CN107779468A - The application of rice NRT1.1A genes and its encoding proteins in plant products breeding is improved - Google Patents
The application of rice NRT1.1A genes and its encoding proteins in plant products breeding is improved Download PDFInfo
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- CN107779468A CN107779468A CN201610680206.2A CN201610680206A CN107779468A CN 107779468 A CN107779468 A CN 107779468A CN 201610680206 A CN201610680206 A CN 201610680206A CN 107779468 A CN107779468 A CN 107779468A
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- 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
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- 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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Abstract
The invention discloses a kind of application of rice NRT1.1A genes and its encoding proteins in plant products breeding is improved.Application provided by the present invention is specially the application of protein or its encoding gene in regulating growth of plants；Described grow is presented as single plant yield and/or plant height and/or grain number per spike and/or flowering time and/or bolting time and/or lotus throne leaf size；The protein is any in following：(1) amino acid sequence is the protein shown in sequence 1 in sequence table；(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing and/or addition and the protein as derived from sequence 1 related to regulating growth of plants.The present invention is significant for cultivating high-yield rice new varieties.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of rice NRT1.1A genes and its encoding proteins and is improving plant
Application in Yield Breeding.
Background technology
Plant needs a variety of mineral nutrient elements to maintain its normal growth, and wherein nitrogen is the mineral that plant has the call
Nutrient.In general, nitrogen can account for the 1.5-2% of plant dry weight.Nitrogen is protein, nucleic acid, phosphatide and plant
The constituent of organonitrogen compound necessary to growing, and these materials structure or function that to be living cells depend on for existence
Composition, therefore nitrogen is also referred to as biological element.In order to pursue the high yield of crop, often apply a large amount of nitrogen in agricultural production
Fertilizer.Wherein, the amount of application of nitrogen fertilizer of rice accounts for the total amount of application of chemical fertilizer considerably beyond other any crops, the loss amount of nitrogenous fertilizer again
70%.China generally existing nitrogenous fertilizer using excessive, nitrogen use efficiency be low and loss of nitrogen fertilizer caused by environmental degradation etc. it is a series of
Problem.
By improving the nitrogen use efficiency of crops and reducing the pass that nitrogen application is then the solution serious environmental problems
Key.Nitrate is one of nitrogen source mostly important in soil, and absorption and utilization of the plant to nitrate largely determine
The nitrogen use efficiencies of crops.And nitrate transport protein is then plant absorption, transhipment and the most direct work(for storing nitrate
Can executor.
The content of the invention
It is an object of the invention to provide a kind of rice NRT1.1A genes and its new application of encoding proteins.
New application provided by the present invention is specially protein or its encoding gene answering in regulating growth of plants
With；It is described grow be presented as single plant yield (including biomass) and/or plant height and/or grain number per spike and/or flowering time and/
Or bolting time and/or lotus throne leaf size；
Wherein, the flowering time is the morning and evening at time point for starting to bloom；The bolting time for start bolting when
Between morning and evening for putting.
The protein is any in following：
(1) amino acid sequence is the protein (i.e. NRT1.1A albumen) shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to regulating growth of plants.
The protein or its encoding gene regulating growth of plants are embodied in：The protein is in the plant
In expression quantity it is higher, the single plant yield (including biomass) of the plant is higher, plant height is higher, grain number per spike is more, when blooming
Between more the early bolting time is more early and/or lotus throne leaf is bigger；Expression quantity of the protein in the plant is lower, the plant
Single plant yield (including biomass) it is lower, plant height is lower, grain number per spike less, flowering time is more late, the bolting time is more late and/or
Lotus throne leaf is smaller.
Protein or its encoding gene increase in seed selection single plant yield (including biomass) and/or plant height increases and/or fringe
Application in the plant variety that grain number increases and/or flowering time shifts to an earlier date and/or bolting time advance and/or lotus throne leaf increase
Belong to protection scope of the present invention；
The protein is any in following：
(1) amino acid sequence is the protein (i.e. NRT1.1A albumen) shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to regulating growth of plants.
In actual applications, when institute's seed selection single plant yield (including biomass) increases and/or plant height increases and/or grain number per spike
, need to be by the egg during plant variety that increase and/or flowering time shift to an earlier date and/or bolting time advance and/or lotus throne leaf increase
The higher plant of white matter expression quantity is hybridized as parent.
Another object of the present invention is cultivation single plant yield (including biomass) increase and/or plant height increases and/or fringe
Grain number increases and/or flowering time shifts to an earlier date and/or the method for the genetically modified plants of bolting time advance and/or the increase of lotus throne leaf.
Cultivation single plant yield (including biomass) increase provided by the present invention and/or plant height increase and/or grain number per spike increases
Add and/or flowering time shifts to an earlier date and/or the method for the genetically modified plants of bolting time advance and/or the increase of lotus throne leaf, it may include
The step of making the expression of purpose plant or overexpressed protein, or including making the step that protein active improves in purpose plant
Suddenly；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to regulating growth of plants；
Further, methods described specifically may include it is following a) and b) the step of：
A) encoding gene of the protein is imported into the purpose plant, obtain expressing the encoding gene turns base
Because of plant；
B) obtained from genetically modified plants obtained by step a) compared with the purpose plant, single plant yield (including biomass)
Increase and/or plant height increase and/or grain number per spike increases and/or flowering time shifts to an earlier date and/or bolting time advance and/or lotus throne leaf
The genetically modified plants of increase.
The application of protein or its encoding gene in following (A) or (B) falls within protection scope of the present invention：
(A) nitrate is promoted to absorb or transport；
(B) expression of nitrate metabolism related gene is promoted；The nitrate metabolism related gene is any in following
Kind：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2；
The protein is any in following：
(1) amino acid sequence is the protein (i.e. NRT1.1A albumen) shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to nitrate transport.
In the present invention, the promotion nitrate in described (A) absorbs or transhipment is specially to promote xenopus leavis oocytes
The absorption or transhipment of middle nitrate, or to promote the absorption or transhipment of nitrate in rice；The promotion nitre in (B)
The expression of hydrochlorate metabolism related gene is specially to promote the expression of nitrate metabolism related gene in rice；The nitric acid salt metabolism
Related gene is any in following：OsNRT1.1B, OsNRT2.1, OsNRT2.3a, OsNAR1 and OsNAR2.
The application of protein or its encoding gene in following (C) or (D) falls within protection scope of the present invention：
(C) plant variety that absorption or turn-over capacity of the seed selection to nitrate improve；
(D) plant variety that the expression quantity of seed selection nitrate metabolism related gene improves；The nitrate is metabolism related
Because any in following：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2；
The protein is any in following：
(1) amino acid sequence is the protein (i.e. NRT1.1A albumen) shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to nitrate transport.
It is a further object to provide a kind of absorption cultivated to nitrate or turn-over capacity improves and/or nitric acid
The method for the genetically modified plants that the expression quantity of salt metabolism related gene improves.
Absorption or turn-over capacity raising and/or nitrate metabolism related gene of the cultivation provided by the present invention to nitrate
The genetically modified plants improved of expression quantity, it may include the step of expressing purpose plant or be overexpressed protein, or including making
The step of protein active improves in purpose plant；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by substitution of the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from sequence 1 related to regulating growth of plants；
Further, methods described specifically may include it is following c) and d) the step of：
C) encoding gene of the protein is imported into the purpose plant, obtain expressing the encoding gene turns base
Because of plant；
D) obtained from genetically modified plants obtained by step c) compared with the purpose plant, absorption or transhipment to nitrate
The genetically modified plants that ability improves and/or the expression quantity of nitrate metabolism related gene improves；
The nitrate metabolism related gene is any in following：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and
NAR2。
In above-mentioned each application and method, the encoding gene can be following (1) any described DNA molecular into (4)：
(1) in sequence table sequence 2 DNA molecular；
(2) in sequence table sequence 3 DNA molecular；
(3) DNA molecular limited under strict conditions with (1) or (2) hybridizes and encoding amino acid sequence is sequence table
Protein DNA molecule shown in middle sequence 1；
(4) DNA molecular limited with (1) or (2) or (3) has more than 90% homology and encoding amino acid sequence is sequence
Protein DNA molecule in list shown in sequence 1.
Above-mentioned stringent condition can be with 6 × SSC, 0.5%SDS solution, hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 2 is made up of 8143 nucleotides, is genome sequence；Sequence 3 is made up of 1812 nucleotides, is
CDNA sequence；Protein in sequence 2 and the polynucleotide of sequence 3 shown in sequence 1, sequence 1 is by 603 amino acid residue groups
Into.
In above-described two methods, the encoding gene can pass through the recombination expression containing the encoding gene
In purpose plant described in vector introduction.
The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture
Bacillus carrier and carrier available for plant micropellet bombardment etc., as pGreen0029, pCAMBIA3301, pCAMBIA1300,
The derivative plant expression vector of pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.The plant expression
Carrier can also include 3 ' end untranslated regions of foreign gene, i.e., processed comprising polyadenylation signals and any other participation mRNA
Or the DNA fragmentation of gene expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Using institute
When stating gene constructed recombinant expression carrier, any enhanced, composing type, tissue can be added before its transcription initiation nucleotides
Idiotype or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin start
Sub (pUbi), stress induced promoter rd29A etc., they can be used alone or are used in combination with other plant promoters；
In addition, when using the gene constructed recombinant expression carrier of the present invention, enhancer, including translational enhancer or transcription also can be used to increase
Hadron, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but required and coded sequence
Reading frame it is identical, to ensure the correct translation of whole sequence.The source of the translation control signal and initiation codon is wide
General, can be natural or synthesis.Translation initiation region can come from transcription initiation region or structural gene.
For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be processed, such as
The coding that adding can express in plant can produce the enzyme of color change or the gene of luminophor, resistant antibiotic
Label or anti-chemical reagent marker gene etc..Also any selected marker can be not added with, is directly screened and converted with adverse circumstance
Plant.
In the present invention, the promoter for starting the encoding gene transcription in the recombinant expression carrier is specially Actin1
The own endogenous promoter (sequence 9) of promoter or rice NRT1.1A genes.More specifically, the recombinant expression carrier is
Small fragment between the restriction enzyme site XbaI and PstI of pCAMBIA2300-Actin carriers is replaced with into the institute of sequence 3 in sequence table
Show the recombinant plasmid obtained after DNA fragmentation；Or it is interleaving in the restriction enzyme site KpnI and EcoRI of pCAMBIA2300 carriers
Enter DNA fragmentation shown in the 1-1814 positions of sequence 9 in sequence table, and the insetion sequence table between restriction enzyme site EcoRI and XmaI
The recombinant plasmid of gained after DNA fragmentation shown in middle sequence 3；Or it is by the restriction enzyme site of pCAMBIA2300-35S-OCS carriers
Small fragment between Bam HI and Sal I replaces with the recombinant plasmid of gained after DNA fragmentation shown in sequence 3 in sequence table.
In above-described two methods, the recombinant expression carrier is imported into the purpose plant, concretely：It is logical
Cross conventional raw using Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated etc.
Thing method converts plant cell or tissue, and the plant tissue of conversion is cultivated into plant.
More than in each application and method, the plant may be either monocotyledon, or dicotyledon.The list
Cotyledon plant concretely rice；The dicotyledon concretely arabidopsis.In one embodiment of the invention, it is described
Plant is specially rice varieties Dong Jin.In another embodiment of the present invention, the plant is specially arabidopsis Columbia-
0。
More than in each application and method, when the plant is specially rice, the nitrate metabolism related gene choosing
It is any in following：OsNRT1.1B, OsNRT2.1, OsNRT2.3a, OsNAR1 and OsNAR2.
In the present invention, the nucleotides sequence of the OsNRT1.1B is classified as sequence 4 in sequence table；The core of the OsNRT2.1
Nucleotide sequence is sequence 5 in sequence table；The nucleotides sequence of the OsNRT2.3a is classified as sequence 6 in sequence table；The OsNAR1's
Nucleotides sequence is classified as sequence 7 in sequence table；The nucleotides sequence of the OsNAR2 is classified as sequence 8 in sequence table.
The present invention proves that NRT1.1A albumen can transport nitric acid by using the external movement system of xenopus leavis oocytes
Salt.Transgenic experiments show that NRT1.1A overexpressions in rice wild type can show obvious growth vigor, and
It is obviously improved grain number per spike and plant height, and then improves rice yield NRT1.1A overexpression Seedling Stage is just in arabidopsis wild type
Certain growth vigor is shown, lotus throne leaf is significantly greater than wild type control；Into after peduncle-growing period for rapeseed, it is notable to be overexpressed strain bolting
Earlier than wild type control.Result above implies that NRT1.1A albumen has great application potential in raising plant products.
Brief description of the drawings
Fig. 1 is expression analysis of the NRT1.1A genes in rice is respectively organized.
Fig. 2 is that NRT1.1A albumen is shown in xenopus leavis oocytes15NO3 -Transport activity.
Fig. 3 is the suppression that rice nrt1.1a mutant shows nitrate transport.Wherein, A is to be transferred to 3h after new nutrient solution
Result；B is the result for being transferred to 24h after new nutrient solution.WT represents wild type control.
Fig. 4 is that the expression of other nitrate transport protein genes under rice nrt1.1a mutant backgrounds is suppressed.
WT represents wild type control.
Fig. 5 is that Transgenic Rice strain OX1-1, OX2-6 of NRT1.1A overexpressions shows obvious growth vigor.
Wherein, A is Seedling Stage phenotype；B is reproductive growth period phenotype.WT represents wild type control.
Fig. 6 is that nitrate utilizes dependency basis under NRT1.1A overexpression Transgenic Rice strain OX1-1, OX2-6 backgrounds
Because expression significantly raises.WT represents wild type control.
Fig. 7 is NRT1.1A overexpression Transgenic Rice strain pNA-2 and pNA-4 in the low nitrogen in field/high nitrogen condition following table
Reveal yield heterosis.Wherein, A is the high nitrogen condition in field；B is the low nitrogen condition in field.
Fig. 8 is that NRT1.1A overexpression arabidopsis transgenic lines OX-3 and OX-17 show obvious growth vigor.
Wherein, NRT1.1A gene expression dose testing results in A WT, OX-3 and OX-17；B is Seedling Stage phenotype；C is peduncle-growing period for rapeseed table
Type.WT represents wild type control (Columbia-0).
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
PCAMBIA2300-Actin carriers：Be recorded in " Wang, K.J., Tang, D., Wang, M., Lu, J.F., Yu,
H.X.,Liu,J.F.,Qian,B.X.,Gong,Z.Y.,Wang,X.,Chen,J.M.,Gu,M.H.and Cheng,Z.K.
(2009)MER3is required for normal meiotic crossover formation,but not for
Presynaptic alignment in rice.J Cell Sci, 122, the texts of 2055-2063. " one, the public can be at applicant
Obtain, can only be used to repeat present invention experiment.
Rice varieties Dong Jin：It is recorded in " Jakyung Yi, Gynheung An.Utilization of T-DNA
Tagging Lines in Rice.J.Plant Biol.(2013)56:" Dongjin " of the texts of 85-90 " one, disclosure can be from Shen
Ask someone to locate to obtain, can only be used to repeat present invention experiment use.
Rice nrt1.1a mutant：From Korea rice mutant library (Crop Biotech Institute, Kyung
Hee University,Republic of Korea,http://www.postech.ac.kr/life/pfg/risd), order
Network address is http://signal.salk.edu/cgi-bin/RiceGE5, the reference numeral of rice nrt1.1a mutant are
PFG_1E-00433.L.Specific construction method is recorded in " Jakyung Yi, Gynheung An.Utilization of T-DNA
Tagging Lines in Rice.J.Plant Biol.(2013)56:The texts of 85-90 " one.
Xenopus leavis oocytes (Xenopus oocyte)：Be recorded in " 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. " one
Text, the public can obtain at applicant, can only be used to repeat present invention experiment.
pCS2+Carrier：It is recorded in " 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. " one texts, the public can be from applicant
Place obtains, and can only be used to repeat present invention experiment.
PCAMBIA2300-35S-OCS carriers：It is recorded in " the .pCAMBIA2300-betA- such as salty ocean, Xiayang, Zhang Jinwen
The structure China agronomy circular of BADH Two gene plant expression vectors, 09 phase in a 2009 " text, the public can obtain at applicant,
It can only be used to repeat present invention experiment.
Embodiment 1, the NRT1.1A expression in rice is respectively organized are analyzed
Involved NRT1.1A gene sources are in rice (Oryza.sativa L.), its genome sequence in the present embodiment
As shown in sequence 2 in sequence table, sequence 2 is made up of 8143 nucleotides, its cDNA sequence be sequence table in sequence 3, sequence 3 by
1812 nucleotides compositions.Protein (NRT1.1A albumen) in sequence 2 and the polynucleotide of sequence 3 shown in sequence 1, sequence 1
It is made up of 603 amino acid residues.
Total serum IgE is extracted respectively from rice varieties Dong Jin root, stem, leaf sheath, leaf and fringe and reverse transcription obtains cDNA.And then
Using gained cDNA as template, real-time quantitative fluorescence PCR, NRT1.1A bases during detection rice is respectively organized are carried out for NRT1.1A genes
Because of the expression quantity on transcriptional level.Experiment is repeated 3 times, results averaged.
Primer sequence for detecting NRT1.1A genes is as follows：
qNRT1.1A-F：5 '-CCGTCTTCTTCGTCGGCTCCATCCT-3 ' (the 1187-1211 positions of sequence 3)；
qNRT1.1A-R：5 '-CCCGTGCTCATCGTCTTCATCCCCT-3 ' (the 1514-1538 positions of sequence 3 it is reverse
Complementary series).
Using OsActin1 as reference gene, its primer sequence is：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’.
The expression quantity of reference gene is considered as 1, calculates the relative expression quantity of NRT1.1A genes.
QRT-PCR analysis shows, NRT1.1A genes have in rice is respectively organized higher level expression (see Fig. 1) and
Highest is expressed in root next to that leaf, leaf sheath and stem, expression quantity is minimum in fringe.This constitutive expression result implies that NRT1.1A can
The maintenance of the basic physiology function of plant can be take part in.
Embodiment 2, NRT1.1A transport nitrate activity checking in vitro
Involved NRT1.1A gene sources are in rice (Oryza.sativa L.), its genome sequence in the present embodiment
As shown in sequence 2 in sequence table, sequence 2 is made up of 8143 nucleotides, its cDNA sequence be sequence table in sequence 3, sequence 3 by
1812 nucleotides compositions.Protein (NRT1.1A albumen) in sequence 2 and the polynucleotide of sequence 3 shown in sequence 1, sequence 1
It is made up of 603 amino acid residues.
First, Xenopus laevis recombinant expression carrier pCS2+/ NRT1.1A structure
Extract japonica rice variety Dong Jin total serum IgE, reverse transcription cDNA.Using the cDNA of acquisition as template, using following primer
Sequence enters performing PCR amplification to NRT1.1A cDNA respectively.The primer both ends that amplification uses introduce restriction enzyme BamHI respectively
With EcoRI recognition site (as shown in underscore), primer sequence：
F：5’-GGATCC(underscore part is BamHI recognition sequence to ATGGTGGGGATGTTGCCGGA-3 ', thereafter
Sequence is the 1-20 positions of sequence 3 in sequence table)；
R：5’-GAATTC(underscore part is EcoRI recognition sequence to TCAGTGGAGGCATGGCTCGG-3 ', thereafter
Sequence is the reverse complementary sequence of the 1793-1812 positions of sequence 3 in sequence table).
The purpose fragment of amplification is connected into xenopus leavis oocytes expression vector pCS2+In, and verified by sequencing.
It will show that the small fragment between by pCS2+ carriers restriction enzyme site BamHI and EcoRI replaces with sequence table through sequencing
The recombinant vector of gained is named as pCS2+/NRT1.1A after DNA fragmentation shown in middle sequence 3.
2nd, in-vitro transcription NRT1.1AmRNA and inject xenopus leavis oocytes checking transhipment nitrate activity
The recombinant vector pCS2 that step 1 structure is obtained+/ NRT1.1A is linearized with restriction enzyme A paI,
Then in-vitro transcription kit (mMESSAGE is usedSP6Kit, Ambion, AM1340) in-vitro transcription goes out band
The cRNA of 5 ' cap sequences and 3 ' poly A structures, concrete operations are referring to kit specification.
By gained cRNA injection xenopus leavis oocytes (Xenopus oocyte).After injection, in ND96 solution (formula：
96mM NaCl, 2mM KCl, 1mM MgCl2, 1.8mM CaCl2, 5mM HEPES, pH 7.4) in culture two days later, be transferred to and contain
There are 10mM K15NO3Absorbent solution (formula：10mM K15NO3(98%atom15N-KNO3,Sigma-Aldrich,
335134), 230mM Mannitol, 0.3mM CaCl2, 10mM MES-Tris, pH 5.5) in, 3h is further cultured for, using element
Mass spectrometer (ICP-MS) measure xenopus leavis oocytes15(specific method is referring to " Kun-Hsiang Liu, Chi- for N content
Ying Huang,Yi-Fang Tsay,et al.CHL1 Is a Dual-Affinity Nitrate Transporter of
Arabidopsis Involved in Multiple Phases of Nitrate Uptake.The Plant Cell,
1999 " one texts).Experiment sets 3 repetitions, and measurement result takes average.
Experiment sets injection same volume ddH simultaneously2O control.
Measurement result shows that injection has NRT1.1A cRNA xenopus leavis oocytes15It is identical that N content is significantly higher than injection
Volume ddH2O compares (P<0.01, see Fig. 2).The result shows that NRT1.1A has transhipment nitrate activity in vitro.
The acquisition and functional verification of embodiment 3, rice nrt1.1a mutant
Involved NRT1.1A gene sources are in rice (Oryza.sativa L.), its genome sequence in the present embodiment
As shown in sequence 2 in sequence table, sequence 2 is made up of 8143 nucleotides, its cDNA sequence be sequence table in sequence 3, sequence 3 by
1812 nucleotides compositions.Protein (NRT1.1A albumen) in sequence 2 and the polynucleotide of sequence 3 shown in sequence 1, sequence 1
It is made up of 603 amino acid residues.
First, the acquisition and identification of rice nrt1.1a mutant
Rice nrt1.1a mutant：From Korea rice mutant library (Crop Biotech Institute, Kyung
Hee University,Republic of Korea,http://www.postech.ac.kr/life/pfg/risd), order
Network address is http://signal.salk.edu/cgi-bin/RiceGE5, the reference numeral of rice nrt1.1a mutant are
PFG_1E-00433.L.Specific construction method is recorded in " Jakyung Yi, Gynheung An.Utilization of T-DNA
Tagging Lines in Rice.J.Plant Biol.(2013)56:The texts of 85-90 " one.
Rice mutant nrt1.1a identification is carried out using " three-primer method ".The sequence of the primer is as follows：
pGA2715L：5’-CTAGAGTCGAGAATTCAGTACA-3’；
NARM14F：5’-AATCCGCAAATGTGTCTTGT-3’；
NARM14R：5’-CTAGGGCCATCTTGTCTTCA-3’.
Verified, NRT1.1A genes are mutated really in rice mutant nrt1.1a, it is impossible to which normal expression is active
The NRT1.1A albumen of energy.
2nd, the nitrate transport functional verification of rice nrt1.1a mutant
In order to specifically understand function inside NRT1.1A, the present inventor is carried out15The nitrate transport of N tracers
Experiment.First, Kimura B nutrient solution of the seedling of wild rice Dong Jin and nrt1.1a mutant in improvement (is formulated：
2mM KNO3, 1.8mM KCl, 0.36mM CaCl2, 0.54mM MgSO4·7H2O, 0.18mM KH2PO4, 40 μM of Na2EDTA-
Fe (II), 13.4 μM of MnCl2·4H2O, 18.8 μM of H3BO3, 0.03 μM of Na2MoO4·2H2O, 0.3 μM of ZnSO4·7H2O,
0.32μM CuSO4·5H2O and 1.6mM Na2SiO3·9H2O culture 10 days, are then transferred to containing 5mM K in)15NO3Improvement
24h is cultivated in Kimura B nutrient solutions.Partly drawn materials above and below the ground for 3h and 24h points after new nutrient solution is transferred to, using element
Mass spectrometer (ICP-MS) determines15(specific method is referring to " Kun-Hsiang Liu, Chi-Ying Huang, Yi- for N content
Fang Tsay,et al.CHL1 Is a Dual-Affinity Nitrate Transporter of Arabidopsis
Involved in Multiple Phases of Nitrate Uptake.The Plant Cell, 1999 " one texts).Experiment
If 4 repetitions, measurement result take average.
By calculating in wild rice Dong Jin and nrt1.1a mutant overground part and root15N content ratio is found, two
In individual sampling time point, nrt1.1a mutant overground part and root15The ratio of N content is substantially less than wild type (P<0.01, see figure
3), illustrate that transhipment of the nitrate from underground to overground part is severely impacted in nrt1.1a mutant.
3rd, nitrate Metabolism-Related Genes Expression determines in rice nrt1.1a mutant
Following nitrate in rice is detected by qRT-PCR and utilizes the expression of related gene：(1) OsNRT1.1B bases
Cause, its cDNA nucleotides sequence are classified as sequence 4 in sequence table, the absorption and transhipment of main responsible nitrate, and participate in nitric acid
The regulation of salt signal；(2) OsNRT2.1 genes, its cDNA nucleotides sequence are classified as sequence 5 in sequence table, are mainly responsible for nitrate
Absorb；(3) OsNRT2.3a genes, its cDNA nucleotides sequence are classified as sequence 6 in sequence table, are responsible for nitrate to aerial part
Transhipment；(4) OsNAR1 genes, its cDNA nucleotides sequence are classified as sequence 7 in sequence table, OsNRT2.1 perform functions it is auxiliary because
Son；(5) OsNAR2 genes, its cDNA nucleotides sequence are classified as sequence 8 in sequence table, the co-factor of OsNRT2.1 perform functions.
Wild rice Dong Jin and nrt1.1a mutant seedlings blade is taken respectively, extracts total serum IgE, and reverse transcription obtains
CDNA, qRT-PCR detections are then carried out using related gene to above-mentioned five kinds of nitrate using following each primer pair.
For detecting the primer pair of OsNRT1.1B genes：
OsNRT1.1B-F：5’-GGCAGGCTCGACTACTTCTA-3’；
OsNRT1.1B-R：5’-AGGCGCTTCTCCTTGTAGAC-3’.
For detecting the primer pair of OsNRT2.1 genes：
OsNRT2.1-F：5’-CTTCACGTCGTCGAGGTACT-3’；
OsNRT2.1-R：5’-CACTCGGAGCCGTAGTAGTG-3’.
For detecting the primer pair of OsNRT2.3a genes：
OsNRT2.3a-F：5’-CGCTGCTGCCGCTCATCCG-3’；
OsNRT2.3a-R：5’-CCGTGCCCATGGCCAGAC-3’.
For detecting the primer pair of OsNAR1 genes：
OsNAR1-F：5’-GTTCAAGAGCGTGAAGGTGA-3’；
OsNAR1-R：5’-CACCACGTAGTCGAACCTG-3’.
For detecting the primer pair of OsNAR2 genes：
OsNAR2-F：5’-TCGTCCTCGAGAACAAGAAG-3’；
OsNAR2-R：5’-TCCGTTGGTTTTGTAGGTTG-3’.
Using OsActin1 as reference gene, its detection primer pair：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’.
The expression quantity of reference gene is considered as 1, calculates the relative expression quantity of each gene.
As a result show, under nrt1.1a mutant backgrounds, compared with wild rice Dong Jin, above-mentioned five kinds of nitrate profits
(P is significantly inhibited with the expression of related gene<0.05, see Fig. 4), imply that NRT1.1A may take part in these genes
The regulation and control of transcriptional level.
Embodiment 4, the acquisition of NRT1.1A transgenic paddy rices and functional verification (allogeneic promoter)
First, recombinant plant expression vector pCAMBIA2300-Actin/NRT1.1A structure
Extract japonica rice Dong Jin total serum IgE, reverse transcription cDNA.Using the cDNA of acquisition as template, using following primer sequence
Performing PCR amplification is entered to NRT1.1A CDS.The primer both ends that amplification uses introduce restriction enzyme XbaI and PstI respectively
Recognition site (as shown in underscore), primer sequence is as follows：
F：5’-TCTAGA(underscore part is XbaI recognition sequence to ATGGTGGGGATGTTGCCGGA-3 ', thereafter
Sequence is the 1-20 positions of sequence 3 in sequence table)；
R：5’-CTGCAG(underscore part is PstI recognition sequence to TCAGTGGAGGCATGGCTCGG-3 ', thereafter
Sequence is the reverse complementary sequence of the 1793-1812 positions of sequence 3 in sequence table).
CDS regions (including terminator codon) using rice varieties Dong Jin cDNA as template amplification NRT1.1A.PCR is produced
After thing connection carrier T pEASY-Blunt (TransGene), plant expression vector is connected into after the checking of XbaI and PstI double digestions
In pCambia2301-Actin.
It will show that the small fragment between by pCAMBIA2300-Actin carriers restriction enzyme site XbaI and PstI replaces through sequencing
The recombinant vector for being changed in sequence table gained after DNA fragmentation shown in sequence 3 is named as pCAMBIA2300-Actin/NRT1.1A.
2nd, the acquisition of NRT1.1A transgenic paddy rices
The recombinant plant expression vector pCAMBIA2300-Actin/NRT1.1A that step 1 is built is transferred to Agrobacterium AGL1
In (coming from ATCC), then infect japonica rice variety Dong Jin callus, specific transformation and selection method referring to document " easily rely on oneself,
Cao Shouyun, Wang Li, what strontium is clean, and storage is become a useful person, Tang Zuoshun, Zhou Piaohua, and Tian Wenzhong improve the research of Agrobacterium-mediated Transformation rice frequency
Acta Genetica Sinica, 2001,28 (4):The texts of 352-358 " one.The control for being transferred to pCAMBIA2300-Actin empty carriers is set simultaneously.Most
Two kinds of transgenic seedlings are obtained eventually, that is, are transferred to pCAMBIA2300-Actin/NRT1.1A rice plant and are transferred to
Rice plant (the T of pCAMBIA2300-Actin empty carriers0)。
3rd, the identification of NRT1.1A transgenic paddy rices
(1) PCR Preliminary Identifications
The T obtained from step 20In generation, is transferred to pCAMBIA2300-Actin/NRT1.1A transgenic paddy rice, and is transferred to
Genomic DNA is extracted respectively in the adjoining tree of pCAMBIA2300-Actin empty carriers.With the primers F 1 for NptII genes
Enter performing PCR identification, identified (PCR primer size is about 500bp) shown containing NptII genes with R1 (primer sequence is as follows)
Plant is the plant of transgenic positive.
F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；
R1：5’-CTGGCGCGAGCCCCTGATGCT-3’.
Through above-mentioned PCR Molecular Identifications, the transgenosis water that is transferred to pCAMBIA2300-Actin/NRT1.1A positive from identification
2 are randomly selected in rice strain and is denoted as OX1-1, OX2-6 respectively.
(2) transcriptional level analysis (rna expression amount)
The T obtained with step (1)0For NRT1.1A transgenic paddy rice strains OX1-1, OX2-6, pCAMBIA2300- is transferred to
The adjoining tree of Actin empty carriers, and wild rice kind Dong Jin are experiment material.Extract the total serum IgE of each material and anti-
Transcription obtains cDNA.And then using gained cDNA as template, real-time quantitative fluorescence PCR is carried out for NRT1.1A genes, detects each material
Expression quantity of the NRT1.1A genes on transcriptional level in material.Experiment is repeated 3 times, results averaged.
Primer sequence for detecting NRT1.1A genes is as follows：
qNRT1.1A-F：5 '-CCGTCTTCTTCGTCGGCTCCATCCT-3 ' (the 1187-1211 positions of sequence 3)；
qNRT1.1A-R：5 '-CCCGTGCTCATCGTCTTCATCCCCT-3 ' (the 1514-1538 positions of sequence 3 it is reverse
Complementary series).
Using OsActin1 as reference gene, its primer sequence is：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’.
The expression quantity of reference gene is considered as 1, calculates the relative expression quantity of NRT1.1A genes.
The real-time quantitative fluorescence PCR testing result of NRT1.1A gene expression amounts is shown in each experiment material, compared to not turning base
The wild rice kind Dong Jin of cause, the T that step (1) obtains0For in NRT1.1A transgenic paddy rice strains OX1-1, OX2-6
The expression quantity of NRT1.1A genes significantly improves on transcriptional level, and for being transferred to pair of pCAMBIA2300-Actin empty carriers
According to plant, the expression quantity of its NRT1.1A gene transcriptional level compared with the wild rice Dong Jin of non-transgenosis it is basically identical,
No difference of science of statistics.
4th, the functional verification of NRT1.1A transgenic paddy rices
1st, experimental method
With the positive T of step 3 identification2For NRT1.1A transgenic paddy rice strains OX1-1, OX2-6, it is transferred to
The adjoining tree of pCAMBIA2300-Actin empty carriers, and the wild rice Dong Jin of non-transgenosis is experiment material.From with
Lower several respects identify its function：
(1) Phenotypic Observation
Observed in Seedling Stage and record the plant height of each genetic stocks, into reproductive growth period after observe and record each again
The plant height of genetic stocks.In experiment, each transgenic line at least selects 30 individual plants to be counted.
(2) qRT-PCR analyzes the transcriptional level of nitrate metabolism related gene
Nitrate metabolism related gene is specifically related to：
OsNRT2.1 genes, its cDNA nucleotides sequence are classified as sequence 5 in sequence table；
OsNRT2.3a genes, its cDNA nucleotides sequence are classified as sequence 6 in sequence table；
OsNIA2 genes, its cDNA nucleotides sequence are classified as sequence 8 in sequence table；
OsNIR1 genes, its cDNA nucleotides sequence are classified as sequence 7 in sequence table.
Specific assay method is carried out referring to the step 3 of embodiment 2.
In experiment, each transgenic line at least selects 5 plants to be tested.
2nd, experimental result
(1) Phenotypic Observation result
T2Certain growth vigor, strain are just shown in Seedling Stage for NRT1.1A transgenic paddy rices strain OX1-1, OX2-6
Height is significantly higher than wild type control；Into florescence, it is overexpressed the phenotype that strain shows florescence and done sth. in advance (see Fig. 5).And turn
Enter its plant height of the adjoining tree of pCAMBIA2300-Actin empty carriers and flowering time basic one compared with wild type control
Cause, no difference of science of statistics.These results illustrate that NRT1.1A has certain application potential in agricultural production, and appropriateness improves
NRT1.1A expression may improve the yield of rice.
(2) qRT-PCR analyzes the transcriptional level of nitrate metabolism related gene
QRT-PCR analysis shows, the positive T of step 3 identification0For NRT1.1A transgenic paddy rice strains OX1-1, OX2-6
In, the transcriptional level of its above-mentioned four kinds of nitrate metabolism related gene obtains significantly raising (P compared to wild type control<
0.01, see Fig. 6).
Embodiment 5, the acquisition of NRT1.1A transgenic paddy rices and functional verification (endogenesis promoter)
In order to confirm that NRT1.1A can reach the effect of increasing production of rice, and constitutive promoter is avoided to Agronomic Traits in Rice
Caused negative effect, the present inventor construct the NRT1.1A overexpression transfer-gen plants of its own promoter driving, and
Further its function is verified.
First, recombinant expression carrier pCAMBIA2300/NRT1.1A structure
Rice varieties Dong Jin genomic DNA and total serum IgE is extracted, and is cDNA by total serum IgE reverse transcription.To the east of Tianjin genome
DNA is template amplification NRT1.1A promoter region (sequence 9), and the primer both ends for expanding use introduce restriction enzyme respectively
Enzyme KpnI and EcoRI recognition site (as shown in underscore), the primer sequence of use are as follows：
NRT1.1Ap-F：5’-GGTACC(underscore part is KpnI identification sequence to TTCGATCTCCCACGTAAGAC-3 '
Row, sequence thereafter are the 1-20 positions of sequence 9)；
NRT1.1Ap-R：5’-GAATTC(underscore part is EcoRI's to TCTCTCTCTCTTCTTCTTCTTCCTC-3 '
Recognition sequence, sequence thereafter for the 1790-1814 positions of sequence 9 reverse complementary sequence).
Meanwhile the CDS regions using rice varieties Dong Jin cDNA as template amplification NRT1.1A, expand the primer two of use
End introduces restriction enzyme EcoRI and XmaI recognition site respectively, and the sequence of primer is as follows：
NRT1.1ACDS-F：5’-GAATTC(underscore part is EcoRI identification to ATGGTGGGGATGTTGCCGGA-3 '
Sequence, sequence thereafter are the 1-20 positions of sequence 3)；
NRT1.1ACDS-R：5’-CCCGGGGTGGAGGCATGGCTCGG-3 ' (underscore part is XmaI recognition sequence,
Thereafter sequence is the reverse complementary sequence of the 1793-1809 positions of sequence 3).
Two PCR fragments that two steps amplify by more than are connected into pBluescript KS (+) respectively at twice
At the corresponding restriction enzyme site of (Stratagene, 212205) carrier.After sequence verification, using KpnI and BamHI double digestions simultaneously
Fragment containing endogenesis promoter and NRT1.1A CDS is connected at binary vector pCAMBIA2300 corresponding restriction enzyme site, will
Gained is named as pCAMBIA2300/NRT1.1A through the correct recombinant vector of sequence verification.
2nd, the acquisition of NRT1.1A transgenic paddy rices
The recombinant plant expression vector pCAMBIA2300/NRT1.1A that step 1 is built is transferred into Agrobacterium AGL1 (to come from
ATCC in), then infect the japonica rice variety Dong Jin specific transformation and selection method of callus referring to document " easily rely on oneself, Cao Shouyun,
Wang Li, what strontium is clean, and storage is become a useful person, Tang Zuoshun, Zhou Piaohua, and Tian Wenzhong improve the research science of heredity of Agrobacterium-mediated Transformation rice frequency
Report, 2001,28 (4):The texts of 352-358 " one.The control for being transferred to pCAMBIA2300 empty carriers is set simultaneously.It is final to obtain two kinds turns
Gene vaccine, that is, it is transferred to pCAMBIA2300/NRT1.1A rice plant and is transferred to the rice plant of pCAMBIA2300 empty carriers
(T0)。
3rd, the identification of NRT1.1A transgenic paddy rices
(1) PCR Preliminary Identifications
The T obtained from step 20In generation, is transferred to pCAMBIA2300/NRT1.1A transgenic paddy rice, and is transferred to
Genomic DNA is extracted respectively in the adjoining tree of pCAMBIA2300 empty carriers.Carried out with primers F 1 and R1 (primer sequence is as follows)
PCR identifies that identified (PCR primer size is about 500bp) plant for showing containing NptII genes is the plant of transgenic positive
Strain.
F1：5’-TCCGGCCGCTTGGGTGGAGAG-3’；
R1：5’-CTGGCGCGAGCCCCTGATGCT-3’.
Through above-mentioned PCR Molecular Identifications, the transgenic paddy rice strain that is transferred to pCAMBIA2300/NRT1.1A positive from identification
In randomly select 2 and be denoted as pNA-2 and pNA-4 respectively.
(2) transcriptional level analysis (rna expression amount)
The identification positive T obtained with step (1)0For NRT1.1A transgenic paddy rice strain pNA-2 and pNA-4, it is transferred to
The adjoining tree of pCAMBIA2300 empty carriers, and wild rice kind Dong Jin are experiment material.Extract the total of each material
Simultaneously reverse transcription obtains cDNA to RNA.And then using gained cDNA as template, real-time quantitative fluorescence PCR is carried out for NRT1.1A genes,
Detect expression quantity of the NRT1.1A genes on transcriptional level in each material.Experiment is repeated 3 times, results averaged.
Primer sequence for detecting NRT1.1A genes is as follows：
qNRT1.1A-F：5 '-CCGTCTTCTTCGTCGGCTCCATCCT-3 ' (the 1187-1211 positions of sequence 3)；
qNRT1.1A-R：5 '-CCCGTGCTCATCGTCTTCATCCCCT-3 ' (the 1514-1538 positions of sequence 3 it is reverse
Complementary series).
Using OsActin1 as reference gene, its primer sequence is：
OsActin1-F：5’-ACCATTGGTGCTGAGCGTTT-3’；
OsActin1-R：5’-CGCAGCTTCCATTCCTATGAA-3’.
The expression quantity of reference gene is considered as 1, calculates the relative expression quantity of NRT1.1A genes.
The real-time quantitative fluorescence PCR testing result of NRT1.1A gene expression amounts is shown in each experiment material, compared to not turning base
The wild rice kind Dong Jin of cause, the T that step (1) obtains0For in NRT1.1A transgenic paddy rice strains pNA-2 and pNA-4
The expression quantity of NRT1.1A genes is significantly improved on transcriptional level, and the control for being transferred to pCAMBIA2300 empty carriers is planted
Strain, the expression quantity of its NRT1.1A gene transcriptional level compared with the wild rice kind Dong Jin of non-transgenosis it is basically identical,
No difference of science of statistics.
4th, the functional verification of NRT1.1A transgenic paddy rices
With the positive T of step 3 identification2For NRT1.1A transgenic paddy rice strain pNA-2 and pNA-4, it is transferred to
The adjoining tree of pCAMBIA2300 empty carriers, and the wild rice Dong Jin of non-transgenosis is experiment material.Field test is set
Put two nitrogenous fertilizer gradients (high nitrogen and low nitrogen).Wherein high nitrogen applies fertilizer according to every 100 square metres of 4.28kg urea, and low nitrogen is according to every
100 square metres of 1.07kg urea fertilisings.3 cells are set under each nitrogenous fertilizer gradient.Field observation and record its phenotype (including
Tiller number, mass of 1000 kernel, grain number per spike and single plant yield), each strain randomly chooses at least ten individual plant in each cell and carried out
Phenotype counts, and amounts to each strain and is no less than 30 individual plants.
The nitrogenous fertilizer test result in field is shown, under the conditions of low nitrogen and high nitrogen, the transgenic line of NRT1.1A overexpressions
What pNA-2 and pNA-4 showed as grain number per spike and single plant yield dramatically increases (P<0.05).Especially under the conditions of low nitrogen,
Being overexpressed the single plant yield of transgenic line increases particularly evident (P<0.01) Fig. 7 is referred to.
Embodiment 6, the acquisition of NRT1.1A transgenic arabidopsis and functional verification (allogeneic promoter)
First, recombinant plant expression vector pCAMBIA2300-35S-OCS/NRT1.1A structure
Extract japonica rice Dong Jin total serum IgE, reverse transcription cDNA.Using the cDNA of acquisition as template, using following primer sequence
Performing PCR amplification is entered to NRT1.1A CDS.The primer both ends that amplification uses introduce restriction enzyme Bam HI and Sal I respectively
Recognition site (as shown in underscore), primer sequence is as follows：
F：5’-GGATCC(underscore part is Bam HI recognition sequence to ATGGTGGGGATGTTGCCGGA-3 ', thereafter
Sequence be sequence table in sequence 3 1-20 positions)；
R：5’-GTCGAC(underscore part is Sal I recognition sequence to TCAGTGGAGGCATGGCTCGG-3 ', thereafter
Sequence is the reverse complementary sequence of the 1793-1812 positions of sequence 3 in sequence table).
CDS regions (including terminator codon) using rice varieties Dong Jin cDNA as template amplification NRT1.1A.PCR is produced
After thing connection carrier T pEASY-Blunt (TransGene), plant expression is connected into after the checking of Bam HI and Sal I double digestions and is carried
In body pCAMBIA2300-35S-OCS.
It will show through sequencing small between restriction enzyme site Bam HI and Sal I by pCAMBIA2300-35S-OCS carriers
The recombinant vector that fragment replaces with gained after DNA fragmentation shown in sequence 3 in sequence table is named as pCAMBIA2300-35S-OCS/
NRT1.1A。
2nd, the acquisition of NRT1.1A transgenic arabidopsis
The recombinant plant expression vector pCAMBIA2300-35S-OCS/NRT1.1A that step 1 is built is passed through into Agrobacterium
The genetic transformation of GV3101 (coming from ATCC) mediations is transferred to arabidopsis Columbia-0 (bibliography：“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”).The control for being transferred to pCAMBIA2300-35S-OCS empty carriers is set simultaneously.Two kinds of transgenic seedlings are finally obtained, that is, are transferred to
PCAMBIA2300-35S-OCS/NRT1.1A Arabidopsis plant and the plan south for being transferred to pCAMBIA2300-35S-OCS empty carriers
Mustard plant (T0)。
3rd, the identification of NRT1.1A transgenic arabidopsis
The T obtained from step 20In generation, is transferred in pCAMBIA2300-35S-OCS/NRT1.1A transgenic arabidopsis random
Two plants are chosen, is designated as respectively as OX-3 and OX-17.By OX-3 and OX-17, it is transferred to pCAMBIA2300-35S-OCS empty carriers
Total serum IgE is extracted respectively in the wildtype Arabidopsis thaliana Columbia-0 of adjoining tree and non-transgenosis and reverse transcription obtains cDNA.
And then using gained cDNA as template, real-time quantitative fluorescence PCR is carried out for NRT1.1A genes, detects NRT1.1A bases in each material
Because of the expression quantity on transcriptional level.Experiment is repeated 3 times, results averaged.
Primer sequence for detecting NRT1.1A genes is as follows：
qNRT1.1A-F：5 '-CCGTCTTCTTCGTCGGCTCCATCCT-3 ' (the 1187-1211 positions of sequence 3)；
qNRT1.1A-R：5 '-CCCGTGCTCATCGTCTTCATCCCCT-3 ' (the 1514-1538 positions of sequence 3 it is reverse
Complementary series).
Using AtActin2 as reference gene, its primer sequence is：
qAtActin2-F：5’-GCACCACCTGAAAGGAAGTACA-3’；
qAtActin2-R：5’-CGATTCCTGGACCTGCCTCATC-3’.
The expression quantity of reference gene is considered as 1, calculates the relative expression quantity of NRT1.1A genes.
The real-time quantitative fluorescence PCR testing result of NRT1.1A gene expression amounts is shown in each experiment material, compared to not turning base
The wildtype Arabidopsis thaliana of cause, the T that step 2 obtains0The transgenosis that generation is transferred to pCAMBIA2300-35S-OCS/NRT1.1A intends south
The expression quantity of NRT1.1A genes significantly improves (see A in Fig. 8) on transcriptional level in mustard strain OX-3 and OX-17.And for turning
Enter the adjoining tree of pCAMBIA2300-35S-OCS empty carriers, the expression quantity of its NRT1.1A gene does not turn in transcriptional level and base
The WT lines of cause compare basically identical, no difference of science of statistics.
4th, the functional verification of NRT1.1A transgenic arabidopsis
1st, experimental method
With the positive T of step 3 identification2For NRT1.1A transgenic arabidopsis strain OX-3 and OX-17, it is transferred to
The adjoining tree of pCAMBIA2300-35S-OCS empty carriers, and the wildtype Arabidopsis thaliana Columbia-0 of non-transgenosis are real
Test material.Observe the lotus throne leaf size of each genetic stocks in Seedling Stage, into peduncle-growing period for rapeseed after when observing the bolting of each genetic stocks
Between.In experiment, each transgenic line at least selects 30 individual plants to be observed.
2nd, experimental result
T2Certain growth vigor is just shown in Seedling Stage for NRT1.1A transgenic arabidopsis strain OX-3 and OX-17,
Lotus throne leaf is significantly greater than wild type control (see B in Fig. 8), into after peduncle-growing period for rapeseed, is overexpressed strain bolting significantly earlier than wild type
Control is (see C in Fig. 8).And for being transferred to the adjoining tree of pCAMBIA2300-35S-OCS empty carriers, its Seedling Stage lotus throne leaf is big
The small and bolting time is basically identical compared with the WT lines of non-transgenosis.
Claims (10)
1. the application of protein or its encoding gene in regulating growth of plants；Described grow is presented as single plant yield
And/or plant height and/or grain number per spike and/or flowering time and/or bolting time and/or lotus throne leaf size；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to regulating growth of plants.
2. protein or its encoding gene increase in seed selection single plant yield and/or plant height increases and/or grain number per spike increase and/or opened
Take time in advance and/or bolting time advance and/or lotus throne leaf increase plant variety in application；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to regulating growth of plants.
3. cultivate single plant yield increase and/or plant height increases and/or grain number per spike increases and/or flowering time shifts to an earlier date and/or bolting
The method of the genetically modified plants of time advance and/or the increase of lotus throne leaf, including make the expression of purpose plant or overexpressed protein
The step of, or including making the step of protein active improves in purpose plant；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to regulating growth of plants；
Specifically, methods described include it is following a) and b) the step of：
A) encoding gene of the protein is imported into the purpose plant, the transgenosis for obtaining expressing the encoding gene is planted
Thing；
B) obtained from genetically modified plants obtained by step a) compared with the purpose plant, single plant yield increase and/or plant height increase
The increase of high and/or grain number per spike and/or flowering time shift to an earlier date and/or the transgenosis plant of bolting time advance and/or the increase of lotus throne leaf
Thing.
4. the application of protein or its encoding gene in following (A) or (B)；
(A) nitrate is promoted to absorb or transport；
(B) expression of nitrate metabolism related gene is promoted；The nitrate metabolism related gene is any in following：
NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to nitrate transport.
5. the application of protein or its encoding gene in following (C) or (D)；
(C) plant variety that absorption or turn-over capacity of the seed selection to nitrate improve；
(B) plant variety that the expression quantity of seed selection nitrate metabolism related gene improves；The nitrate metabolism related gene choosing
It is any in following：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to nitrate transport.
6. cultivate the absorption to nitrate or turn-over capacity improves and/or what the expression quantity of nitrate metabolism related gene improved turns
The method of gene plant, including purpose plant is expressed or the step of overexpressed protein, or including making in purpose plant
The step of protein active improves；
The protein is any in following：
(1) amino acid sequence is the protein shown in sequence 1 in sequence table；
(2) by the amino acid sequence of sequence in sequence table 1 by one or several amino acid residues substitution and/or missing and/
Or addition and the protein as derived from sequence 1 related to regulating growth of plants；
Specifically, methods described include it is following c) and d) the step of：
C) encoding gene of the protein is imported into the purpose plant, the transgenosis for obtaining expressing the encoding gene is planted
Thing；
D) obtained from genetically modified plants obtained by step c) compared with the purpose plant, absorption or turn-over capacity to nitrate
The genetically modified plants that the expression quantity of raising and/or nitrate metabolism related gene improves；
The nitrate metabolism related gene is any in following：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2.
7. according to any described application or method in claim 1-6, it is characterised in that：The encoding gene is following (1)
Any described DNA molecular into (4)：
(1) in sequence table sequence 2 DNA molecular；
(2) in sequence table sequence 3 DNA molecular；
(3) limited under strict conditions with (1) or (2) DNA molecular hybridization and encoding amino acid sequence as sequence table in sequence
Protein DNA molecule shown in row 1；
(4) DNA molecular limited with (1) or (2) or (3) has more than 90% homology and encoding amino acid sequence is sequence table
Protein DNA molecule shown in middle sequence 1.
8. the method according to claim 3 or 6, it is characterised in that：In methods described, the encoding gene be by containing
The recombinant expression carrier of the encoding gene is imported in the purpose plant；
Specifically, the promoter for starting the encoding gene transcription in the recombinant expression carrier is 35S promoter or rice
The own endogenous promoter of NRT1.1A genes.
9. according to any described application or method in claim 1-3 and 5-7, it is characterised in that：The plant is unifacial leaf
Plant or dicotyledon.
10. application according to claim 4, it is characterised in that：In (A), the promotion nitrate absorbs or transhipment
To promote the absorption or transhipment of nitrate in xenopus leavis oocytes, or to promote the absorption or transhipment of nitrate in rice；
In (B), the promotion nitrate metabolism related gene is expressed as promoting nitric acid salt metabolism in xenopus leavis oocytes
The expression of related gene or the expression for promoting nitrate metabolism related gene in rice；The nitrate metabolism related gene is selected from
It is any in following：NRT1.1B, NRT2.1, NRT2.3a, NAR1 and NAR2.
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| CN201610680206.2A CN107779468B (en) | 2016-08-17 | 2016-08-17 | Application of rice NRT1.1A gene and coded protein thereof in breeding for improving plant yield |
| PCT/CN2017/097601 WO2018033083A1 (en) | 2016-08-17 | 2017-08-16 | Applications of rice nrt1.1a gene and encoding protein thereof in breeding for improving productions of plants |
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| CN201610680206.2A CN107779468B (en) | 2016-08-17 | 2016-08-17 | Application of rice NRT1.1A gene and coded protein thereof in breeding for improving plant yield |
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| CN110846322A (en) * | 2018-07-29 | 2020-02-28 | 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） | Corn small-grain mutant and application thereof |
| CN114920810A (en) * | 2021-02-01 | 2022-08-19 | 中国农业大学 | Application of nitrate absorption related protein in regulation and control of nitrate absorption of corn |
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| CN114920810B (en) * | 2021-02-01 | 2023-02-21 | 中国农业大学 | Application of nitrate absorption related protein in regulation and control of nitrate absorption of corn |
| CN116143892A (en) * | 2023-03-30 | 2023-05-23 | 沈阳农业大学 | Application of OsGN11 gene in improving rice grain number per ear character |
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