CN101397565A - Rice high affinity nitrate transport protein gene OsNAR2.1 - Google Patents
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Abstract
The invention discloses a sequence of rice high-affinity nitrate transport protein gene OsNAR2.1 and application thereof, belonging to the gene engineering field. The invention also discloses a cDNA sequence SEQ ID NO.1 of the rice high-affinity nitrate transport protein gene OsNAR2.1 and a coded mino acid sequence SEQ ID NO.2 thereof. The gene function is firstly reported in rice and expressed in frogspawn heterogenous system to determine that the OsNAR2.1 protein can remarkably promote the transportation of nitrate by the rice; and mRNA expression analysis shows that the gene is subject to the induction of low nitrate nitrogen and the inhibition of ammonium and nitrogen metabolin. The knockout mutant of the gene causes the rice to reduce the absorption of nitrate and have obvious anazotic symptoms, the gene also influences the ripening rate of the rice through the field experiments, and therefore, over expression OsNAR2.1 can improve the absorption of nitrate nitrogen of the rice under low-nitrogen condition and the yield of the rice.
Description
Technical field
The invention discloses rice high affinity nitrate transport protein gene OsNAR2.1 sequence and application thereof, belong to the genetically engineered field, the application of this gene has specifically significantly promoted the absorption of paddy rice to nitrate.
Background technology
Nitrogen is one of important macronutrient of crop, participates in the various metabolic processes of organism.It is the moiety of a lot of living matters in the plant materials, such as: amino acid, protein, nucleic acid, enzyme, chlorophyll etc.Nitrogen accounts for 16% (Frink CR., Waggoner PE.and Ausubel JH.Nitrogen fertilizer:retrospect andprospect.Proc.Natl Acad.Sci.USA.1999.96:1175-1180.) of 1.5-2% and plant total protein of plant materials dry weight respectively.
At present, Chinese nitrogen fertilizer amount accounts for 30% (Peng Shaobing, Huang Jianliang of global nitrogen fertilizer amount, Zhong Xuhua, Yang Jianchang, Wang Guanghuo, Zou Yingbin, Zhang Fusuo, Zhu Qingsen, Roland Buresh, Christian Witt. improves the research strategy of Chinese rice field utilization rate of nitrogen fertilizer. Scientia Agricultura Sinica .2002,35 (9): 1095～1103), become the big country of consumption of the first in the world.Wherein the amount of application of nitrogenous fertilizer surpasses other any farm crop in the rice terrace, and the loss amount of nitrogenous fertilizer accounts for 70% of total fertilization amount.China's ubiquity because a series of environmental problems that utilization rate of nitrogen fertilizer is low and a large amount of nitrogens loss causes.Though paddy rice is a happiness ammonium crop, there are some researches show that a certain amount of nitric nitrogen can promote the absorption of paddy rice to ammonium, and the later stage field of rice growth and drought rice mainly based on nitric nitrogen.Therefore high affinity nitrate transport protein OsNAR2.1 can significantly promote paddy rice that nitrate is absorbed, thereby improves paddy rice to the utilization ratio of nitrogenous fertilizer, the output of increase paddy rice.
Summary of the invention:
Technical problem:
Purpose of the present invention provides sequence and the application thereof of rice high affinity nitrate transport protein gene OsNAR2.1, this gene can be used as goal gene overexpression in paddy rice, can improve paddy rice under low nitric nitrogen condition to the absorption of nitric nitrogen and nitrogen utilising efficiency even can improve rice yield.
Technical scheme:
The invention provides rice high affinity nitrate transport protein gene OsNAR2.1, its nucleotides sequence is classified SEQ ID NO.1 as, altogether 621bp.This gene expression product is rice high affinity nitrate transport protein OsNAR2.1, and its aminoacid sequence is SEQ ID NO.2, totally 206 amino acid.The genetically engineered of OsNAR2.1 can be used aspect raising crop nitrogenous fertilizer utilising efficiency and output.
Beneficial effect:
1, open rice high affinity nitrate transport protein gene OsNAR2.1 sequence of the inventor and coded protein thereof.The mRNA expression analysis shows that the OsNAR2.1 gene mainly expresses at rice root, and under the inducing of 0.2mM nitric nitrogen strong expression.
2, the inventor discloses the function of high affinity nitrate transport protein OsNAR2.1 first, its function is significantly to have promoted OsNRT2.1 (AB008519) albumen, OsNRT2.2 (AK109733) albumen (though OsNRT2.1 and OsNRT2.2 gene cDNA sequence difference to some extent, but the sequence of its opening code-reading frame is in full accord, so their protein product is a product) and OsNRT2.3a (AK109776) albumen to the absorption of nitrate, be expected to be applied to the genetic improvement of paddy rice.
3, the inventor obtains the transfer-gen plant of overexpression rice high affinity nitrate transporter gene OsNAR2.1 first, and shows and significantly improve nitrogen utilising efficiency and rice yield 15%.
Description of drawings
Fig. 1: the carrier of frog's egg heterologous expression system-pT7Ts collection of illustrative plates
Fig. 2: frog's egg heterogenous expression OsNAR2.1 albumen shows that this albumen promotes OsNRT2.1 (or OsNRT2.2) albumen that nitrate is absorbed
Fig. 3: frog's egg heterogenous expression OsNAR2.1 albumen shows that this albumen promotes OsNRT2.3a albumen that nitrate is absorbed
Fig. 4: OsNAR2.1 is at paddy rice different sites (blade, stem stalk, lateral root district, apical area) expression characteristic
1: apical area (3-4cm); 2: the lateral root district; 3: the stem stalk; 4: blade
The expression characteristic of Fig. 5: OsNAR2.1 under different nitrogen form
1:0.2mM nitrate; 2:0.2mM ammonium
Fig. 6: OsNAR2.1 and OsNRT2 gene are 1 and 2 and the expression of wild-type (WT) in the strain of RNAi mutant
Fig. 7: overexpression OsNAR2.1 gene is used and contrast wild-type paddy rice setting percentage difference under nitrogen fertilizer application condition not
Specific embodiments
One, the clone of rice high affinity nitrate transport protein gene OsNAR2.1 sequence
1) the extraction paddy rice of total RNA (Japan is fine) seedling grew to for 3 leaf phases, handle with the 0.2mM nitric nitrogen and to get root immediately after 6 hours and place the freezing preservation of liquid nitrogen rapidly, take by weighing root about 0.1g, grind with liquid nitrogen, grind active addition 1.5ml centrifuge tube, add 1ml Trizol reagent rapidly (available from Invitrogen, USA), after fully shaking up vibration, extracted total RNA.
2) clone of OsNAR2.1 full length gene
Utilize the cDNA sequence of the NAR2.1 of barley and Arabidopis thaliana to retrieve the NAR2.1 gene series OsNAR2.1 (AP004023.2) of paddy rice in the gene database of NCBI website (http://www.ncbi.nlm.nih.gov/), it is positioned on the second karyomit(e).Design primer (as follows) angles out the full length sequence of OsNAR2.1 from the tissue cDNA storehouse.Sequence is seen SEQ ID NO.1.
P1:5’-CAATGGCGAGGCTAGCCGGCGTT-3’
P2:5’-CGATCTACTTGTCCTTCTTGCGCTTCT-3’
The total RNA that obtains with step 1) is a template, behind synthetic cDNA first chain of reverse transcription, (Prime Star HSDNA polymerase is available from Takara company) carries out pcr amplification with the high-fidelity enzyme, the PCR program is as follows: 94 ℃ of pre-sex change 2min, 94 ℃ of sex change 30s, 53 ℃ of renaturation 30s, 72 ℃ are extended 30s, after 30 circulations, 72 ℃ of 5min, 4 ℃ of constant temperature.Process adds A reaction 30min (adding the A reaction solution is Time Inc. available from the sky), the PCR product is 621bp.Agarose electrophoresis is separated, is cut glue and reclaims rear clone to pMD-18 carrier (available from Takara company), and the correct back of order-checking just obtains to have the full length sequence (SEQ ID NO.1) of the rice high affinity nitrate transport protein gene OsNAR2.1 of complete coding region.
3) OsNAR2.1 gene expression analysis
The RT-PCR expression analysis shows that this gene mainly expresses in root, do not express in blade, and this gene is subjected to 0.2mM nitric nitrogen induced strong in addition, the inhibition of the metabolite of be subjected to ammonium (Fig. 4,5) and nitrogen.Primer sequence is as follows
NAR2.1-F:5 '-TCCCGTTGGTGCTCGTCTTGC-3 ' is (in the site of cDNA sequence: 29bp)
NAR2.1-R:5 '-GACCTTGAACTGGCACGC-3 ' is (in the site of cDNA sequence: 348bp)
The total RNA that obtains with step 1) is a template, behind synthetic cDNA first chain of reverse transcription, carries out pcr amplification, and the PCR program is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 50 ℃ of renaturation 30s, 72 ℃ are extended 30s, after 30 circulations, 72 ℃ of 5min, 4 ℃ of constant temperature.Race glue detects, and the PCR product size of OsNAR2.1 is 320bp.
Two, frog's egg heterogenous expression system is determined the proteic function of OsNAR2.1
1) frog's egg heterogenous expression vector construction
From cloning vector pCMV-SPORT6 (by KOME website http://cdnaO1.dna.affrc.go.jp/cDNA/) with gene OsNAR2.1 subclone to frog's egg heterogenous expression carrier pT7Ts (Tong Y P, Zhou JJ, Li Z, Miller A is two-component highaffinity nitrate uptake system in barley.The Plant Journal 41 J.2005.A, 442-450.) upward (Fig. 1), 3 multiple clone site (BglII, Eco RV and SpeI) are arranged, the design primer:
VP1:5’-AATC
AGATCTCAATGGCGAGGCTAGCC-3’(SpeI)
VP2:5’-CAGA
ACTAGTCGATCTACTTGTCCTTC-3’(BglII)
94 ℃ of pre-sex change 4min of PCR process, 94 ℃ of sex change 30s, 55 ℃ of renaturation 30s, 72 ℃ are extended 30s, and after 30 circulations, 72 ℃ of 7min run glue and detect.The PCR product size of OsNAR2.1 is 691bp
After PCR product running gel purifying reclaimed, with BglII and the running gel purifying recovery once more of SpeI double digestion, electrophoresis detection was quantitative ,-20 ℃ of preservations.Utilize BglII and SpeI double digestion pT7Ts plasmid rear electrophoresis glue purification to reclaim, electrophoresis detection is quantitative, cuts to be mixed in to be connected under 4 ℃ after product reclaims with the PCR enzyme and spends the night.Heat shock is transformed into 37 ℃ of overnight incubation in the DH5 α intestinal bacteria, utilizes PCR and enzyme to cut screening positive clone, obtains containing the bacterium liquid of the subclone plasmid of gene OsNAR2.1.
2) cRNA's is external synthetic
Extract plasmid (Tong Y P, ZhouJJ, Li Z, Miller A is two-component high affinity nitrate uptakesystem in barley.The Plant Journal 41 J.2005.A, 442-450): get the bacterium liquid 25ml that contains gene OsNAR2.1 subclone plasmid, extract plasmid kit (QIAGEN according to middle amount, UK) extract the plasmid description operation, obtain concentration and be the gene OsNAR2.1 subclone plasmid of OD260=1.56 μ g/ μ l (require plasmid concentration must greater than 1 μ g/ μ l).
Linearization plasmid: with HindIII single endonuclease digestion gene OsNAR2.1 subclone plasmid, 50 μ l reaction systems: 6 μ g plasmid DNA, 2 μ l XbaI (promega), it is 50 μ l that 5 μ l damping fluids, sterilized water add to final volume.37 ℃ of water-baths 2 hours, the electrophoresis checking.Purifying linear plasmid DNA (Tong YP, Zhou JJ, Li Z, Miller AJ.2005.A two-component high affinity nitrateuptake system in barley.The Plant Journal 41,442-450).Obtaining concentration is OD2603.2 μ g/ μ l gene OsNAR2.1 subclone plasmid linear DNA.
CRNA synthesizes (Tong YP, Zhou JJ, Li Z, Miller AJ.2005.A two-component high affinity nitrate uptakesystem in barley.The Plant Journal 41,442-450), the test kit of vitro synthesized RNA is MEGAscript sp6kit, Ambion) obtains 2.5 μ g/ μ l gene OsNAR2.1 subclone plasmid cRNA.
3) the injection of the acquisition of frog's egg and cRNA (Tong YP, Zhou JJ, Li Z, Miller AJ.2005.A two-component highaffinity nitrate uptake system in barley.The Plant Journal 41,442-450)
The injection kapillary is to be that the diameter that Higenberg company produces is 1mm, and internal diameter is the long capillary tube 11cm of the no inner core of 0.8mm.Draw pin instrument (PE-21 type, Japan produces) to draw into about 5 to 6cm long microinjection pins; (CIB is an impellent for air pressure USA.) to the micro-injection instrument for PLI-100Pico-Injector, Harvard Apparatus.Each frog's egg injection 50ng cRNA.Cultivated 2 days for 18 ℃, making the OsNAR2.1 gene of injecting in the frog's egg can heterogenous expression be albumen.
4) detection of OsNAR2.1 protein function
N
15Absorption experiment (Tong YP, Zhou JJ, Li Z, Miller AJ.2005.A two-component high affinity nitrateuptake system in barley.The Plant Journal 41,442-450): the proteic frog's egg of single expression OsNAR2.1 is at 0.5mMNaN
15O
3After spending the night, measure the N that accumulates in the cell
15, the result shows that OsNAR2.1 albumen can not absorb nitrate.But co expression OsNAR2.1 albumen and the proteic frog's egg of OsNRT2.1 (AB008519) (or OsNRT2.2AK109733, OsNRT2.3aAK109776) at 0.5mMNaN
15O
3After spending the night, measure the N that accumulates in the cell
15, the result shows that OsNAR2.1 can significantly promote OsNRT2.1 (or OsNRT2.2) and OsNRT2.3a albumen that nitrate is absorbed (Fig. 2,3).
Three, the OsNAR2.1 gene knocks out
1) structure of RNAi expression vector
1, according to the rice high affinity nitrate transport protein gene OsNAR2.1cDNA sequence that obtains in the step 2, see the complete encoding sequence of SEQ ID NO.1, with cDNA sequence and the whole genome sequence alignment of itself and OsNAR2.2 (AK109571), choose the one section relative OsNAR2 of 350bp family distinguished sequence.And on the upstream and downstream primer, introduce restriction endonuclease sites respectively, design primer such as following table:
| The primer title | Primer sequence (5 '-3 ') | Restriction enzyme site |
| A1F-1F | AATAGGATCCCGTTGGTGCTCGTCTTGC | BamHI |
| A1F-1R | TTTTGGTACCGACCTTGAACTGGCACGC | KpnI |
| A1R-1F | TATTGAGCTCCGTTGGTGCTCGTCTTGC | SacI |
| A1R-1R | CCTGACTAGTGACCTTGAACTGGCACGC | SpeI |
Cloning with the cDNA that obtains is template, carries out PCR, the PCR program with A1R-1F and this a pair of primer of A1R-1R earlier: 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 30s, 58 ℃ of renaturation 30s, 72 ℃ are extended 20s, and after 30 circulations, 72 ℃ of 7min run glue and detect.The size of PCR product is 350bp, the PCR product is cut glue after agarose electrophoresis is separated reclaim, reclaim the back and carry out double digestion with SacI and SpeI, use SacI and SpeI double digestion pTCK303 plasmid (Wang Z simultaneously, Chen CHB, Xu YY, Jiang RX, Han Y, Xu ZHH, Chong K.A practical vector for efficient knockdown of gene expression in rice.PlantMolecular Biology Reporter.200422:409-417), reclaim fragment and carrier then respectively, the recovery back connects under 4 degree by T4 ligase enzyme (promega company) spends the night, and is converted into 37 ℃ of overnight incubation in the DH5 α intestinal bacteria, select positive colony, order-checking; Carry out PCR, the PCR program with A1F-1F and this a pair of primer of A1F-1R again: 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 30s, 56 ℃ of renaturation 30s, 72 ℃ are extended 20s, and after 30 circulations, 72 ℃ of 7min run glue and detect.The size of PCR product is 350bp, the PCR product is cut glue after agarose electrophoresis is separated reclaim, reclaim the back and carry out double digestion with BamHI and KpnI, use the positive colony of the BamHI and the KpnI double digestion the first step simultaneously, the recovery back connects under 4 degree by T4 ligase enzyme (Promega company) spends the night, heat shock is converted into 37 ℃ of overnight incubation in the DH5 α intestinal bacteria, selects positive colony, order-checking.One of OsNAR2.1 section distinguished sequence is cloned into binary expression vector pTCK303 (Wang Z by two pairs of corresponding restriction enzyme sites respectively like this, Chen CHB, Xu YY, JiangRX, Han Y, Xu ZHH, Chong K.A practical vector for efficient knockdown of gene expression in rice.Plant Molecular Biology Reporter.200422:409-417), after order-checking is correct, be converted into EHA105 ((Xu M, ZhuL, Shou H, Wu P.A PIN1family gene, OsPIN1, involved in auxin-dependent adventitious rootemergence and tillering in rice.Plant Cell Physiol.2005 Oct; 46 (10): 1674-81.) in the Agrobacterium.
4) acquisition of RNAi transfer-gen plant
The Agrobacterium that the commentaries on classics that obtains is had expression vector, further be converted into paddy rice, the OsNAR2.1-RNAi transfer-gen plant that obtains is carried out PCR to be detected, analyze the OsNAR2.1 genetic expression of OsNAR2.1-RNAi rice seedling and wild-type rice seedling root with RT-PCR, found that: 1 OsNAR2.1-RNAi transgenic line is no longer expressed the OsNAR2.1 gene, the expression amount of another strain system is compared obvious decline with wild-type, this illustrates that we disturb (RNAi) technology to obtain the mutant of two OsNAR2.1 gene knockouts by RNA.We also find to knock out the also obviously decline (Fig. 6) of OsNRT2.1 gene (AB008519) in the mutant, OsNRT2.2 gene (AK109733), OsNRT2.3a gene (AK109776) expression amount at these two OsNAR2.1 simultaneously.
Use N
15Mark saltpetre comes the absorption of analyzing rice to nitric nitrogen, finds that OsNAR2.1 knocks out the mutant material and significantly reduced absorption and accumulation to nitrate.Occurred tangible nitrogen stress symptom, root/shoot ratio increase simultaneously, the setting percentage comparison descends 15% according to wild-type.
Four, overexpression OsNAR2.1 gene
1) structure of overexpression vector
According to the cDNA sequence of rice high affinity nitrate transport protein gene OsNAR2.1, see the complete encoding sequence of SEQ ID NO.1, on the upstream and downstream primer, introduce restriction endonuclease sites BamHI respectively, the design primer is:
overNAR2.1-F：5’-TTAA
GGATCCCAATGGCGAGGCTAGCCGGCGTT-3’(BamHI)
overNAR2.1-R：5’-CCC
GGATCCCGATCTACTTGTCCTTCTTGCGCT-3’
Cloning with the cDNA that obtains in the above-mentioned step 2 is template, and the PCR program is as follows: 94 ℃ of pre-sex change 2min, and 94 ℃ of sex change 30s, 53 ℃ of renaturation 30s, 72 ℃ are extended 30s, and after 30 circulations, 72 ℃ of 5min, PCR product are 640bp.Behind pcr amplification, the complete encoding sequence of OsNAR2.1 is cloned into pMD-18 carrier (available from Takara company), the correct back of order-checking imports binary expression vector p1390 (Chen TL by corresponding restriction enzyme site, Lin Y L, Lee YL, Yang NS Chan MT 2004 Expression ofbioactive human interferon-gamma in transgenic rice cell suspension cultures Transgenic Research 13:499-510), be converted into EHA 105 (Xu M then, Zhu L, Shou H, Wu P.A PIN1 family gene, OsPIN1, involved inauxin-dependent adventitious root emergence and tillering in rice.Plant Cell Physiol.2005 Oct, 46 (10): 1674-81.) in the Agrobacterium.
2) acquisition of overexpression transfer-gen plant
The commentaries on classics that obtains has the Agrobacterium of expression vector, further be converted into paddy rice, the transfer-gen plant that obtains is carried out PCR detect, with the OsNAR2.1 expression of RT-PCR analyzing rice seedling root, the result shows: OsNAR2.1 induces 1 hour with regard to strong expression at the 0.2mM nitric nitrogen.
Use N after the RT-PCR checking
15Mark saltpetre comes the absorption of analyzing rice to nitric nitrogen, and the overexpression material of finding this gene is obviously compared according to wild-type has increased absorption and accumulation to nitrate.The flooded soils growth can be compared according to wild-type and improve about 15% (Fig. 7) of output (setting percentage).
In sum, the inventor finds that OsNAR2.1 is a very important rice high affinity nitrate transport protein gene, itself does not have the activity of transport of nitrate this gene, but it can promote the expression of OsNRT2.1, OsNRT2.2 and OsNRT2.3a and the activity that has improved them greatly; Can utilize OsNAR2.1 gene of the present invention to make up plant expression vector as goal gene, wherein available any promotor is cauliflower mosaic virus (CAMV) 35S promoter, Ubiquitin promotor or other promotor for example, can comprise enhanser in case of necessity in this expression vector, no matter be transcriptional enhancer or translational enhancer.Can use selected marker for the evaluation of simplifying transformant and comprise enzyme antibiotics resistance, also can utilize the enzyme of the compound that colour-change (for example B-glucuronidase GUS) or luminous (for example luciferase) discern, also available unmarked selection.Used expression vector can use Ti-plasmids, Ri plasmid, plant viral vector etc.Method for transformation can be used through agrobacterium-mediated transformation, particle bombardment, pollen tube passage method or other method and transform plant.This gene of overexpression can improve paddy rice paddy rice absorbing and output nitrogen under the flooded soils condition.
Sequence table
＜110〉Agricultural University Of Nanjing
＜120〉rice high affinity nitrate transport protein gene OsNAR2.1
＜130〉specification sheets
<140>?00
<141>?2008-11-07
<160>?16
<170>?PatentIn?version?3.1
<210>?1
<211>?621
<212>?DNA
＜213〉Oryza sativa (paddy rice)
<220>
＜221〉rice high affinity nitrate transport protein gene OsNAR2.1
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<211>?206
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＜221〉rice high affinity nitrate transport protein OsNAR2.1 aminoacid sequence
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<210>?7
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Claims (5)
1, rice high affinity nitrate transport protein gene OsNAR2.1, its nucleotides sequence is classified SEQ ID NO.1 as.
2, the rice high affinity nitrate transport protein OsNAR2.1 of the described genetic expression of claim 1, its aminoacid sequence is SEQ IDNO.2.
3, the genetically engineered of the described gene OsNAR2.1 of claim 1 is used.
4, the application of the described rice high affinity nitrate transport protein of claim 2 OsNAR2.1.
5, use according to the genetically engineered of the described gene OsNAR2.1 of claim 4, it is characterized in that, the application of this gene aspect raising nitrogenous fertilizer utilising efficiency and output.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103305547A (en) * | 2013-06-08 | 2013-09-18 | 中国科学院遗传与发育生物学研究所 | Application of rice protein CHR1 in adjusting content of plant nitrate |
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| CN108315346A (en) * | 2018-03-02 | 2018-07-24 | 南京农业大学 | Recombinant expression carrier and its application of a kind of OsNAR2.1 containing paddy gene and its promoter |
| CN108341858A (en) * | 2018-03-02 | 2018-07-31 | 南京农业大学 | Applications of the paddy gene OsNAR2.1 in terms of drought resisting |
| CN111748560A (en) * | 2017-12-28 | 2020-10-09 | 南京农业大学 | Application of rice OsNRT2.1 gene in improving manganese content in rice grains |
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2008
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| CN115029356A (en) * | 2021-08-25 | 2022-09-09 | 南京农业大学 | Genetic engineering application of rice nitrate-induced protein gene OsNOI4 |
| CN117904186A (en) * | 2024-01-04 | 2024-04-19 | 南京农业大学 | Application of rice calcium-hydrogen antiport protein gene OsCAX a in improving rice ammonium absorption |
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