinese agronomy report, 2009, 09' article, which can be obtained by the public from the applicant and can only be used for the repeated invention experiments.

Examples 1 and NRT1.1A analysis of expression in various tissues of Rice

The NRT1.1A gene related in the embodiment is derived from rice (oryza. sativa L.), the genome sequence of the gene is shown as a sequence 2 in a sequence table, the sequence 2 consists of 8143 nucleotides, the cDNA sequence of the gene is a sequence 3 in the sequence table, and the sequence 3 consists of 1812 nucleotides. The sequence 2 and the sequence 3 encode the protein (NRT1.1A protein) shown in the sequence 1 in the sequence table, and the sequence 1 consists of 603 amino acid residues.

Total RNA is respectively extracted from roots, stems, leaf sheaths, leaves and ears of Dongjin rice variety and is reversely transcribed to obtain cDNA. Further, real-time quantitative fluorescent PCR was performed on NRT1.1A gene using the obtained cDNA as a template, and the expression level of NRT1.1A gene in rice tissue at the transcription level was detected. The experiment was repeated 3 times and the results averaged.

The primer sequences for detecting NRT1.1A gene were as follows:

qNRT1.1A-F: 5'-CCGTCTTCTTCGTCGGCTCCATCCT-3' (positions 1187-1211 of SEQ ID NO: 3);

qNRT1.1A-R: 5'-CCCGTGCTCATCGTCTTCATCCCCT-3' (reverse complement of position 1514-1538 of SEQ ID NO: 3).

OsActin1 is used as an internal reference gene, and the primer sequence is as follows:

OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；

OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。

the relative expression level of NRT1.1A gene was calculated with the expression level of the reference gene as 1.

qRT-PCR analysis shows that NRT1.1A gene has higher expression level in each tissue of rice (see figure 1), the expression level is highest in root, next to leaf, leaf sheath and stem, and the expression level is lowest in ear. This constitutive expression suggests that NRT1.1A may be involved in maintaining the basic physiological functions of plants.

Example 2, NRT1.1A validation of in vitro transport nitrate Activity

One, Xenopus laevis recombinant expression vector pCS2⁺Construction of/NRT1.1A

Extracting total RNA of Dongjin of japonica rice variety, and reverse transcribing into cDNA. Using the obtained cDNAs as templates, NRT1.1A cDNAs were each amplified by PCR using the following primer sequences. Recognition sites of restriction enzymes BamHI and EcoRI are respectively introduced into two ends of a primer used for amplification (shown by a dashed line), and the primer sequence is as follows:

F：5’-GGATCCATGGTGGGGATGTTGCCGGA-3' (the underlined part is the recognition sequence for BamHI, and the sequence thereafter is the 1 st to 20 th positions of the sequence 3 in the sequence listing);

R：5’-GAATTCTCAGTGGAGGCATGGCTCGG-3' (recognition sequence of EcoRI is underlined, and the sequence following this is the reverse complement of sequence 3 at position 1793-1812 in the sequence listing).

Connecting the amplified target segment into Xenopus laevis oocyte expression vector pCS2⁺And verified by sequencing.

The recombinant vector obtained after replacing a small fragment between the BamHI and EcoRI sites of the pCS2+ vector by a DNA fragment shown in a sequence 3 in a sequence table through sequencing is named as pCS2 +/NRT1.1A.

Secondly, in vitro transcription of NRT1.1AmRNA and injection of Xenopus laevis oocytes to verify activity of transferred nitrate

The recombinant vector pCS2 obtained by the construction of the step one⁺/NRT1.1A linearization with the restriction enzyme ApaI, followed by in vitro transcription kit (mMESSAGE)

SP6Kit, Ambion, AM1340) to transcribe cRNA with 5 'cap structure and 3' poly A structure in vitro, see Kit instructions for specific procedures.

The resultant cRNA was injected into Xenopus oocytes (Xenopus oocytes). After injection, the mixture was dissolved in ND96 solution (formulation: 96mM NaCl, 2mM KCl, 1mM MgCl)₂，1.8mM CaCl₂5mM HEPES, pH 7.4) for two days, transferred into a medium containing 10mM K¹⁵NO₃In an absorbent solution (formulation: 10mM K)¹⁵NO₃(98％atom¹⁵N-KNO₃,Sigma-Aldrich,335134)，230mM Mannitol，0.3mM CaCl₂10mM MES-Tris, pH 5.5),culturing for 3 hr, and determining Xenopus laevis oocyte by element mass spectrum analyzer (ICP-MS)¹⁵N content (see, "Kun-Hsiang Liu, Chi-Ying Huang, Yi-Fan Tsay, et al, CHL1 Is a double-Affinity Nitrate Transporter of Arabidopsis Involuted in Multiple drugs of Nitrate update. the Plant Cell,1999," supra). The experiment was repeated 3 times and the results were averaged.

Experiment set-up injection of same volume ddH₂Control of O.

The determination result shows that the Xenopus laevis oocytes injected with NRT1.1A cRNA¹⁵The content of N is obviously higher than that of ddH injected with the same volume₂O control (P)<0.01, see fig. 2). The results indicate that NRT1.1A has nitrate transport activity in vitro.

Example 3 acquisition and functional verification of Rice nrt1.1a mutant

First, obtaining and identifying rice nrt1.1a mutant

The identification of the rice mutant nrt1.1a is carried out by the three-primer method. The sequences of the primers used were as follows:

pGA2715L：5’-CTAGAGTCGAGAATTCAGTACA-3’；

NARM14F：5’-AATCCGCAAATGTGTCTTGT-3’；

NARM14R：5’-CTAGGGCCATCTTGTCTTCA-3’。

the NRT1.1A gene in the rice mutant nrt1.1a is proved to be mutated, and the functional NRT1.1A protein can not be normally expressed.

Second, nitrate transport function verification of rice nrt1.1a mutant

To specifically understand NRT1.1A function in vivo, the inventors of the present invention conducted¹⁵N-labelled nitrate transport experiments. First, seedlings of the wild type oryza sativa and nrt1.1a mutants were placed in modified Kimura B nutrient solution (formulation: 2mM KNO)₃，1.8mM KCl，0.36mM CaCl₂，0.54mM MgSO₄·7H₂O，0.18mM KH₂PO₄，40μM Na₂EDTA-Fe(II)，13.4μM MnCl₂·4H₂O，18.8μM H₃BO₃，0.03μM Na₂MoO₄·2H₂O，0.3μM ZnSO₄·7H₂O，0.32μM CuSO₄·5H₂O and 1.6mM Na₂SiO₃·9H₂O) for 10 days, then transferred to a medium containing 5mM K¹⁵NO₃The modified Kimura B is cultured in nutrient solution for 24 hours. Collecting materials on the ground and

underground parts

3h and 24h after transferring into new nutrient solution, and measuring with element mass spectrometer (ICP-MS)¹⁵N content (see, "Kun-Hsiang Liu, Chi-Ying Huang, Yi-Fan Tsay, et al, CHL1 Is a double-Affinity Nitrate Transporter of Arabidopsis Involuted in Multiple drugs of Nitrate update. the Plant Cell,1999," supra). The experiment was repeated 4 times and the results were averaged.

The overground part and the middle part of the root of the wild type oryza sativa and nrt1.1a mutant are calculated¹⁵The ratio of N content was found to be in the aerial part and in the root of the nrt1.1a mutant at two sampling time points¹⁵The ratio of N content is significantly lower than that of wild type (P)<0.01, see FIG. 3), indicating that transport of nitrate from underground to above ground in the nrt1.1a mutant is severely affected.

Third, determination of nitrate metabolism related gene expression in rice nrt1.1a mutant

The expression level of the following nitrate utilization related genes in rice is detected by qRT-PCR: (1) the nucleotide sequence of the cDNA of the OsNRT1.1B gene is a sequence 4 in a sequence table, and the OsNRT1.1B gene is mainly responsible for absorption and transportation of nitrate and participates in regulation of nitrate signals; (2) the nucleotide sequence of the cDNA of the OsNRT2.1 gene is a sequence 5 in a sequence table and is mainly responsible for nitrate absorption; (3) the nucleotide sequence of the OsNRT2.3a gene, the cDNA of which is sequence 6 in the sequence table, is responsible for the transport of nitrate to the overground part; (4) the nucleotide sequence of the cDNA of the OsNAR1 gene is a sequence 7 in a sequence table, and the OsNRT2.1 carries out functional cofactors; (5) the nucleotide sequence of the cDNA of the OsNAR2 gene is a sequence 8 in a sequence table, and the OsNRT2.1 carries out functional cofactors.

Respectively taking leaves of wild type oryzanol and nrt1.1a mutant seedlings, extracting total RNA, carrying out reverse transcription to obtain cDNA, and carrying out qRT-PCR detection on the five nitrate utilization related genes by adopting the following primer pairs.

Primer pairs for detecting OsNRT1.1B gene:

OsNRT1.1B-F：5’-GGCAGGCTCGACTACTTCTA-3’；

OsNRT1.1B-R：5’-AGGCGCTTCTCCTTGTAGAC-3’。

primer pairs for detecting OsNRT2.1 gene:

OsNRT2.1-F：5’-CTTCACGTCGTCGAGGTACT-3’；

OsNRT2.1-R：5’-CACTCGGAGCCGTAGTAGTG-3’。

primer pairs for detecting OsNRT2.3a gene:

OsNRT2.3a-F：5’-CGCTGCTGCCGCTCATCCG-3’；

OsNRT2.3a-R：5’-CCGTGCCCATGGCCAGAC-3’。

primer pair for detecting the OsNAR1 gene:

OsNAR1-F：5’-GTTCAAGAGCGTGAAGGTGA-3’；

OsNAR1-R：5’-CACCACGTAGTCGAACCTG-3’。

primer pair for detecting the OsNAR2 gene:

OsNAR2-F：5’-TCGTCCTCGAGAACAAGAAG-3’；

OsNAR2-R：5’-TCCGTTGGTTTTGTAGGTTG-3’。

with OsActin1 as an internal reference gene, the detection primer pair comprises:

OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；

OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。

the expression level of the reference gene was regarded as 1, and the relative expression level of each gene was calculated.

The results show that in the background of the nrt1.1a mutant, the expression of the five nitrate utilization related genes is remarkably inhibited compared with that of wild rice Dongjin (P <0.05, see figure 4), and the fact that NRT1.1A is possibly involved in the regulation of the transcription level of the genes is suggested.

Example 4, NRT1.1A acquisition of transgenic Rice and functional verification (heterologous promoter)

Construction of recombinant plant expression vector pCAMBIA2300-Actin/NRT1.1A

Extracting the total RNA of the Dongjin of the japonica rice, and performing reverse transcription to obtain cDNA. The CDS of NRT1.1A was amplified by PCR using the cDNA obtained as a template and the following primer sequence. Recognition sites of restriction enzymes XbaI and PstI (shown by underlining) are respectively introduced at two ends of a primer used for amplification, and the sequences of the primers are as follows:

F：5’-TCTAGAATGGTGGGGATGTTGCCGGA-3' (the underlined part is the recognition sequence of XbaI, and the sequence thereafter is the 1 st to 20 th positions of the sequence 3 in the sequence listing);

R：5’-CTGCAGTCAGTGGAGGCATGGCTCGG-3' (the underlined part is the recognition sequence of PstI, and the sequence following the recognition sequence is the reverse complement of 1793-1812 of sequence 3 in the sequence listing).

The CDS region (containing a stop codon) of NRT1.1A was amplified using a cDNA of Dongjin, a rice variety, as a template. After the PCR product is connected with a T vector pEASY-Blunt (TransGene), the PCR product is connected into a plant expression vector pCambia2301-Actin after being verified by double enzyme digestion of XbaI and PstI.

The recombinant vector obtained after replacing a small fragment between the enzyme cutting sites XbaI and PstI of the pCAMBIA2300-Actin vector by a DNA fragment shown in a sequence 3 in a sequence table is named as pCAMBIA2300-Actin/NRT1.1A through sequencing.

II, NRT1.1A obtaining transgenic rice

The recombinant plant expression vector pCAMBIA2300-Actin/NRT1.1A constructed in the first step is transferred into Agrobacterium AGL1 (from ATCC), and then the callus of the japonica rice variety Dongjin is infected, and the specific transformation and screening method is disclosed in the literature, "Yi-Li-Zi-Cao-Yun, Wang-Li, which is strontium-Cheng-Shi, Tang 31066shu, Shun, Zhou-Pu-Hua, Tianwen-Zheng-improving the frequency of agrobacterium transformation of rice, Gen-Ci, 2001,28(4): 352-358". Meanwhile, a control for transferring pCAMBIA2300-Actin empty vector is set. Finally obtaining two transgenic seedlings, namely a rice plant transferred with pCAMBIA2300-Actin/NRT1.1A and a rice plant (T) transferred with pCAMBIA2300-Actin empty vector₀)。

Identification of NRT1.1A transgenic rice

(1) Preliminary identification by PCR

T obtained from step two₀Transgenic rice transferred with pCAMBIA2300-Actin/NRT1.1A and a control plant transferred with pCAMBIA2300-Actin empty vector are respectively extracted to obtain genome DNA. The primers F1 and R1 (primer sequences are shown below) aiming at the NptII gene are used for carrying out PCR identification, and the identification shows that the plant containing the NptII gene (the size of the PCR product is about 500bp) is a transgenic positive plant.

F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；

R1：5’-CTGGCGCGAGCCCCTGATGCT-3’。

Through the PCR molecular identification, 2 transgenic rice lines which are positively identified and transferred into pCAMBIA2300-Actin/NRT1.1A are randomly selected and respectively marked as OX1-1 and OX 2-6.

(2) Analysis of transcript level (RNA expression level)

T obtained in step (1)₀Transgenic rice lines OX1-1 and OX2-6 of the generation NRT1.1A, control plants transferred into pCAMBIA2300-Actin empty vectors and wild rice variety Dongjin are experimental materials. Total RNA of each material was extracted and reverse transcribed to obtain cDNA. Further, real-time quantitative fluorescent PCR was performed on NRT1.1A gene using the obtained cDNA as a template, and the expression level of NRT1.1A gene at the transcription level in each material was detected. The experiment was repeated 3 times and the results averaged.

The primer sequences for detecting NRT1.1A gene were as follows:

OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；

OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。

The real-time quantitative fluorescence PCR detection result of NRT1.1A gene expression level in each experimental material shows that compared with the wild rice variety Dongjin without transgenosis, the T obtained in the step (1)₀The expression level of NRT1.1A genes in transgenic rice strains OX1-1 and OX2-6 of generation NRT1.1A is obviously improved on the transcription level, and for a control plant which is transferred into a pCAMBIA2300-Actin empty vector, the expression level of NRT1.1A genes on the transcription level is basically consistent with that of wild rice Dongjin without transgenosis, and no statistical difference exists.

Functional verification of NRT1.1A transgenic rice

1. Experimental methods

Identification of Positive T in step three₂Transgenic rice strains OX1-1 and OX2-6 of the generation NRT1.1A, control plants transferred into pCAMBIA2300-Actin empty vectors and non-transgenic wild rice Dongjin are used as experimental materials. The function of the compound is identified from the following aspects:

(1) phenotypic observations

And observing and recording the plant height of each genetic material in a seedling stage, and observing and recording the plant height of each genetic material again after entering a reproductive growth stage. In the experiment, at least 30 individuals are selected for statistics from each transgenic line.

(2) qRT-PCR analysis of transcript levels of nitrate metabolism-related genes

The nitrate metabolism related gene specifically relates to:

the nucleotide sequence of the OsNRT2.1 gene is sequence 5 in the sequence table;

the nucleotide sequence of the OsNRT2.3a gene is sequence 6 in the sequence table;

the nucleotide sequence of the OsNIA2 gene is shown as sequence 8 in the sequence table;

the nucleotide sequence of the OsNIR1 gene cDNA is the sequence 7 in the sequence table.

The specific determination method is carried out in step three of example 2.

In the experiment, at least 5 strains of each transgenic line were selected for the experiment.

2. Results of the experiment

(1) Phenotypic observations

T₂The generation NRT1.1A transgenic rice strains OX1-1 and OX2-6 show certain growth advantages at the seedling stage, and the plant height is obviously higher than that of a wild type control; entering the flowering phase, the over-expressing lines showed early flowering phenotype (see FIG. 5). The plant height and flowering time of the control plant transferred with the pCAMBIA2300-Actin empty vector are basically consistent with those of a wild control, and no statistical difference exists. These results indicate that NRT1.1A has a certain potential application in agricultural production, and a moderate increase in expression level of NRT1.1A may increase the yield of rice.

(2) qRT-PCR analysis of transcript levels of nitrate metabolism-related genes

qRT-PCR analysis shows that positive T is identified in the third step₀In the generation NRT1.1A transgenic rice lines OX1-1 and OX2-6, the transcription levels of the four nitrate metabolism related genes are obviously up-regulated compared with that of a wild type control (P)<0.01, see fig. 6).

Examples 5, NRT1.1A acquisition of transgenic Rice and functional verification (endogenous promoter)

In order to confirm that NRT1.1A can achieve the effect of increasing the yield of rice and avoid the negative effect of a constitutive promoter on the agronomic traits of rice, the inventor of the invention constructs a NRT1.1A overexpression transgenic plant driven by a self promoter and further verifies the function of the plant.

Construction of recombinant expression vector pCAMBIA2300/NRT1.1A

Extracting genome DNA and total RNA of Dongjin rice variety and reverse transcribing the total RNA into cDNA. The promoter region (sequence 9) of NRT1.1A was amplified using Dongjin genomic DNA as a template, recognition sites for restriction enzymes KpnI and EcoRI were introduced at both ends of the primers used for amplification (shown by underlining), and the sequences of the primers used were as follows:

NRT1.1Ap-F：5’-GGTACCTTCGATCTCCCACGTAAGAC-3' (the underlined part is the recognition sequence for KpnI, followed by the sequence from positions 1-20 of sequence 9);

NRT1.1Ap-R：5’-GAATTCTCTCTCTCTCTTCTTCTTCTTCCTC-3' (recognition sequence of EcoRI is underlined, and the sequence following this is the reverse complement of sequence 9 at positions 1790 and 1814).

Meanwhile, cDNA of rice variety Dongjin is taken as a template to amplify a CDS region of NRT1.1A, recognition sites of restriction enzymes EcoRI and XmaI are respectively introduced into two ends of a primer adopted by amplification, and the sequences of the primers are as follows:

NRT1.1A_CDS-F：5’-GAATTCATGGTGGGGATGTTGCCGGA-3' (recognition sequence for EcoRI is underlined, and the sequence thereafter is 1-20 th position of sequence 3);

NRT1.1A_CDS-R：5’-CCCGGGGTGGAGGCATGGCTCGG-3' (the underlined part is the recognition sequence for XmaI, and the sequence following it is the reverse complement of position 1793-1809 of sequence 3).

The two PCR fragments amplified in the above two steps were ligated to the corresponding cleavage sites of pBluescript KS (+) (Stratagene, 212205) vector in two separate steps. After sequencing verification, adopting KpnI and BamHI double enzyme digestion, connecting the fragment containing the endogenous promoter and NRT1.1A CDS into the corresponding enzyme digestion site of the binary vector pCAMBIA2300, and naming the obtained recombinant vector which is verified to be correct by sequencing as pCAMBIA 2300/NRT1.1A.

II, NRT1.1A obtaining transgenic rice

The recombinant plant expression vector pCAMBIA2300/NRT1.1A constructed in the first step is transferred into Agrobacterium AGL1 (from ATCC), and the callus of japonica rice variety Dongjin is infected by the recombinant plant expression vector, and the specific transformation and screening method is described in the literature "Yi-autogeny, Cao-Li, Wang-Li, and Cheng-Shi, Tang 31066;, Shun, Zhou-Pu-PHua, Tianwenzhong faithful, research for improving the frequency of agrobacterium transformed rice, Gen academic newspaper, 2001,28(4): 352-. Meanwhile, a control for transferring pCAMBIA2300 empty vector is set. Finally obtaining two transgenic seedlings, namely a rice plant transferred with pCAMBIA2300/NRT1.1A and a rice plant (T) transferred with pCAMBIA2300 empty vector₀)。

Identification of NRT1.1A transgenic rice

(1) Preliminary identification by PCR

T obtained from step two₀The transgenic rice transferred into pCAMBIA2300/NRT1.1A and the control plant transferred into pCAMBIA2300 empty vector are respectively extracted to obtain genome DNA. The primers F1 and R1 (the primer sequences are as follows) are used for PCR identification, and the identification shows that the plant containing the NptII gene (the size of the PCR product is about 500bp) is the transgenic positive plant.

F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；

R1：5’-CTGGCGCGAGCCCCTGATGCT-3’。

Through the PCR molecular identification, 2 transgenic rice lines which are positively identified and transferred into pCAMBIA2300/NRT1.1A are randomly selected and respectively marked as pNA-2 and pNA-4.

(2) Analysis of transcript level (RNA expression level)

Identifying positive T obtained in step (1)₀Transgenic rice strains pNA-2 and pNA-4 of generation NRT1.1A, control plants transferred into pCAMBIA2300 empty vector, and wild type rice variety Dongjin are used as experimental materials. Total RNA of each material was extracted and reverse transcribed to obtain cDNA. Further, real-time quantitative fluorescent PCR was performed on NRT1.1A gene using the obtained cDNA as a template, and the expression level of NRT1.1A gene at the transcription level in each material was detected. The experiment was repeated 3 times and the results averaged.

The primer sequences for detecting NRT1.1A gene were as follows:

OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；

OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。

The real-time quantitative fluorescence PCR detection result of NRT1.1A gene expression level in each experimental material shows that compared with the wild rice variety Dongjin without transgenosis, the T obtained in the step (1)₀The expression level of NRT1.1A genes in the generation NRT1.1A transgenic rice strains pNA-2 and pNA-4 is obviously improved on the transcription level, and for a control plant transferred with the pCAMBIA2300 empty vector, the expression level of NRT1.1A genes is basically consistent with that of the wild rice variety Dongjin without the transgenosis on the transcription level, and no statistical difference exists.

Functional verification of NRT1.1A transgenic rice

Identification of Positive T in step three₂Transgenic rice strains pNA-2 and pNA-4 of generation NRT1.1A, control plants transferred into pCAMBIA2300 empty vector, and non-transgenic wild type rice Dongjin are used as experimental materials. Two nitrogen fertilizer gradients (high and low nitrogen) were set up for the field trial. Wherein the high nitrogen is applied according to 4.28kg of urea per 100 square meters, and the low nitrogen is applied according to 1.07kg of urea per 100 square meters. 3 cells are arranged under each nitrogen fertilizer gradient. Observing and recording the phenotype (including tillering number, thousand kernel weight, ear kernel number and single plant yield) in the field, randomly selecting at least 10 single plants in each strain in each cell for phenotype statistics, and totaling not less than 30 single plants in each strain.

The results of nitrogen fertilizer testing in the field show that the NRT1.1A over-expressed transgenic lines pNA-2 and pNA-4 both show a significant increase in grain number per ear and yield per plant (P <0.05) under low nitrogen and high nitrogen conditions. Especially under low nitrogen conditions, the increase in yield per plant of over-expressed transgenic lines is particularly evident (P <0.01) as detailed in FIG. 7.

EXAMPLE 6, NRT1.1A acquisition and functional verification of transgenic Arabidopsis thaliana (heterologous promoter)

Construction of recombinant plant expression vector pCAMBIA2300-35S-OCS/NRT1.1A

Extracting the total RNA of the Dongjin of the japonica rice, and performing reverse transcription to obtain cDNA. The CDS of NRT1.1A was amplified by PCR using the cDNA obtained as a template and the following primer sequence. Recognition sites (shown by underlining) of restriction enzymes Bam HI and Sal I are respectively introduced into two ends of a primer used for amplification, and the sequences of the primers are as follows:

F：5’-GGATCCATGGTGGGGATGTTGCCGGA-3' (the underlined part is the recognition sequence of Bam HI, and the sequence thereafter is the 1 st to 20 th positions of the sequence 3 in the sequence listing);

R：5’-GTCGACTCAGTGGAGGCATGGCTCGG-3' (the recognition sequence of Sal I is underlined, and the sequence following this is the reverse complement of sequence 3 at positions 1793-1812 in the sequence listing).

The CDS region (containing a stop codon) of NRT1.1A was amplified using a cDNA of Dongjin, a rice variety, as a template. After the PCR product is connected with a T vector pEASY-Blunt (TransGene), the PCR product is connected into a plant expression vector pCAMBIA2300-35S-OCS after being verified by double enzyme digestion of Bam HI and Sal I.

The recombinant vector obtained after replacing a small fragment between enzyme cutting sites Bam HI and Sal I of the pCAMBIA2300-35S-OCS vector by a DNA fragment shown in a sequence 3 in a sequence table through sequencing is named as pCAMBIA 2300-35S-OCS/NRT1.1A.

Secondly, NRT1.1A obtaining transgenic Arabidopsis thaliana

The recombinant Plant expression vector pCAMBIA2300-35S-OCS/NRT1.1A constructed in step one was transferred into Arabidopsis thaliana Columbia-0 by Agrobacterium GV3101 (from ATCC) (reference: "Steven J. Cloughhand Andrew F. bent. floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal (1998)16(6), 735-743"). Meanwhile, a control for transferring the pCAMBIA2300-35S-OCS empty vector is set. Finally obtaining two transgenic seedlings, namely an arabidopsis thaliana plant transferred with pCAMBIA2300-35S-OCS/NRT1.1A and an arabidopsis thaliana plant (T) transferred with pCAMBIA2300-35S-OCS empty vector₀)。

Identification of NRT1.1A transgenic arabidopsis thaliana

T obtained from step two₀Transgenic Arabidopsis thaliana transformed into pCAMBIA2300-35S-OCS/NRT1.1A is randomly selectedTwo strains, designated OX-3 and OX-17, respectively. OX-3 and OX-17, a control plant transferred into pCAMBIA2300-35S-OCS empty vector and non-transgenic wild type Arabidopsis thaliana Columbia-0 are respectively extracted with total RNA and reverse transcribed to obtain cDNA. Further, real-time quantitative fluorescent PCR was performed on NRT1.1A gene using the obtained cDNA as a template, and the expression level of NRT1.1A gene at the transcription level in each material was detected. The experiment was repeated 3 times and the results averaged.

The primer sequences for detecting NRT1.1A gene were as follows:

AtActin2 is used as an internal reference gene, and the primer sequence is as follows:

qAtActin2-F：5’-GCACCACCTGAAAGGAAGTACA-3’；

qAtActin2-R：5’-CGATTCCTGGACCTGCCTCATC-3’。

The real-time quantitative fluorescence PCR detection result of NRT1.1A gene expression level in each experimental material shows that compared with the non-transgenic wild type arabidopsis thaliana, the T obtained in the second step₀The expression level of NRT1.1A gene in transgenic Arabidopsis strains OX-3 and OX-17 transformed into pCAMBIA2300-35S-OCS/NRT1.1A was significantly increased at the transcriptional level (see A in FIG. 8). Compared with the wild type plant without transgenosis, the expression level of the NRT1.1A gene of the control plant transferred with the pCAMBIA2300-35S-OCS empty vector is basically consistent at the transcription level and has no statistical difference.

Functional verification of NRT1.1A transgenic Arabidopsis thaliana

1. Experimental methods

Identification of Positive T in step three₂Transgenic Arabidopsis lines OX-3 and OX-17 of the generation NRT1.1A, control plants with pCAMBIA2300-35S-OCS empty vector transferred, and non-transgenic wild type Arabidopsis Columbia-0 as experimental materials. Observing the size of the rosette leaves of each genetic material in the seedling stageAnd observing bolting time of each genetic material after bolting. In the experiment, at least 30 individuals were selected for observation from each transgenic line.

2. Results of the experiment

T₂The transgenic arabidopsis lines OX-3 and OX-17 of the generation NRT1.1A show certain growth advantages at the seedling stage, rosette leaves are obviously larger than wild type controls (shown as B in figure 8), and after bolting, bolting of the overexpression lines is obviously earlier than the wild type controls (shown as C in figure 8). And for the control plant transferred with the pCAMBIA2300-35S-OCS empty vector, the rosette leaf size and bolting time at the seedling stage are basically consistent with those of a wild plant without transgenosis.

Claims

1. The protein or the coding gene thereof is applied to the regulation of the growth and development of plants; the growth and development are embodied as single plant yield and/or plant height and/or spike grain number and/or flowering time and/or bolting time and/or rosette leaf size;

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

2. The protein or the coding gene thereof is applied to breeding of plant varieties with increased single plant yield, increased plant height, increased spike grain number, advanced flowering time, advanced bolting time and/or increased rosette leaves;

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

3. Use according to claim 1 or 2, characterized in that: the encoding gene is a DNA molecule as described in any one of (1) to (2) below:

(1) a DNA molecule of SEQ ID No. 2;

(2) a DNA molecule of SEQ ID No. 3.

4. Use according to claim 1 or 2, characterized in that: the plant is a monocotyledon or a dicotyledon.

5. A method for breeding a transgenic plant with increased yield per plant, increased plant height, increased panicle number, advanced flowering time, advanced bolting time and/or increased rosette leaves, comprising the step of expressing or overexpressing a protein in a target plant, or comprising the step of increasing the activity of a protein in a target plant;

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

6. The method of claim 5, wherein: the method comprises the following steps a) and b):

b) obtaining transgenic plants with increased single plant yield and/or plant height and/or increased spike grain number and/or advanced flowering time and/or advanced bolting time and/or increased rosette leaves compared with the target plants from the transgenic plants obtained in the step a).

7. The method of claim 6, wherein: the encoding gene is a DNA molecule as described in any one of (1) to (2) below:

(1) a DNA molecule of SEQ ID No. 2;

(2) a DNA molecule of SEQ ID No. 3.

8. The method according to claim 6 or 7, characterized in that: in the method, the coding gene is introduced into the target plant by a recombinant expression vector containing the coding gene.

9. The method of claim 8, wherein: the promoter for starting the transcription of the coding gene in the recombinant expression vector is a 35S promoter or a self endogenous promoter of a rice NRT1.1A gene.

10. The method of claim 5, 6 or 7, wherein: the plant is a monocotyledon or a dicotyledon.

11. The use of a protein or a gene encoding the same in (A) or (B) below;

(A) promoting nitrate absorption or transport;

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

12. Use according to claim 11, characterized in that: in the step (A), the nitrate absorption or transport is promoted to be absorbed or transported in Xenopus laevis oocytes, or to be absorbed or transported in rice;

in the step (B), the expression of the nitrate metabolism promoting related gene is the expression of the nitrate metabolism promoting related gene in Xenopus laevis oocytes or the expression of the nitrate metabolism promoting related gene in rice; the nitrate metabolism related gene is selected from any one of the following genes: NRT1.1b, NRT2.1, NRT2.3a, NAR1, and NAR 2.

13. Use of a protein or a gene encoding the same in the following (C) or (D);

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

14. Use according to claim 11 or 13, characterized in that: the encoding gene is a DNA molecule as described in any one of (1) to (2) below:

(1) a DNA molecule of SEQ ID No. 2;

(2) a DNA molecule of SEQ ID No. 3.

15. Use according to claim 11 or 13, characterized in that: the plant is a monocotyledon or a dicotyledon.

16. A method for breeding a transgenic plant with improved nitrate uptake or transport capacity and/or improved expression of a gene associated with nitrate metabolism, comprising the step of expressing or overexpressing a protein in a plant of interest, or comprising the step of increasing the activity of a protein in a plant of interest;

the protein is the protein with an amino acid sequence shown as SEQ ID No. 1.

17. The method of claim 16, wherein: the method comprises the following steps c) and d):

18. The method of claim 17, wherein: the encoding gene is a DNA molecule as described in any one of (1) to (2) below:

(1) a DNA molecule of SEQ ID No. 2;

(2) a DNA molecule of SEQ ID No. 3.

19. The method according to claim 17 or 18, characterized in that: in the method, the coding gene is introduced into the target plant by a recombinant expression vector containing the coding gene.

20. The method of claim 19, wherein: the promoter for starting the transcription of the coding gene in the recombinant expression vector is a 35S promoter or a self endogenous promoter of a rice NRT1.1A gene.

21. The method of claim 16 or 17 or 18, wherein: the plant is a monocotyledon or a dicotyledon.