CN109912702A

CN109912702A - Application of the protein OsARE1 in regulation plant low nitrogen resisting

Info

Publication number: CN109912702A
Application number: CN201711329654.9A
Authority: CN
Inventors: 左建儒; 王青; 粘金沯; 李家洋; 钱前; 谢先芝; 陈凡
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2017-12-13
Filing date: 2017-12-13
Publication date: 2019-06-21
Anticipated expiration: 2037-12-13
Also published as: CN109912702B

Abstract

The invention discloses application of the protein OsARE1 in regulation plant low nitrogen resisting.The protein OsARE1 is that amino acid sequence is protein shown in sequence 1 in sequence table.It is demonstrated experimentally that then the low nitrogen resisting of rice increases by the encoding gene silencing or knockout of protein OsARE1 in rice.Protein OsARE1 and its encoding gene are an effectively hereditary target spots for improveing Genes For Plant Tolerance Low nitrogen stress ability and correlated traits, have market potential value and wide application prospect in agriculture field.The present invention has important application value.

Description

Application of the protein OsARE1 in regulation plant low nitrogen resisting

Technical field

The present invention relates to field of biotechnology, and in particular to protein OsARE1 answering in regulation plant low nitrogen resisting With.

Background technique

The sustainable development of modern agricultural production and the improvement of crop nutrition content utilization efficiency are closely related.Nitrogen is as plant One of macronutrient necessary to growth and development, the influence to crop yield are most important.Plant nitrogen utilization efficiency One of an important factor for being regulated and controled by gene and environmental factor and its interaction, being influence sustainable development of agricultural production, to it Further investigation has important theory significance and application value.

At past more than 50 years, due to the innovation of germ plasm resource, the use of the optimization of cultivation management and commodity chemical fertilizer, make Produce have measured constantly improve.But with population continue increase and consumption demand continuous improvement, especially excessively according to Rely using chemical fertilizer and bring volume increase, so that the yield performance of high-yield crop kind and its stability will face more sternly in future High challenge.Nitrogen is one of macronutrient necessary to plant growth and development, and influences the key of crop yield One of factor.After the industrial revolution, as the large area of nitrogenous fertilizer uses, crop yield increases sharply in worldwide, but The problems such as agriculture production cost increases, soil nutrient is unbalance and ecological environmental pollution is caused simultaneously.Therefore, by including International Rice Including 15 research centers composition International Agriculture research consultation tissue propose with by improvement nutrientuse efficiency mention High crop yield is the Second Green Revolution of target.

Improving crop nitrogen utilization efficiency is to realize one of the effective way of agricultural sustainable development.Plant nitrogen utilizes Efficiency mainly includes that Nitrogen absorption efficiency, nitrogen assimilation efficiency and nitrogen recycle efficiency etc., by a variety of genes and environment The regulation of factor and its interaction.Wherein, Nitrogen absorption efficiency and nitrogen assimilation efficiency are to influence crop nitrogen utilization efficiency Two key factors.The initial source of nitrogen in plant, animals and humans body, almost all from soil.Plant is from soil Middle acquisition nitrogen mainly includes three processes, i.e. Nitrogen Absorption and transhipment, nitrogen assimilation and nitrogen recycles.

It is generally believed that principal mode inorganic nitrogen-sourced in rich oxygen type soil is nitrate ion (NO₃ ^-), and paddy field, wetland Or the principal mode of inorganic nitrogen is ammonium ion (NH in acid soil₄ ⁺).Therefore, high pH and oxidation state soil are grown in (such as drought The oxygen-enriched environments such as field) in favorite plant absorb NO₃ ^-, and it is grown in low pH and reduction-state soil (such as marsh or paddy field anoxic Environment) in favorite plant inhale NH₄ ⁺Or amino acid.It is inorganic nitrogen-sourced in addition to above-mentioned heterogeneous feature around root system of plant, Also present fluctuation feature (nitrogen concentration around root system is usually 100 μM to 10mM), plant therefore develop out compatibility difference Nitrogen movement system, i.e. high-affinity movement system and low compatibility movement system.Plant absorption inorganic nitrogen in soil mainly leads to Ammonium root transport protein and nitrate anion the transport protein collaboration in root on cell membrane is crossed to complete.The master in rice absorbing Inorganic Nitrogen Transport source Wanting form is NH₄ ⁺.In addition, rice root aerating tissue can promote root surface NH to oxygen is discharged around root system₄ ⁺Nitre occurs Change acts on and generates NO₃ ^-.Therefore, NO₃ ^-It is also a kind of inorganic nitrogen-sourced important form of rice absorbing.

After the industrial revolution, with the large-scale production and use of commodity chemical fertilizer, world food yield significantly increases.According to estimating Meter, 40 years futures, agrochemical amount of application need to increase nearly 3 times of population for being just able to satisfy sustainable growth and consumption demand.Therefore, Improving crop nitrogen utilization efficiency (nitrogen use efficiency, NUE) is that reduction agriculture production cost and solution disappear Take one of the effective way of demand.Plant NUE is regulated and controled by a variety of genes and environmental factor and its interaction.The definition of NUE is logical Often refer to the plant total biomass or total output that unit amount of application of nitrogen fertilizer obtains, mainly includes Nitrogen absorption efficiency (NUpE), nitrogen Assimilatory efficiency (NUtE/NAE), nitrogen transfer efficiency (NTE) and nitrogen recycle efficiency (NRE) etc..Usually, plant NUE Mainly determined by NUpE and NAE.Due to the nitrogenous fertilizer that is applied in soil by soil microbial activity, soil types and soil and The factors such as vegetative loss gaseous nitrogen influence, and are difficult to accurately calculate the nitrogenous fertilizer of nitrogenous fertilizer total amount or plant actual absorption obtained by plant Total amount.

Model plant of the rice as monocotyledon fundamental research is also the important cereal crops of human society, It provides staple food for the nearly half population in the whole world.Nitrogen is one of macronutrient necessary to plant growth and development, is Influence one of crop yield and the principal element of ball ecological environment.Plant nitrogen utilization efficiency is a kind of complicated agronomy Shape is regulated and controled by different genes and environmental factor and its interaction, is furtherd investigate to it with important theory significance and application Value.The control gene of plant identification nitrogen utilization efficiency is separated, it can be resistance to low to improve Genes For Plant Tolerance Low nitrogen stress ability and cultivation N stress New Crop Varieties provide effective molecular strategy.

Summary of the invention

The technical problem to be solved by the present invention is to how regulate and control the low nitrogen resisting of plant.

In order to solve the above technical problems, can be following A1 present invention firstly provides the application of protein OsARE1) extremely At least one of A27):

A1) regulate and control plant low nitrogen resisting；A2) regulate and control the nitrogen content of plant organ；A3) regulate and control the nitrogen content of axis sheath； A4) regulate and control the nitrogen content of plant leaf blade；A5) regulate and control the nitrogen content of plant fringe；A6) regulate and control the biomass of plant；A7) regulate and control plant The biomass of root；A8) regulate and control the root/shoot ratio of plant；A9) regulate and control the chlorophyll content of plant；A10) regulate and control the plant height of plant； A11) regulate and control the tiller number of plant；A12) regulate and control the Nitrogen absorption efficiency of plant；A13) regulate and control the nitrogen utilization efficiency of plant； A14) regulate and control the expression quantity of Nitrogen Absorption marker gene in plant；A15) regulate and control the expression of nitrogen assimilation marker gene in plant Amount；A16) regulate and control the yield of plant；A17) regulate and control the single plant yield of plant；A18) regulate and control the grain number per spike of plant；A19) regulation is planted The spike length of object；A20 the Their First Branch for) regulating and controlling plant obstructs number；A21 the second level branch for) regulating and controlling plant obstructs number；A22) regulate and control in plant AMT1；The expression quantity of 1 gene；A23) regulate and control AMT1 in plant；The expression quantity of 2 genes；A24) regulate and control AMT1 in plant；3 genes Expression quantity；A25) regulate and control GS1 in plant；The expression quantity of 2 genes；A26) regulate and control the expression of NADH-GOGAT1 gene in plant Amount；A27) absorption of the regulation plant under low nitrogen growth conditions to nitrogen.

The protein OsARE1 derives from grass family Oryza japonica rice subspecies (Oryza sativa L.), full name ABC1 REPRESSOR1.The protein OsARE1 can be a1) or a2) or a3) or a4):

A1) amino acid sequence is protein shown in sequence 1 in sequence table；

A2) the fused protein that the N-terminal of protein shown in sequence 1 or/and C-terminal connection label obtain in sequence table；

A3) by amino acid sequence shown in sequence 1 in sequence table by one or several amino acid residues substitution and/or The obtained protein with identical biological function is deleted and/or added；

A4) amino acid sequence limited with sequence 1 in sequence table has 80% or 80% or more identity, derives from rice And the protein with identical biological function.

Wherein, sequence 1 is made of 427 amino acid residues in sequence table.

In order to make a1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 1 or Carboxyl terminal connects upper label as shown in Table 1.

The sequence of 1. label of table

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned a3) in protein, the substitution and/or deletion and/or addition of one or several amino acid residues be No more than the substitution and/or deletion and/or addition of 10 amino acid residues.

Above-mentioned a3) in protein, can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain It arrives.

Above-mentioned a3) in protein encoding gene can by by sequence 2 in sequence table from 5 ' end 467-1750 Shown in the codon of one or several amino acid residues is lacked in DNA sequence dna, and/or carry out one or several base-pairs Missense mutation, and/or obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.

The application of the nucleic acid molecules of code for said proteins OsARE1 also belongs to protection scope of the present invention；Encode the egg The application of the nucleic acid molecules of white matter OsARE1 can be following A1) at least one of to A27):

In above-mentioned application, the nucleic acid molecules of the code for said proteins OsARE1 can be following b1) or b2) or b3) or B4 DNA molecular shown in) or b5):

B1) nucleotide sequence is DNA molecular shown in sequence 2 in sequence table；

B2) nucleotide sequence is DNA molecular shown in sequence 3 in sequence table；

B3) code area DNA molecular as shown in sequence 2 467-1750 from 5 ' ends in sequence table；

B4) and b1) or b2) or the nucleotide sequence that b3) limits there is 90% or 90% or more identity, derive from rice And the DNA molecular of code for said proteins OsARE1；

B5) under strict conditions with b1) or b2) or b3) nucleotide sequence hybridization that limits, and code for said proteins The DNA molecular of OsARE1.

Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also To be RNA, such as mRNA or hnRNA.The nucleic acid molecules can be the gene and its regulation sequence of code for said proteins OsARE1 Arrange the nucleic acid molecules formed.

Sequence 2 is made of 2640 nucleotide in sequence table, and sequence 2 is from shown in 5 ' end 467-1750 in sequence table Nucleotide sequence coded sequence table in amino acid sequence shown in sequence 1.

Nucleotide sequence shown in sequence 3 is the genomic DNA of cDNA (in sequence table shown in sequence 2) in sequence table.Sequence Sequence 3 is by 7229 base compositions in list, and in sequence table in sequence 3, the 1st to 1810 is promoter region from 5 ' ends, 1811st to 2276 is the 5 ' areas UTR, and the 6340th to 7229 is the 3 ' areas UTR, and the 2277th to 2883 is first exon, 2884th to 3476 is First Intron, and the 3477th to 3564 is second exon, and the 3565th to 3800 is the Two intrones, the 3801st to 3972 be third exon, the 3973rd to 4116 be third introne, the 4117th to 4232 are the 4th exon, and the 4233rd to 4722 is the 4th introne, and the 4723rd to 4820 aobvious outside for the 5th Son, the 4821st to 5692 is the 5th introne, and the 5693rd to 5828 is the 6th exon, and the 5829th to 6272 is 6th introne, the 6273rd to 6339 is the 7th exon.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding protein OsARE1 of the invention.Those by manually modified, have and this The nucleotide sequence 90% of the protein OsARE1 of invention or the nucleotide of higher identity, as long as coding protein OsARE1 And rice is derived from, it is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.Art used herein Language " identity " refers to the sequence similarity with native sequence nucleic acid." identity " includes and coding protein OsARE1 of the invention Nucleotide sequence have 90% or higher or 95% or higher identity nucleotide sequence.

The nucleic acid molecules of the code for said proteins OsARE1 may be either the cDNA sequence of OsARE1 gene, can also be The genomic gene sequence of OsARE1 gene；There is 90% or more identity and coding protein OsARE1 with OsARE1 gene DNA sequence dna, be that the cDNA of OsARE1 gene is separated and/or modified and/or designed and obtain by known method.This Field it is to be understood by the skilled artisans that the minor alteration of specific gene sequence nucleotide identity may result in the gene effect Can reduction or reinforcement, and in some applications (for example, antisense or co-suppression technology), partial sequence is often and overall length Sequence is equally valid to play a role.Method gene order variation or shortened, and test the gene that these morph The method of validity is well known to those skilled in the art.

The present invention also provides cultivation genetically modified plants method.

The method provided by the present invention for cultivating genetically modified plants, concretely method one, it may include to plant first of setting out It is middle to import the content for improving the protein OsARE1 and/or active substance, the step of obtaining genetically modified plants first；Described turn Gene plant first compared with the plant first of setting out plant low nitrogen resisting reduce and/or plant organ nitrogen content reduce and/or The nitrogen content of axis sheath reduces and/or the nitrogen content of plant leaf blade reduces and/or the nitrogen content of plant fringe reduces and/or plant Biomass reduce and/or the biomass of plant roots reduces and/or the root/shoot ratio of plant reduces and/or the chlorophyll of plant contains Amount reduce and/or plant plant height reduce and/or plant tiller number reduce and/or plant Nitrogen absorption efficiency reduce and/ Or plant nitrogen utilization efficiency reduce and/or plant in Nitrogen Absorption marker gene expression quantity reduce and/or plant in nitrogen The expression quantity of element assimilation marker gene reduces and/or the yield of plant reduces and/or the single plant yield of plant reduces and/or plant Grain number per spike reduce and/or plant spike length reduce and/or plant Their First Branch stalk number reduce and/or plant second level branch obstruct number AMT1 in reduction and/or plant；AMT1 in the expression quantity reduction of 1 gene and/or plant；The expression quantity of 2 genes reduce and/or AMT1 in plant；GS1 in the expression quantity reduction of 3 genes and/or plant；NADH- in the expression quantity reduction of 2 genes and/or plant The expression quantity of GOGAT1 gene reduces and/or plant reduces the absorption of nitrogen under low nitrogen growth conditions.

In the above method one, " content and/or activity that improve the protein OsARE1 " can by multicopy, change Become the methods well known in the art such as promoter, regulatory factor, transgenosis, reaches protein described in expression or overexpression or mention The active effect of the high protein.

It is described " content and/or work for improving the protein OsARE1 to be imported into plant first of setting out in the above method one Property substance " concretely into plant first of setting out import code for said proteins OsARE1 nucleic acid molecules.

In the above method one, described " nucleic acid molecules of code for said proteins OsARE1 are imported into plant first of setting out " is logical Cross plant first of setting out described in recombinant expression carrier importing；The recombinant expression carrier OsARE1 base that concretely embodiment refers to Because of Overexpression vector or OsARE1 complementing vector.

The method provided by the present invention for cultivating genetically modified plants, concretely method two, it may include to plant second of setting out It is middle to import the content for inhibiting the protein OsARE1 and/or active substance, the step of obtaining genetically modified plants second；Described turn Gene plant second compared with the plant second of setting out plant low nitrogen resisting increase and/or plant organ nitrogen content increase and/or The nitrogen content of axis sheath increases and/or the nitrogen content of plant leaf blade increases and/or the nitrogen content of plant fringe increases and/or plant Biomass increase and/or the biomass of plant roots increases and/or the root/shoot ratio of plant increases and/or the chlorophyll of plant contains Amount increase and/or plant plant height increase and/or plant tiller number increase and/or plant Nitrogen absorption efficiency increase and/ Or plant nitrogen utilization efficiency increase and/or plant in Nitrogen Absorption marker gene expression quantity increase and/or plant in nitrogen The expression quantity of element assimilation marker gene increases and/or the yield of plant increases and/or the single plant yield of plant increases and/or plant Grain number per spike increase and/or plant spike length increase and/or plant Their First Branch stalk number increase and/or plant second level branch obstruct number AMT1 in increase and/or plant；AMT1 in the expression quantity increase of 1 gene and/or plant；The expression quantity of 2 genes increases and/or plants AMT1 in object；GS1 in the expression quantity increase of 3 genes and/or plant；NADH- in the expression quantity increase of 2 genes and/or plant The expression quantity of GOGAT1 gene increases and/or plant increases the absorption of nitrogen under low nitrogen growth conditions.

In the above method two, " content and/or activity that inhibit the protein OsARE1 " can by RNA interfere, The methods well known in the art such as homologous recombination, gene site-directed editor, reach the expression quantity for inhibiting the protein and/or active Purpose.

In the above method two, it is described " inhibit the protein OsARE1 content and/or active substance " can be special RNA molecule；Shown in the special RNA molecule such as formula (I): A_Reversely-Y-A_{It is positive}(I)；The A_{It is positive}Sequence be encoding said proteins The single strand RNA molecule that 200-500bp DNA fragmentation in the gene of matter OsARE1 is transcribed；The A_ReverselySequence with it is described A_{It is positive}Sequence reverse complemental；The Y is the A_{It is positive}With the A_ReverselyBetween intervening sequence, in sequence, the Y with it is described A_{It is positive}And the A_ReverselyIt is not complementary.

In the above method two, the implementation method of described " importing special RNA molecule in plant second of setting out " can be as follows: will be special Different DNA molecular first imports plant second of setting out；Shown in the specific DNA molecular first such as formula (II): SEQ_Reversely-X-SEQ_{It is positive}(II)；Institute State SEQ_{It is positive}Sequence be code for said proteins OsARE1 gene in 200-500bp DNA fragmentation；The SEQ_ReverselySequence Column and the SEQ_{It is positive}Sequence reverse complemental；The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence On, the X and the SEQ_{It is positive}And the SEQ_ReverselyIt is not complementary.

In the above method two, the specific DNA molecular first passes through plant second of setting out described in recombinant expression carrier importing；It is described Recombinant expression carrier is the SacI that DNA molecular shown in sequence 5 the 14th to 1138 in sequence table is inserted into pTCK303 carrier The recombinant expression carrier obtained between BamHI recognition site.

In the above method two, it is described " inhibit the protein OsARE1 content and/or active substance " can be plant The carrier of genome editor；The carrier of the Plant Genome editor contains gRNA encoding gene；The gRNA knows in plant Other target DNA is the DNA fragmentation of coding protein OsARE1.

In the above method two, the gRNA encoding gene be in sequence table sequence 2 from 5 ' ends 552-570 and/ Or DNA molecular shown in 1094-1112.

The method provided by the present invention for cultivating genetically modified plants, concretely method three, including the above method will be passed through One or the genetically modified plants that obtain of the above method two and plant hybridization to be improved, the step of obtaining progeny transgenic plant；It is described Progeny transgenic plant and the genetically modified plants (i.e. as the genetically modified plants of parent) phenotype are almost the same.

The present invention also protects special RNA molecule, specific DNA molecular, special recombinant plasmid or the load of Plant Genome editor Body.

Shown in the special RNA molecule such as formula (I): A_Reversely-Y-A_{It is positive}(I)；The A_{It is positive}Sequence be code for said proteins The single strand RNA molecule that 200-500bp DNA fragmentation in the gene of OsARE1 is transcribed；The A_ReverselySequence and the A_{It is positive} Sequence reverse complemental；The Y is the A_{It is positive}With the A_ReverselyBetween intervening sequence, in sequence, the Y and the A_{It is positive} And the A_ReverselyIt is not complementary.

The specific DNA molecular can be specific DNA molecular first or specific DNA molecular second.

Shown in the specific DNA molecular first such as formula (II): SEQ_Reversely-X-SEQ_{It is positive}(II)；The SEQ_{It is positive}Sequence be coding 200-500bp DNA fragmentation in the gene of the protein OsARE1；The SEQ_ReverselySequence and the SEQ_{It is positive}Sequence Reverse complemental；The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive} And the SEQ_ReverselyIt is not complementary.

The specific DNA molecular second may include DNA fragmentation one, intervening sequence and DNA fragmentation two；The DNA fragmentation one Sequence is the 200-500bp DNA fragmentation in the gene of code for said proteins OsARE1；The DNA fragmentation two and DNA The sequence reverse complemental of segment one.

The special recombinant plasmid can be the recombinant plasmid containing the specific DNA molecular.

The carrier of the Plant Genome editor can contain gRNA encoding gene；The target that the gRNA is identified in plant DNA is the DNA fragmentation of coding protein OsARE1.

Any of the above-described A_{It is positive}Sequence can for code for said proteins OsARE1 gene in the 5 ' areas UTR or the 3 ' areas UTR The single strand RNA molecule transcribed of 200-500bp DNA fragmentation.

Any of the above-described SEQ_{It is positive}Sequence or the sequence of any of the above-described DNA fragmentation one can be encoding said proteins The 200-500bp DNA fragmentation in the 5 ' areas UTR or the 3 ' areas UTR in the gene of matter OsARE1.

The nucleotide sequence in the area the 5 ' UTR can be as shown in sequence 2 the 1st to 466 from 5 ' ends in sequence table.It is described The nucleotide sequence in the 3 ' areas UTR can be as shown in sequence 2 the 1751st to 2640 from 5 ' ends in sequence table.

In any of the above-described special RNA molecule, the A_{It is positive}Sequence concretely in sequence table sequence 4 from 5 ' ends It rises shown in the 840th to 1138.Wherein, sequence 2 from the nucleotide sequence of 5 ' end the 567th to 865 transcribes out sequence in sequence table Sequence 4 is from the nucleotide of 5 ' end the 840th to 1138 in list.

Any of the above-described SEQ_{It is positive}Sequence or any of the above-described DNA fragmentation one sequence concretely in sequence table Sequence 5 is from 5 ' ends shown in the 840th to 1138.Wherein, in sequence table sequence 2 from the nucleosides of 5 ' end the 567th to 865 Acid sequence be in sequence table sequence 5 from the nucleotide of 5 ' end the 840th to 1138.

Any of the above-described SEQ_ReverselySequence or any of the above-described DNA fragmentation two sequence concretely in sequence table Sequence 5 is from 5 ' ends shown in the 14th to 312.

The nucleotide sequence of any of the above-described intervening sequence can as the sequence 5 in sequence table from 5 ' ends the 313rd to Shown in 839.

The nucleotide sequence of any of the above-described specific DNA molecular first or any of the above-described specific DNA molecular second can be Following s1) or s2) or s3) or s4) shown in DNA molecular:

S1) nucleotide sequence is sequence 5 in the sequence table DNA molecular shown in the 14th to 1138 from 5 ' ends；

S2) nucleotide sequence is DNA molecular shown in sequence 5 in sequence table；

S3) and s1) or s2) nucleotide sequence that limits has 70% or 70% or more identity, derive from rice while DNA molecular with identical biological function；

S4) the nucleotide sequence hybridization limited under strict conditions with s1) or s2) derives from rice while having identical The DNA molecular of biological function.

In the carrier of any of the above-described Plant Genome editor, the gRNA encoding gene can be sequence 2 in sequence table The DNA molecular shown in 552-570 and/or 1094-1112 from 5 ' ends.

The carrier of any of the above-described Plant Genome editor concretely OsARE1 gene knockout carrier.The OsARE1 Gene knockout carrier be by sequence 2 in sequence table from 5 ' ends shown in the 552nd to 570 and/or the 1094th to 1112 The recombinant expression carrier that DNA molecule insertion pYLCRISPR/Cas9-MH carrier B saI recognition site obtains.OsARE1 clpp gene Except in carrier specific recognition sequence table sequence 3 from 5 ' ends shown in the 2362nd to 2380 and/or the 3497th to 3515 DNA molecular.

Any of the above-described special RNA molecule, any of the above-described specific DNA molecular, any of the above-described special recombinant plasmid, Or the carrier of any of the above-described Plant Genome editor cultivate low nitrogen resisting increase and/or organ nitrogen content increase and/ Or stem sheath nitrogen content increase and/or blade nitrogen content increase and/or fringe nitrogen content increase and/or biomass increase and/ Or the biomass of root increases and/or root/shoot ratio increases and/or chlorophyll content increases and/or plant height increases and/or tiller number increases Add and/or Nitrogen absorption efficiency increases and/or nitrogen utilization efficiency increases and/or the expression quantity of Nitrogen Absorption marker gene increases And/or the expression quantity of nitrogen assimilation marker gene increases and/or yield increases and/or single plant yield increases and/or grain number per spike increases Add and/or spike length increases and/or Their First Branch stalk number increases and/or second level branch stalk number increases and/or AMT1；The expression quantity of 1 gene Increase and/or AMT1；The expression quantity of 2 genes increases and/or AMT1；The expression quantity of 3 genes increases and/or GS1；The table of 2 genes Up to amount increase and/or NADH-GOGAT1 gene expression quantity increase and/or under low nitrogen growth conditions it is increased to the absorption of nitrogen Application in genetically modified plants also belongs to protection scope of the present invention.

The present invention also protects plant breeding method one or plant breeding method two:

The plant breeding method one, it may include following steps: increase plant described in protein OsARE1 content and/ Or activity, so that plant low nitrogen resisting reduces and/or the nitrogen content of plant organ reduces and/or the nitrogen content of axis sheath reduces And/or the nitrogen content of plant leaf blade reduces and/or the nitrogen content of plant fringe reduces and/or the biomass of plant is reduced and/or planted The biomass of object root reduces and/or the root/shoot ratio of plant reduces and/or the chlorophyll content of plant reduces and/or the plant height of plant It reduces and/or the tiller number of plant reduces and/or the Nitrogen absorption efficiency of plant reduces and/or the nitrogen utilization efficiency of plant drop In low and/or plant Nitrogen Absorption marker gene expression quantity reduce and/or plant in nitrogen assimilation marker gene expression quantity It reduces and/or the yield of plant reduces and/or the single plant yield of plant reduces and/or the grain number per spike of plant reduces and/or plant Spike length reduce and/or plant Their First Branch stalk number reduce and/or plant second level branch stalk number reduce and/or plant in AMT1；1 AMT1 in the expression quantity reduction of gene and/or plant；AMT1 in the expression quantity reduction of 2 genes and/or plant；The expression of 3 genes GS1 in amount reduction and/or plant；The expression quantity of NADH-GOGAT1 gene reduces in the expression quantity reduction of 2 genes and/or plant And/or plant reduces the absorption of nitrogen under low nitrogen growth conditions.

The plant breeding method two, it may include following steps: reduce plant described in protein OsARE1 content and/ Or activity, so that plant low nitrogen resisting increases and/or the nitrogen content of plant organ increases and/or the nitrogen content of axis sheath increases And/or the nitrogen content of plant leaf blade increases and/or the nitrogen content of plant fringe increases and/or the biomass of plant increases and/or plants The biomass of object root increases and/or the root/shoot ratio of plant increases and/or the chlorophyll content of plant increases and/or the plant height of plant Increase and/or the tiller number of plant increases and/or the Nitrogen absorption efficiency of plant increases and/or the nitrogen utilization efficiency of plant increases Add and/or plant in Nitrogen Absorption marker gene expression quantity increase and/or plant in nitrogen assimilation marker gene expression quantity Increase and/or the yield of plant increases and/or the single plant yield of plant increases and/or the grain number per spike of plant increases and/or plant Spike length increase and/or plant Their First Branch stalk number increase and/or plant second level branch stalk number increase and/or plant in AMT1；1 AMT1 in the expression quantity increase of gene and/or plant；AMT1 in the expression quantity increase of 2 genes and/or plant；The expression of 3 genes GS1 in amount increase and/or plant；The expression quantity of NADH-GOGAT1 gene increases in the expression quantity increase of 2 genes and/or plant And/or plant increases the absorption of nitrogen under low nitrogen growth conditions.

The biomass of any of the above-described plant is the fresh weight of plant.

The biomass of any of the above-described plant roots is the fresh weight of plant roots.

Any of the above-described plant can be following c1) any one of to c9): c1) dicotyledon；C2) unifacial leaf is planted Object；C3) gramineae plant；C4) rice；C5) rice varieties OryzasativaLcv.Nipponbare；C6) rice varieties Nanjing 6；C7) rice varieties are bright Extensive No. 63；C8) rice varieties no loadtransformer；C9) rice varieties Huang Huazhan.

Concretely nitrogen content is lower than 120kg/ha (such as 120kg/ha, 60kg/ha or 0kg/ to any of the above-described low nitrogen ha)。

Any of the above-described low nitrogen growth conditions is that nitrogen content is lower than 120kg/ha (such as 120kg/ha, 60kg/ha or 0kg/ Ha growth conditions).

Low nitrogen resisting stress transgenic plant is cultivated using protein OsARE1 or its encoding gene as RNA disturbance target point Using also belonging to the scope of protection of the invention.

The plant expression vector for carrying OsARE1 gene of the present invention or other homologous sequences can be by using protoplast- Chemistry mediation method (Ca²⁺, PEG), Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube, microinjection, electricity Swash, any or several method combination conversion plant cell in the conventional biology methods such as particle gun, mediated by agriculture bacillus, Tissue or organ, and the plant cell, tissue or organ of conversion are cultivated into plant；The tissue and organ may include that host plants Fruit pod, callus, stem apex, blade and seed of object etc..Transgenic plant includes plant to the change of Low nitrogen stress resistance Interior nitrogen metabolism channel marking gene (including Nitrogen Absorption marker gene AMT and nitrogen assimilation marker gene GS/GOGAT etc.) and In Different Organs (including stem sheath, blade and fringe etc.) nitrogen content increase or decrease and plant nitrogen regulation character (including Biomass, root biomass, root/shoot ratio, chlorophyll content, plant height, tiller number, single plant yield, grain number per spike, Their First Branch obstruct number With second level branch stalk number etc.) change.

Any of the above-described dicotyledon can also for arabidopsis, rape, peanut, cotton, soybean, sunflower, palm, Chinese olive tree, castor-oil plant, potato or tobacco.Any of the above-described monocotyledon can be also corn, wheat, barley, oat, black Wheat, jowar or turfgrass.

It is demonstrated experimentally that resistance of the protein OsARE1 negative regulation plant to Low nitrogen stress, overexpression OsARE1 gene drop Low plant lowers expression or knocks out OsARE1 gene and improve plant to the resistance of Low nitrogen stress to the resistance of Low nitrogen stress.are1- 1 site is the effective site for improveing Rice Resistance Low nitrogen stress.OsARE1 gene is pinpointed using plant gene engineering technology Transformation provides effective molecular strategy to cultivate low nitrogen resisting stress New Crop Varieties.

Detailed description of the invention

Fig. 1 is the genetic screening of abc1-1 are1 double-mutant；

A is phenotype (scale 15cm) of the rice in tillering stage；

B is phenotype (scale 15cm) of the rice in the jointing stage；

C is phenotype (scale 15cm) of the rice at heading stage；

D is the phenotype (scale 15cm) of rice in the watery stage；

E be rice respectively in Seedling Stage, tillering stage, jointing stage, heading stage and pustulation period plant height quantitative analysis；

F be rice respectively in Seedling Stage, tillering stage, jointing stage, heading stage and pustulation period tiller number quantitative analysis；

G is the rice opposite chlorophyll of the top layer blade in Seedling Stage, tillering stage, jointing stage, heading stage and pustulation period respectively The quantitative analysis of content (SPAD)；

E, numerical value indicates that average value ± standard deviation, the sample size of each growthdevelopmental stage are 50 plants of plants in F and G；

WT is wild rice (rice varieties OryzasativaLcv.Nipponbare), and abc1-1 is abc1-1 mutant, and abc1-1 are1-1 is Abc1-1 are1-1 double-mutant, abc1-1 are1-2 are abc1-1 are1-2 double-mutant, F₁For abc1-1 are1-1 Double-mutant hybridizes the hybrid F obtained with abc1-1 are1-2 double-mutant₁For plant.

Fig. 2 is the phenotypic analysis of are1-1 single mutant；

A-E is that at Seedling Stage, tillering stage, jointing stage, heading stage and maturity period, (scale is rice for the phenotype of plant respectively 15cm)；

Quantitative analysis of the F for relative chlorophyll content (SPAD) in 0-60d boot leaf after Rice Flowering, numerical value expression average value ± standard deviation, sample size are 40 plants of plants；

G is quantitative analysis of the rice in maturity period plant height, and numerical value indicates that average value ± standard deviation, sample size are 40 plants Plant；

H is the quantitative analysis of the main grain number per spike of rice, and numerical value indicates that average value ± standard deviation, sample size are 40 plants of plants Object；

I is the quantitative analysis of glutelin content in rice paddy seed, and numerical value indicates average value ± standard deviation, by 3 technologies Reproducible results statistics, each sample include 20 seeds；

J is the quantitative analysis of content of prolamine in rice paddy seed, and numerical value indicates average value ± standard deviation, by 3 skills Art reproducible results statistics, each sample include 20 seeds；

* indicates that it is horizontal (P < 0.01) to reach extremely significant property for the two difference in t test；

WT is wild rice (rice varieties OryzasativaLcv.Nipponbare), and are1-1 is are1-1 single mutant.

Fig. 3 is Analysis of Resistance of the are1-1 single mutant to Low nitrogen stress；

A is rice in normal (+NH₄NO₃) and nitrogen stress (- NH₄NO₃) quantitative point of root weight after 20d is grown under growth conditions Analysis；Numerical value indicates that average value ± standard deviation, sample size are 40 plants of plants；

B is rice in normal (+NH₄NO₃) and nitrogen stress (+NH₄NO₃) quantitative point of root/shoot ratio after 20d is grown under growth conditions Analysis；Numerical value indicates that average value ± standard deviation, sample size are 40 plants of plants；

C is rice in normal (+NH₄NO₃) and nitrogen stress (+NH₄NO₃) grow under growth conditions first after 20d, 25d and 30d The phenotypic analysis (scale 1cm) of piece true leaf；

D is the quantitative analysis of chlorophyll content in leaf blades in C, and numerical value indicates average value ± standard deviation, sample size 12 Strain plant；

E-I is rice Nitrogen Absorption mark in the root under normal (300kg/ha) and low nitrogen (120kg/ha) growth conditions Gene OsAMT1；1,OsAMT1；2 and OsAMT1；3 and nitrogen assimilation marker gene OsGS1；2 and OsNADH-GOGAT1 table Up to the quantitative analysis of amount.Specific steps are as follows: rice is planted in normal growing conditions respectively (urea amount of application is 300kg/ha) Under low nitrogen growth conditions (urea amount of application is 120kg/ha), after transplanting 50 days, fresh adventitious root is taken to detect above-mentioned nitrogen metabolism Mark of correlation gene expression amount.Numerical value indicates average value ± standard deviation, from 3 technology reproducible results statistics, each sample Product include 3 plants of plants；

J and K is rice Nitrogen absorption efficiency (NUpE) and nitrogen utilization efficiency (NUE) under the conditions of different nitrogen amount applieds Quantitative analysis.Specific steps are as follows: using total nitrogen content in Kjeldahl nitrogen determination rice stem sheath, blade and fringe；NUpE is contained by fringe Nitrogen total amount and external source amount of nitrogen ratio calculation, and NUE is from cell total output and external source amount of nitrogen ratio calculation；Numerical value Indicate average value ± standard deviation, from 3 independent biological reproducible results statistics, each sample includes 6 plants of plants；

* be illustrated respectively in * * t test in both difference reach the level of signifiance (P < 0.05) and extremely significant property level (P < 0.01)；

The mill water culture nutrient solution of normal growing conditions includes 1.46mM NH₄NO₃, and the mill water culture nutrient solution under nitrogen stress growth conditions Not comprising any nitrogen source；

Fig. 4 is Nitrogen Regulation character analysis of are1-1 single mutant under the conditions of different nitrogen amount applieds；

A is maturity period field photo of rice under the conditions of different nitrogen amount applieds；

B is maturity period plant phenotype (scale 15cm) of rice under the conditions of different nitrogen amount applieds；

C-J is rice in the test of field nitrogenous fertilizer boot leaf relative chlorophyll content (SPAD), plant height, tiller number, master in 2014 The quantitative analysis of spike length, Their First Branch stalk number, second level branch stalk number, main grain number per spike and mass of 1000 kernel, numerical value indicate average value ± standard Deviation, sample size are 40 plants of plants.

K-N is the statistical analysis of continuous 4 year cell production of rice under the conditions of different nitrogen amount applieds；Each cell includes 200 plants of plants.Numerical value indicates average value ± standard deviation, is repeated from statistics by 3-5 biology.

Fig. 5 is the map based cloning of OsARE1 gene；

A is to be constructed respectively with abc1-1 are1-1 double-mutant using rice rice variety bright extensive No. 63 and Nanjing 6 F₂170 exchange single plants in Genes location group, OsARE1 gene are short in No. 8 chromosome (Chr.8) of rice by Primary Location Between two SSR molecular markers RM3374 and RM3481 of arm；N represents the number of exchange single plant；

B is to utilize widened F₂The 1233 exchange single plants separated in target group, OsARE1 gene is by further fine It is located between two SNP markers M79 and M81 in the section about 410kb；Digital representation OsARE1 gene under straight line with should The number of single plant is exchanged between molecular labeling；

C is the relative position and direction of 13 candidate genes in OsARE1 gene finely positioning section；Black arrow instruction OsARE1 gene position and direction；

D is the gene coding region OsARE1 schematic diagram；Black box indicates that exon, filament indicate introne；Black triangle Symbol indicates that the mutational site that single nucleotide deletion is mutated, gray triangles symbol occur for OsARE1 gene in are1-1 mutant Indicate the mutational site of OsARE1 gene generation mononucleotide Substitution in are1-2 mutant.

Fig. 6 is the Genetic complementation analysis of OsARE1 gene；

A is the phenotype (scale 15cm) of rice in the watery stage；

B is phenotype (scale 15cm) of the rice in the maturity period；

C is the phenotype (scale 2cm) in B in the middle part of plant boot leaf；

Quantitative analysis of the D for relative chlorophyll content (SPAD) in 0-60d boot leaf after Rice Flowering, numerical value expression average value ± standard deviation, each time point sample size are 40 plants of plants；

WT is wild rice, and abc1-1 is abc1-1 mutant, and are1-1 is are1-1 single mutant, abc1-1 Are1-1 is abc1-1 are1-1 double-mutant, and the pARE1 in A is that OsARE1 full length gene sequence is transferred to abc1-1 The T that are1-1 double-mutant obtains₁For genetically modified plants, the pARE1 in B, C and D is to be transferred to OsARE1 full length gene sequence The T that are1-1 single mutant obtains₁For genetically modified plants.

Fig. 7 is Analysis of Resistance of the OsARE1 genetically modified plants to Low nitrogen stress；

A is rice varieties OryzasativaLcv.Nipponbare (WT), abc1-1 mutant, abc1-1 are1-1 double-mutant and abc1-1 are mutated 3 independent T of body background₁(scale is phenotype for OsARE1 gene silencing plant (#29, #10 and #34) in dough stage 15cm)；

B is the quantitative analysis of OsARE1 gene relative expression quantity in plant boot leaf in A, and numerical value indicates average value ± standard deviation Difference, from 3 technology reproducible results statistics, each sample includes 6 plants of plants；

C is the quantitative analysis of plant plant height in A；

D is the quantitative analysis of relative chlorophyll content (SPAD) in plant boot leaf in A；

E is the rice varieties OryzasativaLcv.Nipponbare and 3 independent T of (urea amount of application is 180kg/ha) under low nitrogen growth conditions₁ For OsARE1 gene silencing OryzasativaLcv.Nipponbare (#1, #37 and #24) dough stage phenotype (scale 15cm)；

F is the quantitative analysis of OsARE1 gene relative expression quantity in plant boot leaf in E, and numerical value indicates average value ± standard deviation Difference, from 3 technology reproducible results statistics, each sample includes 6 plants of plants；

G is the quantitative analysis of plant plant height in E；

H is the quantitative analysis of yield per plant in E；

I is rice varieties OryzasativaLcv.Nipponbare and 3 independent T₁The OryzasativaLcv.Nipponbare (#55, #77 and #36) that generation turns OsARE1 gene exists The phenotype (scale 15cm) of dough stage；

J is the quantitative analysis of OsARE1 gene relative expression quantity in plant boot leaf in I, and numerical value indicates average value ± standard deviation Difference, from 3 technology reproducible results statistics, each sample includes 6 plants of plants；

K is the quantitative analysis of plant plant height in I；

L is the quantitative analysis of yield per plant in I；

C, numerical value indicates that average value ± standard deviation, sample size are 30-40 plants of plants in D, G, H, K and L.B-D, * * indicates that it is horizontal (P < 0.01) to reach extremely significant property for the two difference in t test in F-H and J-L.

Fig. 8 is Analysis of Resistance of the OsARE1 knockout plants to Low nitrogen stress；

A is 5 kinds of allelic variation form analyses that OsARE1 gene knocks out generation through CRISPR/Cas9 system；Of the invention Inventor choose respectively OsARE1 gene two target sequences carry out gene editing, target sequence 1 be from 5 ' ends the 94th to 111 nucleotide, target sequence 2 are from the nucleotide of 5 ' end the 622nd to 649 (transcription initiation site is considered as+1)；Grey Font indicates that insertion mutation, grey dot indicate deletion mutation；

B carries the Huang Huazhan plant of difference OsARE1 genotype in A in high nitrogen (urea amount of application for 240kg/ha) and low The phenotype (scale 15cm) in the maturity period under nitrogen (urea amount of application is 150kg/ha) growth conditions；

C-F is quantifying for plant plant height in B, relative chlorophyll content (SPAD) in boot leaf, main grain number per spike and single plant yield Analysis；Numerical value indicates that average value ± standard deviation, sample size are 30 plants of plants；

* be illustrated respectively in * * t test in both difference reach significance (P < 0.05) and extremely significant property level (P < 0.01)。

Fig. 9 is Analysis of Resistance of the are1-1 segment substitution line plant to Low nitrogen stress；

A is the are1-1 segment substitution line (MH63 of bright extensive No. 63 (MH63) and bright extensive No. 63 genetic background^are1-1) plant exists Plant phenotype (scale 15cm) under the conditions of different nitrogen amount applieds；

B-E is plant relative chlorophyll content (SPAD), main grain number per spike and single plant in the plant height of dough stage, boot leaf in A The quantitative analysis of yield；

F is the are1-1 segment substitution line (KY131 of no loadtransformer (KY131) and no loadtransformer genetic background^are1-1) plant exists Plant phenotype (scale 15cm) under the conditions of different nitrogen amount applieds；

G and H is that plant in dough stage, divide in boot leaf by relative chlorophyll content (SPAD) and the quantitative of main grain number per spike in F Analysis；

Numerical value indicates that average value ± standard deviation, sample size are 30 plants of plants in B-E, G and H；

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.

The information of primer involved in following embodiments is shown in Table the 2, the 1st and is classified as Primer, and the 2nd is classified as the nucleotide of primer Sequence, the 3rd is classified as purposes in embodiment.

Table 2

The genetic screening of embodiment 1, abc1-1 are1 double-mutant

The present inventor is screened one thin by chemical mutagenesis rice varieties OryzasativaLcv.Nipponbare (Nipponbare, NPB) The mutant of born of the same parents' mitogen response abnormality, and it is named as abnormal cytokinin reponse1 mutant (abc1-1 Mutant) (Yang et al., 2016).ABC1 gene encodes the Fd-GOGAT albumen of chloroplaset positioning, is catalysis nitrogen One key enzyme of plain assimilation process.Abc1-1 mutant has apparent growth and development defect phenotype, including plant is downgraded, divides Tiller number reduces and the nitrogen assimilations abnormal phenotypes such as yellowing leaf.The present inventor has further screened abc1-1 mutant Inhibit mutation abc1-1 repressors (are mutation).In the serial abc1-1 are double-mutant of separation identification, there is 2 A double-mutant phenotype is similar.This 2 double-mutants are returned 3 times with abc1-1 mutant respectively, phenotypic analysis finds BC₂F₂ Still stable abc1-1 phenotype part is isolated in group restores mutant.Allelic analysis hair is carried out to this 2 double-mutants It is existing, the hybrid F in different growth and development periods₁For plant cannot complementation abc1-1 mutation type surface (A, B, C and D in Fig. 1), table It is bright they be caused by two allelic variations of the same gene, therefore we to be respectively designated as abc1-1 are1-1 bis- Mutant and abc1-1 are1-2 double-mutant.

The present inventor by the field phenotypic analysis of multiple spot for years experiments have shown that: in the entire Life Cycle of rice Interim, it includes that plant height reduces, tiller number is reduced that abc1-1 are1 double-mutant, which can partly restore abc1-1 mutant, With chlorophyll content reduce etc. nitrogen metabolism abnormal phenotype (E, F and G in Fig. 1).It follows that are1 Mutational part inhibits abc1-1 Mutant nitrogen assimilation abnormal phenotype implies that are1 mutation may enhance the resistance to Low nitrogen stress ability of rice.

The phenotypic analysis of embodiment 2, are1-1 single mutant

In order to obtain are1 single mutant, the present inventor is by abc1-1 are1-1 double-mutant and abc1-1 Are1-2 double-mutant is returned 3 times with rice varieties OryzasativaLcv.Nipponbare respectively, obtains BC₂F₂Segregating population.Then to BC₂F₂Separate group Body carries out phenotypic analysis, obtains are1-1 single mutant and are1-2 single mutant.Are1-1 single mutant and are1-2 are mono- prominent Variant has similar phenotype, and the present invention chooses are1-1 single mutant and carries out subsequent representative experimental analysis.

Under normal growing conditions, compared with rice varieties OryzasativaLcv.Nipponbare, are1-1 single mutant vegetative growth phase not With apparent phenotypic difference (A, B and C in Fig. 2), are1-1 single mutant heading stage postpones about 3 to 5d, has apparent Evening declines phenotype (D, E and F in Fig. 2), and are1-1 single mutant boot leaf chlorophyll content 35d after heading nearly doubles (Fig. 2 Middle F), into after heading stage, are1-1 single mutant plant height gradually increases (D, E and G in Fig. 2), and grain number per spike dramatically increases (Fig. 2 Middle E and H).In addition, there is no significant changes for are1-1 single mutant seed size, but plant compared with rice varieties OryzasativaLcv.Nipponbare Sub- major storage protein glutelin and content of prolamine dramatically increase (I and J in Fig. 2).Above-mentioned analysis the result shows that, are1-1 Mutation improves rice series Nitrogen Regulation character, and are1-1 is mutated the biological function that may have resistance to Low nitrogen stress.

Embodiment 3, are1-1 single mutant are to the Analysis of Resistance of Low nitrogen stress

Whether change rice to the tolerance of Low nitrogen stress for clear are1-1 mutation, the present inventor analyzes Phenotype of the are1-1 single mutant under low nitrogen growth conditions.For growth inhibition caused by response environment nutrient dificiency, plant Developing out, various countermeasures are to maintain normal vital movement, including the root/shoot ratio increased under low nitrogen growth conditions.

Are1-1 single mutant and rice wild-type variety OryzasativaLcv.Nipponbare are planted in normal growing conditions respectively and nitrogen stress is raw Under elongate member, when 20d statistics root weight and and root/shoot ratio, the table of rough leaf is observed when 20d, when 25d and when 30d Type simultaneously detects blade Determination of Chlorophyll content.

The result shows that compared with rice varieties OryzasativaLcv.Nipponbare, are1-1 single mutant root increases under nitrogen stress growth conditions again (A in Fig. 3), root/shoot ratio increase (B in Fig. 3), are1-1 single mutant blade Determination of Chlorophyll content significantly improve (in Fig. 3 C and D), show are1-1 single mutant to nitrogen hunger processing resistance with higher.

The present inventor also uses the different nitrogen amount applied Rice under Condition kind OryzasativaLcv.Nipponbare of Kjeldahl nitrogen determination With the nitrogen content in are1-1 single mutant Different Organs (including stem sheath, blade and fringe).Experimental result is by 3 secondary pollutants It repeats to count, each biology repeats to include 6 plants of plants.

The result shows that (table 3), under the conditions of different nitrogen amount applieds, are1-1 single mutant Different Organs nitrogen content is high In rice varieties OryzasativaLcv.Nipponbare, and under low nitrogen growth conditions, increase ratio is more obvious, shows are1-1 mutation enhancing rice benefit With the ability of nitrogen.

Nitrogen absorption efficiency (NUpE) and nitrogen utilization efficiency (NUE) are calculated according to Different Organs nitrogen content.As a result Show that NUpE and NUE of the are1-1 single mutant under low nitrogen growth conditions are all remarkably higher than rice varieties OryzasativaLcv.Nipponbare (in Fig. 3 J and K).Correspondingly, under low nitrogen growth conditions, Nitrogen Absorption marker gene OsAMT1 in are1-1 single mutant root；1, OsAMT1；2 and OsAMT1；3 and assimilation marker gene OsGS1；2 and OsNADH-GOGAT1 expression quantity increase (E, F in Fig. 3, G, H and I).The above results show are1-1 mutation enhancing rice to the resistance of Low nitrogen stress, and the Nitrogen absorption efficiency of enhancing is One of the reason of are1-1 single mutant resistance to low nitrogen growth conditions.

Table 3

The Nitrogen Regulation character analysis of embodiment 4, are1-1 mutant under the conditions of different nitrogen amount applieds

It is above-mentioned the experimental results showed that, are1-1 mutation enhancing rice implies are1-1 single mutation to the resistance of Low nitrogen stress Body may have certain growth vigor under low nitrogen growth conditions.Therefore, using RANDOMIZED BLOCK DESIGN scheme, hair of the invention Bright people has carried out field nitrogenous fertilizer test in continuous 4 years (2013-2016), applies nitrogen to analyze are1-1 single mutant in difference Nitrogen Regulation character under level conditions, stresses analysing output Related Agronomic Characters.It selects urea as only nitrogen source, sets respectively Set six concentration for the treatment of of 300 kg/ha, 240kg/ha, 180kg/ha, 120kg/ha, 60kg/ha and 0kg/ha.In rice seedling 30%, 40% and 30% urea total amount applied nitrogen is pressed in phase, tillering stage and boot stage, each processing respectively.Select potassium sulfate Exclusive source with double superhosphate as potash fertilizer and phosphate fertilizer, and by the pure potassium of 30kg/ha and the pure phosphorus standard of 120kg/ha in rice shoot The application of transplanting time equivalent.Rice varieties OryzasativaLcv.Nipponbare and are1-1 single mutant are distinguished equivalent and are diagonally planted in 3.8-4.0 × 4.0m Cell in, spacing in the rows × line-spacing be 25 × 25cm (A in Fig. 4).

In vegetative growth phase, are1-1 single mutant does not show apparent phenotype compared with rice varieties OryzasativaLcv.Nipponbare Difference.After heading, are1-1 single mutant all had under the conditions of different nitrogen amount applieds apparent evening decline phenotype (in Fig. 4 A and B), this is along with the enhancing (C in Fig. 4) of photosynthetic capacity effective during grouting and the increase (K in Fig. 3) of nitrogen utilization efficiency.With Rice varieties OryzasativaLcv.Nipponbare is compared, although are1-1 single mutant plant height and tiller number are slightly changed (D and E in Fig. 4), its fringe Long, Their First Branch stalk number and second level branch stalk number increase, and increase more obvious (F, G and H in Fig. 4) under low nitrogen growth conditions.Phase Ying Di, are1-1 single mutant grain number per spike dramatically increase (I in Fig. 4).However, are1-1 single mutant thousand grain weigth slightly drops Low (J in Fig. 4).Finally, cell production measurement result shows under the conditions of the normal nitrogen amount applied of 40%-60%, are1-1 Single mutant increases production about 10%-20% (K, L, M and N in Fig. 4), and under the conditions of the nitrogen amount applied, are1-1 single mutant with NUpE the and NUE difference of rice varieties OryzasativaLcv.Nipponbare is especially pronounced (J and K in Fig. 3).In addition, when amount of application of nitrogen fertilizer is reduced to normally When dose half or so, are1-1 single mutant yield and yield of rice varieties OryzasativaLcv.Nipponbare under the conditions of normal nitrogen amount applied Quite (K, L, M and N in Fig. 4).The above results show that are1-1 mutation improves rice varieties OryzasativaLcv.Nipponbare in Low nitrogen stress condition Under Nitrogen Regulation character, are1-1 single mutant for breeding low nitrogen resisting coerce rice varieties provide ideal genetic stocks.

The map based cloning of embodiment 5, OsARE1 gene

In order to determine the property of are1-1 mutation, the present inventor is Japanese by are1-1 single mutant and rice varieties Fine carry out backcross analysis.It is returned the 18 plants of hybrid F obtained₁The phenotype that rice varieties OryzasativaLcv.Nipponbare is all shown as plant, has The plant of the normal and late phenotype that declines is in F₂Phenotypic segregation ratio (the 561:196=2.86:1 close to 3:1 is showed in group；χ²c< χ² _0.05=3.84).It can be seen that are1-1 single mutant is caused by the recessive mutation of single karyogene.

The present inventor's OsARE1 gene candidate using the separation identification of map based cloning method.Utilize abc1-1 Are1-1 double-mutant and rice rice variety bright extensive No. 63 and Nanjing 6 construct genetic mapping group respectively.With blade evening The phenotype that declines utilizes F as index is selected₂170 there is similar abc1-1 are1-1 double-mutant evening to decline phenotype in segregating population Exchange single plant, OsARE1 gene by Primary Location No. 8 the short arm of a chromosome two SSR molecular marker RM3374 with Between RM3481 (A in Fig. 5).Utilize widened F₂1233 exchange single plants in genetic mapping group, OsARE1 gene is by into one Step finely positioning is between two SNP markers M79 and M81 in the section of about 410kb (B in Fig. 5).By comparing rice base Because a group annotations database (Rice Genome Annotion Project) has found, it includes 13 functional annotations which, which has altogether, Candidate gene (C in Fig. 5), by its all sequencing discovery only have Loc_Os08g12780 gene mutate.In abc1-1 In are1-1 double-mutant and are1-1 single mutant, single-base deletion mutation occurs for the 4th exon of the gene, causes Frameshift mutation occurs for its coded product protein OsARE1, generates the truncated protein of C-terminal；It is bis- in abc1-1 are1-2 In mutant and are1-2 single mutant, single base Substitution (C sports T) occurs for the 6th exon of the gene, leads Cause a highly conserved amino acid that replacement (proline replaces with leucine) (D in Fig. 5) occurs.

Embodiment 6, the verifying of the genetic complement of OsARE1 gene

One, the extraction of oryza sativa genomic dna

1) about 400mg rice varieties OryzasativaLcv.Nipponbare blade is taken, is fitted into the centrifuge tube (specification 2mL) containing steel ball, is added 400 μ L DNA extracting solution (pH7.5,100mM Tris- of NaCl containing 500mM, 50mM EDTA and 1% (1mg/100mL) SDS HCl buffer).

2) after completing step 1), the centrifuge tube is loaded to mixing grind away instrument (product of RETSCH company, model MM400), 3min is vibrated with maximum frequency, then 65 DEG C of water-bath 30min, are cooled to room temperature.

3) after completing step 2), the centrifuge tube is taken, isometric chloroform is added, oscillation mixes, 13000r/min centrifugation 10min。

4) after completing step 3), the centrifuge tube is taken, supernatant is shifted to another new centrifuge tube (specification 1.5mL), adds Enter isometric isopropanol, mixes, -20 DEG C of standing 1h.

5) after completing step 4), the centrifuge tube is taken, 13000r/min is centrifuged 10min, abandons supernatant, 70% (v/v) ethyl alcohol It aqueous solution washing precipitating 2 times, dries.

6) after completing step 5), the centrifuge tube is taken, 400 μ L deionized waters are added, mixes, obtains oryza sativa genomic dna. - 20 DEG C save backup.

Two, the building of OsARE1 complementing vector

1) it using oryza sativa genomic dna as template, is carried out using primer pair first (ARE1Comp1F and ARE1Comp1R are formed) PCR amplification, then using DNA agarose gel purification QIAquick Gel Extraction Kit, (product of Biomed company, catalog number are DH101) purification and recovery obtains the product first of about 5000bp；Take cloning vector pBluescript SK II (-) (Alting- Mees et al., 1992), with restriction enzyme EcoRV digestion, obtain carrier framework；Carrier framework and product first are connected It connects, obtains intermediate vector first.

According to above-mentioned steps, primer pair first is replaced with into primer pair B (ARE1Comp2F and ARE1Comp2R are formed), Its step is constant, obtains intermediate vector second.

According to above-mentioned steps, primer pair first is replaced with into primer pair third (ARE1Comp3F and ARE1Comp3R are formed), Its step is constant, obtains intermediate vector third.

2) intermediate vector first, intermediate vector second and intermediate vector third are converted into bacillus coli DH 5 alpha respectively, it is big to obtain recombination Enterobacteria bacterial strain first, bacterial strain second and bacterial strain third；Using Wizard Plus Minipreps DNA Purification System Kit (product of Promega company, catalog number A9280) extracts Plasmid DNA in a small amount, is then sequenced.

Sequencing result shows in intermediate vector first containing sequence 3 in ordered list from 5 ' ends shown in the 1st to 3860 Nucleotide sequence, containing the nucleotide shown in the 3761st to 5760 from 5 ' ends of sequence 3 in ordered list in intermediate vector second Sequence, containing the nucleotide sequence shown in the 5041st to 7229 from 5 ' ends of sequence 3 in ordered list in intermediate vector third.

3) restriction enzyme PstI and KpnI digestion intermediate vector first are used, the digestion products first of about 3809bp is recycled.With Restriction enzyme KpnI digestion intermediate vector second recycles the digestion products second of about 1880bp.With restriction enzyme KpnI and SalI digestion intermediate vector third recycles the digestion products third of about 1540bp.

PCAMBIA1300 carrier (Roberts et al., 1997) is taken, with restriction enzyme PstI and SalI digestion, Obtain carrier framework first；Carrier framework first, digestion products first, digestion products second and digestion products third are connected, OsARE1 is obtained Complementing vector.

OsARE1 complementing vector is sequenced.According to sequencing result, structure description is carried out such as to OsARE1 complementing vector Under: to DNA shown in sequence 3 divides in insetion sequence table between the restriction enzyme PstI and SalI of pCAMBIA1300 carrier Sub (the full-length genome sequence of Loc_Os08g12780 gene), obtained recombinant plasmid.

Three, Electroporation conversion and Agrobacterium tumefaciens mediated rice transformation

1) Electroporation conversion is used, OsARE1 complementing vector is imported into Agrobacterium tumefaciems EHA105, obtains recombinational agrobacterium, It is named as EHA105/OsARE1-Comp.

2) Agrobacterium tumefaciens mediated rice transformation method is used, EHA105/OsARE1-Comp is imported into abc1-1 Are1-1 double-mutant, obtains T₁In generation, turns the abc1-1 are1-1 double-mutant of OsARE1 gene.

3) Agrobacterium tumefaciens mediated rice transformation method is used, EHA105/OsARE1-Comp is imported into are1-1 Single mutant obtains T₁In generation, turns the are1-1 single mutant of OsARE1 gene.

Four, the phenotypic analysis of OsARE1 transgenosis complementation plant

T₁Generation turn OsARE1 gene double-mutant can complete complementary abc1-1 are1-1 double-mutant it is prominent to abc1-1 Variant phenotype, including plant height reduction, tiller number reduction, chlorophyll content reduction and entire biomass reduction etc. are (in Fig. 6 A)；T₁Generation turn OsARE1 gene are1-1 single mutant can complete complementary are1-1 single mutant to rice wild-type variety OryzasativaLcv.Nipponbare phenotype (B in Fig. 6), especially chlorophyll content in leaf blades reduce and senescence phenotype (C and D in Fig. 6).

Rice genetic complementation test the result shows that, Loc_Os08g12780 gene is exactly OsARE1 gene.OsARE1 gene Full-length genome sequence nucleotide sequence as shown in sequence 3 in sequence table, the nucleotide sequence of the cDNA of OsARE1 gene As shown in sequence 2 in sequence table, the nucleotide sequence coded sequence shown in 467-1750 from the end 5' of sequence 2 in sequence table Protein OsARE1 shown in sequence 1 in list.

Embodiment 7, OsARE1 genetically modified plants are to the Analysis of Resistance of Low nitrogen stress

One, the acquisition of the cDNA of OryzasativaLcv.Nipponbare

1) using RNAprep Pure plant total RNA extraction reagent box, (product of TIANGEN company, catalog number are DP432 total serum IgE in rice varieties OryzasativaLcv.Nipponbare blade) is extracted, OryzasativaLcv.Nipponbare total serum IgE is obtained.

2) 1-2 μ g OryzasativaLcv.Nipponbare total serum IgE is taken, using TransScript First-Strand cDNA Synthesis SuperMix kit (product of TransGen Biotech company, catalog number AT301) synthesizes the first chain cDNA, obtains To the cDNA of OryzasativaLcv.Nipponbare.

Two, the building of OsARE1 gene silencing vector

It is compared by Rice Genome Sequence, chooses OsARE1 gene transcription start site downstream 101-400bp and (transcribe Beginning site is considered as+1) target sequence of the sequence as OsARE1 gene silencing.

1) using the cDNA of OryzasativaLcv.Nipponbare as template, PCR expansion is carried out using primer pair (ARE1RNAiF and ARE1RNAiR are formed) Increase, then uses DNA agarose gel purification QIAquick Gel Extraction Kit purification and recovery, obtain the product of about 323bp；Take cloning vector PBluescript SK II (-) obtains carrier framework with restriction enzyme EcoRV digestion；Carrier framework and product are connected It connects, obtains intermediate vector.

Intermediate vector is sequenced.Sequencing result shows in intermediate vector containing sequence 5 in ordered list from 5 ' ends the Nucleotide sequence shown in 840 to 1138.

2) restriction enzyme SpeI and SacI digestion intermediate vector are used, the DNA fragmentation 1 of about 305bp is recycled；With restricted Restriction endonuclease SpeI and SacI digestion pTCK303 carrier (Wang et al., 2004) recycles the carrier framework 1 of about 14.6kb；It will DNA fragmentation 1 and carrier framework 1 connect, and obtain recombinant plasmid 1；With restriction enzyme KpnI and BamHI digestion intermediate vector, Recycle the DNA fragmentation 2 of about 311bp；With restriction enzyme KpnI and BamHI digestion recombinant plasmid 1, the load of about 14.9kb is recycled Body skeleton 2；DNA fragmentation 2 and carrier framework 2 are connected, OsARE1 gene silencing vector is obtained.

OsARE1 gene silencing vector is sequenced.According to sequencing result, OsARE1 gene silencing vector is tied Structure is described as follows: by the DNA molecular insertion pTCK303 carrier shown in the 5 ' end the 14th to 312 of the sequence 5 in sequence table Between SpeI and SacI recognition site, while by the sequence 5 in sequence table DNA points shown in the 5 ' end the 840th to 1138 The recombinant expression carrier obtained between KpnI the and BamHI recognition site of son insertion pTCK303 carrier.OsARE1 gene silencing carries Body surface is up to the RNA molecule shown in the 5 ' end the 14th to 1138 of sequence 4 in sequence table.

Three, the building of OsARE1 gene overexpression carrier

1) it using the cDNA of rice varieties OryzasativaLcv.Nipponbare as template, is carried out using primer pair (ARE1OEF and ARE1OER are formed) Then PCR amplification uses DNA agarose gel purification QIAquick Gel Extraction Kit purification and recovery, obtains the product of about 1281bp；Take pSK- FLAG carrier (Alting-Mees et al., 1992), with restriction enzyme PstI and XmaI digestion, obtains the load of about 3kb Body skeleton；Carrier framework is connected with product, obtains intermediate vector first.

Intermediate vector first is sequenced.Sequencing result shows last from 5' containing sequence 2 in ordered list in intermediate vector first Nucleotide sequence shown in having held 467-1747.

2) restriction enzyme PstI and SpeI digestion intermediate vector first are used, the DNA fragmentation 1 of about 1293bp is recycled；With limit Property restriction endonuclease PstI and SpeI digestion pCAMBIA1300-NOS carrier processed (Roberts et al., 1997), recycles about 9.2kb Carrier framework 1；DNA fragmentation 1 and carrier framework 1 are connected, intermediate vector second is obtained.

3) with pCAMBIA1300 carrier (Roberts et al., 1997) for template, using primer pair (35sF and 35sR Composition) PCR amplification is carried out, obtain the pcr amplification product of about 861bp.

4) then the pcr amplification product obtained with restriction enzyme PstI digestion step 3) uses DNA Ago-Gel The recycling of purification and recovery kits, obtains DNA fragmentation 2；With restriction enzyme PstI digestion intermediate vector second, recycling is about The carrier framework 2 of 10.5kb；DNA fragmentation 2 and carrier framework 2 are connected, OsARE1 gene overexpression carrier is obtained.

Four, the Analysis of Resistance to Low nitrogen stress of OsARE1 genetically modified plants

1) Electroporation conversion is used, OsARE1 gene silencing vector is imported into Agrobacterium tumefaciems EHA105, obtains recombination agriculture Bacillus is named as EHA105/OsARE1-RNAi.

OsARE1 gene overexpression vector introduction Agrobacterium tumefaciems EHA105 is recombinated using Electroporation conversion Agrobacterium is named as EHA105/OsARE1-OE.

2) Agrobacterium tumefaciens mediated rice transformation method is used, EHA105/OsARE1-RNAi is imported into abc1-1 Mutant obtains 3 independent T₁For the abc1-1 mutant of OsARE1 gene silencing.

Using Agrobacterium tumefaciens mediated rice transformation method, by EHA105/OsARE1-RNAi Introduced into Rice kind OryzasativaLcv.Nipponbare obtains 3 independent T₁For the OryzasativaLcv.Nipponbare of OsARE1 gene silencing.

Using Agrobacterium tumefaciens mediated rice transformation method, by EHA105/OsARE1-OE Introduced into Rice kind day This is fine, obtains 3 independent T₁For the OryzasativaLcv.Nipponbare of OsARE1 gene overexpression.

As a result as follows: 3 independent T₁For OsARE1 gene table in the abc1-1 mutant flag leaf of OsARE1 gene silencing It is significantly reduced up to amount, plant height, tiller number, boot leaf Determination of Chlorophyll content and biomass (being Nitrogen Regulation character) are significant Increase (A and B in Fig. 7), and plant height and the increased degree of chlorophyll content and gene expression amount reduce degree and be positively correlated (in Fig. 7 B, C and D)；3 independent T₁It is significantly reduced for OsARE1 gene expression amount in the OryzasativaLcv.Nipponbare boot leaf of OsARE1 gene silencing, strain High and single plant yield (being Nitrogen Regulation character) dramatically increases (E, F, G and H in Fig. 7)；3 independent T₁For OsARE1 base Because OsARE1 gene expression amount dramatically increases in the OryzasativaLcv.Nipponbare boot leaf of overexpression, but plant height and single plant yield significantly reduce (i.e. inhibition Nitrogen Regulation character) (I, J, K and L in Fig. 7).It is above-mentioned the experimental results showed that, OsARE1 gene participate in negative regulation plant Object low nitrogen resisting stress procedure is the excellent genetic locus of the resistance to low nitrogen plant variety of breeding.

Embodiment 8, OsARE1 knockout plants are to the Analysis of Resistance of Low nitrogen stress

The gene editing technology that CRISPR/Cas9 is mediated provides effective ways for improvement plant specific trait.It is clear Whether OsARE1 gene can be knocked out and its be knocked out whether plant has low nitrogen resisting, the present inventor's structure by the technology It has built OsARE1 gene knockout carrier and has analyzed phenotype of the OsARE1 knockout plants under low nitrogen growth conditions.

One, the building of OsARE1 gene knockout carrier

In order to improve gene editing efficiency, by comparing rice genome database, OsARE1 gene extron sub-district 2 is selected (target sequence 1 to be edited is located in the 1st exon a 5 '-ATGTTCGACACTGGTTCC-3 ' of high specific sequence, turns The initiation site downstream position 94-111bp is recorded, transcription initiation site is considered as+1) and 5 '- (target sequence 2 to be edited is located in the 2nd exon, transcription initiation TTATTGATTGTGATTCCATGGGTGTTGG-3 ' The site downstream position 622-649bp, transcription initiation site are considered as+1), potential target spot (Fig. 8 as OsARE1 gene editing Middle A).

1) (contain transcription initiation site downstream using primer pair a (U6aF and U6aR are formed) synthesis gRNAa connector The OsARE1 genetic fragment of 86-104bp, transcription initiation site are considered as+1)；With restriction enzyme BsaI digestion gRNA table Up to carrier pYLgRNA-U6a (Ma et al., 2016), the carrier framework a of about 3kb is recycled；GRNAa and carrier framework a is connected It connects, obtains recombinant plasmid a；

2) using primer pair b (U6bF and U6bR are formed), synthesis gRNAb connector (contains transcription initiation site downstream The OsARE1 genetic fragment of 628-646bp, transcription initiation site are considered as+1)；With restriction enzyme BsaI digestion gRNA table Up to carrier pYLgRNA-U6b (Ma et al., 2016), the carrier framework b of about 3kb is recycled；GRNAb and carrier framework b is connected It connects, obtains recombinant plasmid b；

3) with restriction enzyme BsaI distinguish digestion recombinant plasmid a and recombinant plasmid b, be separately recovered about 800bp and (U6b promoter drives gRNAb to the gRNA expression cassette a (U6a promoter drives gRNAa expression) and gRNA expression cassette b of 600bp Expression)；With restriction enzyme BsaI digestion monocot genes group editor's carrier pYLCRISPR/Cas9-MH (Ma et Al., the carrier framework of about 16.5kb 2016), is recycled；GRNA expression cassette a and gRNA expression cassette b is connect with carrier framework, is obtained Obtain OsARE1 gene knockout carrier.

OsARE1 gene knockout carrier is sequenced.According to sequencing result, OsARE1 gene knockout carrier is tied Structure is described as follows: by sequence 2 in sequence table from 5 ' ends DNA shown in the 552nd to 570 and/or the 1094th to 1112 The recombinant expression carrier that molecule insertion pYLCRISPR/Cas9-MH carrier B saI recognition site obtains.OsARE1 gene knockout carries The DNA shown in the 2362nd to 2380 and/or the 3497th to 3515 from 5 ' ends of sequence 3 in body specific recognition sequence table Molecule.

Two, the Molecular Detection of OsARE1 gene knockout effect

1) Electroporation conversion is used, OsARE1 gene knockout carrier is imported into Agrobacterium tumefaciems EHA105, obtains recombination agriculture Bacillus is named as EHA105/OsARE1-KO.

2) Agrobacterium tumefaciens mediated rice transformation method is used, by EHA105/OsARE1-KO Introduced into Rice kind Huang Huazhan (HHZ, big, the adaptable rice varieties of a popularizing area, with certain senescence phenotype), obtains OsARE1 Knockout plants.

Whether edited and its edit effect to analyze OsARE1 gene, OsARE1 gene is had detected by sequencing approach Knock out the integrality of OsARE1 gene in plant.The result shows that equal in OsARE1 gene target sequence 1 and 2 region of target sequence Generation 1 obtains the T of 5 class OsARE1 Allelic Variation forms to the missing or insertion mutation of multiple bases altogether₁It is planted for transgenosis Object (A in Fig. 8).With wild type Huang Huazhan (genotype HHZ^ARE1) compare, HHZ^are1-24 bases are lacked at target sequence 1 TCGA；HHZ^are1-103 base CGA are lacked at target sequence 1, increase by 1 base T at target sequence 2；HHZ^are1-12? 2 bases G A are lacked at target sequence 1, and 1 bases G is lacked at target sequence 2； HHZ^are1-18Increase at target sequence 1 1 bases G lacks 37 bases at target sequence 2；HHZ^are1-25Increase by 1 base T (A in Fig. 8) at target sequence 1. Hair is compared with protein OsARE1 sequence in OsARE1 mutein sequence derived from 5 class OsARE1 Allelic Variations Existing, 5 class allelic variations cause amino acid frameshift mutation and protein translation to terminate in advance, show the mutability of 5 class allelic variations Matter is the mutation of OsARE1 gene lacks functionality type.Therefore, the present inventor is to this 5 class OsARE1 knockout plants Phenotype is analyzed, and is stressed to the phenotypic analysis under low nitrogen growth conditions, to detect OsARE1 knockout plants to low nitrogen The resistance of stress.

Three, Analysis of Resistance of the OsARE1 knockout plants to Low nitrogen stress

Under high nitrogen and low nitrogen growth conditions, compared with wild type Huang China accounts for, OsARE1 knockout plants tiller number becomes Change unobvious, plant height is increased slightly (B and C in Fig. 8).

One of an important factor for nitrogen is influence chlcrophyll biosynthesis, therefore the present inventor has detected The chlorophyll content of OsARE1 knockout plants boot leaf.Analysis is found, under the conditions of different nitrogen amount applieds, OsARE1 clpp gene Except plant boot leaf Determination of Chlorophyll content increases nearly 1 times (D in Fig. 8) compared with wild type Huang Hua Zhanjun.Chlorophyll content in leaf blades height with Plant photosynthetic capability power is closely related, implies that OsARE1 gene knockout may influence rice yield.Spike number, grain number per spike and grain weight It is three principal elements for determining rice yield.Analysis is found, under the conditions of different nitrogen amount applieds, the independent OsARE1 base of 5 classes Because the Huang Huazhan grain number per spike of knockout increases (E in Fig. 8).Correspondingly, the Huang Huazhan of OsARE1 gene knockout grows item in high nitrogen Under part, single plant yield is slightly increased, but increase ratio is more obvious (F in Fig. 8) under low nitrogen growth conditions.The above result shows that Knocking out OsARE1 gene can be improved rice to the resistance of Low nitrogen stress, for improvement plant low nitrogen resisting ability provide effective site and Method.

Embodiment 9, are1-1 segment substitution line are to the Analysis of Resistance of Low nitrogen stress

For application potential of the detection are1-1 mutation in improvement Nitrogen Use Efficiency in Rice, the present inventor's building A series of are1-1 segment substitution lines.Choose Indica Rice kind bright extensive No. 63 (Minghui63, MH63) and the main cultivation product of japonica rice Kind no loadtransformer (Kongyu131, KY131) two receptoroid kind is analyzed.By are1-1 single mutant respectively with MH63 and KY131 hybridization obtains hybrid F₁For plant, continues to be returned 6 times with receptor kind, obtain the BC of are1-1 mutation₅F₃For segment generation Changing is MH63^are1-1And KY131^are1-1。

Under the conditions of different nitrogen amount applieds, compared with receptor parent MH63, MH63^are1-1Plant plant height, leaf chlorophyll contain The Nitrogen Regulations character such as amount and main grain number per spike obviously improves (A, B, C and D in Fig. 9).It is consistent with this, MH63^are1-1Plant is single Strain yield increases, and under low nitrogen growth conditions, effect is more obvious (E in Fig. 9).Rice includes two Asias of japonica rice and long-grained nonglutinous rice Kind, compared with japonica rice, long-grained nonglutinous rice usually has higher nitrogen utilization efficiency.Are1-1 segment can further increase Indica Rice Kind MH63 shows that are1-1 segment is to improve the important genetic targets of Rice Resistance Low nitrogen stress ability to the resistance of Low nitrogen stress Point.Same policy is taken to have detected are1-1 mutation to the improved effect of japonica rice main breed KY131 low nitrogen resisting stress ability. KY131^are1-1Genes For Plant Tolerance Low nitrogen stress ability dramatically increases, the increase of increase and main grain number per spike including chlorophyll content in leaf blades (F, G and H in Fig. 9).The above result shows that are1-1 mutation is the effective site for cultivating low nitrogen resisting stress rice varieties, utilize Hereditary Backcrossing methods building are1-1 segment substitution line is the effective ways for improveing rice receptor kind low nitrogen resisting stress ability.

It is above-mentioned the experimental results showed that, OsARE1 gene be regulate and control Genes For Plant Tolerance Low nitrogen stress important gene.Are1-1 mutation Inhibit the extremely caused defect phenotype of Nitrogen Metabolism of Rice, increases Rice Resistance Low nitrogen stress ability, improve under low nitrogen growth conditions NUE and rice yield are the effective genetic locus for improveing Rice Resistance Low nitrogen stress ability；Inhibit OsARE1 gene expression or knockout OsARE1 gene, increase Rice Resistance Low nitrogen stress ability, are the effective ways for cultivating resistance to Low nitrogen stress plant new material.

<110>Inst. of Genetics and Development Biology, CAS

<120>application of the protein OsARE1 in regulation plant low nitrogen resisting

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 427

<212> PRT

<213>rice Oryza sativa L.

<400> 1

Met Ser Arg Ser Ala Val Ser Ser Glu Gly Gly Ile Ala Leu Arg Leu

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Phe Val Asp Trp Arg Ile Arg Arg Arg Arg Val Cys Ala Cys Lys Met

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Phe Asp Thr Gly Ser Gln Arg Gly Arg Val Lys Gln Leu Val Ala Phe

35 40 45

Ala Lys Lys Arg Arg Arg Pro Lys Lys Gln Pro Ser Arg Arg Pro Trp

50 55 60

Trp Lys Ala Trp Phe Ser Asp Trp Asn Asp Glu Glu Glu Ser Leu Ser

65 70 75 80

Gly Trp Arg Glu Asp Glu Glu Leu Leu Glu Glu Val Gly Gly Glu Glu

85 90 95

Gly Leu Ser Asp Asp Glu Lys Phe Glu Thr Trp Lys Arg Lys Ala Glu

100 105 110

Ala Ile Val Glu Leu Arg Glu Ala Arg Gln Asp Ala Met Asn Ala Glu

115 120 125

Gly Arg Ser Trp Glu Asp Trp Ile Gly Gly Gly Ser Ser Thr Ala Gly

130 135 140

Asp Gly Gly Gly Asp Trp Gly Gly Asp Leu Asp Val Ser Asp Leu Ile

145 150 155 160

Thr Asp Asp Pro Thr Glu Ile Val Arg Asp Lys Gly Leu Ile Glu Thr

165 170 175

Phe Arg Asp Ser Val Asp Glu Asp Tyr Asn Asp Met Leu Phe Glu Asp

180 185 190

Arg Val Phe Leu Tyr Ala Ser Thr Asn Ser Ala Lys Phe Leu Ala Leu

195 200 205

Leu Ile Val Ile Pro Trp Val Leu Asp Phe Leu Val His Asp Tyr Val

210 215 220

Leu Met Pro Phe Leu Glu Arg Tyr Val Gln Lys Val Pro Leu Ala Ala

225 230 235 240

Glu Leu Leu Asp Val Arg Arg Ser Gln Lys Leu Leu Met Val Lys Asp

245 250 255

Ile Asn Thr Glu Lys Ala Arg Tyr Arg Phe Glu Val Glu Ile Gly Lys

260 265 270

Ser Pro Pro Leu Ser Asp Asp Glu Leu Trp Ser Glu Leu Arg Glu Lys

275 280 285

Ala Ile Glu Leu Arg Asp Glu Trp Arg Leu Glu Asn Arg Lys Ala Phe

290 295 300

Ala Asn Ile Trp Ser Asp Met Val Tyr Gly Ile Ser Leu Phe Leu Leu

305 310 315 320

Met Tyr Phe Asn Gln Ser Lys Val Ala Met Leu Lys Phe Thr Gly Tyr

325 330 335

Lys Leu Leu Asn Asn Ile Ser Asp Ser Gly Lys Ala Phe Leu Ile Ile

340 345 350

Leu Val Ser Asp Ile Leu Leu Gly Tyr His Ser Glu Ser Gly Trp His

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Ser Leu Val Glu Val Ile Leu Glu His Tyr Gly Leu Glu Ala Asp Gln

370 375 380

Ala Ala Ile Thr Phe Phe Val Cys Leu Val Pro Val Ala Leu Asp Val

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Phe Ile Lys Phe Trp Val Tyr Lys Tyr Leu Pro Arg Leu Ser Pro Ser

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Val Gly Asn Ile Leu Asp Glu Ile Lys Arg His

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<210> 2

<211> 2640

<212> DNA

<213>rice Oryza sativa L.

<400> 2

cgggcgtccc cttcccgtgt cttatccact cccaacgttc cattctgtgg atgcttccgc 60

caccgccacc gcccaacgac cagcagcaag cagctcagcc aaggggctcc ccatcgtatc 120

gatccccatt cgcgccgtcc tcttcttctt cttcttcttc tcgattcata ggtgtggttt 180

ccatttcttg ctgtggttgc tggttgaact agcttttgct ttgcttggat tgtgttttga 240

ttgaggggtt tgttagtgtt agcaatctaa cccaagatat ctttgcaatt atatgctcca 300

gttaatccgt caggcttttg ttgcttgctg ctgctgctgc ggtggcgggt ggggaggaag 360

gatgctgatt gcggctgatt tcccgggaga ttcgacggtg tcccgatgcg cattccccgg 420

tttgttctga gtgatgatgc ggtctccgtt tcgtgatctt tgattgatga gccgttccgc 480

ggtaagctct gaaggcggca ttgcgctccg gctcttcgtg gactggagga tccggcgccg 540

gagggtctgc gcatgcaaga tgttcgacac tggttcccaa aggggtaggg tgaagcagct 600

ggtggctttt gccaagaaga ggaggcggcc caagaagcag ccgtcgcggc ggccatggtg 660

gaaggcgtgg ttctcggact ggaacgacga ggaggagagc ttgtctggct ggagggagga 720

tgaggagctg ctcgaggagg ttggcggcga ggaagggctg tcggacgacg agaagtttga 780

gacgtggaag aggaaggccg aggctattgt cgagctgcgg gaggcgcggc aggatgccat 840

gaacgcggaa gggcgctcgt gggaggattg gattggcggg ggcagtagca cagcggggga 900

tggcggcggt gactggggtg gggatttgga tgtgtcagac ctgataacgg atgatcctac 960

agagatagtg agggataaag gtttgattga aactttcaga gactctgtag atgaagatta 1020

caatgacatg ctgtttgaag accgggtttt tctgtatgct tcaacgaatt cggccaaatt 1080

tctagcatta ttgattgtga ttccatgggt gttggatttt ctggttcatg actatgttct 1140

gatgccattt ttggaaaggt acgtccagaa ggtaccactt gctgctgagc tgcttgatgt 1200

aaggcgcagc cagaaactcc tgatggtaaa ggacataaat actgagaaag caagatatcg 1260

ttttgaagta gagattggta aatctcctcc actttctgat gatgagctct ggtcagagtt 1320

acgggaaaaa gcgatagagt tgagagatga atggagatta gaaaaccgaa aagcgtttgc 1380

aaatatctgg tctgacatgg tttatggaat ttccctattc cttcttatgt acttcaacca 1440

gagtaaagtt gcgatgctga aattcacagg atataagtta ctaaataata tttcagacag 1500

tgggaaagca ttccttataa ttctagtttc agacatcctt ctagggtacc attcagagtc 1560

aggttggcat tctttggtag aagtcattct cgagcactac ggccttgaag ccgatcaagc 1620

tgcaatcacc ttctttgttt gtctggttcc tgttgccttg gatgtattta taaaattttg 1680

ggtatacaaa taccttccaa gattatcacc tagtgtgggt aacattttgg atgaaataaa 1740

gcgtcattag agactttcca tcaggaattt gatctgctta attcattcca ttaagaattt 1800

gaaggaaaga ccatccttcc aggtatgtat tcggtgttat atacctttgg cttgtattgt 1860

gttgttatca aaggttcatt agcaactttt tgtgatggtg tttgcgaggt gcttcttgat 1920

tctttcttaa attgttgaaa tattggcttt ctttctttct ttctttcttt ctttttgaac 1980

cagactgtga atgaaacaga gataaacatg tctgattgac aaaagaacta gttacatatt 2040

gctgtgagca gttaatcgtt cattgagaaa gcaaatttga atctgagttt ctgaagaaag 2100

ctactctgct gcagggctca ctcggggcta gtgtctgatt gatgaaatgc caacatagtt 2160

gccatggatt gcaatttcgg aggtcgccct aaccagtgct taccctacaa aaggcataca 2220

taacattctg tagtatatat actctccaca taaaaaaaat gtgctgatta cacatatcat 2280

ccaggaatat tcaggttgat gcggtattca ccgataatga gcgattttgt cccaggatgc 2340

tcacatatcg aaccttctgt gttttcatct ttttgttatg tgaaattcga aacaactatg 2400

tatatgtata caaacagcag aggctgcagt aggaatttgc ctttccccct tttttctcca 2460

ttgtagttcc ttgtggcatt tgtaatgtta tcaaggaatt taggccagtc taagaaaata 2520

atttaggtcc aatttggtgt gcgggatgag actattttct tgttcctgca ggaaattatt 2580

caattcatgc atgtgaggaa atacctatgc ttcaatcaga aattgctccc ttttttacaa 2640

<210> 3

<211> 7229

<212> DNA

<213>rice Oryza sativa L.

<400> 3

ctgcagggga ggagggtgcc gccgcgccag ccaggaggag gatgttggga aggaggaggc 60

ctaccgggga ggagtgtgac gggcagggag gagggcgcca cccgggatga gggcgtcggc 120

aggggaggag ggcgccggcc aggaggagcg gcgtagatcc gtggaggcgc agctagcggg 180

atatttcacg tgaaaaaaga agtgcccgcg tgtctctgcc gcggatgggg gctgcggaga 240

aagcccctgg gactgggggc ctgaggggat aatttggagg ccttcctatt ttaggggctt 300

tagtaggggc cttattggac ctgatttttt agtctcaact ctcaaaatta gtataggggc 360

tgtgtttaga acctctactg gagatgctct aagggaaaat atatggaatg gagcctctgc 420

aaccctaatt agtgtggatc catcttatat atattgtcat cacataatat ttcatgatag 480

gaaaaactat aagctataga actttaacac cgctgaggtc gataacatag cacacctcaa 540

agtttaggtg gatcaaacac gttagccact tgccactagg tcatggctga tcagtgacag 600

tacatggtgg attggtgccg ccctgatgct tagaccattg ctaagatttt cagcccgtgt 660

caaaagacaa aaaaagaggt ttctatttaa caaatactaa ataaacagag tacattttac 720

ttcaatctat ataaatatag aaaaattgca tgaatttcat gtgatcaacg tcaactagag 780

ctataccaaa ggaacttact aactgacaac gatggtgatg atcatgacta agtaggaact 840

aagaagaatt atcatatcat aaagcagtga ttcagttctc cttactaagc gaacaaatat 900

agtggaacct ttactgcctg gaatgcacat tcacatcatc atcatgagct ttagttcttt 960

gaggtagctg tagccgagaa aaaacaagct tgttctagct aaataagtga aagagaaaaa 1020

gagactttaa aaaatagaac aagaagatct taaaattata ttaaaagtgc atttggcttt 1080

gactggatta taaaataaga ggttattttt tgaagggaag atgttgggtt ctggacagca 1140

aagttgggtc acgtgttcca ctatgtcata taggacgctc cgtctttggt tgaggtagtg 1200

cactatgatt aaatttacaa tttttgtaac ctgttattga atttaggttt cgtagaactc 1260

ccatccggtt aatgaaatgg tcgacaattt tgtcgacaag agtttttttc tttaagaatt 1320

ttgtgtaaca caatatataa tatatgtttt gagataggag atacagtagg tagcaaggcc 1380

ctcgtagcga tgtcgcaaac ttacaataga aggtcaggcc aaagttataa aatttgataa 1440

tttgattgta ttaacacttg tggcaatgac cacgtgggac cttctcgagt ctacgacggt 1500

aagtcaggat gtcaaataat caaagtactc cctccgtctc aaaaaaagta aacctaggag 1560

gggatgtgac attttctggt acaacaaatc tagataaagg gctgtccaga tttattgtat 1620

taggagatgt cacatatttg cttgagtttt tttttttgac ggagtacagt ttatatagga 1680

tgttaaaaaa agaagagaga gagagagaaa gatatctctg ggccaaaggt agggtcggac 1740

aaatgggccc aacagccaat acggtactag ttacgtagcc cagcaacatg gcgccatccg 1800

tggaacggaa cgggcgtccc cttcccgtgt cttatccact cccaacgttc cattctgtgg 1860

atgcttccgc caccgccacc gcccaacgac cagcagcaag cagctcagcc aaggggctcc 1920

ccatcgtatc gatccccatt cgcgccgtcc tcttcttctt cttcttcttc tcgattcata 1980

ggtgtggttt ccatttcttg ctgtggttgc tggttgaact agcttttgct ttgcttggat 2040

tgtgttttga ttgaggggtt tgttagtgtt agcaatctaa cccaagatat ctttgcaatt 2100

atatgctcca gttaatccgt caggcttttg ttgcttgctg ctgctgctgc ggtggcgggt 2160

ggggaggaag gatgctgatt gcggctgatt tcccgggaga ttcgacggtg tcccgatgcg 2220

cattccccgg tttgttctga gtgatgatgc ggtctccgtt tcgtgatctt tgattgatga 2280

gccgttccgc ggtaagctct gaaggcggca ttgcgctccg gctcttcgtg gactggagga 2340

tccggcgccg gagggtctgc gcatgcaaga tgttcgacac tggttcccaa aggggtaggg 2400

tgaagcagct ggtggctttt gccaagaaga ggaggcggcc caagaagcag ccgtcgcggc 2460

ggccatggtg gaaggcgtgg ttctcggact ggaacgacga ggaggagagc ttgtctggct 2520

ggagggagga tgaggagctg ctcgaggagg ttggcggcga ggaagggctg tcggacgacg 2580

agaagtttga gacgtggaag aggaaggccg aggctattgt cgagctgcgg gaggcgcggc 2640

aggatgccat gaacgcggaa gggcgctcgt gggaggattg gattggcggg ggcagtagca 2700

cagcggggga tggcggcggt gactggggtg gggatttgga tgtgtcagac ctgataacgg 2760

atgatcctac agagatagtg agggataaag gtttgattga aactttcaga gactctgtag 2820

atgaagatta caatgacatg ctgtttgaag accgggtttt tctgtatgct tcaacgaatt 2880

cggtgagctc tttacctatc ttgttttctg tcaccaaatg ttataaccca ctgtccagtg 2940

ataaccatga gatacttgta atgctttttg aatatgatga actacttcct ccgtttcata 3000

ttttaagtcg ttttgacttt tttcttcatc aaacttcttt aaatttgacc aaatttgtag 3060

aaaaaattag caacacctat cacaccaaat tagtttcatt aaatatagca ttgaatatat 3120

tttgatagta cgtttgtttt atgttggaaa tgttaccata tttttctata aacttggtca 3180

aactaaaaaa agtttgactg gtaaaaaagc caaaacgacg tataatatga aatagaggta 3240

gtactatata atattggagt gcataatata gcaacaaact ttaatcttta caccttacaa 3300

tattatcgaa cgctgagggt ccattttgat ggtaatgatt ttgcatctgg tggctggtgt 3360

attttgacca ttttgatgat taaaaattaa aaatgttaaa aagtgtagca gcattatatt 3420

gttatattaa gaccctagca aattggatct aacaaaagaa agtttgcact ttcaggccaa 3480

atttctagca ttattgattg tgattccatg ggtgttggat tttctggttc atgactatgt 3540

tctgatgcca tttttggaaa ggtaaatcct ctcacagtgt tccattttta ctttcttaac 3600

tttttcccct cacttcttaa tgtaagaaaa tgcattccat gtacacccaa tctgatgttt 3660

aggaatttat aaggtgaagt ttcaagcact gatgatattc tcagtttctc actctctgtg 3720

cttctgaaat tacatgcgat caataatcat ctatgctaaa tgcagaattg attacatgga 3780

acattacaca ataacaggta cgtccagaag gtaccacttg ctgctgagct gcttgatgta 3840

aggcgcagcc agaaactcct gatggtaaag gacataaata ctgagaaagc aagatatcgt 3900

tttgaagtag agattggtaa atctcctcca ctttctgatg atgagctctg gtcagagtta 3960

cgggaaaaag cgtgagaaac actctaacta ttatactttc ttgccttgtt gtaacttctg 4020

ttgatgcact agcattgtct gacatgagca atggaccttc tatcctgcaa tttcatatgt 4080

tcaatcttaa cagatctgct ctgctacttg atcaggatag agttgagaga tgaatggaga 4140

ttagaaaacc gaaaagcgtt tgcaaatatc tggtctgaca tggtttatgg aatttcccta 4200

ttccttctta tgtacttcaa ccagagtaaa gtatgtttta tcagataagc agctttgttt 4260

tttttttcca tctttgattg tgttccaata aatttttcag ctttctgagt gtgcatgtga 4320

gctaagtgat tggttataaa taaacacccc acaagttgac aaatttctgc actattaggc 4380

ctctctcaga gttatccgat ttctgctgtt aatacatagt acatgatgat tttgtgccaa 4440

aagaactgca gtgatgaact gaccagagta ttcccccatt ctatttgtac ctggttatac 4500

atcatacttt tctgacctag tgcagtagta agaccacctc agtttctgca atatgtttga 4560

ttgttagtat catttagtaa cacatacctg gagtccatga tttatgtaca atgaattcat 4620

caatctatct tatccatcat cgtagcctat ttttctctcc cattgaatgc ttagtgttat 4680

cctgcgtaca acttattctt attgcctgct aagctttcag gttgcgatgc tgaaattcac 4740

aggatataag ttactaaata atatttcaga cagtgggaaa gcattcctta taattctagt 4800

ttcagacatc cttctagggt gagttacaat tcatttttct tgagttcata tatacatgtg 4860

gttttagtgt gaacctagtt tgtaacaaag catacactaa acaattgtgc tatttctttt 4920

gctggataat ggatgtcagc aacctaacta aaggcaaata acatgagttc taacctacct 4980

ctctcatgtg ttacctgctt atctaaaaaa tcatacattt aaataagtaa ttcttgttaa 5040

aagaagattc gtccaaaatg ttcaaagaag ttaaagagga ggccagagct ccctccaggg 5100

gagagtgcct tttcttttca gaaactaata tcagggggat actcagcagc atgttttaga 5160

aagcaattca ttatatcttg tcatcatgat tcatgagcga ttttttaaat attctctcat 5220

aagtatggat aagtttgtaa aaatgcatgg aagaaatatt atagctattt tttttgtcag 5280

gaatgaggca gatggttctg tgtttcttct tcgttcgcac ttcaaatatg atcttaatat 5340

ttcttcattt ttctctctct tcaaagatgt ttgtagcatt cgtgtggcag aatcttattt 5400

atcatctagt tagaaactta gaatagtgaa cctaagtggt acagtatccc gttagaagtt 5460

aattcacaca aatgcatatg aactgctttg cgaataatgg aattcttcct gttcatgctt 5520

atgttataca tcatgacatc ttttctgttt caaacaaact atggatagtt tgtaattttg 5580

tttctgcttt ttttcttgtt ttcatgtaaa cgtaacacaa ggttaacatt gttttggttt 5640

gtctgtgatc ttgttgacta tattaacatc atttagctgc attttcacag gtaccattca 5700

gagtcaggtt ggcattcttt ggtagaagtc attctcgagc actacggcct tgaagccgat 5760

caagctgcaa tcaccttctt tgtttgtctg gttcctgttg ccttggatgt atttataaaa 5820

ttttgggtca gtccacttct tgccttcaca caatcaagct aatagggcta tgaacattta 5880

ttttattctt taaaaaacat ttatttcatt ttatcttttt ttaaaaaaga atataagtgt 5940

ttgttaaggc ctcataattt atgactagtt tcacccaaag ttgataacat tatagattca 6000

cgtttggcta ctcagcgcta acatttcaga taatattaac tgaaagtttt atagtctttc 6060

tacaaagtga tttgaacttt attgctgggc cgacagttat taggaagaca accttttgta 6120

gatacaattt catttcctga cactgctctt aaatcaattg ttcaccttgc atgtgctaga 6180

acccattgca ttttttaata actggaaata catgttctct tgataagatc aacaattcga 6240

cgtacttaat tttatttcct tattttccag gtatacaaat accttccaag attatcacct 6300

agtgtgggta acattttgga tgaaataaag cgtcattaga gactttccat caggaatttg 6360

atctgcttaa ttcattccat taagaatttg aaggaaagac catccttcca ggtatgtatt 6420

cggtgttata tacctttggc ttgtattgtg ttgttatcaa aggttcatta gcaacttttt 6480

gtgatggtgt ttgcgaggtg cttcttgatt ctttcttaaa ttgttgaaat attggctttc 6540

tttctttctt tctttctttc tttttgaacc agactgtgaa tgaaacagag ataaacatgt 6600

ctgattgaca aaagaactag ttacatattg ctgtgagcag ttaatcgttc attgagaaag 6660

caaatttgaa tctgagtttc tgaagaaagc tactctgctg cagggctcac tcggggctag 6720

tgtctgattg atgaaatgcc aacatagttg ccatggattg caatttcgga ggtcgcccta 6780

accagtgctt accctacaaa aggcatacat aacattctgt agtatatata ctctccacat 6840

aaaaaaaatg tgctgattac acatatcatc caggaatatt caggttgatg cggtattcac 6900

cgataatgag cgattttgtc ccaggatgct cacatatcga accttctgtg ttttcatctt 6960

tttgttatgt gaaattcgaa acaactatgt atatgtatac aaacagcaga ggctgcagta 7020

ggaatttgcc tttccccctt ttttctccat tgtagttcct tgtggcattt gtaatgttat 7080

caaggaattt aggccagtct aagaaaataa tttaggtcca atttggtgtg cgggatgaga 7140

ctattttctt gttcctgcag gaaattattc aattcatgca tgtgaggaaa tacctatgct 7200

tcaatcagaa attgctccct tttttacaa 7229

<210> 4

<211> 1145

<212> RNA

<213>artificial sequence

<220>

<223>

<400> 4

ggauccgagc ucccucccac gagcgcccuu ccgcguucau ggcauccugc cgcgccuccc 60

gcagcucgac aauagccucg gccuuccucu uccacgucuc aaacuucucg ucguccgaca 120

gcccuuccuc gccgccaacc uccucgagca gcuccucauc cucccuccag ccagacaagc 180

ucuccuccuc gucguuccag uccgagaacc acgccuucca ccauggccgc cgcgacggcu 240

gcuucuuggg ccgccuccuc uucuuggcaa aagccaccag cugcuucacc cuaccccuuu 300

gggaaccagu guacuagugg uacccucgag gucgacagau cugcuagcgg uaaguuacua 360

caaaccuuuu uguacuuaug uuccagugac aauuauuugu guucucaugu uccacguauc 420

acuuuaaugu ucaugguuga ucauuguacc gccucaucuc uuuuagagga ucaagaguau 480

augccugucu uaacuuuuuc uuucucuggu ccagucuuuc cgcugauauu aagaugaauu 540

uuacaacaaa aaaugugcug ccuguguaug aagguucaga ggcauaguuc auaauuuuac 600

ccuguucuca auuaggaaau guauuuugca aggucauaaa gucuugacau ugaugaucaa 660

auauuuucua gagcuaaaau uucauaauca aauaugacag uuccacggca guagauaaag 720

aguacccacu guauauauua guaugaagau uaacacuuga aaaaaccuuu gauuguuccu 780

auaacaccua augauugacu augacacggc uguuucgaga uuuucagauc gauacuagua 840

cacugguucc caaaggggua gggugaagca gcugguggcu uuugccaaga agaggaggcg 900

gcccaagaag cagccgucgc ggcggccaug guggaaggcg ugguucucgg acuggaacga 960

cgaggaggag agcuugucug gcuggaggga ggaugaggag cugcucgagg agguuggcgg 1020

cgaggaaggg cugucggacg acgagaaguu ugagacgugg aagaggaagg ccgaggcuau 1080

ugucgagcug cgggaggcgc ggcaggaugc caugaacgcg gaagggcgcu cgugggaggg 1140

agcuc 1145

<210> 5

<211> 1145

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 5

ggatccgagc tccctcccac gagcgccctt ccgcgttcat ggcatcctgc cgcgcctccc 60

gcagctcgac aatagcctcg gccttcctct tccacgtctc aaacttctcg tcgtccgaca 120

gcccttcctc gccgccaacc tcctcgagca gctcctcatc ctccctccag ccagacaagc 180

tctcctcctc gtcgttccag tccgagaacc acgccttcca ccatggccgc cgcgacggct 240

gcttcttggg ccgcctcctc ttcttggcaa aagccaccag ctgcttcacc ctaccccttt 300

gggaaccagt gtactagtgg taccctcgag gtcgacagat ctgctagcgg taagttacta 360

caaacctttt tgtacttatg ttccagtgac aattatttgt gttctcatgt tccacgtatc 420

actttaatgt tcatggttga tcattgtacc gcctcatctc ttttagagga tcaagagtat 480

atgcctgtct taactttttc tttctctggt ccagtctttc cgctgatatt aagatgaatt 540

ttacaacaaa aaatgtgctg cctgtgtatg aaggttcaga ggcatagttc ataattttac 600

cctgttctca attaggaaat gtattttgca aggtcataaa gtcttgacat tgatgatcaa 660

atattttcta gagctaaaat ttcataatca aatatgacag ttccacggca gtagataaag 720

agtacccact gtatatatta gtatgaagat taacacttga aaaaaccttt gattgttcct 780

ataacaccta atgattgact atgacacggc tgtttcgaga ttttcagatc gatactagta 840

cactggttcc caaaggggta gggtgaagca gctggtggct tttgccaaga agaggaggcg 900

gcccaagaag cagccgtcgc ggcggccatg gtggaaggcg tggttctcgg actggaacga 960

cgaggaggag agcttgtctg gctggaggga ggatgaggag ctgctcgagg aggttggcgg 1020

cgaggaaggg ctgtcggacg acgagaagtt tgagacgtgg aagaggaagg ccgaggctat 1080

tgtcgagctg cgggaggcgc ggcaggatgc catgaacgcg gaagggcgct cgtgggaggg 1140

agctc 1145

Claims

1. it is following A1 the application of protein OsARE1) at least one of to A27):

A1) regulate and control plant low nitrogen resisting；A2) regulate and control the nitrogen content of plant organ；A3) regulate and control the nitrogen content of axis sheath；A4 it) adjusts Control the nitrogen content of plant leaf blade；A5) regulate and control the nitrogen content of plant fringe；A6) regulate and control the biomass of plant；A7) regulate and control plant roots Biomass；A8) regulate and control the root/shoot ratio of plant；A9) regulate and control the chlorophyll content of plant；A10) regulate and control the plant height of plant；A11 it) adjusts Control the tiller number of plant；A12) regulate and control the Nitrogen absorption efficiency of plant；A13) regulate and control the nitrogen utilization efficiency of plant；A14) regulate and control The expression quantity of Nitrogen Absorption marker gene in plant；A15) regulate and control the expression quantity of nitrogen assimilation marker gene in plant；A16 it) adjusts Control the yield of plant；A17) regulate and control the single plant yield of plant；A18) regulate and control the grain number per spike of plant；A19) regulate and control the spike length of plant； A20 the Their First Branch for) regulating and controlling plant obstructs number；A21 the second level branch for) regulating and controlling plant obstructs number；A22) regulate and control AMT1 in plant；1 gene Expression quantity；A23) regulate and control AMT1 in plant；The expression quantity of 2 genes；A24) regulate and control AMT1 in plant；The expression quantity of 3 genes；A25) Regulate and control GS1 in plant；The expression quantity of 2 genes；A26) regulate and control the expression quantity of NADH-GOGAT1 gene in plant；A27) regulation is planted Absorption of the object under low nitrogen growth conditions to nitrogen.

2. application as described in claim 1, it is characterised in that: the protein OsARE1 is a1) or a2) or a3) or a4):

A1) amino acid sequence is protein shown in sequence 1 in sequence table；

A3) amino acid sequence shown in sequence 1 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues And/or the protein with identical biological function that addition obtains；

A4) amino acid sequence limited with sequence 1 in sequence table has 80% or 80% or more identity, derives from rice and tool There is the protein of identical biological function.

3. encode claims 1 or 2 described in protein OsARE1 nucleic acid molecules, be following A1) into A27) at least one Kind:

4. application as claimed in claim 3, it is characterised in that: protein OsARE1 described in described coding claims 1 or 2 Nucleic acid molecules be following b1) b2) or b3) b4) or b5) shown in DNA molecular:

B3) code area DNA molecular as shown in sequence 2 467-1750 from the end 5' in sequence table；

B4) and b1) or b2) or the nucleotide sequence that b3) limits there is 90% or 90% or more identity, derive from rice and volume The DNA molecular of protein OsARE1 described in code claims 1 or 22；

B5) under strict conditions with b1) or b2) or b3) nucleotide sequence hybridization that limits, and encode institute in claims 1 or 2 State the DNA molecular of protein OsARE1.

5. cultivating the method one of genetically modified plants or cultivating the method two of genetically modified plants:

The method one for cultivating genetically modified plants, including imported into plant first of setting out and improve egg described in claims 1 or 2 The content of white matter OsARE1 and/or active substance, the step of obtaining genetically modified plants first；The genetically modified plants first with it is described Plant first of setting out is reduced compared to plant low nitrogen resisting and/or the nitrogen content of plant organ reduces and/or the nitrogen content of axis sheath Reduce and/or plant leaf blade nitrogen content reduce and/or plant fringe nitrogen content reduce and/or plant biomass reduce and/ Or the biomass of plant roots reduces and/or the root/shoot ratio of plant reduces and/or the chlorophyll content of plant reduces and/or plant Plant height reduces and/or the tiller number of plant reduces and/or the Nitrogen absorption efficiency of plant reduces and/or the Nitrogen utility of plant effect Rate reduce and/or plant in Nitrogen Absorption marker gene expression quantity reduce and/or plant in nitrogen assimilation marker gene table Up to amount reduce and/or plant yield reduce and/or plant single plant yield reduce and/or plant grain number per spike reduce and/or The spike length of plant reduces and/or the Their First Branch of plant stalk number reduces and/or the second level branch of plant obstructs in number reduction and/or plant AMT1；AMT1 in the expression quantity reduction of 1 gene and/or plant；AMT1 in the expression quantity reduction of 2 genes and/or plant；3 genes Expression quantity reduce and/or plant in GS1；The expression of NADH-GOGAT1 gene in the expression quantity reduction of 2 genes and/or plant Amount reduces and/or plant reduces the absorption of nitrogen under low nitrogen growth conditions；

The method two for cultivating genetically modified plants, including imported into plant second of setting out and inhibit egg described in claims 1 or 2 The content of white matter OsARE1 and/or active substance, the step of obtaining genetically modified plants second；The genetically modified plants second with it is described Plant second of setting out increases compared to plant low nitrogen resisting and/or the nitrogen content of plant organ increases and/or the nitrogen content of axis sheath Increase and/or plant leaf blade nitrogen content increase and/or plant fringe nitrogen content increase and/or plant biomass increase and/ Or the biomass of plant roots increases and/or the root/shoot ratio of plant increases and/or the chlorophyll content of plant increases and/or plant Plant height increases and/or the tiller number of plant increases and/or the Nitrogen absorption efficiency of plant increases and/or the Nitrogen utility of plant effect Rate increase and/or plant in Nitrogen Absorption marker gene expression quantity increase and/or plant in nitrogen assimilation marker gene table Up to amount increase and/or plant yield increase and/or plant single plant yield increase and/or plant grain number per spike increase and/or The spike length of plant increases and/or the Their First Branch of plant stalk number increases and/or the second level branch of plant obstructs in number increase and/or plant AMT1；AMT1 in the expression quantity increase of 1 gene and/or plant；AMT1 in the expression quantity increase of 2 genes and/or plant；3 genes Expression quantity increase and/or plant in GS1；The expression of NADH-GOGAT1 gene in the expression quantity increase of 2 genes and/or plant Amount increases and/or plant increases the absorption of nitrogen under low nitrogen growth conditions.

6. method as claimed in claim 5, it is characterised in that: described " to inhibit the content and/or work of the protein OsARE1 Property substance " be special RNA molecule；Shown in the special RNA molecule such as formula (I): A_Reversely-Y-A_{It is positive}(I)；The A_{It is positive}Sequence The single strand RNA molecule transcribed for the 200-500bp DNA fragmentation in the gene of code for said proteins OsARE1；It is described A_ReverselySequence and the A_{It is positive}Sequence reverse complemental；The Y is the A_{It is positive}With the A_ReverselyBetween intervening sequence, in sequence On, the Y and the A_{It is positive}And the A_ReverselyIt is not complementary.

7. method as claimed in claim 6, it is characterised in that:

The implementation method of described " importing special RNA molecule in plant second of setting out " is as follows: the importing of specific DNA molecular first is set out Plant second；Shown in the specific DNA molecular first such as formula (II): SEQ_Reversely-X-SEQ_{It is positive}(II)；The SEQ_{It is positive}Sequence be coding 200-500bp DNA fragmentation in the gene of the protein OsARE1；The SEQ_ReverselySequence and the SEQ_{It is positive}Sequence Reverse complemental；The X is the SEQ_{It is positive}With the SEQ_ReverselyBetween intervening sequence, in sequence, the X and the SEQ_{It is positive} And the SEQ_ReverselyIt is not complementary.

8. method as claimed in claim 5, it is characterised in that: described " to inhibit the content and/or work of the protein OsARE1 Property substance " be Plant Genome editor carrier；The carrier of the Plant Genome editor contains gRNA encoding gene；It is described The target DNA that gRNA is identified in plant is the DNA fragmentation of coding protein OsARE1.

9. being planted described in special RNA molecule, specific DNA molecular, special recombinant plasmid or claim 8 described in claim 6 The carrier of object genome editor；

The specific DNA molecular is specific DNA molecular first or specific DNA molecular second described in claim 7；

The specific DNA molecular second includes DNA fragmentation one, intervening sequence and DNA fragmentation two；The sequence of the DNA fragmentation one is 200-500bp DNA fragmentation in the gene of code for said proteins OsARE1；The DNA fragmentation two and the DNA fragmentation one Sequence reverse complemental；

The special recombinant plasmid is the recombinant plasmid containing the specific DNA molecular.

10. plant breeding method one or plant breeding method two:

The plant breeding method one includes the following steps: to increase protein OsARE1 described in claims 1 or 2 in plant Content and/or activity, so that plant low nitrogen resisting reduces and/or the nitrogen content of plant organ reduces and/or axis sheath Nitrogen content reduces and/or the nitrogen content of plant leaf blade reduces and/or the nitrogen content of plant fringe reduces and/or the biomass of plant drop Low and/or plant roots biomass reduce and/or the root/shoot ratio of plant reduces and/or the chlorophyll content of plant reduces and/or The plant height of plant reduces and/or the tiller number of plant reduces and/or the Nitrogen absorption efficiency of plant reduces and/or the nitrogen of plant Utilization efficiency reduce and/or plant in Nitrogen Absorption marker gene expression quantity reduce and/or plant in nitrogen assimilation mark base The expression quantity of cause reduces and/or the yield of plant reduces and/or the single plant yield of plant reduces and/or the grain number per spike of plant reduces And/or the spike length of plant reduces and/or the Their First Branch of plant stalk number reduces and/or the second level branch of plant stalk number is reduced and/or planted AMT1 in object；AMT1 in the expression quantity reduction of 1 gene and/or plant；AMT1 in the expression quantity reduction of 2 genes and/or plant；3 GS1 in the expression quantity reduction of gene and/or plant；NADH-GOGAT1 gene in the expression quantity reduction of 2 genes and/or plant Expression quantity reduces and/or plant reduces the absorption of nitrogen under low nitrogen growth conditions；

The plant breeding method two includes the following steps: to reduce protein OsARE1 described in claims 1 or 2 in plant Content and/or activity, so that plant low nitrogen resisting increases and/or the nitrogen content of plant organ increases and/or axis sheath Nitrogen content increases and/or the nitrogen content of plant leaf blade increases and/or the nitrogen content of plant fringe increases and/or the biomass of plant increases Add and/or the biomass of plant roots increase and/or plant root/shoot ratio increase and/or plant chlorophyll content increase and/or The plant height of plant increases and/or the tiller number of plant increases and/or the Nitrogen absorption efficiency of plant increases and/or the nitrogen of plant Utilization efficiency increase and/or plant in Nitrogen Absorption marker gene expression quantity increase and/or plant in nitrogen assimilation mark base The expression quantity of cause increases and/or the yield of plant increases and/or the single plant yield of plant increases and/or the grain number per spike of plant increases And/or the spike length of plant increases and/or the Their First Branch of plant stalk number increases and/or the second level branch of plant stalk number increases and/or plants AMT1 in object；AMT1 in the expression quantity increase of 1 gene and/or plant；AMT1 in the expression quantity increase of 2 genes and/or plant；3 GS1 in the expression quantity increase of gene and/or plant；NADH-GOGAT1 gene in the expression quantity increase of 2 genes and/or plant Expression quantity increases and/or plant increases the absorption of nitrogen under low nitrogen growth conditions.