WO2018033083A1 - 水稻nrt1.1a基因及其编码蛋白在提高植物产量育种中的应用 - Google Patents

水稻nrt1.1a基因及其编码蛋白在提高植物产量育种中的应用 Download PDF

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WO2018033083A1
WO2018033083A1 PCT/CN2017/097601 CN2017097601W WO2018033083A1 WO 2018033083 A1 WO2018033083 A1 WO 2018033083A1 CN 2017097601 W CN2017097601 W CN 2017097601W WO 2018033083 A1 WO2018033083 A1 WO 2018033083A1
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protein
plant
seq
gene
amino acid
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储成才
王威
胡斌
李华
张志华
刘永强
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中国科学院遗传与发育生物学研究所
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention belongs to the field of biotechnology and relates to the application of a rice NRT1.1A gene and a protein encoded thereby for improving plant yield breeding.
  • Nitrogen is a constituent of organic nitrogen compounds necessary for the growth and development of proteins, nucleic acids, phospholipids, and plants. These substances are structural or functional components on which living cells depend. Therefore, nitrogen is also called a living element.
  • a large amount of nitrogen fertilizer is often applied in agricultural production. Among them, the application rate of nitrogen fertilizer in rice is far more than any other crops, and the loss of nitrogen fertilizer accounts for 70% of the total application rate of chemical fertilizer.
  • a series of problems such as excessive use of nitrogen fertilizer, low nitrogen utilization efficiency and environmental degradation caused by nitrogen fertilizer loss are common in China.
  • Nitrate is one of the most important nitrogen sources in the soil. The absorption and utilization of nitrate by plants largely determines the nitrogen use efficiency of crops. Nitrate transporters are the most direct function performers of plant uptake, transport and storage of nitrates.
  • novel use provided by the present invention is specifically the application of a protein or a gene encoding the same in regulating plant growth and development; the growth and development is manifested as yield per plant and/or plant height and/or kernel number and/or flowering time and/or Or convulsion time and / or rosette leaf size and / or biomass;
  • the flowering time is the morning and evening of the time point at which the flowering starts; the time of the convulsion is the morning and evening of the time point at which the convulsion is started.
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the sequence listing (ie, NRT1.1A protein);
  • the protein or its coding gene regulates plant growth and development: the higher the expression level of the protein in the plant, the higher the yield per plant, the higher the plant height, and the more the number of grains per ear, The earlier the flowering time, the earlier the twitching time and/or the larger the rosette leaves and/or the larger the biomass; the lower the expression level of the protein in the plant, the lower the yield per plant and the lower the plant height. The smaller the number of kernels per ear, the later the flowering time, the later the convulsion time and/or the smaller the rosette leaves and/or the smaller the biomass.
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the sequence listing (ie, NRT1.1A protein);
  • a further object of the invention is to cultivate an increase in yield per plant and/or an increase in plant height and/or an increase in the number of kernels and/or an advance in flowering time and/or an advance in convulsion time and/or an increase in rosette leaves and/or biomass.
  • a method of transgenic plants is to cultivate an increase in yield per plant and/or an increase in plant height and/or an increase in the number of kernels and/or an advance in flowering time and/or an advance in convulsion time and/or an increase in rosette leaves and/or biomass.
  • the present invention provides a transgenic plant with increased yield and/or increased plant height and/or increased kernel number and/or increased flowering time and/or advanced convulsion time and/or increased rosette leaf and/or increased biomass.
  • a method comprising the steps of expressing or overexpressing a protein of a plant of interest, or comprising the step of increasing the activity of the protein in the plant of interest;
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the Sequence Listing;
  • the method may specifically include the following steps a) and b):
  • transgenic plants obtained in step a an increase in yield per plant and/or an increase in plant height and/or an increase in the number of kernels and/or an advance in flowering time and/or an advance in the time of withdrawal and/or compared to the plant of interest.
  • transgenic plants with increased rosettes and/or increased biomass are particularly useful for transgenic plants with increased rosettes and/or increased biomass.
  • nitrate metabolism-related gene is selected from any one of the following: NRT1.1B, NRT2.1, NRT2.3a, NAR1, and NAR2;
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the sequence listing (ie, NRT1.1A protein);
  • the promoting nitrate absorption or transport in (A) specifically promotes absorption or transport of nitrate in Xenopus oocytes, or promotes absorption or transport of nitrate in rice;
  • the expression of the gene for promoting nitrate metabolism in (B) is specifically for promoting the expression of a gene related to nitrate metabolism in rice; the gene related to nitrate metabolism is selected from any one of the following: OsNRT1.1B, OsNRT2.1 , OsNRT2.3a, OsNAR1 and OsNAR2.
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the sequence listing (ie, NRT1.1A protein);
  • Another object of the present invention is to provide a method of cultivating a transgenic plant having an increased ability to absorb or transport nitrates and/or an increased expression level of a gene related to nitrate metabolism.
  • the transgenic plant provided by the present invention for cultivating an increase in the absorption or transport ability of a nitrate and/or an increase in the expression level of a gene related to nitrate metabolism may include a step of expressing or overexpressing a protein of a plant of interest, or comprising: a step of increasing protein activity;
  • the protein is any of the following:
  • amino acid sequence is the protein shown in SEQ ID NO: 1 in the Sequence Listing;
  • the method may specifically comprise the steps of c) and d) below:
  • transgenic plant obtained in the step c) obtaining, from the transgenic plant obtained in the step c), a transgenic plant having an increased ability to absorb or transport nitrate and/or an expression of a gene related to nitrate metabolism as compared with the plant of interest;
  • the nitrate metabolism-related gene is selected from any one of the following: NRT1.1B, NRT2.1, NRT2.3a, NAR1, and NAR2.
  • the coding gene may be the DNA molecule of any one of (1) to (4) below:
  • the above stringent conditions may be that the solution is mixed with a solution of 6 x SSC, 0.5% SDS at 65 ° C, and then washed once with 2 x SSC, 0.1% SDS and 1 x SSC, 0.1% SDS.
  • sequence 2 consists of 8143 nucleotides, which is a genomic sequence
  • sequence 3 consists of 1812 nucleotides, which is a cDNA sequence
  • sequence 2 and sequence 3 encode a protein represented by sequence 1 in the sequence listing, sequence 1 consists of Composition of 603 amino acid residues.
  • the coding gene can be introduced into the plant of interest by a recombinant expression vector containing the coding gene.
  • the recombinant expression vector can be constructed using existing plant expression vectors.
  • the plant expression vector includes a dual Agrobacterium vector and 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 further comprise a 3' untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal directs the addition of polyadenylation to the 3' end of the mRNA precursor.
  • any enhanced, constitutive, tissue-specific or inducible promoter may be added before the transcription initiation nucleotide, for example, cauliflower mosaic virus (CAMV) 35S promoter a ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A, etc., which can be used alone or in combination with other plant promoters; in addition, when a recombinant expression vector is constructed using the gene of the present invention, it can also be used.
  • Enhancers including translational enhancers or transcriptional enhancers, may be ATG start codons or contiguous region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the recombinant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound that can be expressed in plants, and a resistant antibiotic marker. Or anti-chemical reagents, etc. Transformed plants can also be screened directly under adverse conditions without any selectable marker genes.
  • the promoter for initiating transcription of the coding gene in the recombinant expression vector is specifically an Actin1 promoter or a self-endogenous promoter of the rice NRT1.1A gene (SEQ ID NO: 9). More specifically, the recombinant expression vector is a recombinant plasmid obtained by replacing a small fragment between the restriction sites XbaI and PstI of the pCAMBIA2300-Actin vector with the DNA fragment shown in SEQ ID NO: 3 in the sequence table; or the vector in pCAMBIA2300 The DNA fragment shown in 1-1814 of SEQ ID NO: 9 in the sequence listing was inserted between the cleavage site KpnI and EcoRI, and the DNA fragment shown in SEQ ID NO: 3 in the sequence listing was inserted between the restriction sites EcoRI and XmaI.
  • the recombinant plasmid or a recombinant plasmid obtained by replacing the small fragment between the cleavage sites Bam HI and Sal I of the pCAMBIA2300-35S-OCS vector with the DNA fragment shown in SEQ ID NO: 3 in the Sequence Listing.
  • the recombinant expression vector is introduced into the plant of interest, specifically by using a Ti plasmid, a Ri plasmid, a plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium
  • the conventional biological methods are mediated to transform plant cells or tissues, and the transformed plant tissues are grown into plants.
  • the plant can be either a monocot or a dicot.
  • the monocot may specifically be rice; the dicot may specifically be Arabidopsis thaliana.
  • the plant is specifically a rice variety Dongjin.
  • the plant is specifically Arabidopsis Columbia-0.
  • the nitrate metabolism-related gene is selected from any one of the following: OsNRT1.1B, OsNRT2.1, OsNRT2.3a, OsNAR1, and OsNAR2.
  • the nucleotide sequence of the OsNRT1.1B is the sequence 4 in the sequence listing; the nucleotide sequence of the OsNRT2.1 is the sequence 5 in the sequence listing; the nucleotide sequence of the OsNRT2.3a is Sequence 6 in the Sequence Listing; the nucleotide sequence of OsNAR1 is Sequence 7 in the Sequence Listing; the nucleotide sequence of OsNAR2 is Sequence 8 in the Sequence Listing.
  • Figure 1 shows the expression of NRT1.1A gene in various tissues of rice.
  • FIG 2 is a protein exhibiting NRT1.1A in Xenopus oocytes 15 NO 3 - transport activity.
  • Figure 3 shows that the rice nrt 1.1a mutant exhibits inhibition of nitrate transport.
  • A is the result of 3 hours after the transfer of the new nutrient solution;
  • B is the result of 24 hours after the transfer of the new nutrient solution.
  • WT represents a wild type control.
  • Figure 4 shows the inhibition of the expression levels of other nitrate transporter genes in the context of rice nrt1.1a mutants.
  • WT represents a wild type control.
  • Figure 5 shows that rice transgenic lines OX1-1 and OX2-6 overexpressed by NRT1.1A showed significant growth advantages. Among them, A is the phenotype of seedling stage; B is the phenotype of reproductive growth period. WT represents a wild type control.
  • Figure 6 shows that the expression level of nitrate utilization-related genes was significantly up-regulated in the context of NRT1.1A overexpressing rice transgenic lines OX1-1 and OX2-6.
  • WT represents a wild type control.
  • Figure 7 shows that NRT1.1A overexpressing rice transgenic lines pNA-2 and pNA-4 showed yield advantages under low nitrogen/high nitrogen conditions in the field.
  • A is a high nitrogen condition in the field
  • B is a low nitrogen condition in the field.
  • FIG 8 shows that NRT1.1A overexpressing Arabidopsis transgenic lines OX-3 and OX-17 showed significant growth advantages.
  • A is the detection result of NRT1.1A gene expression level in WT, OX-3 and OX-17;
  • B is the seedling stage phenotype;
  • C is the convulsion stage phenotype.
  • WT represents the wild type control (Columbia-0).
  • pCAMBIA2300-Actin vector described in "Wang, KJ, Tang, D., Wang, M., Lu, JF, Yu, HX, Liu, JF, Qian, BX, Gong, ZY, Wang, X., Chen, JM , Gu, MHand Cheng, ZK (2009) MER3is required for normal meiotic crossover formation, but not for presynaptic alignment in rice. J Cell Sci, 122, 2055-2063.”, the public is available from the applicant, only available The experiment of the present invention was repeated.
  • Rice nrt1.1a mutant from the Korean rice mutant library (Crop Biotech Institute, Kyung Hee University, Republic of Korea, http://www.postech.ac.kr/life/pfg/risd), the ordering address is http://signal.salk.edu/cgi-bin/RiceGE5, the rice nrt1.
  • the corresponding number of the 1a mutant is PFG_1E-00433.L.
  • a specific construction method is described in "Jakyung Yi, Gynheung An. Utilization of T-DNA Tagging Lines in Rice. J. Plant Biol. (2013) 56: 85-90".
  • Xenopus oocyte described in "Suhong Xu, Feng Cheng, Juan Liang, et al. Maternal x Norrin, a Canonical Wnt Signaling Agonist and TGF- ⁇ Antagonist, Controls Early Neuroectoderm Specification in Xenopus. PloS Biology, 2012. The article, available to the public from the applicant, can only be used to repeat the experiments of the present invention.
  • pCS2 + vector described in "Suhong Xu, Feng Cheng, Juan Liang, et al. Maternal x Norrin, a Canonical Wnt Signaling Agonist and TGF- ⁇ Antagonist, Controls Early Neuroectoderm Specification in Xenopus. PloS Biology, 2012." Obtained by the Applicant, can only be used to repeat the experiments of the present invention.
  • pCAMBIA2300-35S-OCS vector recorded in "Xianyang, Xiayang, Zhang Jinwen et al. Construction of pCAMBIA2300-betA-BADH bivalent plant expression vector. Chinese Agricultural Science Bulletin, 2009 09", the public can obtain from the applicant, It can only be used to repeat the experiments of the present invention.
  • NRT1.1A gene involved in the present example is derived from rice (Oryza.sativa L.), and its genomic sequence is shown in SEQ ID NO: 2 in the sequence listing, and sequence 2 is composed of 8143 nucleotides, and its cDNA sequence is a sequence listing. In sequence 3, sequence 3 consists of 1812 nucleotides. Sequence 2 and Sequence 3 encode the protein shown in SEQ ID NO: 1 in the Sequence Listing (NRT 1.1A protein), and Sequence 1 consists of 603 amino acid residues.
  • RNA was extracted from the roots, stems, sheaths, leaves and ears of the rice variety Dongjin and reverse transcribed to obtain cDNA. Furthermore, using the obtained cDNA as a template, real-time quantitative fluorescent PCR was performed on the NRT1.1A gene to detect the expression level of NRT1.1A gene in the rice tissues. The experiment was repeated 3 times and the results were averaged.
  • the primer sequences used to detect the NRT1.1A gene are as follows:
  • qNRT1.1A-F 5'-CCGTCTTCTTCGTCGGCTCCATCCT-3' (positions 1187-1211 of sequence 3);
  • qNRT1.1A-R 5'-CCCGTGCTCATCGTCTTCATCCCCT-3' (reverse complement of positions 1514-1538 of SEQ ID NO: 3).
  • OsActin1 was used as an internal reference gene, and its primer sequence was:
  • 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 amount of the NRT1.1A gene was calculated.
  • NRT1.1A gene was highly expressed in all tissues of rice (see Figure 1) and the highest in roots followed by leaves, sheaths and stems, and the lowest expression in ears. This constitutive expression suggests that NRT1.1A may be involved in the maintenance of the basic physiological functions of plants.
  • NRT1.1A gene involved in the present example is derived from rice (Oryza.sativa L.), and its genomic sequence is shown in SEQ ID NO: 2 in the sequence listing, and sequence 2 is composed of 8143 nucleotides, and its cDNA sequence is a sequence listing. In sequence 3, sequence 3 consists of 1812 nucleotides. Sequence 2 and Sequence 3 encode the protein shown in SEQ ID NO: 1 in the Sequence Listing (NRT 1.1A protein), and Sequence 1 consists of 603 amino acid residues.
  • the total RNA of the indica variety Dongjin was extracted and reverse transcribed into cDNA.
  • the obtained cDNA was used as a template, and the NRT1.1A cDNA was subjected to PCR amplification using the following primer sequences.
  • the ends of the primers used for amplification were introduced into the recognition sites of restriction endonucleases BamHI and EcoRI (shown below), and the primer sequences were:
  • R 5'- GAATTC TCAGTGGAGGCATGGCTCGG-3' (the underlined portion is the recognition sequence of EcoRI, and the subsequent sequence is the reverse complement of positions 1793-1812 of SEQ ID NO: 3 in the sequence listing).
  • the amplified fragment of interest was ligated into the Xenopus oocyte expression vector pCS2 + and verified by sequencing.
  • the recombinant vector obtained by sequencing after the replacement of the small fragment between the cleavage sites BamHI and EcoRI of the pCS2+ vector into the DNA fragment shown in SEQ ID NO: 3 in the sequence listing was designated as pCS2+/NRT1.1A.
  • the recombinant vector pCS2 + /NRT1.1A obtained in the first step was linearized with the restriction endonuclease ApaI, and then the in vitro transcription kit (mMESSAGE) was used.
  • mMESSAGE in vitro transcription kit
  • the resulting cRNA was injected into Xenopus oocyte. After the injection, after culturing for two days in ND96 solution (formulation: 96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1.8 mM CaCl 2 , 5 mM HEPES, pH 7.4), the solution was transferred to an absorption solution containing 10 mM K 15 NO 3 (formulation: 10 mM).
  • the experiment was simultaneously set to inject a control of the same volume of ddH 2 O.
  • Example 3 Obtainment and functional verification of rice nrt1.1a mutant
  • NRT1.1A gene involved in the present example is derived from rice (Oryza.sativa L.), and its genomic sequence is shown in SEQ ID NO: 2 in the sequence listing, and sequence 2 is composed of 8143 nucleotides, and its cDNA sequence is a sequence listing. In sequence 3, sequence 3 consists of 1812 nucleotides. Sequence 2 and Sequence 3 encode the protein shown in SEQ ID NO: 1 in the Sequence Listing (NRT 1.1A protein), and Sequence 1 consists of 603 amino acid residues.
  • Rice nrt1.1a mutant from the Korean rice mutant library (Crop Biotech Institute, Kyung Hee University, Republic of Korea, http://www.postech.ac.kr/life/pfg/risd), the ordering URL is http: //signal.salk.edu/cgi-bin/RiceGE5, the corresponding number of the rice nrt1.1a mutant is PFG_1E-00433.L.
  • a specific construction method is described in "Jakyung Yi, Gynheung An. Utilization of T-DNA Tagging Lines in Rice. J. Plant Biol. (2013) 56: 85-90".
  • the identification of the rice mutant nrt1.1a was carried out using the "three primer method".
  • the sequences of the primers used are as follows:
  • pGA2715L 5'-CTAGAGTCGAGAATTCAGTACA-3';
  • NARM14F 5'-AATCCGCAAATGTGTCTTGT-3';
  • NARM14R 5'-CTAGGGCCATCTTGTCTTCA-3'.
  • NRT1.1A In order to specifically understand the in vivo function of NRT1.1A, the inventors of the present invention conducted a 15 N tracer nitrate transport experiment.
  • seedlings of wild-type rice Dongjin and nrt1.1a mutants were in modified Kimura B nutrient solution (recipe: 2 mM KNO 3 , 1.8 mM KCl, 0.36 mM CaCl 2 , 0.54 mM MgSO 4 ⁇ 7H 2 O, 0.18 mM KH 2 PO 4 , 40 ⁇ M Na 2 EDTA-Fe(II), 13.4 ⁇ M MnCl 2 ⁇ 4H 2 O, 18.8 ⁇ M H 3 BO 3 , 0.03 ⁇ M Na 2 MoO 4 ⁇ 2H 2 O, 0.3 ⁇ M ZnSO 4 ⁇ 7H 2 O
  • the cells were cultured for 10 days in 0.32 ⁇ M CuSO 4 ⁇ 5H 2 O and 1.6 mM Na 2 SiO 3 ⁇ 9H 2 O
  • OsNRT1.1B gene the nucleotide sequence of which is the sequence 4 in the sequence listing, which is mainly responsible for the absorption and transport of nitrates, and participation.
  • OsNRT2.1 gene the nucleotide sequence of the cDNA is sequence 5 in the sequence listing, which is mainly responsible for nitrate uptake
  • OsNRT2.3a gene the nucleotide sequence of which is Sequence 6 in the sequence listing is responsible for the transport of nitrate to the aerial part
  • OsNAR1 gene the nucleotide sequence of which is the sequence 7 of the sequence listing, the cofactor of the function of OsNRT2.1
  • OsNAR2 gene The nucleotide sequence of the cDNA is a cofactor for the function of sequence 8 and OsNRT2.1 in the sequence listing.
  • the leaves of wild type rice Dongjin and nrt1.1a mutants were extracted, total RNA was extracted, and cDNA was reverse transcribed. Then, the above five nitrate utilization related genes were detected by qRT-PCR using the following primer pairs.
  • OsNRT1.1B-F 5'-GGCAGGCTCGACTACTTCTA-3';
  • OsNRT1.1B-R 5'-AGGCGCTTCTCCTTGTAGAC-3'.
  • OsNRT2.1-F 5'-CTTCACGTCGTCGAGGTACT-3';
  • OsNRT2.1-R 5'-CACTCGGAGCCGTAGTAGTG-3'.
  • OsNRT2.3a-F 5'-CGCTGCTGCCGCTCATCCG-3';
  • OsNRT2.3a-R 5'-CCGTGCCCATGGCCAGAC-3'.
  • OsNAR1-F 5'-GTTCAAGAGCGTGAAGGTGA-3';
  • OsNAR1-R 5'-CACCACGTAGTCGAACCTG-3'.
  • OsNAR2-F 5'-TCGTCCTCGAGAACAAGAAG-3';
  • OsNAR2-R 5'-TCCGTTGGTTTTGTAGGTTG-3'.
  • OsActin1 As an internal reference gene, its detection primer pair:
  • 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 amount of each gene was calculated.
  • RNA of indica rice Dongjin was extracted and reverse transcribed into cDNA.
  • the obtained cDNA was used as a template, and the CDS of NRT1.1A was subjected to PCR amplification using the following primer sequences.
  • the ends of the primers used for amplification were introduced into the recognition sites of restriction endonucleases XbaI and PstI (shown below), and the primer sequences were as follows:
  • R 5'- CTGCAG TCAGTGGAGGCATGGCTCGG-3 ' ( underlined PstI recognition sequence, followed by a sequence of 1793-1812 reverse complement sequence at position 3 of the sequence table).
  • the CDS region of NRT1.1A (including the stop codon) was amplified using the cDNA of the rice variety Dongjin as a template.
  • the PCR product was ligated into the T vector pEASY-Blunt (TransGene), and then digested with XbaI and PstI to be ligated into the plant expression vector pCambia2301-Actin.
  • the recombinant vector obtained by sequencing after replacing the small fragment between the restriction sites XbaI and PstI of the pCAMBIA2300-Actin vector with the DNA fragment shown in SEQ ID NO: 3 in the sequence listing was designated as pCAMBIA2300-Actin/NRT1.1A.
  • the recombinant plant expression vector pCAMBIA2300-Actin/NRT1.1A constructed in the first step was transferred into Agrobacterium AGL1 (from ATCC), and then infected into the callus of the indica rice variety Dongjin.
  • Agrobacterium AGL1 from ATCC
  • a control transferred to the pCAMBIA2300-Actin empty vector was also set.
  • T obtained from step 20 into the generation of transgenic rice pCAMBIA2300-Actin / NRT1.1A and into pCAMBIA2300-Actin empty vector control plants were extracted genomic DNA. PCR was carried out using primers F1 and R1 (primer sequences as follows) against the NptII gene, and it was confirmed that the plant containing the NptII gene (the PCR product was about 500 bp in size) was a transgenic-positive plant.
  • F1 5'-TCCGGCCGCTTGGGTGGAGAG-3';
  • R1 5'-CTGGCGCGAGCCCCTGATGCT-3'.
  • the T 0 generation NRT1.1A transgenic rice lines OX1-1 and OX2-6 obtained in the step (1), the control plants transferred into the pCAMBIA2300-Actin empty vector, and the wild type rice variety Dongjin were used as experimental materials.
  • Total RNA from each material was extracted and reverse transcribed to obtain cDNA.
  • real-time quantitative fluorescent PCR was performed on the NRT1.1A gene, and the expression level of the NRT1.1A gene in each material at the transcription level was examined. The experiment was repeated 3 times and the results were averaged.
  • the primer sequences used to detect the NRT1.1A gene are as follows:
  • qNRT1.1A-F 5'-CCGTCTTCTTCGTCGGCTCCATCCT-3' (positions 1187-1211 of sequence 3);
  • qNRT1.1A-R 5'-CCCGTGCTCATCGTCTTCATCCCCT-3' (reverse complement of positions 1514-1538 of SEQ ID NO: 3).
  • OsActin1 was used as an internal reference gene, and its primer sequence was:
  • 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 amount of the NRT1.1A gene was calculated.
  • the plant height of each genetic material was observed and recorded at the seedling stage, and the plant height of each genetic material was observed and recorded again after entering the reproductive growth period. In the experiment, at least 30 individual plants were selected for each transgenic line for statistics.
  • genes related to nitrate metabolism are specifically related to:
  • the nucleotide sequence of the cDNA is the sequence 5 in the sequence listing;
  • the nucleotide sequence of the cDNA is the sequence 6 in the sequence listing;
  • the nucleotide sequence of the cDNA is the sequence 8 in the sequence listing;
  • the OsNIR1 gene whose nucleotide sequence is the sequence 7 in the sequence listing.
  • the specific measurement method is carried out in the third step of Example 2.
  • the T 2 generation NRT1.1A transgenic rice lines OX1-1 and OX2-6 showed certain growth advantages at seedling stage, and the plant height was significantly higher than that of the wild type control; when entering the flowering stage, the overexpressing lines showed early flowering stage.
  • the phenotype see Figure 5).
  • the plant height and flowering time of the control plants transferred to pCAMBIA2300-Actin empty vector were basically the same as those of the wild type control, and there was no statistical difference.
  • NRT1.1A can achieve the effect of rice yield increase and avoid the negative effect of constitutive promoter on rice agronomic traits
  • the inventors of the present invention constructed a self-promoter-driven NRT1.1A overexpression transgenic plant, and further Its functionality has been verified.
  • the genomic DNA and total RNA of the rice variety Dongjin were extracted, and the total RNA was reverse transcribed into cDNA.
  • the promoter region of NRT1.1A was amplified by using genomic DNA of Dongjin as a template (sequence 9), and the recognition sites of restriction endonucleases KpnI and EcoRI were introduced at both ends of the primers for amplification (shown below).
  • the primer sequences are as follows:
  • NRT1.1Ap-F 5'- GGTACC TTCGATCTCCCACGTAAGAC-3' (the underlined portion is the recognition sequence of KpnI, and the subsequent sequence is position 1-20 of sequence 9);
  • NRT1.1Ap-R 5'- GAATTC TCTCTCTCTTCTTCTTCTTCCTC-3' (the underlined portion is the recognition sequence of EcoRI, and the subsequent sequence is the reverse complement of positions 1790-1814 of SEQ ID NO: 9).
  • the cDNA of NRT1.1A was amplified by using the cDNA of rice variety Dongjin as a template.
  • the ends of the primers used for amplification were introduced into the recognition sites of restriction endonucleases EcoRI and XmaI.
  • the sequences of the primers were as follows:
  • NRT1.1A CDS- F 5'- GAATTC ATGGTGGGGATGTTGCCGGA-3' (the underlined part is the recognition sequence of EcoRI, and the subsequent sequence is the 1st to 20th position of sequence 3);
  • NRT1.1A CDS- R 5'- CCCGGG GTGGAGGCATGGCTCGG-3' (the underlined portion is the recognition sequence of XmaI, and the subsequent sequence is the reverse complement of positions 1793-1809 of SEQ ID NO: 3).
  • the two PCR fragments amplified in the above two steps were ligated into the corresponding cleavage sites of the pBluescript KS(+) (Stratagene, 212205) vector, respectively.
  • the fragments containing the endogenous promoter and NRT1.1A CDS were ligated into the corresponding restriction sites of the binary vector pCAMBIA2300 by KpnI and BamHI, and the obtained recombinant vectors confirmed by sequencing were named.
  • pCAMBIA2300/NRT1.1A For pCAMBIA2300/NRT1.1A.
  • the recombinant plant expression vector pCAMBIA2300/NRT1.1A constructed in step 1 was transferred into Agrobacterium AGL1 (from ATCC), and the callus of indica rice cultivar Dongjin was transformed. See the literature "Yi Zili, Cao Shouyun, Wang Li, He Yujie, Chu Chengcai, Tang Yi, Zhou Puhua, Tian Wenzhong. Study on improving the frequency of Agrobacterium-mediated transformation of rice. Chinese Journal of Genetics, 2001, 28(4): 352-358. A control that was transferred to the pCAMBIA2300 empty vector was also set. The final vaccine is obtained both transgenes, i.e. into rice plants pCAMBIA2300 / NRT1.1A and empty vector pCAMBIA2300 into rice plants (T 0).
  • T obtained from step 20 into the generation of transgenic rice pCAMBIA2300 / NRT1.1A, and transferred to the empty vector control pCAMBIA2300 plant genomic DNA was extracted.
  • PCR was carried out using primers F1 and R1 (primer sequences as follows), and it was confirmed that the plant containing the NptII gene (the PCR product was about 500 bp in size) was a transgenic-positive plant.
  • F1 5'-TCCGGCCGCTTGGGTGGAGAG-3';
  • R1 5'-CTGGCGCGAGCCCCTGATGCT-3'.
  • pNA-2 and pNA-4 two randomly selected transgenic rice lines transformed into pCAMBIA2300/NRT1.1A were designated as pNA-2 and pNA-4, respectively.
  • step (1) Identification of the obtained positive T 0 generation of transgenic rice NRT1.1A pNA-2, and pNA-4, into pCAMBIA2300 empty vector control plants, and wild type rice cultivars Dongjin as experimental materials. Total RNA from each material was extracted and reverse transcribed to obtain cDNA. Further, using the obtained cDNA as a template, real-time quantitative fluorescent PCR was performed on the NRT1.1A gene, and the expression level of the NRT1.1A gene in each material at the transcription level was examined. The experiment was repeated 3 times and the results were averaged.
  • the primer sequences used to detect the NRT1.1A gene are as follows:
  • qNRT1.1A-F 5'-CCGTCTTCTTCGTCGGCTCCATCCT-3' (positions 1187-1211 of sequence 3);
  • qNRT1.1A-R 5'-CCCGTGCTCATCGTCTTCATCCCCT-3' (reverse complement of positions 1514-1538 of SEQ ID NO: 3).
  • OsActin1 was used as an internal reference gene, and its primer sequence was:
  • 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 amount of the NRT1.1A gene was calculated.
  • the positive T 2 generation NRT1.1A transgenic rice lines pNA-2 and pNA-4 were identified in step 3, the control plants transformed into pCAMBIA2300 empty vector, and the untransgenic wild type rice Dongjin were used as experimental materials.
  • the field trial set two nitrogen fertilizer gradients (high nitrogen and low nitrogen). Among them, high nitrogen is fertilized according to 4.28 kg of urea per 100 square meters, and low nitrogen is fertilized according to 1.07 kg of urea per 100 square meters. Three cells were set under each nitrogen fertilizer gradient. Observe and record the phenotype (including number of tillers, 1000-grain weight, grain number per ear, and yield per plant) in the field. Each line randomly selects at least 10 individual plants in each plot for phenotypic statistics, totaling a large number of each strain. In 30 individual plants.
  • NRT1.1A overexpressing transgenic lines pNA-2 and pNA-4 showed a significant increase in kernel number per ear and yield per plant (P ⁇ 0.05). . Especially under low nitrogen conditions, the yield per plant of overexpressing transgenic lines was particularly significant (P ⁇ 0.01).
  • RNA of indica rice Dongjin was extracted and reverse transcribed into cDNA.
  • the obtained cDNA was used as a template, and the CDS of NRT1.1A was subjected to PCR amplification using the following primer sequences.
  • the ends of the primers used for amplification were introduced into the recognition sites of restriction endonucleases Bam HI and Sal I (shown below), and the primer sequences were as follows:
  • the CDS region of NRT1.1A was amplified using the cDNA of the rice variety Dongjin as a template.
  • the PCR product was ligated into the T vector pEASY-Blunt (TransGene), and then digested with Bam HI and Sal I and ligated into the plant expression vector pCAMBIA2300-35S-OCS.
  • the recombinant vector obtained by sequencing after sequencing the small fragment between the cleavage sites Bam HI and Sal I of the pCAMBIA2300-35S-OCS vector into the DNA fragment shown in SEQ ID NO: 3 in the sequence table was named pCAMBIA2300-35S-OCS/ NRT1.1A.
  • the recombinant plant expression vector pCAMBIA2300-35S-OCS/NRT1.1A constructed in step 1 was transformed into Arabidopsis Columbia-0 by Agrobacterium tumefaciens GV3101 (from ATCC)-mediated genetic transformation (Reference: "Steven J. Clough and Andrew F. Bent. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal. (1998) 16(6), 735-743").
  • a control transferred to the pCAMBIA2300-35S-OCS empty vector was also set.
  • Arabidopsis plants were finally obtained seedlings both transgenes, i.e. into Arabidopsis plants pCAMBIA2300-35S-OCS / NRT1.1A pCAMBIA2300-35S-OCS and into an empty vector (T 0).
  • T obtained from step 20 into the generation of transgenic Arabidopsis pCAMBIA2300-35S-OCS / NRT1.1A two randomly selected, as denoted as OX-3 and OX-17.
  • Total RNA was extracted from OX-3 and OX-17, control plants transformed into pCAMBIA2300-35S-OCS empty vector, and wild-type Arabidopsis Columbia-0, which was not transgenic, and reverse transcribed to obtain cDNA. Further, using the obtained cDNA as a template, real-time quantitative fluorescent PCR was performed on the NRT1.1A gene, and the expression level of the NRT1.1A gene in each material at the transcription level was examined. The experiment was repeated 3 times and the results were averaged.
  • the primer sequences used to detect the NRT1.1A gene are as follows:
  • qNRT1.1A-F 5'-CCGTCTTCTTCGTCGGCTCCATCCT-3' (positions 1187-1211 of sequence 3);
  • qNRT1.1A-R 5'-CCCGTGCTCATCGTCTTCATCCCCT-3' (reverse complement of positions 1514-1538 of SEQ ID NO: 3).
  • the primer sequence is:
  • qAtActin2-F 5'-GCACCACCTGAAAGGAAGTACA-3';
  • qAtActin2-R 5'-CGATTCCTGGACCTGCCTCATC-3'.
  • the expression level of the reference gene was regarded as 1, and the relative expression amount of the NRT1.1A gene was calculated.
  • the real-time quantitative PCR analysis of the expression level of NRT1.1A gene in each experimental material showed that the T 0 generation obtained in step 2 was transferred to the transgene of pCAMBIA2300-35S-OCS/NRT1.1A compared to the untransgenic wild-type Arabidopsis thaliana.
  • the expression level of the NRT1.1A gene in the Arabidopsis lines OX-3 and OX-17 was significantly increased at the transcriptional level (see A in Figure 8).
  • the expression level of NRT1.1A gene was basically the same at the transcription level as that of the non-transgenic wild-type plants, and there was no statistical difference.
  • the positive T 2 generation NRT1.1A transgenic Arabidopsis lines OX-3 and OX-17 were identified in step 3, the control plants transferred into the pCAMBIA2300-35S-OCS empty vector, and the untransformed wild-type Arabidopsis Columbia- 0 is the experimental material.
  • the size of the rosette leaves of each genetic material was observed at the seedling stage, and the convulsion time of each genetic material was observed after entering the twitching stage. In the experiment, at least 30 individual plants were selected for each transgenic line for observation.
  • the T 2 generation NRT1.1A transgenic Arabidopsis lines OX-3 and OX-17 showed certain growth advantages at the seedling stage, and the rosette leaves were significantly larger than the wild type control (see Figure B, B). After entering the convulsion stage, Overexpression lines were significantly earlier than wild type controls (see Figure 8 C). For the control plants transferred to the pCAMBIA2300-35S-OCS empty vector, the lotus leaf size and the time of twitching at the seedling stage were basically the same as those of the non-transgenic wild type plants.
  • the present invention demonstrates that the NRT 1.1A protein is capable of transporting nitrate by utilizing an in vitro transport system of Xenopus oocytes.
  • Transgenic experiments showed that overexpression of NRT1.1A in wild type of rice can show obvious growth advantage, and significantly increase the number of kernels and plant height, and thus increase rice yield.
  • NRT1.1A overexpresses seedling stage in Arabidopsis wild type. On the basis of showing certain growth advantages, the rosette leaves were significantly larger than the wild type control; after entering the twitching stage, the overexpressed lines were significantly earlier than the wild type control.

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Abstract

一种水稻NRT1.1A基因及其编码蛋白在提高植物产量育种中的应用。所述应用具体为蛋白质或其编码基因在调控植物生长发育中的应用；所述生长发育体现为单株产量和/或株高和/或穗粒数和/或开花时间和/或抽薹时间和/或莲座叶大小；所述蛋白质为如下中任一种：(1)氨基酸序列为序列表中序列1所示的蛋白质；(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。所述应用对于培育高产水稻新品种具有重要意义。

Description

水稻NRT1.1A基因及其编码蛋白在提高植物产量育种中的应用 技术领域
本发明属于生物技术领域，涉及一种水稻NRT1.1A基因及其编码蛋白在提高植物产量育种中的应用。
背景技术
植物需要多种矿质营养元素来维持其正常生长，其中氮是植物需求量最大的矿质营养元素。一般而言，氮元素能占植物体干重的1.5-2％。氮是蛋白质、核酸、磷脂以及植物生长发育所必需的有机氮化合物的构成成分，而这些物质是活细胞赖以生存的结构或功能成分，因此氮元素也被称为生命元素。为了追求作物的高产，在农业生产中往往施加大量氮肥。其中，水稻的氮肥施用量又远远超过其他任何农作物，氮肥的损失量占化肥总施用量的70％。我国普遍存在氮肥使用过量、氮利用效率低以及氮肥损失导致的环境恶化等一系列问题。
通过提高农作物的氮利用效率以及减少氮肥施用则是解决该严重环境问题的关键。硝酸盐是土壤中最为重要的氮源之一，植物对硝酸盐的吸收和利用在很大程度上决定了农作物的氮利用效率。而硝酸盐转运蛋白则是植物吸收、转运和储存硝酸盐的最直接功能执行者。
发明公开
本发明的目的是提供一种水稻NRT1.1A基因及其编码蛋白的新用途。
本发明所提供的新用途具体为蛋白质或其编码基因在调控植物生长发育中的应用；所述生长发育体现为单株产量和/或株高和/或穗粒数和/或开花时间和/或抽薹时间和/或莲座叶大小和/或生物量；
其中，所述开花时间为开始开花的时间点的早晚；所述抽薹时间为开始抽薹的时间点的早晚。
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质(即NRT1.1A蛋白)；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
所述蛋白质或其编码基因调控植物生长发育具体体现在：所述蛋白质在所述植物中的表达量越高，所述植物的单株产量越高、株高越高、穗粒数越多、开花时间越早抽薹时间越早和/或莲座叶越大和/或生物量越大；所述蛋白质在所述植物中的表达量越低，所述植物的单株产量越低、株高越低、穗粒数越少、开花时间越晚、抽薹时间越晚和/或莲座叶越小和/或生物量越小。
蛋白质或其编码基因在选育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的植物品种中的应用也属于本发明的保护范围；
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质(即NRT1.1A蛋白)；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
在实际应用中，当所选育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的植物品种时，需将所述蛋白质表达量较高的植株作为亲本进行杂交。
本发明的再一个目的是培育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量减少的转基因植物的方法。
本发明所提供的培育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的转基因植物的方法，可包括使目的植物表达或超量表达蛋白质的步骤，或者包括使目的植物中蛋白质活性提高的步骤；
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质；
进一步，所述方法具体可包括如下a)和b)的步骤：
a)向所述目的植物中导入所述蛋白质的编码基因，得到表达或超量表达所述编码基因的转基因植物；
b)从步骤a)所得转基因植物中得到与所述目的植物相比，单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的转基因植物。
蛋白质或其编码基因在如下(A)或(B)中的应用也属于本发明的保护范围：
(A)促进硝酸盐吸收或转运；
(B)促进硝酸盐代谢相关基因的表达；所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2；
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质(即NRT1.1A蛋白)；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与硝酸盐转运相关的由序列1衍生的蛋白质。
在本发明中，所述(A)中的所述促进硝酸盐吸收或转运具体为促进爪蟾卵母细胞中硝酸盐的吸收或转运，或者为促进水稻中硝酸盐的吸收或转运；所述(B)中的所述促进硝酸盐代谢相关基因的表达具体为促进水稻中硝酸盐代谢相关基因的表达；所述硝酸盐代谢相关基因选自如下中任一种：OsNRT1.1B、OsNRT2.1、OsNRT2.3a、OsNAR1和OsNAR2。
蛋白质或其编码基因在如下(C)或(D)中的应用也属于本发明的保护范围：
(C)选育对硝酸盐的吸收或转运能力提高的植物品种；
(D)选育硝酸盐代谢相关基因的表达量提高的植物品种；所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2；
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质(即NRT1.1A蛋白)；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与硝酸盐转运相关的由序列1衍生的蛋白质。
本发明的另一个目的是提供一种培育对硝酸盐的吸收或转运能力提高和/或硝酸盐代谢相关基因的表达量提高的转基因植物的方法。
本发明所提供的培育对硝酸盐的吸收或转运能力提高和/或硝酸盐代谢相关基因的表达量提高的转基因植物，可包括使目的植物表达或过表达蛋白质的步骤，或者包括使目的植物中蛋白质活性提高的步骤；
所述蛋白质为如下中任一种：
(1)氨基酸序列为序列表中序列1所示的蛋白质；
(2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质；
进一步，所述方法具体可包括如下c)和d)的步骤：
c)向所述目的植物中导入所述蛋白质的编码基因，得到表达所述编码基因的转基因植物；
d)从步骤c)所得转基因植物中得到与所述目的植物相比，对硝酸盐的吸收或转运能力提高和/或硝酸盐代谢相关基因的表达量提高的转基因植物；
所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2。
上述各应用和方法中，所述编码基因均可为如下(1)至(4)中任一所述的DNA分子：
(1)序列表中序列2的DNA分子；
(2)序列表中序列3的DNA分子；
(3)在严格条件下与(1)或(2)所限定的DNA分子杂交且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子；
(4)与(1)或(2)或(3)限定的DNA分子具有90％以上同源性且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子。
上述严格条件可为用6×SSC，0.5％SDS的溶液，在65℃下杂交，然后用2×SSC，0.1％SDS和1×SSC，0.1％SDS各洗膜一次。
其中，序列2由8143个核苷酸组成，为基因组序列；序列3由1812个核苷酸组成，为cDNA序列；序列2和序列3编码序列表中序列1所示的蛋白质，序列1由 603个氨基酸残基组成。
在以上所述的两种方法中，所述编码基因均可通过含有所述编码基因的重组表达载体导入所述目的植物中。
所述重组表达载体可用现有的植物表达载体构建。所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等，如pGreen0029、pCAMBIA3301、pCAMBIA1300、pBI121、pBin19、pCAMBIA2301、pCAMBIA1301-UbiN或其它衍生植物表达载体。所述植物表达载体还可包含外源基因的3’端非翻译区域，即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端。使用所述基因构建重组表达载体时，在其转录起始核苷酸前可加上任何一种增强型、组成型、组织特异型或诱导型启动子，例如花椰菜花叶病毒(CAMV)35S启动子、泛素基因Ubiquitin启动子(pUbi)、胁迫诱导型启动子rd29A等，它们可单独使用或与其它的植物启动子结合使用；此外，使用本发明的基因构建重组表达载体时，还可使用增强子，包括翻译增强子或转录增强子，这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等，但必需与编码序列的阅读框相同，以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的，可以是天然的，也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用重组表达载体进行加工，如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、具有抗性的抗生素标记物或是抗化学试剂标记基因等。也可不加任何选择性标记基因，直接以逆境筛选转化植株。
在本发明中，所述重组表达载体中启动所述编码基因转录的启动子具体为Actin1启动子或水稻NRT1.1A基因的自身内源启动子(序列9)。更加具体的，所述重组表达载体为将pCAMBIA2300-Actin载体的酶切位点XbaI和PstI之间的小片段替换为序列表中序列3所示DNA片段后得到的重组质粒；或者为在pCAMBIA2300载体的酶切位点KpnI和EcoRI之间插入序列表中序列9的第1-1814位所示DNA片段，且在酶切位点EcoRI和XmaI之间插入序列表中序列3所示DNA片段后所得的重组质粒；或者为将pCAMBIA2300-35S-OCS载体的酶切位点Bam HI和Sal I之间的小片段替换为序列表中序列3所示DNA片段后所得的重组质粒。
在以上所述的两种方法中，将所述重组表达载体导入所述目的植物，具体可为：通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织，并将转化的植物组织培育成植株。
在以上各应用和方法中，所述植物既可为单子叶植物，也可为双子叶植物。所述单子叶植物具体可为水稻；所述双子叶植物具体可为拟南芥。在本发明的一个实施例中，所述植物具体为水稻品种东津。在本发明的另一个实施例中，所述植物具体为拟南芥Columbia-0。
在以上各应用和方法中，当所述植物具体为水稻时，所述硝酸盐代谢相关基因选自如下中任一种：OsNRT1.1B、OsNRT2.1、OsNRT2.3a、OsNAR1和OsNAR2。
在本发明中，所述OsNRT1.1B的核苷酸序列为序列表中序列4；所述OsNRT2.1的核苷酸序列为序列表中序列5；所述OsNRT2.3a的核苷酸序列为序列表中序列6；所述OsNAR1的核苷酸序列为序列表中序列7；所述OsNAR2的核苷酸序列为序列表中序列8。
附图说明
图1为NRT1.1A基因在水稻各组织中的表达情况分析。
图2为NRT1.1A蛋白在爪蟾卵母细胞中表现出15NO3 -转运活性。
图3为水稻nrt1.1a突变体表现出硝酸盐转运的抑制。其中，A为转入新营养液后3h的结果；B为转入新营养液后24h的结果。WT表示野生型对照。
图4为水稻nrt1.1a突变体背景下其他硝酸盐转运蛋白基因的表达水平受到抑制。WT表示野生型对照。
图5为NRT1.1A过量表达的水稻转基因株系OX1-1、OX2-6表现出明显的生长优势。其中，A为幼苗期表型；B为生殖生长时期表型。WT表示野生型对照。
图6为在NRT1.1A过量表达水稻转基因株系OX1-1、OX2-6背景下硝酸盐利用相关基因表达水平显著上调。WT表示野生型对照。
图7为NRT1.1A过量表达水稻转基因株系pNA-2和pNA-4在田间低氮/高氮条件下表现出产量优势。其中，A为田间高氮条件；B为田间低氮条件。
图8为NRT1.1A过量表达拟南芥转基因株系OX-3和OX-17表现出明显的生长优势。其中，A为WT、OX-3和OX-17中NRT1.1A基因表达水平检测结果；B为幼苗期表型；C为抽薹期表型。WT表示野生型对照(Columbia-0)。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
pCAMBIA2300-Actin载体：记载于“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 meiotic crossover formation,but not for presynaptic alignment in rice.J Cell Sci,122,2055-2063.”一文，公众可从申请人处获得，仅可用于重复本发明实验。
水稻品种东津：记载于“Jakyung Yi,Gynheung An.Utilization of T-DNA Tagging Lines in Rice.J.Plant Biol.(2013)56:85-90”一文的“Dongjin”，公开可从申请人处获得，仅可用于重复本发明实验使用。
水稻nrt1.1a突变体：来自韩国水稻突变体库(Crop Biotech Institute, Kyung Hee University,Republic of Korea,http://www.postech.ac.kr/life/pfg/risd)，订购网址为http://signal.salk.edu/cgi-bin/RiceGE5，该水稻nrt1.1a突变体的对应编号为PFG_1E-00433.L。具体构建方法记载于“Jakyung Yi,Gynheung An.Utilization of T-DNA Tagging Lines in Rice.J.Plant Biol.(2013)56:85-90”一文。
爪蟾卵母细胞(Xenopus oocyte)：记载于“Suhong Xu,Feng Cheng,Juan Liang,et al.Maternal xNorrin,a Canonical Wnt Signaling Agonist and TGF-βAntagonist,Controls Early Neuroectoderm Specification in Xenopus.PloS Biology,2012.”一文，公众可从申请人处获得，仅可用于重复本发明实验。
pCS2+载体：记载于“Suhong Xu,Feng Cheng,Juan Liang,et al.Maternal xNorrin,a Canonical Wnt Signaling Agonist and TGF-βAntagonist,Controls Early Neuroectoderm Specification in Xenopus.PloS Biology,2012.”一文，公众可从申请人处获得，仅可用于重复本发明实验。
pCAMBIA2300-35S-OCS载体：记载于“咸洋,夏阳,张金文等.pCAMBIA2300-betA-BADH双价植物表达载体的构建.中国农学通报,2009年09期”一文，公众可从申请人处获得，仅可用于重复本发明实验。
实施例1、NRT1.1A在水稻各组织中表达情况分析
本实施例中所涉及的NRT1.1A基因来源于水稻(Oryza.sativa L.)，其基因组序列如序列表中序列2所示，序列2由8143个核苷酸组成，其cDNA序列为序列表中序列3，序列3由1812个核苷酸组成。序列2和序列3编码序列表中序列1所示的蛋白质(NRT1.1A蛋白)，序列1由603个氨基酸残基组成。
从水稻品种东津的根、茎、叶鞘、叶和穗中分别提取总RNA并反转录得到cDNA。进而以所得cDNA为模板，针对NRT1.1A基因进行实时定量荧光PCR，检测水稻各组织中NRT1.1A基因在转录水平上的表达量。实验重复3次，结果取平均值。
用于检测NRT1.1A基因的引物序列如下：
qNRT1.1A-F：5’-CCGTCTTCTTCGTCGGCTCCATCCT-3’(序列3的第1187-1211位)；
qNRT1.1A-R：5’-CCCGTGCTCATCGTCTTCATCCCCT-3’(序列3的第1514-1538位的反向互补序列)。
以OsActin1作为内参基因，其引物序列为：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。
将内参基因的表达量视为1，计算NRT1.1A基因的相对表达量。
qRT-PCR分析表明，NRT1.1A基因在水稻各组织中均有较高水平表达(见图1)而在根中表达最高其次是叶、叶鞘和茎，穗中表达量最低。这种组成型表达结果暗示，NRT1.1A可能参与了植物体的基础生理功能的维持。
实施例2、NRT1.1A体外转运硝酸盐活性验证
本实施例中所涉及的NRT1.1A基因来源于水稻(Oryza.sativa L.)，其基因组序列如序列表中序列2所示，序列2由8143个核苷酸组成，其cDNA序列为序列表中序列3，序列3由1812个核苷酸组成。序列2和序列3编码序列表中序列1所示的蛋白质(NRT1.1A蛋白)，序列1由603个氨基酸残基组成。
一、爪蟾重组表达载体pCS2+/NRT1.1A的构建
提取粳稻品种东津的总RNA，反转录为cDNA。以获得的cDNA为模板，采用下述引物序列分别对NRT1.1A cDNA进行PCR扩增。扩增采用的引物两端分别引入限制性内切酶BamHI和EcoRI的识别位点(如下划线所示)，引物序列：
F：5’-GGATCCATGGTGGGGATGTTGCCGGA-3’(下划线部分为BamHI的识别序列，其后的序列为序列表中序列3的第1-20位)；
R：5’-GAATTCTCAGTGGAGGCATGGCTCGG-3’(下划线部分为EcoRI的识别序列，其后的序列为序列表中序列3的第1793-1812位的反向互补序列)。
将扩增的目的片段连接入爪蟾卵母细胞表达载体pCS2+中，并通过测序进行验证。
将经测序表明将pCS2+载体的酶切位点BamHI和EcoRI之间的小片段替换为序列表中序列3所示DNA片段后所得的重组载体命名为pCS2+/NRT1.1A。
二、体外转录NRT1.1A mRNA并注射爪蟾卵母细胞验证转运硝酸盐活性
将步骤一构建获得的重组载体pCS2+/NRT1.1A用限制性内切酶ApaI进行线性化，然后采用体外转录试剂盒(mMESSAGE
Figure PCTCN2017097601-appb-000001
SP6Kit,Ambion,AM1340)体外转录出带5’帽子结构和3’poly A结构的cRNA，具体操作参见试剂盒说明书。
将所得cRNA注射爪蟾卵母细胞(Xenopus oocyte)。注射后，在ND96溶液(配方：96mM NaCl，2mM KCl，1mM MgCl2，1.8mM CaCl2，5mM HEPES，pH 7.4)中培养两天后，转移入含有10mM K15NO3的吸收溶液(配方：10mM K15NO3(98％atom 15N-KNO3,Sigma-Aldrich,335134)，230mM Mannitol，0.3mM CaCl2，10mM MES-Tris，pH 5.5)中，再培养3h，采用元素质谱分析仪(ICP-MS)测定爪蟾卵母细胞的15N含量(具体方法参见“Kun-Hsiang Liu,Chi-Ying Huang,Yi-Fang Tsay,et al.CHL1Is a Dual-Affinity Nitrate Transporter of Arabidopsis Involved in Multiple Phases of Nitrate Uptake.The Plant Cell,1999”一文)。实验设3次重复，测定结果取均值。
实验同时设置注射相同体积ddH2O的对照。
测定结果显示，注射有NRT1.1A cRNA的爪蟾卵母细胞的15N含量显著高于 注射相同体积ddH2O对照(P<0.01，见图2)。该结果表明NRT1.1A在体外具有转运硝酸盐活性。
实施例3、水稻nrt1.1a突变体的获得及功能验证
本实施例中所涉及的NRT1.1A基因来源于水稻(Oryza.sativa L.)，其基因组序列如序列表中序列2所示，序列2由8143个核苷酸组成，其cDNA序列为序列表中序列3，序列3由1812个核苷酸组成。序列2和序列3编码序列表中序列1所示的蛋白质(NRT1.1A蛋白)，序列1由603个氨基酸残基组成。
一、水稻nrt1.1a突变体的获得及鉴定
水稻nrt1.1a突变体：来自韩国水稻突变体库(Crop Biotech Institute,Kyung Hee University,Republic of Korea,http://www.postech.ac.kr/life/pfg/risd)，订购网址为http://signal.salk.edu/cgi-bin/RiceGE5，该水稻nrt1.1a突变体的对应编号为PFG_1E-00433.L。具体构建方法记载于“Jakyung Yi,Gynheung An.Utilization of T-DNA Tagging Lines in Rice.J.Plant Biol.(2013)56:85-90”一文。
水稻突变体nrt1.1a的鉴定采用“三引物法”进行。所用引物的序列如下：
pGA2715L：5’-CTAGAGTCGAGAATTCAGTACA-3’；
NARM14F：5’-AATCCGCAAATGTGTCTTGT-3’；
NARM14R：5’-CTAGGGCCATCTTGTCTTCA-3’。
经证实，水稻突变体nrt1.1a中NRT1.1A基因确实发生了突变，不能正常表达有功能的NRT1.1A蛋白。
二、水稻nrt1.1a突变体的硝酸盐转运功能验证
为了具体了解NRT1.1A的体内功能，本发明的发明人进行了15N示踪的硝酸盐转运实验。首先，将野生型水稻东津和nrt1.1a突变体的幼苗在改良的Kimura B营养液(配方：2mM KNO3，1.8mM 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和1.6mM Na2SiO3·9H2O)中培养10天，然后转入含有5mM K15NO3的改良Kimura B营养液中培养24h。在转入新营养液后3h和24h分地上和地下部分取材，采用元素质谱分析仪(ICP-MS)测定15N含量(具体方法参见“Kun-Hsiang Liu,Chi-Ying Huang,Yi-Fang Tsay,et al.CHL1Is a Dual-Affinity Nitrate Transporter of Arabidopsis Involved in Multiple Phases of Nitrate Uptake.The Plant Cell,1999”一文)。实验设4次重复，测定结果取均值。
通过计算野生型水稻东津和nrt1.1a突变体地上部与根中15N含量比值发现，在两个取样时间点，nrt1.1a突变体地上部与根中15N含量的比值显著低于野生型(P<0.01，见图3)，说明nrt1.1a突变体内硝酸盐由地下向地上部的转 运受到严重影响。
三、水稻nrt1.1a突变体中硝酸盐代谢相关基因表达测定
通过qRT-PCR检测水稻中如下硝酸盐利用相关基因的表达水平：(1)OsNRT1.1B基因，其cDNA的核苷酸序列为序列表中序列4，主要负责硝酸盐的吸收和转运，以及参与硝酸盐信号的调节；(2)OsNRT2.1基因，其cDNA的核苷酸序列为序列表中序列5，主要负责硝酸盐吸收；(3)OsNRT2.3a基因，其cDNA的核苷酸序列为序列表中序列6，负责硝酸盐向地上部分转运；(4)OsNAR1基因，其cDNA的核苷酸序列为序列表中序列7，OsNRT2.1执行功能的辅因子；(5)OsNAR2基因，其cDNA的核苷酸序列为序列表中序列8，OsNRT2.1执行功能的辅因子。
分别取野生型水稻东津和nrt1.1a突变体幼苗叶片，提取总RNA，并反转录得到cDNA，然后采用如下各引物对对上述五种硝酸盐利用相关基因进行qRT-PCR检测。
用于检测OsNRT1.1B基因的引物对：
OsNRT1.1B-F：5’-GGCAGGCTCGACTACTTCTA-3’；
OsNRT1.1B-R：5’-AGGCGCTTCTCCTTGTAGAC-3’。
用于检测OsNRT2.1基因的引物对：
OsNRT2.1-F：5’-CTTCACGTCGTCGAGGTACT-3’；
OsNRT2.1-R：5’-CACTCGGAGCCGTAGTAGTG-3’。
用于检测OsNRT2.3a基因的引物对：
OsNRT2.3a-F：5’-CGCTGCTGCCGCTCATCCG-3’；
OsNRT2.3a-R：5’-CCGTGCCCATGGCCAGAC-3’。
用于检测OsNAR1基因的引物对：
OsNAR1-F：5’-GTTCAAGAGCGTGAAGGTGA-3’；
OsNAR1-R：5’-CACCACGTAGTCGAACCTG-3’。
用于检测OsNAR2基因的引物对：
OsNAR2-F：5’-TCGTCCTCGAGAACAAGAAG-3’；
OsNAR2-R：5’-TCCGTTGGTTTTGTAGGTTG-3’。
以OsActin1为内参基因，其检测引物对：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。
将内参基因的表达量视为1，计算各基因的相对表达量。
结果表明，在nrt1.1a突变体背景下，与野生型水稻东津相比，上述五种硝酸盐利用相关基因的表达均受到显著抑制(P<0.05，见图4)，暗示NRT1.1A可能参与了对这些基因转录水平的调控。
实施例4、NRT1.1A转基因水稻的获得及功能验证(异源启动子)
一、重组植物表达载体pCAMBIA2300-Actin/NRT1.1A的构建
提取粳稻东津的总RNA，反转录为cDNA。以获得的cDNA为模板，采用下述引物序列对NRT1.1A的CDS进行PCR扩增。扩增采用的引物两端分别引入限制性内切酶XbaI和PstI的识别位点(如下划线所示)，引物序列如下：
F：5’-TCTAGAATGGTGGGGATGTTGCCGGA-3’(下划线部分为XbaI的识别序列，其后的序列为序列表中序列3的第1-20位)；
R：5’-CTGCAGTCAGTGGAGGCATGGCTCGG-3’(下划线部分为PstI的识别序列，其后的序列为序列表中序列3的第1793-1812位的反向互补序列)。
以水稻品种东津的cDNA为模板扩增NRT1.1A的CDS区域(包含终止密码子)。PCR产物连接T载体pEASY-Blunt(TransGene)后，经XbaI和PstI双酶切验证后连入植物表达载体pCambia2301-Actin中。
将经测序表明将pCAMBIA2300-Actin载体的酶切位点XbaI和PstI之间的小片段替换为序列表中序列3所示DNA片段后所得的重组载体命名为pCAMBIA2300-Actin/NRT1.1A。
二、NRT1.1A转基因水稻的获得
将步骤一构建的重组植物表达载体pCAMBIA2300-Actin/NRT1.1A转入农杆菌AGL1(来自ATCC)中，再侵染粳稻品种东津的愈伤组织，具体的转化筛选方法参见文献“易自力,曹守云,王力,何锶洁,储成才,唐祚舜,周朴华,田文忠.提高农杆菌转化水稻频率的研究.遗传学报,2001,28(4):352-358”一文。同时设置转入pCAMBIA2300-Actin空载体的对照。最终获得两种转基因苗，即转入pCAMBIA2300-Actin/NRT1.1A的水稻植株和转入pCAMBIA2300-Actin空载体的水稻植株(T0)。
三、NRT1.1A转基因水稻的鉴定
(1)PCR初步鉴定
从步骤二获得的T0代转入pCAMBIA2300-Actin/NRT1.1A的转基因水稻，以及转入pCAMBIA2300-Actin空载体的对照植株中分别提取基因组DNA。用针对NptII基因的引物F1和R1(引物序列如下)进行PCR鉴定，经鉴定表明含有NptII基因的(PCR产物大小约为500bp)植株即为转基因阳性的植株。
F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；
R1：5’-CTGGCGCGAGCCCCTGATGCT-3’。
经上述PCR分子鉴定，从鉴定阳性的转入pCAMBIA2300-Actin/NRT1.1A的转基因水稻株系中随机选取2个分别记作OX1-1、OX2-6。
(2)转录水平分析(RNA表达量)
以步骤(1)获得的T0代NRT1.1A转基因水稻株系OX1-1、OX2-6，转入pCAMBIA2300-Actin空载体的对照植株，以及野生型水稻品种东津为实验材料。提取各材料的总RNA并反转录得到cDNA。进而以所得cDNA为模板，针对NRT1.1A基因进行实时定量荧光PCR，检测各材料中NRT1.1A基因在转录水平上的表达量。 实验重复3次，结果取平均值。
用于检测NRT1.1A基因的引物序列如下：
qNRT1.1A-F：5’-CCGTCTTCTTCGTCGGCTCCATCCT-3’(序列3的第1187-1211位)；
qNRT1.1A-R：5’-CCCGTGCTCATCGTCTTCATCCCCT-3’(序列3的第1514-1538位的反向互补序列)。
以OsActin1作为内参基因，其引物序列为：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。
将内参基因的表达量视为1，计算NRT1.1A基因的相对表达量。
各实验材料中NRT1.1A基因表达量的实时定量荧光PCR检测结果显示，相比未转基因的野生型水稻品种东津，步骤(1)获得的T0代NRT1.1A转基因水稻株系OX1-1、OX2-6中NRT1.1A基因的表达量在转录水平上显著提高，而对于转入pCAMBIA2300-Actin空载体的对照植株，其NRT1.1A基因的表达量在转录水平与未转基因的野生型水稻东津相比基本一致，无统计学差异。
四、NRT1.1A转基因水稻的功能验证
1、实验方法
以步骤三鉴定阳性的T2代NRT1.1A转基因水稻株系OX1-1、OX2-6，转入pCAMBIA2300-Actin空载体的对照植株，以及未转基因的野生型水稻东津为实验材料。从以下几方面鉴定其功能：
(1)表型观察
于幼苗期观察并记录各遗传材料的株高，进入生殖生长时期后再次观察并记录各遗传材料的株高。实验中，每个转基因株系至少选择30个单株进行统计。
(2)qRT-PCR分析硝酸盐代谢相关基因的转录水平
硝酸盐代谢相关基因具体涉及：
OsNRT2.1基因，其cDNA的核苷酸序列为序列表中序列5；
OsNRT2.3a基因，其cDNA的核苷酸序列为序列表中序列6；
OsNIA2基因，其cDNA的核苷酸序列为序列表中序列8；
OsNIR1基因，其cDNA的核苷酸序列为序列表中序列7。
具体测定方法参见实施例2步骤三进行。
实验中，每个转基因株系至少选择5株进行实验。
2、实验结果
(1)表型观察结果
T2代NRT1.1A转基因水稻株系OX1-1、OX2-6在幼苗期就表现出一定的生长优势，株高显著高于野生型对照；进入开花期，过表达株系表现出开花期提早的表型(见图5)。而转入pCAMBIA2300-Actin空载体的对照植株其株高和开花时间与野生型对照相比均基本一致，无统计学差异。这些结果说明，NRT1.1A在 农业生产上具有一定的应用潜力，适度提高NRT1.1A的表达水平可能提高水稻的产量。
(2)qRT-PCR分析硝酸盐代谢相关基因的转录水平
qRT-PCR分析表明，步骤三鉴定阳性的T0代NRT1.1A转基因水稻株系OX1-1、OX2-6中，其上述四种硝酸盐代谢相关基因的转录水平相比于野生型对照均得到显著上调(P<0.01，见图6)。
实施例5、NRT1.1A转基因水稻的获得及功能验证(内源启动子)
为了确认NRT1.1A能达到水稻增产的效果，并避免组成型启动子对水稻农艺性状产生的负效应，本发明的发明人构建了自身启动子驱动的NRT1.1A过量表达转基因植株，并进一步对其功能进行了验证。
一、重组表达载体pCAMBIA2300/NRT1.1A的构建
提取水稻品种东津的基因组DNA和总RNA，并将总RNA反转录为cDNA。以东津基因组DNA为模板扩增NRT1.1A的启动子区域(序列9)，扩增采用的引物两端分别引入限制性内切酶KpnI和EcoRI的识别位点(如下划线所示)，采用的引物序列如下：
NRT1.1Ap-F：5’-GGTACCTTCGATCTCCCACGTAAGAC-3’(下划线部分为KpnI的识别序列，其后的序列为序列9的第1-20位)；
NRT1.1Ap-R：5’-GAATTCTCTCTCTCTCTTCTTCTTCTTCCTC-3’(下划线部分为EcoRI的识别序列，其后的序列为序列9的第1790-1814位的反向互补序列)。
同时，以水稻品种东津的cDNA为模板扩增NRT1.1A的CDS区域，扩增采用的引物两端分别引入限制性内切酶EcoRI和XmaI的识别位点，引物的序列如下：
NRT1.1ACDS-F：5’-GAATTCATGGTGGGGATGTTGCCGGA-3’(下划线部分为EcoRI的识别序列，其后的序列为序列3的第1-20位)；
NRT1.1ACDS-R：5’-CCCGGGGTGGAGGCATGGCTCGG-3’(下划线部分为XmaI的识别序列，其后的序列为序列3的第1793-1809位的反向互补序列)。
将以上两步扩增出的两个PCR片段分两次分别连入pBluescript KS(+)(Stratagene，212205)载体的对应酶切位点处。经测序验证后，采用KpnI和BamHI双酶切并将含有内源启动子及NRT1.1A CDS的片段连入双元载体pCAMBIA2300的对应酶切位点处，将所得经测序验证正确的重组载体命名为pCAMBIA2300/NRT1.1A。
二、NRT1.1A转基因水稻的获得
将步骤一构建的重组植物表达载体pCAMBIA2300/NRT1.1A转入农杆菌AGL1(来自ATCC)中，再侵染粳稻品种东津的愈伤组织具体的转化筛选方法参见文献“易自力,曹守云,王力,何锶洁,储成才,唐祚舜,周朴华,田文忠.提高农杆菌转化水稻频率的研究.遗传学报,2001,28(4):352-358”一文。同时设置转入pCAMBIA2300空载体的对照。最终获得两种转基因苗，即转入 pCAMBIA2300/NRT1.1A的水稻植株和转入pCAMBIA2300空载体的水稻植株(T0)。
三、NRT1.1A转基因水稻的鉴定
(1)PCR初步鉴定
从步骤二获得的T0代转入pCAMBIA2300/NRT1.1A的转基因水稻，以及转入pCAMBIA2300空载体的对照植株中分别提取基因组DNA。用引物F1和R1(引物序列如下)进行PCR鉴定，经鉴定表明含有NptII基因的(PCR产物大小约为500bp)植株即为转基因阳性的植株。
F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；
R1：5’-CTGGCGCGAGCCCCTGATGCT-3’。
经上述PCR分子鉴定，从鉴定阳性的转入pCAMBIA2300/NRT1.1A的转基因水稻株系中随机选取2个分别记作pNA-2和pNA-4。
(2)转录水平分析(RNA表达量)
以步骤(1)获得的鉴定阳性T0代NRT1.1A转基因水稻株系pNA-2和pNA-4，转入pCAMBIA2300空载体的对照植株，以及野生型水稻品种东津为实验材料。提取各材料的总RNA并反转录得到cDNA。进而以所得cDNA为模板，针对NRT1.1A基因进行实时定量荧光PCR，检测各材料中NRT1.1A基因在转录水平上的表达量。实验重复3次，结果取平均值。
用于检测NRT1.1A基因的引物序列如下：
qNRT1.1A-F：5’-CCGTCTTCTTCGTCGGCTCCATCCT-3’(序列3的第1187-1211位)；
qNRT1.1A-R：5’-CCCGTGCTCATCGTCTTCATCCCCT-3’(序列3的第1514-1538位的反向互补序列)。
以OsActin1作为内参基因，其引物序列为：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’。
将内参基因的表达量视为1，计算NRT1.1A基因的相对表达量。
各实验材料中NRT1.1A基因表达量的实时定量荧光PCR检测结果显示，相比未转基因的野生型水稻品种东津，步骤(1)获得的T0代NRT1.1A转基因水稻株系pNA-2和pNA-4中NRT1.1A基因的表达量在转录水平上显著提高，而对于转入pCAMBIA2300空载体的对照植株，其NRT1.1A基因的表达量在转录水平与未转基因的野生型水稻品种东津相比基本一致，无统计学差异。
四、NRT1.1A转基因水稻的功能验证
以步骤三鉴定阳性的T2代NRT1.1A转基因水稻株系pNA-2和pNA-4，转入pCAMBIA2300空载体的对照植株，以及未转基因的野生型水稻东津为实验材料。田间试验设置两个氮肥梯度(高氮和低氮)。其中高氮按照每100平方米4.28kg尿素施肥，低氮按照每100平方米1.07kg尿素施肥。每个氮肥梯度下设置3个小区。在田间观察和记录其表型(包括分蘖数、千粒重、穗粒数以及单株产 量)，每个株系在各个小区随机选择至少10个单株进行表型统计，总计每个株系不少于30个单株。
田间的氮肥测试结果显示，在低氮和高氮条件下，NRT1.1A过表达的转基因株系pNA-2和pNA-4均表现为穗粒数以及单株产量的显著增加(P<0.05)。尤其是在低氮条件下，过表达转基因株系的单株产量增加尤为明显(P<0.01)详见图7。
实施例6、NRT1.1A转基因拟南芥的获得及功能验证(异源启动子)
一、重组植物表达载体pCAMBIA2300-35S-OCS/NRT1.1A的构建
提取粳稻东津的总RNA，反转录为cDNA。以获得的cDNA为模板，采用下述引物序列对NRT1.1A的CDS进行PCR扩增。扩增采用的引物两端分别引入限制性内切酶Bam HI和Sal I的识别位点(如下划线所示)，引物序列如下：
F：5’-GGATCCATGGTGGGGATGTTGCCGGA-3’(下划线部分为Bam HI的识别序列，其后的序列为序列表中序列3的第1-20位)；
R：5’-GTCGACTCAGTGGAGGCATGGCTCGG-3’(下划线部分为Sal I的识别序列，其后的序列为序列表中序列3的第1793-1812位的反向互补序列)。
以水稻品种东津的cDNA为模板扩增NRT1.1A的CDS区域(包含终止密码子)。PCR产物连接T载体pEASY-Blunt(TransGene)后，经Bam HI和Sal I双酶切验证后连入植物表达载体pCAMBIA2300-35S-OCS中。
将经测序表明将pCAMBIA2300-35S-OCS载体的酶切位点Bam HI和Sal I之间的小片段替换为序列表中序列3所示DNA片段后所得的重组载体命名为pCAMBIA2300-35S-OCS/NRT1.1A。
二、NRT1.1A转基因拟南芥的获得
将步骤一构建的重组植物表达载体pCAMBIA2300-35S-OCS/NRT1.1A通过农杆菌GV3101(来自ATCC)介导的遗传转化转入拟南芥Columbia-0(参考文献：“Steven J.Clough and Andrew F.Bent.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The Plant Journal.(1998)16(6),735-743”)。同时设置转入pCAMBIA2300-35S-OCS空载体的对照。最终获得两种转基因苗，即转入pCAMBIA2300-35S-OCS/NRT1.1A的拟南芥植株和转入pCAMBIA2300-35S-OCS空载体的拟南芥植株(T0)。
三、NRT1.1A转基因拟南芥的鉴定
从步骤二获得的T0代转入pCAMBIA2300-35S-OCS/NRT1.1A的转基因拟南芥中随机选取两株，分别记为为OX-3和OX-17。将OX-3和OX-17、转入pCAMBIA2300-35S-OCS空载体的对照植株以及未转基因的野生型拟南芥Columbia-0中分别提取总RNA并反转录得到cDNA。进而以所得cDNA为模板，针对NRT1.1A基因进行实时定量荧光PCR，检测各材料中NRT1.1A基因在转录水平上的表达量。实验重复3次，结果取平均值。
用于检测NRT1.1A基因的引物序列如下：
qNRT1.1A-F：5’-CCGTCTTCTTCGTCGGCTCCATCCT-3’(序列3的第1187-1211位)；
qNRT1.1A-R：5’-CCCGTGCTCATCGTCTTCATCCCCT-3’(序列3的第1514-1538位的反向互补序列)。
以AtActin2作为内参基因，其引物序列为：
qAtActin2-F：5’-GCACCACCTGAAAGGAAGTACA-3’；
qAtActin2-R：5’-CGATTCCTGGACCTGCCTCATC-3’。
将内参基因的表达量视为1，计算NRT1.1A基因的相对表达量。
各实验材料中NRT1.1A基因表达量的实时定量荧光PCR检测结果显示，相比未转基因的野生型拟南芥，步骤二获得的T0代转入pCAMBIA2300-35S-OCS/NRT1.1A的转基因拟南芥株系OX-3和OX-17中NRT1.1A基因的表达量在转录水平上显著提高(见图8中A)。而对于转入pCAMBIA2300-35S-OCS空载体的对照植株，其NRT1.1A基因的表达量在转录水平与未转基因的野生型植株相比基本一致，无统计学差异。
四、NRT1.1A转基因拟南芥的功能验证
1、实验方法
以步骤三鉴定阳性的T2代NRT1.1A转基因拟南芥株系OX-3和OX-17，转入pCAMBIA2300-35S-OCS空载体的对照植株，以及未转基因的野生型拟南芥Columbia-0为实验材料。于幼苗期观察各遗传材料的莲座叶大小，进入抽薹期后观察各遗传材料的抽薹时间。实验中，每个转基因株系至少选择30个单株进行观察。
2、实验结果
T2代NRT1.1A转基因拟南芥株系OX-3和OX-17在幼苗期就表现出一定的生长优势，莲座叶明显大于野生型对照(见图8中B)，进入抽薹期后，过表达株系抽薹显著早于野生型对照(见图8中C)。而对于转入pCAMBIA2300-35S-OCS空载体的对照植株，其幼苗期莲座叶大小以及抽薹时间与未转基因的野生型植株相比基本一致。
工业应用
本发明通过利用爪蟾卵母细胞的体外转运系统证明NRT1.1A蛋白能够转运硝酸盐。转基因实验表明，NRT1.1A在水稻野生型中过量表达能够表现出明显的生长优势，并且显著提升穗粒数和株高，进而提高水稻产量NRT1.1A在拟南芥野生型中过量表达幼苗期就表现出一定的生长优势，莲座叶明显大于野生型对照；进入抽薹期后，过表达株系抽薹显著早于野生型对照。以上结果暗示NRT1.1A蛋白在提高植物产量具有极大的应用潜力。

Claims (20)

  1. 蛋白质或其编码基因在调控植物生长发育中的应用；所述生长发育体现为单株产量和/或株高和/或穗粒数和/或开花时间和/或抽薹时间和/或莲座叶大小和/或生物量；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
  2. 蛋白质或其编码基因在选育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的植物品种中的应用；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
  3. 根据权利要求1或2所述的应用，其特征在于：所述编码基因是如下(1)至(4)中任一所述的DNA分子：
    (1)序列表中序列2的DNA分子；
    (2)序列表中序列3的DNA分子；
    (3)在严格条件下与(1)或(2)所限定的DNA分子杂交且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子；
    (4)与(1)或(2)或(3)限定的DNA分子具有90％以上同源性且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子。
  4. 根据权利要求1或2所述的应用，其特征在于：所述植物为单子叶植物或双子叶植物。
  5. 培育单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的转基因植物的方法，包括使目的植物表达或超量表达蛋白质的步骤，或者包括使目的植物中蛋白质活性提高的步骤；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
  6. 根据权利要求5所述的方法，其特征在于：所述方法包括如下a)和b)的步骤：
    a)向所述目的植物中导入所述蛋白质的编码基因，得到表达或超量表达所 述编码基因的转基因植物；
    b)从步骤a)所得转基因植物中得到与所述目的植物相比，单株产量增加和/或株高增高和/或穗粒数增加和/或开花时间提前和/或抽薹时间提前和/或莲座叶增大和/或生物量增加的转基因植物。
  7. 根据权利要求6所述的方法，其特征在于：所述编码基因是如下(1)至(4)中任一所述的DNA分子：
    (1)序列表中序列2的DNA分子；
    (2)序列表中序列3的DNA分子；
    (3)在严格条件下与(1)或(2)所限定的DNA分子杂交且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子；
    (4)与(1)或(2)或(3)限定的DNA分子具有90％以上同源性且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子。
  8. 根据权利要求6或7所述的方法，其特征在于：所述方法中，所述编码基因是通过含有所述编码基因的重组表达载体导入所述目的植物中的。
  9. 根据权利要求8所述的方法，其特征在于：所述重组表达载体中启动所述编码基因转录的启动子为35S启动子或水稻NRT1.1A基因的自身内源启动子。
  10. 根据权利要求5-7中任一所述的方法，其特征在于：所述植物为单子叶植物或双子叶植物。
  11. 蛋白质或其编码基因在如下(A)或(B)中的应用；
    (A)促进硝酸盐吸收或转运；
    (B)促进硝酸盐代谢相关基因的表达；所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与硝酸盐转运相关的由序列1衍生的蛋白质。
  12. 根据权利要求11所述的应用，其特征在于：所述(A)中，所述促进硝酸盐吸收或转运为促进爪蟾卵母细胞中硝酸盐的吸收或转运，或为促进水稻中硝酸盐的吸收或转运；
    所述(B)中，所述促进硝酸盐代谢相关基因的表达为促进爪蟾卵母细胞中硝酸盐代谢相关基因的表达或促进水稻中硝酸盐代谢相关基因的表达。
  13. 蛋白质或其编码基因在如下(C)或(D)中的应用；
    (C)选育对硝酸盐的吸收或转运能力提高的植物品种；
    (D)选育硝酸盐代谢相关基因的表达量提高的植物品种；所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与硝酸盐转运相关的由序列1衍生的蛋白质。
  14. 根据权利要求11-13中任一所述的应用，其特征在于：所述编码基因是如下(1)至(4)中任一所述的DNA分子：
    (1)序列表中序列2的DNA分子；
    (2)序列表中序列3的DNA分子；
    (3)在严格条件下与(1)或(2)所限定的DNA分子杂交且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子；
    (4)与(1)或(2)或(3)限定的DNA分子具有90％以上同源性且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子。
  15. 培育对硝酸盐的吸收或转运能力提高和/或硝酸盐代谢相关基因的表达量提高的转基因植物的方法，包括使目的植物表达或超量表达蛋白质的步骤，或者包括使目的植物中蛋白质活性提高的步骤；
    所述蛋白质为如下中任一种：
    (1)氨基酸序列为序列表中序列1所示的蛋白质；
    (2)将序列表中序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与调控植物生长发育相关的由序列1衍生的蛋白质。
  16. 根据权利要求15所述的方法，其特征在于：所述方法包括如下c)和d)的步骤：
    c)向所述目的植物中导入所述蛋白质的编码基因，得到表达所述编码基因的转基因植物；
    d)从步骤c)所得转基因植物中得到与所述目的植物相比，对硝酸盐的吸收或转运能力提高和/或硝酸盐代谢相关基因的表达量提高的转基因植物；
    所述硝酸盐代谢相关基因选自如下中任一种：NRT1.1B、NRT2.1、NRT2.3a、NAR1和NAR2。
  17. 根据权利要求16所述的方法，其特征在于：所述编码基因是如下(1)至(4)中任一所述的DNA分子：
    (1)序列表中序列2的DNA分子；
    (2)序列表中序列3的DNA分子；
    (3)在严格条件下与(1)或(2)所限定的DNA分子杂交且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子；
    (4)与(1)或(2)或(3)限定的DNA分子具有90％以上同源性且编码氨基酸序列为序列表中序列1所示的蛋白质的DNA分子。
  18. 根据权利要求16或17所述的方法，其特征在于：所述方法中，所述编码基因是通过含有所述编码基因的重组表达载体导入所述目的植物中的。
  19. 根据权利要求18所述的方法，其特征在于：所述重组表达载体中启动所述编码基因转录的启动子为35S启动子或水稻NRT1.1A基因的自身内源启动 子。
  20. 根据权利要求15-17中任一所述的方法，其特征在于：所述植物为单子叶植物或双子叶植物。
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652423A (zh) * 2018-12-07 2019-04-19 沈阳农业大学 一种水稻开花期调控蛋白及其在育种中的应用
CN112626115A (zh) * 2020-12-22 2021-04-09 福建农林大学 OsELF4基因在控制水稻抽穗期中的应用
CN113647322A (zh) * 2021-09-13 2021-11-16 江苏丘陵地区镇江农业科学研究所 一种培育氮素高效利用粳稻品种的育种方法
CN115109865A (zh) * 2022-06-16 2022-09-27 四川农业大学 一种丹参花药的内参基因及其用途
CN115852014A (zh) * 2022-08-12 2023-03-28 广东省林业科学研究院 木荷生长与开花相关内参基因筛选方法、引物及应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110846322B (zh) * 2018-07-29 2020-12-22 山东省农业科学院玉米研究所（山东省农业科学院玉米工程技术研究中心） 一种玉米小籽粒突变体及其应用
CN110734484B (zh) * 2019-11-26 2021-04-13 中国农业大学 Nrt2_5蛋白在调控植物苞叶宽度中的应用
CN114920810B (zh) * 2021-02-01 2023-02-21 中国农业大学 硝酸盐吸收相关蛋白在调控玉米对硝酸盐吸收中的应用
CN116143892B (zh) * 2023-03-30 2024-01-12 沈阳农业大学 OsGN11基因在改良水稻每穗粒数性状中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101765660A (zh) * 2007-05-22 2010-06-30 巴斯夫植物科学有限公司 具有提高的环境胁迫耐受性和/或抗性和提高的生物量生产的植物细胞和植物
WO2013067289A1 (en) * 2011-11-02 2013-05-10 University Of North Texas MtNIP REGULATED PLANTS WITH SIGNIFICANTLY INCREASED SIZE AND BIOMASS
CN104204210A (zh) * 2012-03-20 2014-12-10 英美烟草(投资)有限公司 具有叶中改变的硝酸盐水平的转基因植物
CN104277101A (zh) * 2014-09-24 2015-01-14 中国科学院遗传与发育生物学研究所 水稻硝酸盐转运蛋白nrt1.1b在提高植物氮利用效率中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101027397A (zh) * 2004-04-23 2007-08-29 西尔斯公司 用于修饰植物中氮利用效率特征的核苷酸序列和其编码的多肽
CN101392257B (zh) * 2008-11-10 2013-03-13 南京农业大学 水稻硝酸盐运输蛋白基因组OsNRT2.3的基因工程应用
CN105385696A (zh) * 2015-11-17 2016-03-09 江苏省农业科学院 茶树nrt1基因、蛋白及基因表达方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101765660A (zh) * 2007-05-22 2010-06-30 巴斯夫植物科学有限公司 具有提高的环境胁迫耐受性和/或抗性和提高的生物量生产的植物细胞和植物
WO2013067289A1 (en) * 2011-11-02 2013-05-10 University Of North Texas MtNIP REGULATED PLANTS WITH SIGNIFICANTLY INCREASED SIZE AND BIOMASS
CN104204210A (zh) * 2012-03-20 2014-12-10 英美烟草(投资)有限公司 具有叶中改变的硝酸盐水平的转基因植物
CN104277101A (zh) * 2014-09-24 2015-01-14 中国科学院遗传与发育生物学研究所 水稻硝酸盐转运蛋白nrt1.1b在提高植物氮利用效率中的应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [O] 1 March 2016 (2016-03-01), "PREDICTED: Protein NRT1/PTR FAMILY 6.3 [Oryza Sativa Japonica Group", XP055466991, Database accession no. XP_015650127 .1 *
DATABASE nucleotide [O] 1 March 2016 (2016-03-01), "PREDICTED: Oryza Sativa Japonica Group Protein NRT1/PTR FAMILY 6.3 (LOC4344690", XP055466987, retrieved from ncbi Database accession no. XM_015794641.1 *
FAN, X.R. ET AL.: "A Putative 6-Transmembrane Nitrate Transporter OsNRTl .lb Plays a Key Role in Rice under Low Nitrogen", JOURNAL OF INTEGRATIVE PLANT BIOLOGY, vol. 58, no. 6, 30 June 2016 (2016-06-30), pages 590 - 599, XP055467001 *
LIN, C.M. ET AL.: "Cloning and Functional Characterization of a Constitutively Expressed Nitrate Transporter Gene , OsNRTl, from Rice", PLANT PHYSIOLOGY, vol. 122, 29 February 2000 (2000-02-29), pages 379 - 388, XP055466995 *
MIAO, QISONG: "Functional Analysis of Two Rice Nitrate Transporter Genes OsNRT1.1a and OsNRT1.1b", CHINA MASTER'S THESES FULL-TEXT DATABASE, AGRICULTURE SCIENCE AND TECHNOLOGY, 15 June 2012 (2012-06-15) *
WANG, H. ET AL.: "Effects of Salt Stress on Ion Balance and Nitrogen Metabolism in Rice", PLANT SOIL ENVIRON, vol. 2, no. 58, 31 December 2012 (2012-12-31), pages 62 - 67, XP055467005 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652423A (zh) * 2018-12-07 2019-04-19 沈阳农业大学 一种水稻开花期调控蛋白及其在育种中的应用
CN109652423B (zh) * 2018-12-07 2023-08-11 沈阳农业大学 一种水稻开花期调控蛋白及其在育种中的应用
CN112626115A (zh) * 2020-12-22 2021-04-09 福建农林大学 OsELF4基因在控制水稻抽穗期中的应用
CN113647322A (zh) * 2021-09-13 2021-11-16 江苏丘陵地区镇江农业科学研究所 一种培育氮素高效利用粳稻品种的育种方法
CN113647322B (zh) * 2021-09-13 2022-05-20 江苏丘陵地区镇江农业科学研究所 一种培育氮素高效利用粳稻品种的育种方法
CN115109865A (zh) * 2022-06-16 2022-09-27 四川农业大学 一种丹参花药的内参基因及其用途
CN115109865B (zh) * 2022-06-16 2024-04-09 四川农业大学 一种丹参花药的内参基因及其用途
CN115852014A (zh) * 2022-08-12 2023-03-28 广东省林业科学研究院 木荷生长与开花相关内参基因筛选方法、引物及应用
CN115852014B (zh) * 2022-08-12 2023-08-18 广东省林业科学研究院 木荷生长与开花相关内参基因筛选方法、引物及应用

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