CN107937433B - Application of OsNPF8.13 gene in promotion of rice growth under high nitrogen - Google Patents
Application of OsNPF8.13 gene in promotion of rice growth under high nitrogen Download PDFInfo
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Abstract
The invention discloses a geneOsNPF8.13The application of promoting rice growth under high nitrogen belongs to the field of plant gene engineering.OsNPF8.13The amino acid and cDNA sequence of the gene coding protein are shown in SEQ ID NO.3 or 4, 1 or 2. The invention constructs riceOsNPF8.13Over-expression of the gene, found to be elevated under high nitrogenOsNPF8.13The expression of the gene can increase the root length, the root number, the plant height, the fresh weight and the free amino acid content. By constructionOsNPF8.13Gene interference and mutant plants, found by reducingOsNPF8.13The gene expression can reduce the root length, root number, plant height, fresh weight and free amino acid content of rice. Thus, it is possible to provideOsNPF8.13The fertilizer can be used for promoting the growth of rice under high nitrogen to improve the biomass and the yield of the rice; can also be used for the aspect of high soil fertility adaptation of rice.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to a plant genetic engineering geneOsNPF8.13The gene is applied to promoting the growth of rice under high nitrogen.
Background
Plants obtain nitrogen by absorbing ammonium, nitrate, amino acids, soluble peptides, etc. in soil; nitrogen uptake and transport is mainly accomplished by Transporters such as ammonium transport protein (AMT), nitrate transport protein (NRT), amino acid transport protein (AAT), peptide transport Protein (PTR) (Williams L, Miller A. Transporters for the uptake and purification of nitrogenes sources, Annual Review of Plant Physiology and Plant Molecular Biology, 2001, 52: 659. sup. 688.). Ammonium is taken up by the Plant AMT and then glutamine and glutamate are synthesized by Glutamine Synthetase (GS) and glutamate synthase (GOGAT), which in turn further form other amino acids (Sonoda Y, Ikeda A, Saiki S, et al. Feedback regulation of the ammonium transporter gene family AMT1 by glutamine in rice. Plant cell physiology, 2003, 44: 1396. sup. 1402.). Plants can absorb environmental nitrates via NRT2 of the High Affinity Transport System (HATS) and NRT1 of the Low Affinity Transport System (LATS), form ammonium upon reduction by Nitrate Reductase (NR) and nitrite reductase (NiR), and further form amino acids (Paungfoo-Lonhienne C, Lonhienne T G, Rentsch D, et al. plant can use protein as a nitrogen source with a sodium out of organic chemistry. Proceedings of the National Academy of Sciences, 2008, 105: 4524-4529.).
The NPF family of nitrogen transporters includes the NRT1 and PTR subfamilies, where different members transport nitrate, oligopeptides, amino acids, etc. at different sites of the plant and play different roles in plant growth and development (Rentsch D, Schmidt S, Tegeter M. transporters for uptake and allocation of organic nitrogen compounds in plants Febs Letters, 2007, 581: 2281-2289.). OsNPF2.2 mediates the unloading of xylem nitrate and affects rice plant growth (Li Y, Ouyang J, Wang Y, et al, precipitation of the rice transporter OsNPF2.2 promoters root-to-shoot nitrate and Scientific replacementrts, 2015, 5: 9635.）。OsNPF7.2Has low affinity transport for nitrate and can affect plant growth (Hu R, Qiu D, Chen Y, et al, Knock-down of aeroplast localized low-affinity nitrate transportOsNPF7.2affects ricegrowth under high nitrate supply. Frontiers in plant science, 2016, 7.）。
While nitrogen nutrition is known to promote plant growth and development, there is currently no systematic understanding of what types of plants nitrogen nutrition affects growth and development. In addition, more than 80 members of the rice NPF family exist, nitrogen nutrition responds through which member of the NPF gene family, nitrogen nutrition transportation is mediated at what position, and therefore what type of plant growth and development are influenced, and the existence of nitrogen nutrition is almost unknown at present. Therefore, the excavated NPF family may have nitrogen efficient transport genes, especially nitrogen transport key genes capable of controlling the agronomic characters of rice, and is beneficial to the cultivation of high-yield rice varieties. The invention discovers that NPF family through long-term researchOsNPF8.13After transcription of the gene, there are two kinds of splicing, respectivelyOsNPF8.13aAndOsNPF8.13bthe fertilizer has important effects on the influence of rice biomass under low nitrogen and high nitrogen, can be applied to improving the utilization efficiency of plant nitrogen, and particularly prevents further environmental pollution caused by overhigh fertility of soil or excessive artificial consumption of chemical fertilizers.
Disclosure of Invention
The invention aims to solve the problems in the prior art and providesOsNPF8.13The gene is applied to promoting the growth of rice under high nitrogen.
The purpose of the invention is realized by the following technical scheme:
the invention relates to a rice geneOsNPF8.13As a subject, a clone from rice flower 11OsNPF8.13The cDNA sequence of (1). By constructionOsNPF8.13The overexpression vector of the gene is obtained by introducing the overexpression vector into the normal japonica rice variety middle flower 11 by adopting an agrobacterium EHA105 mediated genetic transformation methodOsNPF8.13The root length, the root number, the plant height, the fresh weight, the free amino acid content and the like of the over-expressed plant of the gene are compared with those of a control under the high-nitrogen (the nitrogen concentration is more than 2 mM) cultureFlower 11 was significantly improved in the wild type compared to the wild type. At the same time constructOsNPF8.13The gene interference and CRISPR gene knockout vector is obtained by respectively introducing the interference vector and the CRISPR gene knockout vector into the Zhonghua 11OsNPF8.13The root length, the root number, the plant height, the fresh weight, the free amino acid content and the like of the gene interference plant and the mutant plant are obviously reduced compared with those of the medium flower 11 under the high-nitrogen culture. These results show that by increasingOsNPF8.13The gene expression can promote the growth of rice, increase biomass and promote the accumulation of nitrogen concentration in the rice;OsNPF8.13the gene has application value in the aspect of adapting to high soil fertility of rice, and can reduce negative effects on the environment possibly caused by overhigh soil fertility and artificial too much fertilization; can also be applied to the rice variety improvement through molecular breeding.
Based on the discovery of the present inventionOsNPF8.13The function of the gene can be used for promoting the growth of rice, improving the biomass of the rice and increasing the content of free amino acid under the condition of high nitrogen. In particular, it can be achieved by genetic engineering, i.e.overexpressionOsNPF8.13The expression of the gene increases the root length, the root number, the plant height, the fresh weight, the content of free amino acid and the like of the rice, and achieves the purpose of improving the biomass of the rice. SaidOsNPF8.13The gene includes two kinds of splicing, respectivelyOsNPF8.13aAndOsNPF8.13b，OsNPF8.13a、OsNPF8.13bthe cDNA sequence of (A) is preferably as shown in SEQ ID NO.1, 2.OsNPF8.13a、OsNPF8.13bThe amino acid sequences of the coded OsNPF8.13a protein and OsNPF8.13b protein are shown as SEQ ID NO.3 and SEQ ID NO. 4.
It is understood that amino acid sequences having equivalent functions can be obtained by those skilled in the art by variously substituting, adding and/or deleting one or several amino acids of the amino acid sequences shown in SEQ ID NO.3 or 4 without affecting the activity of the OsNPF8.13a or OsNPF8.13b protein (i.e., without the active center of the protein). Therefore, the OsNPF8.13 protein also includes proteins with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequences shown in SEQ ID NO.3 or 4. Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.
The invention has the advantages and effects that:
(1) cloned according to the inventionOsNPF8.13After the gene is improved and expressed, the root length, the root number, the plant height, the fresh weight and the free amino acid content of the rice can be increased under high nitrogen, which indicates thatOsNPF8.13The gene has obvious effect on increasing the biomass of the rice, so the gene is improved by the gene engineering technologyOsNPF8.13The expression of the gene can improve the biomass of the plant and the organic nitrogen content in the plant.
（2）OsNPF8.13The successful cloning of the gene further proves the important function of nitrogen absorption of the plant to growth and development, has important significance for clarifying the biological function of the nitrogen gene, and has great promotion effect on further understanding the nitrogen metabolic pathway of the plant and improving the nitrogen absorption efficiency.
(3) Although some genes affecting plant growth in the nitrogen nutrient pathway have been cloned to date, the molecular mechanisms underlying plant growth and development are unclear. And cloned according to the inventionOsNPF8.13The gene can improve the biomass and the amino acid content of rice, and has great promotion effect on determining the key factors of plant yield increase.
Drawings
FIG. 1 shows control medium flower 11 (WT) in low nitrogen (0.5 mM ammonium nitrate) culture,OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13overall plant phenotype map of gene mutant (OsNPF8.13b-C).
FIG. 2 shows control medium flower 11 (WT) in medium nitrogen (2.0 mM ammonium nitrate) culture,OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13overall plant phenotype map of gene mutant (OsNPF8.13b-C).
FIG. 3 shows control medium flower 11 (WT) in high nitrogen (5.0 mM ammonium nitrate) culture,OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13overall plant phenotype map of gene mutant (OsNPF8.13b-C).
FIG. 4 shows control middle flower 11 (WT) cultured with different nitrogen concentrations (A, B, C, low nitrogen concentration, medium nitrogen concentration, and high nitrogen concentration, respectively),OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13statistical histogram of root length of gene mutant (OsNPF8.13b-C). Data were analyzed for variable analysis (ANOVA) using SPSS software using Duncan's to analyze differential significance at three levels, 0.05, 0.01, and 0.001, with three significant levels of rice indicators labeled as x, and x at each concentration compared to the control. The low nitrogen concentration was 0.5mM ammonium nitrate, the medium nitrogen concentration was 2mM ammonium nitrate and the high nitrogen concentration was 5mM ammonium nitrate, and the following phenotypic statistics were performed in the same manner.
FIG. 5 shows control middle flower 11 (WT) cultured with different nitrogen concentrations (A, B, C, low nitrogen concentration, medium nitrogen concentration, and high nitrogen concentration, respectively),OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13statistical histogram of the number of gene mutants (OsNPF8.13b-C).
FIG. 6 shows control middle flower 11 (WT) cultured with different nitrogen concentrations (A, B, C, low nitrogen concentration, medium nitrogen concentration, and high nitrogen concentration, respectively),OsNPF8.133 strains (OsNPF8.13a-OE) of the first splicing OsNPF8.13a overexpression plant of the gene and the second splicing OsNPF8.13b overexpression3 strains of plants (OsNPF8.13b-OE),OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13statistical histogram of plant height of gene mutant (OsNPF8.13b-C).
FIG. 7 shows control middle flower 11 (WT) cultured with different nitrogen concentrations (A, B, C, low nitrogen concentration, medium nitrogen concentration, and high nitrogen concentration, respectively),OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13statistical histogram of fresh weight of gene mutant (OsNPF8.13b-C).
FIG. 8 shows control middle flower 11 (WT) cultured with different nitrogen concentrations (A, B, C, low nitrogen concentration, medium nitrogen concentration, and high nitrogen concentration, respectively),OsNPF8.133 strains (OsNPF8.13a-OE) of a first splicing OsNPF8.13a overexpression plant of the gene, 3 strains (OsNPF8.13b-OE) of a second splicing OsNPF8.13b overexpression plant of the gene, and a promoter,OsNPF8.13Gene-interfering plants 3 lines (OsNPF8.13-Ri) andOsNPF8.13statistical histogram of free amino acid content in roots of gene mutant (OsNPF8.13b-C).
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to recombinant techniques already described (cf. molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.; Ma X et al, A robust CRISPR/Cas9 system for containment, high-efficiency multiplex gene evaluation in monocot and dicotplants. Mol plant 2015, 8(8): 1274. sup. 1284.); the materials, reagents and the like used are all commercially available.
Example 1OsNPF8.13Construction of Gene-overexpressing plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F1：5'-AGATCTATGGAGGCAGGGGCAGCAGATGAG-3'（BglII），
R1：5'-CTTAAGAGACGACAGCGTGTTGTTATTGGC-3'（AflII）；
F2：5'-AGATCTATGCTCATTGTGACGGTTTCATCA-3'（BglII），
R2：5'-CTTAAGAGACGACAGCGTGTTGTTATTGGC-3'（AflII）
amplification by PCR Using F1/R1 and F2/R2 primers, respectivelyOsNPF8.13Two kinds of splicing of genesOsNPF8.13aAndOsNPF8.13bafter cDNA of (3), byBglII andAflII cleaved with an enzyme, ligated into pCAMBIA-1301 vector (pCAMBIA-1301 vector was purchased from Cambia, Inc.), and constructedOsNPF8.13Overexpression vector of geneOsNPF8.13aP1301 andOsNPF8.13bp 1301. The overexpression vectors are respectively introduced into the flowers 11 of the normal rice variety by adopting an agrobacterium EHA105 mediated genetic transformation method.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, soaking 50 rice transgenic seedlings into a hygromycin solution with the concentration of 50mg/L prepared by 500mL of distilled water for 48 hours when the seedlings are about 10cm long, and then taking plants with green leaves and good growth states as positive transgenic plants; while plants with withered and yellow leaves and curly leaves were negative plants and died immediately. Planting positive plants individually and harvesting until T2 generation identifies homozygous transgenic plants without any withered and yellow leaves and curly leaves in the hygromycin solution, namely obtaining the positive plantsOsNPF8.13Over-expression plants with two splicing modes of genes. Soaking the over-expression plant and the seeds of the middle flower 11 in distilled water for 3 days on a culture dish, culturing for 7 days, transferring to a rice nutrient solution for culturing, wherein the formula of the nutrient solution refers to the formula of the international rice institute, but ammonium nitrate is adjusted to 0.5mM (low nitrogen), 2mM (medium nitrogen) and 5mM (high nitrogen), culturing for 40 days respectively, observing the phenotype, counting the root length, the root number, the plant height and the fresh weight, and measuring the amino acid in the roots of the planted plants, wherein the results are shown in figures 1-8. Under the condition of high-nitrogen culture,OsNPF8.13compared with the control flower 11 plant, the gene over-expression plant has long root, number of roots, plant height andthe content of free amino acid in roots is increased, and the difference is obvious; whereas the difference was not significant at medium and low nitrogen with high nitrogen and no significant increase in amino acid concentration. Statistical data of root length, root number, plant height, fresh weight and free amino acid content in roots of the over-expressed plants and the control under different nitrogen culture are shown in FIGS. 4 to 8. At the later stage of the process,OsNPF8.13the yield of the gene over-expressed plants is significantly higher than that of the control flower 11.
Example 2OsNPF8.13Gene interference and construction of mutant plants
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F3：5'-GGTACCGTGCCACGGTCTACACAGGGCT-3'（KpnI），
R3：5'-GGATCCTGCAACCACCACCTGGGACA-3'（BamH I）；
F4：5'-ACTAGTGTGCCACGGTCTACACAGGGCT-3'（SpeI），
R4：5'-GAGCTCTGCAACCACCACCTGGGACA-3'（SacI）；
respective PCR amplificationOsNPF8.13The cDNA fragment 347bp of the gene is cut by the corresponding restriction enzyme and then is connected into a pTCK303 vector to constructOsNPF8.13Interfering expression vector of geneOsNPF8.13-pTCK 303. The interference expression vector is introduced into the normal japonica rice variety middle flower 11 by adopting an agrobacterium EHA105 mediated genetic transformation method.
F5：5'-AGACCGGGAACATCCGCAGCAGG-3'，
F6：5'-AGGCGGCCGGAAGGGTGAACGGG-3'；
The two target sequences of F5 and F6 are utilized to constructOsNPF8.13Gene knockout vectorOsNPF8.13C (method reference Ma X et al, A robust CRISPR/Cas9 system for meeting, high-efficiency multiplex gene editing in monocot and dicot plants. Molplant. 2015, 8(8): 1274-. The gene knockout vector is introduced into the flower 11 of the normal japonica rice variety by adopting an agrobacterium EHA105 mediated genetic transformation method.
Transplanting all the obtained interference and mutant plant seedlings into a basket with soil, and periodicallyWatering and fertilizing, when the height of the seedlings is about 10cm, soaking 50 rice transgenic seedlings in a hygromycin solution with the concentration of 50mg/L prepared by 500mL of distilled water for 48 hours, and then taking plants with green leaves, relaxation and good growth states as positive transgenic plants; while plants with withered and yellow leaves and curly leaves were negative plants and died immediately. The interfered positive plants are planted and harvested individually until T2 generation identifies the homozygous transgenic plants without any withered and yellow leaves and curly leaves in the hygromycin solution, namely obtaining the transgenic plantsOsNPF8.13And (3) sequencing the gene interference plant and the mutant plant at the T1 generation to confirm that the gene is knocked out. The interfering plant, the mutant plant and the seed of the middle flower 11 are soaked in distilled water for 3 days on a culture dish and cultured for 7 days, then the seeds are transferred to a rice nutrient solution for culture, the formula of the nutrient solution refers to the formula of international rice, but ammonium nitrate is adjusted to 0.5mM (low nitrogen), 2mM (medium nitrogen) and 5mM (high nitrogen), the seeds are cultured for 40 days respectively, the phenotype is observed, the root length, the root number, the plant height and the fresh weight are counted, and the amino acid in the root of the permanent planting plant is measured, and the result is shown in figures 1-8. Under the culture of low nitrogen, medium nitrogen and high nitrogen,OsNPF8.13compared with the flower 11 plants in the control, the gene interference plants and the mutant plants have the advantages of reduced root length, root number, plant height and content of free amino acid in roots, and achieve obvious difference. Statistical data for root length, root number, plant height, fresh weight and free amino acid content in roots of interfering, mutant and control plants under different nitrogen cultures are shown in FIGS. 4-8. At the later stage of the process,OsNPF8.13the yield of both gene interfering plants and mutant plants was also significantly lower than that of control flower 11.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Wuhan bioengineering college
Application of <120> OsNPF8.13 gene in promotion of rice growth under high nitrogen
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>1701
<212>DNA
<213>Oryza sativa
<400>1
atggaggcag gggcagcaga tgaggagacg ccactgattc agcagctgcc tccggaggaa 60
caatgttcac aatacacatg tgatggaaca gttaatagtg acaaaaagcc tgcactgaag 120
cagagtacag ggcattggag agcatgcttc ttcattttag gtgctcaatt cgctgaaacc 180
ttgtgcttct tcatggtttc gaagaactta gtcatgtacc tcacgagtgc gctgcacgaa 240
agcaacatcg acgctgcaca aagtgtgtct atttggatcg gcacttcctt cttcacacca 300
ctcattggag ccttcttggc tgatacatat tggggaagat actggacgac agttatttcc 360
ctctttatta tcatcatcgg aatgctcatt gtgacggttt catcatcacc attgttcctg 420
aattcttctt actacaattg gaacatttgc cgtgccacgg tctacacagg gctctacctt 480
actgccgttg gaagtggatg tatgaagccc tgcattccag cctttggagc cgatcaattt 540
gacagtgctg acccggtgga acggctggcg aagggctcat tcttcaactg gtattacttc 600
tcaatgaacg tcggctcact gctgtcgacg actctgcttg tctgggtggt ggccaacata 660
gggtggagcg tcggttttgc gatcccgatg ctactctcgg ggttcggcct cgccctgttt 720
ttcgctggta ggaaggttta caggtacaag aaacagggag ggagtccact gacaagggtg 780
tcccaggtgg tggttgcagc tgtaaggaat cataggctga aattgcctga cgatagctca 840
ctcctgcatg aggtttcaaa agtgactgaa gatgattaca ggactcaact caccactcaa 900
ttcaggttct ttgacaaggc tgccatcttg tccgacgaga tctcgccggc gcagtggagc 960
ccgtggaggc tgtgcacggt ttcgcaggtg gaggagctga agatcctgct gcggatgttc 1020
ccggtctggg tgtccatggt cgtcttcttc gtggtgaccg cgcagattac gtcgacgctg 1080
atcgagcagg gcatggccat ggacggccgc gtcggcccgt tcacccttcc ggccgcctca 1140
atcgccacct tcgacgtcat cagcgtcctc gtctgggtcc ccgtctacga caccgtgctg 1200
gtgccactgg cacggcgcgt caccggcaag gaccgtggca tctcccacct gcagcgcatt 1260
ggcgtcggcc tcgcgctcgc cgcggtggcc atggcgtact cggcggtggt cgaggcacgg 1320
cggctgggga cggcgccagc gccggtgagc atcatgtggc aggcgccgtc gtacctggtg 1380
ctgggcgtgg cggaggcgtt cagcgtgatc ggcatgatgg agttcttcta cgagcagtcg 1440
ccggagtcga tgaagagcct gtgcacggcg ctcgggcagc tcgccatcgc ggtcgccaac 1500
tacctcaact ccggcgtgct cgtcgtggtg gcggcggcca ccacgcgcgg cggcggggcc 1560
ggctggatcc cggacaacct cgacgagggg cacctggact acttcttctg gatgatggct 1620
gttgttagcg tcctcaacct gctgcacttc ttgcattgct caatcagata tagagccaat 1680
aacaacacgc tgtcgtcttg a 1701
<210>2
<211>1320
<212>DNA
<213>Oryza sativa
<400>2
atgctcattg tgacggtttc atcatcacca ttgttcctga attcttctta ctacaattgg 60
aacatttgcc gtgccacggt ctacacaggg ctctacctta ctgccgttgg aagtggatgt 120
atgaagccct gcattccagc ctttggagcc gatcaatttg acagtgctga cccggtggaa 180
cggctggcga agggctcatt cttcaactgg tattacttct caatgaacgt cggctcactg 240
ctgtcgacga ctctgcttgt ctgggtggtg gccaacatag ggtggagcgt cggttttgcg 300
atcccgatgc tactctcggg gttcggcctc gccctgtttt tcgctggtag gaaggtttac 360
aggtacaaga aacagggagg gagtccactg acaagggtgt cccaggtggt ggttgcagct 420
gtaaggaatc ataggctgaa attgcctgac gatagctcac tcctgcatga ggtttcaaaa 480
gtgactgaag atgattacag gactcaactc accactcaat tcaggttctt tgacaaggct 540
gccatcttgt ccgacgagat ctcgccggcg cagtggagcc cgtggaggct gtgcacggtt 600
tcgcaggtgg aggagctgaa gatcctgctg cggatgttcc cggtctgggt gtccatggtc 660
gtcttcttcg tggtgaccgc gcagattacg tcgacgctga tcgagcaggg catggccatg 720
gacggccgcg tcggcccgtt cacccttccg gccgcctcaa tcgccacctt cgacgtcatc 780
agcgtcctcg tctgggtccc cgtctacgac accgtgctgg tgccactggc acggcgcgtc 840
accggcaagg accgtggcat ctcccacctg cagcgcattg gcgtcggcct cgcgctcgcc 900
gcggtggcca tggcgtactc ggcggtggtc gaggcacggc ggctggggac ggcgccagcg 960
ccggtgagca tcatgtggca ggcgccgtcg tacctggtgc tgggcgtggc ggaggcgttc 1020
agcgtgatcg gcatgatgga gttcttctac gagcagtcgc cggagtcgat gaagagcctg 1080
tgcacggcgc tcgggcagct cgccatcgcg gtcgccaact acctcaactc cggcgtgctc 1140
gtcgtggtgg cggcggccac cacgcgcggc ggcggggccg gctggatccc ggacaacctc 1200
gacgaggggc acctggacta cttcttctgg atgatggctg ttgttagcgt cctcaacctg 1260
ctgcacttct tgcattgctc aatcagatat agagccaata acaacacgct gtcgtcttga 1320
<210>3
<211>566
<212>PRT
<213>Oryza sativa
<400>3
Met Glu Ala Gly Ala Ala Asp Glu Glu Thr Pro Leu Ile Gln Gln Leu
1 5 10 15
Pro Pro Glu Glu Gln Cys Ser Gln Tyr Thr Cys Asp Gly Thr Val Asn
20 25 30
Ser Asp Lys Lys Pro Ala Leu Lys Gln Ser Thr Gly His Trp Arg Ala
35 40 45
Cys Phe Phe Ile Leu Gly Ala Gln Phe Ala Glu Thr Leu Cys Phe Phe
50 55 60
Met Val Ser Lys Asn Leu Val Met Tyr Leu Thr Ser Ala Leu His Glu
65 70 75 80
Ser Asn Ile Asp Ala Ala Gln Ser Val Ser Ile Trp Ile Gly Thr Ser
85 90 95
Phe Phe Thr Pro Leu Ile Gly Ala Phe Leu Ala Asp Thr Tyr Trp Gly
100 105 110
Arg Tyr Trp Thr Thr Val Ile Ser Leu Phe Ile Ile Ile Ile Gly Met
115 120 125
Leu Ile Val Thr Val Ser Ser Ser Pro Leu Phe Leu Asn Ser Ser Tyr
130 135 140
Tyr Asn Trp Asn Ile Cys Arg Ala Thr Val Tyr Thr Gly Leu Tyr Leu
145 150 155 160
Thr Ala Val Gly Ser Gly Cys Met Lys Pro Cys Ile Pro Ala Phe Gly
165 170 175
Ala Asp Gln Phe Asp Ser Ala Asp Pro Val Glu Arg Leu Ala Lys Gly
180 185 190
Ser Phe Phe Asn Trp Tyr Tyr Phe Ser Met Asn Val Gly Ser Leu Leu
195 200 205
Ser Thr Thr Leu Leu Val Trp Val Val Ala Asn Ile Gly Trp Ser Val
210 215 220
Gly Phe Ala Ile Pro Met Leu Leu Ser Gly Phe Gly Leu Ala Leu Phe
225 230 235 240
Phe Ala Gly Arg Lys Val Tyr Arg Tyr Lys Lys Gln Gly Gly Ser Pro
245 250 255
Leu Thr Arg Val Ser Gln Val Val Val Ala Ala Val Arg Asn His Arg
260 265 270
Leu Lys Leu Pro Asp Asp Ser Ser Leu Leu His Glu Val Ser Lys Val
275 280 285
Thr Glu Asp Asp Tyr Arg Thr Gln Leu Thr Thr Gln Phe Arg Phe Phe
290 295 300
Asp Lys Ala Ala Ile Leu Ser Asp Glu Ile Ser Pro Ala Gln Trp Ser
305 310 315 320
Pro Trp Arg Leu Cys Thr Val Ser Gln Val Glu Glu Leu Lys Ile Leu
325 330 335
Leu Arg Met Phe Pro Val Trp Val Ser Met Val Val Phe Phe Val Val
340 345 350
Thr Ala Gln Ile Thr Ser Thr Leu Ile Glu Gln Gly Met Ala Met Asp
355 360 365
Gly Arg Val Gly Pro Phe Thr Leu Pro Ala Ala Ser Ile Ala Thr Phe
370 375 380
Asp Val Ile Ser Val Leu Val Trp Val Pro Val Tyr Asp Thr Val Leu
385 390 395 400
Val Pro Leu Ala Arg Arg Val Thr Gly Lys Asp Arg Gly Ile Ser His
405 410 415
Leu Gln Arg Ile Gly Val Gly Leu Ala Leu Ala Ala Val Ala Met Ala
420 425 430
Tyr Ser Ala Val Val Glu Ala Arg Arg Leu Gly Thr Ala Pro Ala Pro
435 440 445
Val Ser Ile Met Trp Gln Ala Pro Ser Tyr Leu Val Leu Gly Val Ala
450 455 460
Glu Ala Phe Ser Val Ile Gly Met Met Glu Phe Phe Tyr Glu Gln Ser
465 470 475 480
Pro Glu Ser Met Lys Ser Leu Cys Thr Ala Leu Gly Gln Leu Ala Ile
485 490 495
Ala Val Ala Asn Tyr Leu Asn Ser Gly Val Leu Val Val Val Ala Ala
500 505 510
Ala Thr Thr Arg Gly Gly Gly Ala Gly Trp Ile Pro Asp Asn Leu Asp
515 520 525
Glu Gly His Leu Asp Tyr Phe Phe Trp Met Met Ala Val Val Ser Val
530 535 540
Leu Asn Leu Leu His Phe Leu His Cys Ser Ile Arg Tyr Arg Ala Asn
545 550 555 560
Asn Asn Thr Leu Ser Ser
565
<210>4
<211>439
<212>PRT
<213>Oryza sativa
<400>4
Met Leu Ile Val Thr Val Ser Ser Ser Pro Leu Phe Leu Asn Ser Ser
1 5 10 15
Tyr Tyr Asn Trp Asn Ile Cys Arg Ala Thr Val Tyr Thr Gly Leu Tyr
20 25 30
Leu Thr Ala Val Gly Ser Gly Cys Met Lys Pro Cys Ile Pro Ala Phe
35 40 45
Gly Ala Asp Gln Phe Asp Ser Ala Asp Pro Val Glu Arg Leu Ala Lys
50 55 60
Gly Ser Phe Phe Asn Trp Tyr Tyr Phe Ser Met Asn Val Gly Ser Leu
65 70 75 80
Leu Ser Thr Thr Leu Leu Val Trp Val Val Ala Asn Ile Gly Trp Ser
85 90 95
Val Gly Phe Ala Ile Pro Met Leu Leu Ser Gly Phe Gly Leu Ala Leu
100 105 110
Phe Phe Ala Gly Arg Lys Val Tyr Arg Tyr Lys Lys Gln Gly Gly Ser
115 120 125
Pro Leu Thr Arg Val Ser Gln Val Val Val Ala Ala Val Arg Asn His
130 135 140
Arg Leu Lys Leu Pro Asp Asp Ser Ser Leu Leu His Glu Val Ser Lys
145 150 155 160
Val Thr Glu Asp Asp Tyr Arg Thr Gln Leu Thr Thr Gln Phe Arg Phe
165 170 175
Phe Asp Lys Ala Ala Ile Leu Ser Asp Glu Ile Ser Pro Ala Gln Trp
180 185 190
Ser Pro Trp Arg Leu Cys Thr Val Ser Gln Val Glu Glu Leu Lys Ile
195 200 205
Leu Leu Arg Met Phe Pro Val Trp Val Ser Met Val Val Phe Phe Val
210 215 220
Val Thr Ala Gln Ile Thr Ser Thr Leu Ile Glu Gln Gly Met Ala Met
225 230 235 240
Asp Gly Arg Val Gly Pro Phe Thr Leu Pro Ala Ala Ser Ile Ala Thr
245 250 255
Phe Asp Val Ile Ser Val Leu Val Trp Val Pro Val Tyr Asp Thr Val
260 265 270
Leu Val Pro Leu Ala Arg Arg Val Thr Gly Lys Asp Arg Gly Ile Ser
275 280 285
His Leu Gln Arg Ile Gly Val Gly Leu Ala Leu Ala Ala Val Ala Met
290 295 300
Ala Tyr Ser Ala Val Val Glu Ala Arg Arg Leu Gly Thr Ala Pro Ala
305 310 315 320
Pro Val Ser Ile Met Trp Gln Ala Pro Ser Tyr Leu Val Leu Gly Val
325 330 335
Ala Glu Ala Phe Ser Val Ile Gly Met Met Glu Phe Phe Tyr Glu Gln
340 345 350
Ser Pro Glu Ser Met Lys Ser Leu Cys Thr Ala Leu Gly Gln Leu Ala
355 360 365
Ile Ala Val Ala Asn Tyr Leu Asn Ser Gly Val Leu Val Val Val Ala
370 375 380
Ala Ala Thr Thr Arg Gly Gly Gly Ala Gly Trp Ile Pro Asp Asn Leu
385 390 395 400
Asp Glu Gly His Leu Asp Tyr Phe Phe Trp Met Met Ala Val Val Ser
405 410 415
Val Leu Asn Leu Leu His Phe Leu His Cys Ser Ile Arg Tyr Arg Ala
420 425 430
Asn Asn Asn Thr Leu Ser Ser
435
Claims (2)
1.OsNPF8.13The application of the gene in improving the rice biomass under high nitrogen is characterized in that: the rice biomass is rice root length, root number, plant height, fresh weight and free amino acid content; saidOsNPF8.13The amino acid sequence of the OsNPF8.13 protein coded by the gene is shown as SEQ ID NO.3 or 4; by increasingOsNPF8.13Expression of the gene enables the use.
2. Use according to claim 1, characterized in that: saidOsNPF8.13The cDNA sequence of the gene is shown as SEQ ID NO.1 or 2.
Priority Applications (1)
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|---|---|---|---|
| CN201711221530.9A CN107937433B (en) | 2017-11-22 | 2017-11-22 | Application of OsNPF8.13 gene in promotion of rice growth under high nitrogen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711221530.9A CN107937433B (en) | 2017-11-22 | 2017-11-22 | Application of OsNPF8.13 gene in promotion of rice growth under high nitrogen |
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| CN107937433B true CN107937433B (en) | 2020-05-29 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027397A (en) * | 2004-04-23 | 2007-08-29 | 西尔斯公司 | Nucleotide sequences and polypeptides encoded thereby useful for modifying nitrogen use efficiency characteristics in plants |
| CN102604962A (en) * | 2011-01-20 | 2012-07-25 | 中国科学院华南植物园 | Gene OsPTR9 capable of improving nitrogen absorption efficiency and yield of rice and application thereof |
| CN104277101A (en) * | 2014-09-24 | 2015-01-14 | 中国科学院遗传与发育生物学研究所 | Application of rice nitrate transporter NRT1.1B in enhancing nitrogen utilization efficiency of plants |
| CN106222180A (en) * | 2016-07-28 | 2016-12-14 | 武汉生物工程学院 | Improve rice yield and the gene OsNPF7.3 of grain of rice protein content and purposes |
| CN106434666A (en) * | 2016-10-20 | 2017-02-22 | 武汉生物工程学院 | Application of promoter achieving specific expression in rice tiller bud base and panicle |
| CN107012153A (en) * | 2017-03-30 | 2017-08-04 | 武汉生物工程学院 | Applications of the nitrogen nutrition transporter gene OsNPF8.1 in rice tillering number is improved |
-
2017
- 2017-11-22 CN CN201711221530.9A patent/CN107937433B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027397A (en) * | 2004-04-23 | 2007-08-29 | 西尔斯公司 | Nucleotide sequences and polypeptides encoded thereby useful for modifying nitrogen use efficiency characteristics in plants |
| CN102604962A (en) * | 2011-01-20 | 2012-07-25 | 中国科学院华南植物园 | Gene OsPTR9 capable of improving nitrogen absorption efficiency and yield of rice and application thereof |
| CN104277101A (en) * | 2014-09-24 | 2015-01-14 | 中国科学院遗传与发育生物学研究所 | Application of rice nitrate transporter NRT1.1B in enhancing nitrogen utilization efficiency of plants |
| CN106222180A (en) * | 2016-07-28 | 2016-12-14 | 武汉生物工程学院 | Improve rice yield and the gene OsNPF7.3 of grain of rice protein content and purposes |
| CN106434666A (en) * | 2016-10-20 | 2017-02-22 | 武汉生物工程学院 | Application of promoter achieving specific expression in rice tiller bud base and panicle |
| CN107012153A (en) * | 2017-03-30 | 2017-08-04 | 武汉生物工程学院 | Applications of the nitrogen nutrition transporter gene OsNPF8.1 in rice tillering number is improved |
Non-Patent Citations (5)
| Title |
|---|
| ACCESSION NO.XM_015757529，PREDICTED: Oryza sativa Japonica Group protein NRT1/ PTR FAMILY 8.3 (LOC9272029),transcript variant X1, mRNA;无;《GenBank》;20160301;FEATURES,ORIGIN * |
| Disruption of the Rice Nitrate Transporter OsNPF2.2 Hinders Root-To-Shoot Nitrate Transport and Vascular Development;Y Li et al.;《Scientific Reports》;20150429;第1-10页 * |
| Knock-Down of a Tonoplast Localized Low-Affinity Nitrate Transporter OsNPF7.2 Affects Rice Growth Under High Nitrate Supply;Hu R et al;《Front Plant Sci》;20161025;第7卷;第1-13页 * |
| Transporters for uptake and allocation of organic nitrogen compounds in plants;Doris Rentsch et al;《FEBS Letters》;20070418;第2281-2289页 * |
| 植物寡肽运输与硝酸根运输基因家族的研究进展;蔡昭艳 等;《热带亚热带植物学报》;20110115;第19卷(第1期);第91-96页 * |
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| Publication number | Publication date |
|---|---|
| CN107937433A (en) | 2018-04-20 |
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