CN117567576A

CN117567576A - NAR2.1 protein variants and uses thereof

Info

Publication number: CN117567576A
Application number: CN202311440750.6A
Authority: CN
Inventors: 傅向东; 李翔宇; 刘慧�; 高秀华; 次仁吉; 刘学英
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2023-11-01
Filing date: 2023-11-01
Publication date: 2024-02-20

Abstract

The invention discloses NAR2.1 protein variants for improving plant nitrogen utilization efficiency, biomass and seed yield and application thereof, wherein the amino acid sequences of the NAR2.1 protein variants are shown as SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30. Serine at 186 th and 189 th positions of NAR2.1 protein in arabidopsis and serine (OsNAR 2.1 is S186, S189, taNAR2.1 is S178 and S181) which is conserved and corresponds to homologous NAR2.1 protein in wheat and rice are mutated into a simulated non-phosphorylated form, such as mutated into alanine or phenylalanine, and the generated NAR2.1 protein variant can remarkably increase NAR2.1 protein stability and membrane localization thereof, enhance high affinity nitrate absorption transporter activity, and further improve nitrogen absorption and utilization efficiency.

Description

NAR2.1 protein variants and uses thereof

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a NAR2.1 protein variant and application thereof, wherein the NAR2.1 protein variant is introduced into plants through transgenosis or is generated in the plants through gene editing, so that nitrate absorption and carbon nitrogen metabolism balance of the plants are promoted, and photosynthesis, nitrogen fertilizer utilization efficiency, biomass and seed yield of the plants are improved.

Background

Nitrogen (N) is a macronutrient element necessary for plant growth and development and resistance to biotic and abiotic stresses, and is also a constituent of amino acids, proteins, nucleic acids, chlorophyll and various primary and secondary metabolites. The nitrogen content is critical to the growth and development of plants and the formation of crop yield. Nitrogen deficiency results in altered root system architecture, reduced photosynthesis, reduced plant biomass and yield (kishakumar et al 2020). In order to increase crop yield, a large amount of nitrogen fertilizer needs to be applied in agricultural production, however, nitrogen fertilizer utilization efficiency (NUE) of many plants is only 30-50% (Guo et al, 2010), and most of nitrogen fertilizer is not effectively utilized by plants, which increases not only production cost (Sutton et al, 2011), but also causes serious environmental pollution problems (Ju et al, 2009). Therefore, increasing NUE in plants is a key strategy to reduce nitrogen fertilizer input and promote sustainable development in agriculture. NUE refers to the ratio of grain yield to soil nitrogen supply, and can be specifically classified into nitrogen uptake efficiency (NUpE) and nitrogen physiological utilization efficiency (NUtE). NUpE refers to the removal of NO from soil by plants ₃ ^- And NH ₄ ⁺ The ability of ions, NUtE, refers to the ability of a plant to produce grain yield using N.

Plant nitrogen metabolism includes the overall process of uptake, transport, assimilation and reuse of nitrogen (McAllister et al 2012; krapp et al 2015). Plants rely mainly on two high affinity nitrate uptake systems (HATS) and one low affinity nitrate uptake system (LATS) to uptake nitrate. HATS are in turn classified into combined high affinity nitrate uptake systems (cha ts) and induced high affinity nitrate uptake systems (iHATS). cHATS and iHATS operate in low nitrate concentration external media in the saturation range of 0.2-0.5mM, while LATS operates in higher external nitrate media. Nitrate uptake from HATS and LATS is achieved by nitrate transporters of the NRT2 and NRT1 families, respectively (Forde et al 2000;Miller et al, 2007). The nitrogen state in the plant body can negatively feed back and regulate the intake of HATS and LATS to nitrate, and further regulate the expression and protein activity of nitrate intake genes.

The arabidopsis NRT2 family has 7 members (attrt 2.1 to attrt 2.7), of which attrt 2.1 is an important component of the inducible high affinity nitrate transport system and is also the root most important high affinity nitrate transporter. The high affinity nitrate transport capacity of the nrt2.1 single mutant was reduced by 56% and that of the nrt2.1-nrt2.2 double mutant was reduced by 75% (Li et al, 2007). High concentrations of nitrate lead to phosphorylation of the S501 site of NRT2.1, thereby inhibiting its high affinity nitrate transport activity, but do not affect the interaction with NAR2.1, and NRT2.1 localized on the membrane is reduced, while total protein abundance is unchanged (Jacquot et al 2020). The PP 2C-class phosphatase CEPH (CEPD-induced phosphatase) is capable of dephosphorylating S501 of NRT2.1, thereby activating its high affinity transport activity (Ohkubo et al, 2021). Nitrate at low concentrations can cause phosphorylation of the S28 site of NRT2.1, making NRT2.1 protein more stable (Zou et al, 2019). HPCAL1 (hydro-oxide-reduced Ca) ²⁺ The invents like 1) is able to phosphorylate the S21, S28 sites of NRT2.1 and thus regulate the interaction of NRT2.1 with NAR2.1 (Li et al 2020). The G119R mutation of NRT2.1 can affect NRT2.1 mediated initiation and elongation of the root at the high carbon/nitrogen ratio downside (Little et al 2005). The L85Q mutation of NRT2.1 inhibits NRT2.1 interaction with NAR2.1 (kotus et al 2016). In addition, NRT2.1 interacts with PIN7 and antagonizes PIN 7-regulated auxin efflux, thereby regulating root growth at very low nitrogenLong (Wang et al 2023).

Previous studies have shown that NRT2 proteins mostly require a relatively small chaperone NAR2 (Nitrate assimilation related genes 2) to assist in functioning (Tong et al 2005;Okamoto et al, 2006). AtNAR2.1 is a single transmembrane small protein with 210 amino acids, the protein structure being mostly extracellular. NAR2.1 is capable of promoting film formation on NRT2.1 and maintaining the stability of NRT2.1 on film (Wirth et al, 2007), both of which may form tetrameric complexes on film (Tsay et al, 2007; yong et al, 2010). Mutation of aspartic acid (D) at position 105 of atnar2.1 to asparagine (N) reduces its interaction with NRT2.1, resulting in reduced nitrate uptake (Kawachi et al, 2006). The extracellular domain of osnar2.1 is critical for its interaction with osnrt2.3a, and the R100G and D109N mutations of osnar2.1 inhibit its interaction with osnrt2.3a (Liu et al, 2014).

There are 4 NRT2s genes (osnrt 2.1-2.4) and 2 NAR2s genes (osnar 2.1-2.2) in the rice genome (Katayama et al, 2009; yan et al, 2011; wei et al, 2018). OsNAR2.1 is a chaperone protein for OsNRT2.1, osNRT2.2 and OsNRT2.3a, is mainly expressed in rice root epidermal cells and stems, and may regulate the transport of auxin from stems to roots, affecting lateral root formation, while functioning in nitrate signaling (Araki and Hasegawa et al.,2006; cai et al.,2008, feng et al.,2011; huang et al., 2015). It has now been found that 14 NRT2 family members and 9 NAR2 family members out of the 3 subgenomic groups of wheat are found. TaNAR2 can be classified into three types, named TaNAR2.1/2.2/2.3, respectively, and only TaNAR2.1-6B, taNAR2.2-5B and TaNAR2.3-6B have been reported to date, wherein TaNAR2.1-6B can interact with TaNRT2.5-3B and transport nitrate (Li et al 2020).

Nitrate transporters as a "gate" for controlling plant uptake, transport of nitrate are critical to Nitrogen Use Efficiency (NUE) and growth and development of plants. On the basis of fully knowing the structure, the regulation and control functions and the signal transduction network involved in the nitrate transporter, a certain strategy is adopted to apply the nitrate transporter to crop high-yield and nitrogen high-efficiency utilization breeding, so that the nitrate transporter is a direction worthy of exploration. Studies have shown that overexpression of OsNRT2.1 or OsNRT2.3 alone in rice does not increase yield (possibly because constitutive overexpression consumes significant amounts of energy and disrupts nitrogen transport in leaves), whereas overexpression of OsNAR2.1, co-overexpression of OsNRT2.3a and OsNAR2.1, and driving expression of OsNAR2.1, osNRT2.1 or OsNRT2.3a with the own promoter of OsNAR2.1 all increase rice yield and NUE (Chen et al, 2016; chen et al, 2017; chen et al, 2020). In addition, overexpression of OsNAR2.1 in rice can affect the flowering phase and drought tolerance of rice in addition to increasing yield and NUE (Chen et al, 2019; chen et al, 2020). Overexpression of tanrt2.5-3B in wheat can increase yield and nitrogen accumulation (Li et al 2020).

Nitrogen uptake is the initiation of plant nitrogen metabolism and is finely regulated by carbon metabolism. Precise regulation of nitrogen uptake by carbon metabolites is important for maintaining carbon-nitrogen balance in plants. Researchers have discovered by a series of physiological experiments as early as twenty years that photosynthesis-generated sugar and transportation thereof to root systems can regulate nitrate absorption of root systems, and increase CO in the air ₂ The concentration also promotes the absorption of nitrate (Gastal and Saugier,1989;Delhon et al, 1996), but the molecular mechanism therein is not clear. NRT2.1 is one of the important high affinity nitrate transporters in roots of plants under external nitrate stress conditions. Previous studies have shown that NRT2.1 is finely regulated by carbon and nitrogen status in plants at both mRNA and protein levels. The NRT2.1 is taken as a target gene, and analysis of regulation and control of photosynthesis products on nitrogen absorption is an important entry point for researching carbon-nitrogen metabolic balance and cooperative regulation and control of plants.

There is currently little research on the molecular mechanisms by which photosynthesis products regulate high affinity nitrate absorption and is focused primarily on the transcriptional level (Lejay et al, 2008; gilin et al, 2007;de Jong et al, 2014; chen et al, 2016). Laugier et al found that in the 35 S:NRT 2.1 transgenic line, although mRNA for NRT2.1 was constitutively highly expressed, its protein levels and high affinity nitrate transport activity were still significantly reduced in the dark, suggesting that regulation of NRT2.1 protein levels by photosynthesis products plays a major role in controlling high affinity nitrate uptake (Laugier et al 2012). However, the specific molecular mechanism therein is not yet known. Our earlier studies also showed that NRT2.1 had a significant sugar response at the mRNA and protein levels, and that an increase in sucrose concentration in the medium could significantly promote the accumulation of mRNA and protein for NRT 2.1.

In summary, current studies on the regulation of NRT2.1-NAR2.1 complex by photosynthesis products (sugar) have focused on the transcriptional level, while the molecular mechanism of regulation of NRT2.1 protein stability at the posttranscriptional level is not yet known, and there are no reports on the study of regulation of NAR2.1 protein by photosynthesis products (sugar). The molecular mechanism of regulating and controlling NRT2.1 protein stability and high affinity nitrate absorption by NAR2.1 protein mediated photosynthesis products (sugar) can provide theoretical and technical support for crop high yield and nitrogen efficient breeding.

Disclosure of Invention

The inventor uses a mutant screening system based on a Luciferase reporter gene (Luciferase), genetically mutagenizes and screens an arabidopsis mutant sine1 which enables AtNRT2.1 to no longer respond to sugar level change at the protein level, and experiments such as map cloning and genetic complementation prove that the sine1 is caused by the mutation of the AtNAR2.1 (Nitrate assimilation related genes 2.1) gene. The invention proves that the sugar can promote the stability and membrane localization of the AtNAR2.1 protein. The AtNAR2.1 protein is phosphorylated by plant energy receptor SnRK1.1 (sub non-interfering-1-related protein kinases 1.1), and serine at positions 186 and 189 of the AtNAR2.1 transmembrane domain mediates this phosphorylation; phosphorylation at the S186, S189 sites results in degradation of the atnar2.1 protein via the 26S ubiquitinated proteasome pathway; mutation of both sites S186, S189 to alanine (A) in non-phosphorylated form, resulting in protein variant AtNAR2.1 ^{S186A S189A} (abbreviated as AtNAR2.1 ^2A ) Enhanced protein stability from the cytoplasm to the cell membrane and enhanced interaction with NRT 2.1; mutation of both sites S186 and S189 to the phosphorylated form of glutamic acid (E) gives rise to protein variant AtNAR2.1 ^S186E ^S189E (abbreviated as AtNAR2.1 ^2E ) Is decreased, the localization from the cytoplasm to the cell membrane is diminished, and the interaction with NRT2.1 is diminished. The invention is thatFurther, it was demonstrated that AtNAR2.1 was overexpressed in Arabidopsis thaliana ^2A The response of NRT2.1 protein level to sugar concentration is obviously enhanced, namely the promotion effect of sugar on NRT2.1 protein accumulation and membrane localization is enhanced, and AtNAR2.1 is over-expressed ^2E The result of (2) is the opposite. In addition, the S186 and S189 sites are highly conserved in higher and lower plants and are identical to Arabidopsis AtNAR2.1 ^2A Similarly, protein variant crnar2.1 ^2A (Chlamydomonas reinhardtii), mpNAR2.1 ^2A (liverwort), osNAR2.1 ^2A (Rice), taNAR2.1 ^2A Protein stability and membrane localization of (wheat) are enhanced, and it is speculated that this conserved site is highly conserved in function. The present inventors have found that AtNAR2.1 is overexpressed ^2A The growth of the root system of the arabidopsis thaliana and the absorption of nitrate can be promoted, and the biomass and the seed yield of plants can be increased under the condition of reducing the application amount of nitrogen fertilizer; gene editing rice line OsNAR2.1 ^S189A 、OsNAR2.1 ^2A The root system nitrate absorption rate, the tiller number, the biomass and the seed yield of the plant are obviously increased compared with the wild type plant; osNAR2.1 self promoter drives OsNAR2.1 ^2A Compared with the transgenic rice driven by the OsNAR2.1 self promoter to express, the expressed transgenic rice has higher biomass and seed yield; overexpression of TaNAR2.1 ^2A Or AtNAR2.1 ^2A Can promote the absorption of nitrate by the root system of the wheat and improve the biomass and the seed yield; root-specific promoters drive TaNAR2.1 ^2A The expression can also promote the absorption of nitrate by the wheat root system and can obviously increase the yield.

In summary, the research of the inventor analyzes the Sugar-SnRK1.1-NAR2.1-NRT2.1 signal transduction path, and discovers that S186 and S189 sites conserved by NAR2.1 protein in the path are important sites for regulating NAR2.1 protein stability and subcellular localization, are key sites for coordinating carbon and nitrogen metabolism and interaction of carbon and nitrogen signal transduction, and are potential target sites for improving plant nitrogen fertilizer utilization efficiency and seed yield by using NAR 2.1.

In general, the present invention aims to provide a method for promoting nitrate uptake by plant root systems (including but not limited to Arabidopsis thaliana, rice, wheat, chlamydomonas reinhardtii, dipalea, etc.) and increasing plant biomass and seed production An amount of a protein variant and uses thereof. In particular, the invention relates to protein variants NAR2.1 ^2A 、NAR2.1 ^S186A 、NAR2.1 ^S189A (including but not limited to AtNAR2.1 ^2A 、AtNAR2.1 ^S186A 、AtNAR2.1 ^S189A 、OsNAR2.1 ^2A 、OsNAR2.1 ^S186A 、OsNAR2.1 ^S189A 、TaNAR2.1 ^2A 、TaNAR2.1 ^S186A 、TaNAR2.1 ^S189A 、CrNAR2.1 ^2A 、CrNAR2.1 ^S186A 、CrNAR2.1 ^S189A 、MpNAR2.1 ^2A 、MpNAR2.1 ^S186A 、MpNAR2.1 ^S189A ) The application in improving the carbon-nitrogen metabolism efficiency of plants and increasing biomass and seed yield.

In one embodiment, protein variants AtNAR2.1 are provided that promote high affinity nitrate transport in plants and increase biomass and seed yield in plants ^2A 、AtNAR2.1 ^S186A 、AtNAR2.1 ^S189A 、OsNAR2.1 ^2A 、OsNAR2.1 ^S186A 、OsNAR2.1 ^S189A 、TaNAR2.1 ^2A 、TaNAR2.1 ^S186A 、TaNAR2.1 ^S189A 、CrNAR2.1 ^2A 、CrNAR2.1 ^S186A 、CrNAR2.1 ^S189A 、MpNAR2.1 ^2A 、MpNAR2.1 ^S186A 、MpNAR2.1 ^S189A The corresponding amino acid sequences are shown as SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30, and the corresponding nucleotide sequences are shown as SEQ ID NO. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27 and 29.

The present invention provides in a first aspect the use of a protein variant which promotes high affinity nitrate transport in plants and increases biomass and seed yield in plants, wherein the protein comprises one of the amino acid sequences selected from the group consisting of:

1) 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

2) Amino acid sequences which differ from the amino acid sequences shown in SEQ ID NOs 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 by substitution, deletion and/or insertion of one or more (e.g. 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) amino acid residues, but which are active as proteins consisting of the amino acid sequences shown in SEQ ID NOs 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

3) Amino acid sequences which are at least 70%, preferably at least 80%, more preferably at least 90% identical, in particular at least 95% or 98% or 99% identical, to the amino acid sequences shown in SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and which are active in the same manner as proteins consisting of the amino acid sequences shown in SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

4) An active fragment comprising any one of the amino acid sequences 1) to 3).

In some embodiments of the invention, the nucleotide sequence encoding a protein variant that promotes nitrate uptake by plant roots and increases plant biomass and seed yield comprises one selected from the group consisting of:

1) 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29;

2) Nucleotide sequences which differ from the nucleotide sequences shown in SEQ ID NO 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 by substitution, deletion and/or insertion of one or more (e.g., 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) nucleotide sequences, but which encode a protein having the same activity as the nucleotide sequences shown in 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 encode a protein;

3) A nucleotide sequence which is at least 70%, preferably at least 80%, more preferably at least 90% identical, in particular at least 95% or 98% or 99% identical to the nucleotide sequence shown in SEQ ID No. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and which encodes a protein whose activity is identical to the activity of a protein encoded by the nucleotide sequence shown in SEQ ID No. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29;

4) A nucleotide sequence which differs in sequence from SEQ ID NO. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 due to the degeneracy of the genetic code;

5) An active fragment comprising any one of nucleotide sequences 1) to 4).

6) Nucleotide sequences which hybridize under medium stringency conditions, preferably high stringency hybridization conditions, with the complement of the nucleotide sequence shown in SEQ ID No. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29.

7) A nucleotide sequence complementary to any one of the nucleotide sequences 1) to 5).

A second aspect of the present invention provides a method for improving nitrogen uptake, biomass and seed yield in plant roots by gene editing techniques, the method comprising targeting NAR2.1 gene, site-directed substitution of serine (S) at conserved sites 186 and 189 of NAR2.1 for alanine (A) by gene editing techniques to produce NAR2.1 mutated to alanine at two sites ^2A Protein variants, and NAR2.1 mutated to alanine at a single site ^S186A 、NAR2.1 ^S189A Protein variants, thereby promoting nitrogen uptake by plants, including but not limited to rice, and increasing biomass and seed yield in plants, including but not limited to prime-directed editing techniques.

In some embodiments of the invention, the gene-edited rice strain with enhanced nitrate uptake and increased yield obtained by gene editing techniques is OsNAR2.1 ^S189A And OsNAR2.1 ^2A . Specifically, the 186 th and 189 th silk amino acids of OsNAR2.1 are replaced to alanine by gene editing technology, so as to obtain single point mutated gene editing strain OsNAR2.1 ^S189A And a gene editing strain OsNAR2.1 with double site mutation ^2A Compared with the wild type, the root systems of the two gene editing strains are more developed, the absorption of nitrate is stronger, and the plant height, tillering, the number of primary branches, the number of secondary branches, the number of grains per ear and the yield of single plant seeds are all increased.

A third aspect of the invention provides a method of enhancing nitrogen uptake and yield by genetic transformation techniquesA method of increasing the amount of a crop comprising overexpressing tanar2.1 in the crop, respectively ^2A And AtNAR2.1 ^2A Or specifically express tanar2.1 in roots of crops ^2A Cultivating a crop with enhanced nitrogen uptake and increased yield, wherein the crop is wheat.

In a preferred embodiment of the present invention, the wheat with enhanced nitrogen uptake and increased yield is tanar2.1 ^2A Or AtNAR2.1 ^2A Is characterized by over-expression of transgenic wheat and specific expression of TaNAR2.1 in root ^2A Is a transgenic wheat of (a). Specifically, the overexpression vector pUbi:TaNAR2.1 is used by wheat genetic transformation technology ^2A 、pUbi::AtNAR2.1 ^2A A root-specific expression vector pSAD1:: taNAR2.1 ^2A Transferring into wheat respectively to obtain transgenic wheat. pUbi:TaNAR2.1 compared with the wild type ^2A 、pUbi::AtNAR2.1 ^2A pSAD 1:TaNAR2.1 ^2A The transgenic wheat has developed root system, strong nitrate absorption, and increased plant height, biomass and seed yield.

In a fourth aspect, the present invention provides a method for transforming AtNAR2.1 by genetic transformation techniques ^2A A method for transferring into a plant to obtain a plant with enhanced nitrogen uptake and increased biomass and seed yield, said method comprising introducing AtNAR2.1 by genetic transformation techniques ^2A The over-expression vector of (2) is transferred into a plant to obtain a transgenic plant with enhanced nitrogen uptake and increased biomass and seed yield, wherein the plant is Arabidopsis thaliana.

In a preferred embodiment of the invention, the transgenic Arabidopsis thaliana with enhanced nitrogen uptake and increased biomass and seed yield is over-expressed AtNAR2.1 ^2A Is a transgenic Arabidopsis thaliana. Specifically, p35S:: atNAR2.1 is obtained by Arabidopsis genetic transformation technique ^2A The GFP overexpression vector is transferred into nar2.1-c1 (CRISPR/cas 9 knockout mutant of AtNAR2.1 gene) to obtain transgenic arabidopsis thaliana. Compared with the wild type, the transgenic arabidopsis thaliana has the advantages of more developed root system, stronger absorption of nitrate, and early ripening, and increased biomass and seed yield.

In a fifth aspect, the invention provides a method of transforming CrNAR2.1 by genetic transformation techniques ^2A And MpNAR2.1 ^2A A method for transferring into a plant to obtain a plant with enhanced nitrogen uptake and increased biomass and seed yield, said method comprising introducing CrNAR2.1 by genetic transformation techniques ^2A And MpNAR2.1 ^2A The over-expression vectors of (2) are respectively transferred into plants to obtain plants with obviously promoted growth and development, wherein the plants are arabidopsis thaliana.

In a preferred embodiment of the invention, the transgenic Arabidopsis thaliana with enhanced nitrogen uptake and increased biomass and seed yield is over-expressed CrNAR2.1 ^2A And MpNAR2.1 ^2A Is a transgenic Arabidopsis thaliana. Specifically, pNAR2.1:CrNAR2.1 is obtained by Arabidopsis genetic transformation technique ^2A GFP and pNAR2.1:: mpNAR2.1 ^2A The GFP overexpression vectors are respectively transferred into nar2.1-c1 (CRISPR/cas 9 knockout mutant of AtNAR2.1 gene) to obtain transgenic arabidopsis thaliana. The growth and development of transgenic arabidopsis thaliana is significantly promoted compared to wild type.

In a preferred embodiment, atNAR2.1 ^2A 、OsNAR2.1 ^2A 、TaNAR2.1 ^2A 、CrNAR2.1 ^2A 、MpNAR2.1 ^2A The related sequences are shown in SEQ ID NO. 1,2,7,8, 13, 14, 19, 20, 25, 26, and are specifically shown in the following Table 1.

TABLE 1 AtNAR2.1 ^2A 、OsNAR2.1 ^2A 、TaNAR2.1 ^2A 、CrNAR2.1 ^2A 、MpNAR2.1 ^2A Related sequence names and sources thereof

SEQ ID NOs:	Name of the name	Source
			1	AtNAR2.1 ^2A CDS sequences	Arabidopsis Col-0
2	AtNAR2.1 ^2A Amino acid sequence	Arabidopsis Col-0
			7	OsNAR2.1 ^2A CDS sequences	Wuyun japonica No. 7 (WYJ 7)
8	OsNAR2.1 ^2A Amino acid sequence	Wuyun japonica No. 7 (WYJ 7)
			13	TaNAR2.1 ^2A CDS sequences	Wheat field
14	TaNAR2.1 ^2A Amino acid sequence	Wheat field
			19	CrNAR2.1 ^2A CDS sequences	Chlamydomonas reinhardtii
20	CrNAR2.1 ^2A Amino acid sequence	Chlamydomonas reinhardtii
			25	MpNAR2.1 ^2A CDS sequences	Liverwort with money
26	MpNAR2.1 ^2A Amino acid sequence	Liverwort with money

The present invention provides a recombinant host cell comprising a recombinant construct or a gene editing vector according to the invention or having integrated in its genome a protein variant AtNAR2.1 according to the invention encoding a protein that promotes nitrate uptake and increases biomass and seed yield in plants ^2A 、OsNAR2.1 ^2A 、TaNAR2.1 ^2A 、CrNAR2.1 ^2A 、MpNAR2.1 ^2A Is a polynucleotide sequence of (a). The host cell may be selected from plant cells or microbial cells, such as e.coli cells or agrobacterium cells, preferably plant cells, most preferably rice cells. The cells may be isolated, cultured or part of a plant.

The polynucleotides provided by the invention (i.e., encoding protein variants AtNAR2.1 that promote nitrate uptake in plants and increase biomass and seed yield in plants) ^2A 、OsNAR2.1 ^2A 、TaNAR2.1 ^2A 、CrNAR2.1 ^2A 、MpNAR2.1 ^2A OsNAR2.1 modified by polynucleotide or gene editing technology ^S189A And OsNAR2.1 ^2A ) Or the use of the polypeptide or the recombinant construct of the invention or the gene editing vector of the invention or the recombinant host cell of the invention for improving plant traits (e.g., increasing crop seed yield) and nitrogen fertilizer utilization efficiency.

The invention provides the AtNAR2.1 ^2A 、OsNAR2.1 ^2A 、TaNAR2.1 ^2A 、CrNAR2.1 ^2A 、MpNAR2.1 ^2A For promoting nitrate uptake and increasing biomass and seed yield in plants, but is not limited thereto.

The invention provides a method for cultivating rice with high yield and high nitrogen utilization, which comprises the following steps: 1. guide editing technique using prime editingDirectly carrying out T-to-G single base substitution editing on a corresponding codon TCC of serine at 186 th position and 189 rd position of OsNAR2.1 protein on a rice genome to obtain a protein variant OsNAR2.1 with single amino acid site mutation ^S189A And double amino acid site mutated OsNAR2.1 ^2A Gene editing rice; 2. the rice genetic transformation technology is utilized to obtain pOsNAR2.1:OsNAR2.1 ^2A And stabilizing the transgenic rice. The rice obtained by these two methods is preferably rice having improved nitrogen utilization efficiency and seed yield.

The invention provides a method for high-yield and nitrogen-efficient wheat variety, comprising the following steps: 1. obtaining over-expression TaNAR2.1 by using wheat genetic transformation technology ^2A Or AtNAR2.1 ^2A Is a transgenic wheat strain; 2. obtaining the specific expression of TaNAR2.1 in root by wheat genetic transformation technology ^2A The transgenic line pSAD 1:TaNAR2.1 ^2A . The wheat plants obtained are preferably wheat plants with improved nitrogen utilization efficiency and seed yield.

The following are definitions of some of the terms used in the present invention. Unless otherwise indicated, the terms used herein have meanings known to those of ordinary skill in the art.

In the invention, "AtNAR2.1" and "AtNAR2.1" refer to NAR2.1 genes and encoding proteins thereof in arabidopsis thaliana, "OsNAR2.1" and "OsNAR2.1" refer to NAR2.1 genes and encoding proteins thereof in rice, and "TaNAR2.1" refer to NAR2.1 genes and encoding proteins thereof in wheat; "AtNAR2.1 ^2A And AtNAR2.1 ^2A "refers to protein variants of NAR2.1 protein in Arabidopsis thaliana with serine at positions 186 and 189 mutated to alanine and corresponding genes," OsNAR2.1 ^2A Sum OsNAR2.1 ^2A "refers to protein variant of NAR2.1 protein 186 th and 189 th serine mutated into alanine in rice and corresponding gene thereof," TaNAR2.1 ^2A And TaNAR2.1 ^2A "refers to protein variants of NAR2.1 protein in wheat in which serine at 178 and 181 is mutated into alanine and corresponding genes. "CrNAR2.1 ^2A Sum CrNAR2.1 ^2A "refers to protein obtained by mutating serine at 229 and 232 of NAR2.1 protein in Chlamydomonas reinhardtii into alanineVariants and their corresponding genes. "MpNAR2.1 ^2A Sum MpNAR2.1 ^2A "refers to a protein variant in which serine at positions 189 and 193 of NAR2.1 protein in liverwort is mutated into alanine and the corresponding gene. In addition, the serine of the present invention can be mutated to phenylalanine, and the same technical effects as those of the mutation to alanine can be obtained.

"related"/"operably linked" refers to two nucleic acid sequences that are physically or functionally related. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence encoding an RNA or protein if the promoter or regulatory DNA sequence and the DNA sequence encoding the RNA or protein are operably linked or positioned such that the regulatory DNA sequence will affect the expression level of the coding or structural DNA sequence.

A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to or associated with a nucleic acid sequence encoding mRNA or expressed as a protein such that the regulatory nucleic acid sequence is capable of regulating transcription or expression of the associated nucleic acid sequence. The regulatory nucleic acid sequences of the chimeric gene are not normally operably linked to the relevant nucleic acid sequence as found in nature.

A "coding sequence" is a nucleic acid sequence transcribed into RNA, such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably, the RNA is subsequently translated in an organism to produce the protein.

In the context of the present invention, "corresponding to" means that when nucleic acid coding sequences or amino acid sequences of NAR2.1 genes or proteins of different origin are aligned with each other, the nucleic acids or amino acids that "correspond" to some counting positions are aligned with these positions, but not necessarily in these precise digital positions with respect to the specific NAR2.1 nucleic acid sequence or coding amino acid sequence. Likewise, when a nucleic acid sequence or amino acid encoding sequence of a particular NAR2.1 is aligned with a nucleic acid sequence or amino acid encoding sequence of a reference NAR2.1, the nucleic acid or amino acid in the particular NAR2.1 sequence "corresponding" to some counted positions of the reference NAR2.1 sequence is aligned with these positions of the reference NAR2.1 sequence, but not necessarily the nucleic acid or amino acid in these precise numerical positions of the respective nucleic acid encoding sequence or amino acid sequence of the particular NAR2.1 protein.

As used herein, an "expression cassette" means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operably linked to a nucleotide sequence of interest operably linked to a termination signal. Typically, it also contains sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be naturally occurring, but obtained in recombinant form for heterologous expression. However, in general, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell, and must be introduced into the host cell or precursor to the host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, wherein the inducible promoter initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, such as plants, the promoter may also be specific for a particular tissue, or organ or stage of development.

A "gene" is a defined region located within the genome that comprises, in addition to the aforementioned coding nucleic acid sequences, other predominantly regulatory nucleic acid sequences responsible for expression of the coding portion, i.e., transcriptional and translational control. Genes may also contain other 5 'and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

A "heterologous" nucleic acid sequence is a nucleic acid sequence that is not naturally associated with the host cell into which it is introduced, comprising multiple copies of the naturally occurring nucleic acid sequence that are not naturally occurring.

A "homologous" nucleic acid sequence is a nucleic acid sequence that is naturally associated with the host cell into which it is introduced.

An "isolated" nucleic acid molecule or isolated protein is one that exists artificially separated from its natural environment and is therefore not a natural product. The isolated nucleic acid molecule or protein may be present in purified form or may be present in a non-natural environment such as, for example, a recombinant host cell or a transgenic plant.

"native gene" refers to a gene that is present in the genome of an untransformed cell.

The term "naturally occurring" is used to describe objects that may be found in nature, as opposed to artificially created objects. For example, proteins or nucleotide sequences present in organisms (including viruses) that may be isolated from natural sources and that have not been intentionally modified by the laboratory are "naturally occurring".

A "nucleic acid molecule" or "nucleic acid sequence" is a linear fragment of single or double stranded DNA or RNA that can be isolated from any source. In the context of the present invention, preferably, the nucleic acid molecule is a DNA fragment. "nucleic acid molecule" is also known as a polynucleotide molecule.

A "plant" is any plant at any stage of development, in particular a seed plant.

"plant cells" are the structural and physiological units of plants, including protoplasts and cell walls. The plant cells may be in the form of isolated individual cells or cultured cells, or as higher organized units such as, for example, plant tissues, plant organs or parts of whole plants.

"plant material" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cells or tissue cultures, or any other part or product of a plant.

"plant organ" is a well-defined and distinctly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

"plant tissue" as used herein means a group of plant cells organized into structural and functional units. Including any tissue of the plant in the plant or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and/or functional units. The use of this term in combination with or alone with any particular type of plant tissue as listed above or encompassed by this definition is not meant to exclude any other type of plant tissue.

A "promoter" is an untranslated DNA sequence upstream of a coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also contain other elements as modulators of gene expression.

A "protoplast" is an isolated plant cell that has no cell wall or only a portion of a cell wall.

"regulatory element" refers to a sequence involved in controlling the expression of a nucleotide sequence. The regulatory element comprises a promoter operably linked to the nucleotide sequence of interest and a termination signal. They also typically contain sequences required for proper translation of the nucleotide sequence.

The phrase "substantially identical" in two nucleic acid or protein sequence alignments refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90%, even more preferably 95% and most preferably at least 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or visual inspection. Preferably, substantial identity exists over a region of the sequence that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences in at least about 150 residues are substantially identical. In a particularly preferred embodiment, the sequences are substantially identical throughout the length of the coding region. Furthermore, substantially identical nucleic acid or protein sequences have substantially identical functions.

For sequence comparison, typically, a sequence is compared to the test sequence as a reference sequence. When using the sequence comparison algorithm, the detection and reference sequences are input into a computer, the coordinates of the subsequences are designated if necessary, and the parameters of the sequence algorithm program are designated. The sequence comparison algorithm will then calculate the percent sequence identity of the test sequence relative to the reference sequence based on the selected program parameters.

For example, optimal alignment of sequences for comparison can be performed by the localized homology algorithm of Smith & Waterman, adv.appl.Math.2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J.mol.biol.48:443 (1970), by the similarity search method of Pearson & Lipman, proc.Nat' l.Acad.Sci.USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA, genetics Computer Group,575Science Dr., madison, wis. In Wisconsin Genetics software package) or by visual inspection (see generally Ausubel et al, infra).

An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J.mol.biol.215:403-410 (1990). Software for BLAST analysis is publicly available through the national center for Biotechnology information (http:// www.Ncbi.nlm.nih.gov /). The algorithm comprises the following steps: high scoring sequence pairs (HSPs) are first identified by identifying short words of length W in the search sequence that match or meet some positive-valued threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al, 1990). These initial neighborhood word hits act as clues to begin searches to find longer HSPs containing them. These word hits will then extend as far as possible in both directions of each sequence until the accumulated alignment score is no longer increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for matching residues in pairs; always greater than zero) and N (penalty score for mismatched residues; always less than zero). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Word hit extension in each direction stops when the cumulative alignment score falls back by an amount X from the maximum value obtained, the cumulative score reaches or falls below zero due to one or more negative-scoring residue alignments, or either of the two sequences reaches the end. The parameters W, T and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length value (W) 11, an expected value (E) 10, a cut-off value of 100, m= 5,N = -4 and a comparison of the two strands as default values. For amino acid sequences, the BLASTP program uses the word Length value (W) 3, the expected value (E) 10 and the BLOSUM62 scoring matrix as default values (see, henikoff & Henikoff, proc. Natl. Acad. Sci. USA 89:10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., karlin & Altschul, proc. Nat' l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability of a match between two nucleotide or amino acid sequences occurring by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability of a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Another indicator that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase "specifically hybridizes" refers to the binding of a molecule to only a specific nucleotide sequence under stringent conditions to form a duplex or hybridization when the sequence is present in a complex mixture (e.g., of total cells) of DNA or RNA. "substantial binding" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and involves fewer mismatches that can be tolerated by decreasing the stringency of the hybridization medium to achieve the desired detection of the target nucleic acid sequence.

"stringent hybridization conditions" and "stringent hybridization rinse conditions" in the context of nucleic acid hybridization assays such as Southern and Northern hybridization are sequence dependent and differ under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A great deal of guidance for nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic AcidProbes, section I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, new York. Generally, for a particular sequence at a defined ionic strength and pH, high stringency hybridization and rinse conditions are selected to be about 5℃below the thermal melting point (Tm). Typically, under "stringent conditions" a probe will hybridize to its target subsequence, but not to other sequences.

Tm is the temperature (under defined ionic strength and pH conditions) at which 50% of the target sequence hybridizes to a perfectly matched probe. For a particular probe, very stringent conditions are chosen to be equal to Tm. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1mg heparin at 42℃overnight. An example of high stringency rinse conditions is 72℃with 0.15M NaCl for about 15 minutes. An example of stringent rinse conditions is a rinsing in 0.2 XSSC at 65℃for 15 minutes (see, sambrook, infra, description of SSC buffers). Typically, a low stringency rinse is performed prior to a high stringency rinse to remove background probe signals. For double helices of, for example, more than 100 nucleotides, an example of a medium stringency rinse is 45℃with 1 XSSC rinse for 15 minutes. For double helices of, for example, more than 100 nucleotides, an example of a low stringency rinse is a 40℃4-6 XSSC rinse for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically include a salt concentration of less than about 1.0M Na ion, typically about 0.01 to 1.0M Na ion concentration (or other salt), at pH7.0 to 8.3, typically at a temperature of at least about 30 ℃. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, in a specific hybridization assay, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe indicates detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions remain substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created with the maximum codon degeneracy permitted by the genetic code.

The following are examples of hybridization/rinse conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention: the reference nucleotide sequence and the homologous nucleotide sequence are preferably at 50℃with 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization in 1mM EDTA, rinsing in 50 ℃,2 XSSC, 0.1% SDS, more desirably in 50 ℃,7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization in 1mM EDTA, rinsing in 50 ℃,1 XSSC, 0.1% SDS, more desirably in 50 ℃,7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization in 1mM EDTA, rinsing in 50℃0.5 XSSC, 0.1% SDS, preferably 7% sodium dodecyl sulfate (SDS at 50℃)，0.5M NaPO ₄ Hybridization in 1mM EDTA, rinsing in 50 ℃,0.1 XSSC, 0.1% SDS, more preferably, 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO at 50 DEG ₄ Hybridization in 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃.

Another indicator that two nucleic acid sequences or amino acid sequences are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross-reactive or specifically binding to the protein encoded by the second nucleic acid. Thus, a protein is typically substantially identical to a second protein, e.g., where the two proteins differ only by conservative substitutions.

"synthetic" refers to a nucleotide sequence that contains structural features that are not found in the native sequence. For example, artificial sequences more closely resembling the g+c content and normal codon distribution of dicotyledonous and/or monocotyledonous genes are considered synthetic.

"transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism, in particular "transformation" means the stable integration of a DNA molecule into the genome of the organism of interest.

"transformed/transgenic/recombinant" refers to a host organism, such as a bacterium or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the host genome or the nucleic acid molecule may be present as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues, or plants are understood to include not only the end product of the transformation process but also the transgenic progeny thereof. "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain a heterologous nucleic acid molecule.

The terms "polynucleotide", "polynucleotide molecule", "polynucleotide sequence", "coding sequence", "Open Reading Frame (ORF)" and the like as used herein include single or double stranded DNA and RNA molecules, and may comprise one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences comprising exons and introns, chemically synthesized DNA and RNA sequences, and sense and corresponding antisense strands.

Methods for producing and manipulating the polynucleotide molecules and oligonucleotide molecules disclosed herein are known to those of skill in the art and can be accomplished according to the described recombinant techniques (see Maniatis et al, 1989, molecular cloning, laboratory Manual, cold spring harbor laboratory Press, cold spring harbor, N.Y., ausubel et al, 1989, current techniques in molecular biology, greene Publishing Associates & Wiley Interscience, NY; sambrook et al, 1989, molecular cloning, laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, N.Y., innis et al (eds.), 1995, PCR strategies, academic Press, inc., san Diego, and Erlich (Ind.), 1992, PCR techniques, oxford university Press, new York).

"plant transformation" refers to the expression of at least one exogenous gene in a plant in order to confer one or more desirable phenotypic traits on the transformed plant.

In a particularly preferred embodiment, a variant of a gene according to the invention which regulates the rate of high affinity nitrate uptake as well as biomass and seed yield of plants is expressed in higher organisms such as plants. In particular, the nucleotide sequence of the gene variant of the invention which regulates the high affinity nitrate uptake rate as well as biomass and seed yield of plants can be inserted into an expression cassette, which is then preferably stably integrated in the genome of said plants.

The plant transformed according to the invention may be a monocot or dicot plant including, but not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, summer squash, apple, pear, wen, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, beet, sunflower, canola, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, arabidopsis and woody plants such as conifers and deciduous trees. Particularly preferred are rice, wheat, arabidopsis.

Once the desired nucleotide sequence has been transformed into a particular plant species, it can be propagated in that species or transferred into other varieties of the same species, including in particular commercial varieties, using conventional breeding techniques.

Preferably, the nucleotide sequences of the invention are expressed in transgenic plants, thereby causing biosynthesis of proteins in transgenic plants that correspondingly control the plant's high affinity nitrate uptake rate as well as biomass and seed yield traits. In this way, transgenic plants with improved traits of interest can be produced. In order to express the nucleotide sequences of the present invention in transgenic plants, the nucleotide sequences of the present invention may require modification and optimization. All organisms have a specific codon usage preference, as is known in the art, which can be altered to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the invention unchanged. Moreover, high levels of expression in plants can be best achieved from coding sequences having a GC content of at least about 35%, preferably greater than about 45%, more preferably greater than 50%, and most preferably greater than about 60%. Although the preferred gene sequences can be expressed adequately in monocot and dicot species, the sequences can be modified to accommodate the specific codon bias and GC content bias of monocot or dicot species, as these bias have been shown to be different (Murray et al, nucl. Acids Res.17:477-498 (1989)). In addition, the nucleotide sequence may be screened for the presence of non-conventional splice sites that cause truncation of the message. All changes required in these nucleotide sequences, such as those described above, were made by PCR and synthetic gene construction using site-directed mutagenesis techniques well known in the art using the methods described in published patent applications EP 0 385 962 (Monsanto), EP 0 359 472 (Lubrizol) and WO 93/07278 (Ciba-Geigy).

Drawings

The above technical features of the present invention will be described in detail below with reference to the accompanying drawings.

FIG. 1 identification of Arabidopsis mutant sine1 with impaired high sugar response of the NRT2.1 gene. (A) LUC signal was decreased in the sine1 mutant. Comparison of endogenous NRT2.1 expression levels in (B) #11-13 and sine 1. (C) #11Comparison of endogenous NRT2.1 and LUC protein content in-13 and sine 1. (D) SINE1 gene structure schematic. HSP82 served as an internal reference for loading. Seed with #11-13 and sine1 in low nitrogen (LN, 0.2mM KNO) containing 0%, 1%, 3% Suc (sucrose) ₃ ) Germinating on the culture medium for 6 days, and taking the root of the seedling for experiment. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

Figure 2.Nrt2.1 protein stability and membrane localization response to sugar is dependent on NAR2.1. (A) The sugar promotes localization of the NAR2.1-GFP fusion protein from the cytoplasm to the cell membrane. (B) The sugar promotes localization of the NRT2.1-GFP fusion protein from the cytoplasm to the cell membrane. (C) The fluorescent intensity and membrane localization of NRT2.1-GFP in the nar2.1-c1 background lost the sugar response. (D) The sugar enhances the stability of the NAR2.1 protein and promotes its accumulation on the cell membrane. (E) Sugar enhances the stability of NRT2.1 protein and promotes its accumulation on cell membranes. (F) The total protein amount of NRT2.1-GFP and membrane localized protein amounts lost the sugar response in the nar2.1-c1 background. Transgenic plants were enriched with LN (0.2 mM KNO) at 0%, 0.5%, 3% sucrose concentration ₃ ) Roots were stained with FM-4-64 (a membrane selective red fluorescent dye) and the GFP signal from the maturation zone was observed under a laser scanning confocal microscope while root tissues were taken to detect NRT2.1-GFP and NAR2.1-GFP total protein and membrane-localized protein levels, respectively, for 6 days on the medium. T stands for NRT2.1-GFP, NAR2.1-GFP Total protein (Total), M stands for NRT2.1-GFP, NAR2.1-GFP Membrane localization protein (Membrane). Actin is an internal reference for total protein loading, bip is an internal reference for membrane protein loading.

FIG. 3.SnRK1.1 is able to phosphorylate NAR2.1 and negatively regulate the protein stability of NAR2.1 and NRT 2.1. (A) Two-photon fluorescence complementation (BiFC) experiments of tobacco prove that SnRK1.1 interacts with NAR2.1 and does not interact with NRT 2.1. The scale is 20. Mu.m. (B) SnRK1.1 is capable of phosphorylating NAR2.1 in vitro. ATP-gamma-p 32 labeled autoradiography (top) and protein gel electrophoresis after dye-stripping (bottom). (C) SnRK1.1 reduces NRT2.1 and NAR2.1 protein content. Ler, p 35S-analysis of NRT2.1 and NAR2.1 protein content in the SnRK1.1 transgenic Arabidopsis thaliana lines and the SnRK1.1-RNAi transgenic Arabidopsis thaliana lines. HSP82 serves as an internal reference for protein loading. Plants were grown on HN containing 1% Suc10mM KNO ₃ )、LN(0.2mM KNO ₃ ) The medium was germinated directly for 6 days, and root tissue was taken for experiments.

FIG. 4.SnRK1.1 phosphorylates the NAR2.1 protein at positions S186 and S189 to regulate the stability of NAR2.1 protein. (A) The phosphorylation state of the S186, S189 sites affects the content of NAR2.1 protein and subcellular localization. A number of possible phosphorylation sites of NAR2.1 protein were mutated to mimic the phosphorylated form and dephosphorylated form, respectively, and subcellular localization of each protein variant was analyzed. The fusion proteins of the different protein variants and GFP are expressed by using a tobacco epidermal cell transient expression system. The scale is 20. Mu.m. (B) The S186 and S189 sites are key phosphorylation sites of SnRK1.1 for regulating the stability and the protein localization of NAR 2.1. NAR2.1-GFP and NAR2.1 were expressed separately in the context of the p35S: snRK1.1 transgenic line ^2A GFP protein, GFP signal intensity and subcellular localization analysis of the fusion protein, scale 50 μm. (C) Ubiquitination degradation of NAR2.1 relies on phosphorylation at positions S186 and S189. NAR2.1, p35S ^2E NAR2.1 after treatment of GFP transgenic line MG132 ^2E GFP protein content assay. Plants were high nitrogen at 0% sucrose (HN, 10mM KNO) ₃ ) The roots were treated with 50. Mu.M MG132 for 8 hours after germination and growth on the medium for 6 days. (D) The phosphorylation states of the S186 and S189 sites mediate the regulation of NAR2.1 protein stability by photosynthetic carbon assimilation products. The transgenic lines were first grown in high nitrogen medium (HN, 10mM KNO) with 1% sucrose ₃ ) Germinating for 4 days, and transplanting to low nitrogen medium (LN, 0.2mM KNO ₃ ) And treated with 25 μm DCMU for various times.

FIG. 5 function of double site phosphorylation at S186, S189. (A) - (D) S186, S189 dual site phosphorylation significantly affects NAR2.1 protein stability and membrane localization. (A) NAR2.1 in tobacco epidermal cells ^2A GFP and NAR2.1 ^2E GFP signal intensity and subcellular localization analysis. Expression of NAR2.1-GFP and NAR2.1 by using transient expression system of tobacco epidermal cells ^2A -GFP、NAR2.1 ^2E GFP. The scale is 20. Mu.m. (B) NAR2.1, p35S ^2A GFP and p 35S::: NAR2.1 ^2E NAR2.1 in root cells of GFP transgenic lines ^2A GFP and NAR2.1 ^2E GFP signal of GFP fusion protein. The scale is 50 μm。(C),(D)p35S::NAR2.1 ^2A GFP and p 35S::: NAR2.1 ^2E NAR2.1 in GFP transgenic lines ^2A GFP and NAR2.1 ^2E Analysis of the content of GFP total protein (C) and membrane protein (D). Plants were grown in high nitrogen medium (HN, 10mM KNO) with 0% sucrose ₃ ) The cells were germinated and grown for 6 days, and GFP signal and protein content were measured. T represents Total protein (Total), and M represents Membrane protein (Membrane). Actin is an internal reference for total protein loading, bip is an internal reference for membrane protein loading. (E) S186, S189 dual site phosphorylation significantly affects NAR2.1 interaction with NRT 2.1. NAR2.1 in tobacco BiFC experiment ^2A 、NAR2.1 ^2E Interaction with NRT2.1, respectively. The scale is 20. Mu.m.

FIG. 6S 186, S189 double site phosphorylation affects self and the sugar response of NRT2.1 proteins. (A) S186, S189 dual site phosphorylation significantly affected the fluorescence intensity and subcellular localization of NAR 2.1-GFP. NAR2.1-c1 background p35S:: NAR2.1-GFP, p35S:: NAR2.1 ^2A -GFP、p35S::NAR2.1 ^2E GFP transgenic lines were grown for 6 days on LN (0.2 mM KNO3) medium containing 0%, 1%, 3% sucrose and assayed for GFP signal. The scale is 50. Mu.m. (B) S186, S189 double site phosphorylation significantly affected the sugar response of NRT 2.1. NAR2.1, p35S ^2A -GFP、p35S::NAR2.1 ^2E LUC signal analysis of pNRT2.1:gNRT2.1-LUC transgenic lines against GFP background. Plants were grown on LN (0.2 mM KNO) with 0%, 1%, 3% sucrose ₃ ) LUC signal was detected after 6 days of growth on the medium.

FIG. 7 NAR2.1 ^2A Promote the growth and development of the root system of the arabidopsis thaliana. And (A) plant root phenotype analysis. The scale is 1cm. And (B) performing phenotype analysis on the overground parts of plants. The scale is 0.5cm. (C) analysis of plant biomass in the upper part. (D) - (F) statistical analysis of plant main root length (D), lateral root number (E), total lateral root length (F). NAR2.1-GFP, p35S:: NAR2.1 in the context of Col-0, NAR2.1-c1 mutant, NAR2.1-c1 ^2A GFP and p35S NAR2.1 ^2E GFP transgenic lines, first in 1% sucrose HN (10 mM KNO ₃ ) The seedlings were grown on the medium for 4 days and transferred to LN (0.2 mM KNO) with 1% sucrose ₃ ) The culture medium was grown for 10 days. Values in the figures are mean ± standard deviation (n=6). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 8 NAR2.1 ^2A The nitrate absorption rate of the root system of the arabidopsis is obviously improved. (A) HN of 1% sucrose (10 mM KNO) ₃ )、LN(0.2mM KNO ₃ ) Nitrate uptake rate analysis of plants on the medium. (B) LN of 0%, 3% sucrose (0.2 mM KNO) ₃ ) Nitrate uptake rate analysis of plants on the medium. NAR2.1-GFP, p35S:: NAR2.1 in the context of Col-0, NAR2.1-c1, and NAR2.1-c1 ^2A GFP, and p 35S::: NAR2.1 ^2E GFP transgenic plants were first treated with 1% sucrose HN (10 mM KNO) ₃ ) After growing on the culture medium for 4 days, transplanting seedlings to different culture mediums respectively, and growing on the different culture mediums for 10 days, and measuring the nitrate absorption rate. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 9.NAR2.1 regulates carbon/nitrogen ratio at low nitrogen in Arabidopsis. C/N ratio analysis of seedlings. Plants were first grown on HN (10 mM KNO) ₃ ) The seedlings were transferred to HN (10 mM KNO) after growing on the medium for 4 days ₃ )、LN(0.2mM KNO ₃ ) Growing on the culture medium for 10 days, measuring the carbon and nitrogen content, and calculating the C/N ratio. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 10 NAR2.1 ^2A Influence on the growth and development of Arabidopsis thaliana. (A) - (C) NAR2.1 ^2A Promote early flowering and maturing of arabidopsis thaliana. (A) phenotypic analysis of Arabidopsis thaliana at different growth phases. The scale is 5cm. (C) analysis of the number of rosette leaves of Arabidopsis. NAR2.1-GFP, p35S:: NAR2.1 in the context of Col-0, NAR2.1-c1, and NAR2.1-c1 ^2A GFP and p 35S::: NAR2.1 ^2E GFP transgenic plants were first grown on LN (0.2 mM KNO) ₃ ) The culture medium is germinated for 4 days, and then the seedlings are transplanted to a greenhouse with long sunlight (16 h light/8 h dark) for 20 days (A) and 40 days (B) for soil culture growth. Values in the figures are mean ± standard deviation (n=5). The significance analysis adopts a Tukey multiple comparison test method. (D) (E) NAR2.1 ^2A Improving biomass and seed yield of Arabidopsis thaliana. Biomass (D) and seed yield (E) analysis. NAR2.1-GFP, p35S:: NAR2.1 in the context of Col-0, NAR2.1-c1, and NAR2.1-c1 ^2A GFP and p 35S::: NAR2.1 ^2E GFP transgenic plants were grown on long days (16 h light/8 h black)Dark) in the greenhouse, the soil cultures with different nitrogen concentrations are grown to maturity, and the biomass of the overground parts and the weight of seeds are measured. The different nitrogen concentrations are 15% N, 30% N, 60% N and 100% N, each watering is correspondingly weighed 0.3g, 0.6g, 1.2g and 2.0g of potassium nitrate fertilizer of the Israel sea Baolifeng brand are dissolved in 2L of water, and the water is poured for 5 times in the whole growth period. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 11 NAR2.1 homologous protein sequence alignment and phylogenetic tree analysis. (A) NAR2.1 homologous protein sequence alignment in different species. Red arrows indicate conserved S186, S189 sites. (B) NAR2.1 homologous protein phylogenetic tree analysis of different species.

FIG. 12 effects of phosphorylation status at S186, S189 on NAR2.1 protein stability, subcellular localization and function are highly conserved in different plants. (A) Effect of phosphorylation status of the S186, S189 sites of NAR2.1 homologous proteins of different species on NAR2.1 protein stability and subcellular localization. And expressing NAR2.1 proteins and protein variants of different plants by using a tobacco epidermal cell transient expression system to fuse GFP, and observing GFP signals under a confocal microscope. The scale is 20. Mu.m. (B) (C) phenotypic analysis of CrNAR2.1 and MpNAR2.1 transgenic complementation lines. MpNAR2.1 and CrNAR2.1 transgenic complementary plants in LN (0.2 mM KNO) against nar2.1-c1 background ₃ ) And growing on the culture medium for 5-6 days. The scale is 1cm.

FIG. 13 overexpression of TaNAR2.1 ^2A And AtNAR2.1 ^2A Improving photosynthesis of wheat, nitrogen fertilizer utilization efficiency and yield. And (C) and (D) phenotype analysis of wheat seedlings. Fielder, pUbi:: taNAR2.1 ^2A pUbi:: atNAR2.1 ^2A The transgenic wheat is firstly cultured in clear water to a two-leaf one-heart period, and then is respectively transplanted to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) After 2 weeks of growth in culture, phenotyping (A), upper and lower biomass analysis (C), and root-cap ratio analysis (D) were performed. The scale is 10cm. (B) Wheat root system ¹⁵ NO ₃ ^- Absorption rate analysis. Fielder, pUbi:: taNAR2.1 ^2A pUbi:: atNAR2.1 ^2A The transgenic wheat is firstly grown in clear water until two leaves and one heart stageThen transplanting the seedlings to HN (2 mM KNO) ₃ ) Measurement after 6 days of growth in culture ¹⁵ NO ₃ ^- Absorption rate. (E) C/N ratio analysis. Fielder, pUbi:: taNAR2.1 ^2A pUbi:: atNAR2.1 ^2A The transgenic wheat is firstly cultured in clear water to a two-leaf one-heart period, and then is respectively transplanted to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) The culture medium was grown for 2 weeks. (F) - (I) overexpression of TaNAR2.1, taNAR2.1 ^2A 、AtNAR2.1 ^2A Phenotype analysis of the transgenic lines in maturity (F), plant height (G), biomass (H) and seed yield analysis (I). The scale is 15cm. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 14 root-specific expression of TaNAR2.1 ^2A Improving photosynthesis of wheat, nitrogen fertilizer utilization efficiency and yield. The test materials are Fielder and pSAD 1:TaNAR2.1 ^2A 、pSAD1::TaNAR2.1 ^2E Transgenic wheat. (A), (C) and (D) phenotyping of the hydroponic seedlings. Firstly, raising seedlings of different materials in clear water to a two-leaf one-heart period, and respectively transplanting the seedlings to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) Phenotype analysis (A), upper and lower biomass analysis (C), root-cap ratio analysis (D) were performed after growth in culture for 2 weeks. The scale is 10cm. (B) ¹⁵ NO ₃ ^- Absorption rate analysis. Firstly, raising seedlings in clear water to a two-leaf one-heart period, and then transplanting the seedlings to HN (2 mM KNO) ₃ ) The culture medium was grown for 6 days. (E) C/N ratio analysis. Firstly, raising seedlings of different materials in clear water to a two-leaf one-heart period, and respectively transplanting the seedlings to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) The culture medium was grown for 2 weeks. (F) - (I) plant maturity phenotyping (F), plant height (G), biomass (H) and seed yield analysis (I). The scale is 15cm. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 15 Gene editing OsNAR2.1 ^2A Improving the utilization efficiency and the yield of the nitrogen fertilizer of the rice. The test materials are Wuyunjing 7 (WYJ 7) and WYJ7 background gene editing mutant OsNAR2.1 ^S189A And OsNAR2.1 ^2A . (A) Schematic of editing of single base substitution at S186 and S189 sites of OsNAR 2.1. (B) Plants and methods of making the sameAnd (3) analyzing the protein content of the OsNAR2.1. Actin serves as an internal reference for protein loading. (C) phenotype analysis of the hydroponic seedlings. The scale is 10cm. (D) Rice root system ¹⁵ NO ₃ ^- Absorption rate analysis. (E) analysis of the biomass of the aerial parts and root systems of the rice. (F) root cap ratio analysis of rice. And (G) analyzing the carbon-nitrogen ratio value. Seeds of different rice materials are first grown on HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) Germinating in nutrient solution for 1 week, and transplanting to HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) And LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) The culture medium was grown for 4 weeks. Values in the figures are mean ± standard deviation (n=3). The significance analysis adopts a Tukey multiple comparison test method. And (H) - (N) analysis of agronomic traits in maturity. (H) agronomic trait analysis. The scale is 10cm. (I) plant height comparison. (J) comparison of tiller number. (K) comparing the number of the primary branches. (L) secondary branch number comparison. (M) number of particles per cluster comparison. (N) comparison of individual yield. Values in the figures are mean ± standard deviation (n=6). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 16 self-promoter-driven OsNAR2.1 ^2A Expression can improve rice yield. The rice materials tested were WYJ7, osnar2.1 and pOsNAR2.1 in the WYJ7 background:: osNAR2.1, pOsNAR2.1::: osNAR2.1 ^2A 、pOsNAR2.1::OsNAR2.1 ^2E Transgenic rice of transgenic plants. (A) analysis of rice plant types. The scale is 10cm. And (B) comparing the plant heights in the mature period. (C) comparison of tiller number. (D) comparing the number of the primary branches. (E) comparing the secondary branch number. (F) comparing the number of particles per ear. (G) comparison of seed yield of individual plants. Values in the figures are mean ± standard deviation (n=6). The significance analysis adopts a Tukey multiple comparison test method.

FIG. 17 OsNAR2.1 ^2A Site-directed substitution vector (modified PE-P3) Gene synthesis template 1 and Gene synthesis template 2 schematic.

FIG. 18 OsNAR2.1 ^2A Site-directed replacement vector (modified PE-P3) final vector construction schematic.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

The experimental methods in the following examples are conventional experimental methods unless otherwise specified. Reagents, kits and laboratory instruments used in the experiments are all available from biological instruments and reagent companies unless otherwise specified.

Example 1: identification of nar2.1 as a mutant with impaired high sugar response of the NRT2.1 Gene

The pNRT2.1:: gNRT2.1-LUC vector, genetic transformation of Col-0 Arabidopsis, resulted in single copy insertion and stable inheritance of transgenic line #11-13 (FIG. 1A). The NRT2.1 promoter sequence, genomic sequence, and luciferase reporter gene (LUC) sequence were cloned, respectively, using genomic DNA of Col Arabidopsis and a laboratory-maintained vector containing a luciferase reporter gene (LUC) as templates according to the following primers:

pNRT2.1-EcoR I-F:cggaattctgatagtctttgtagataggattcgag(SEQ ID NO:35)

pNRT2.1-BamH I-R:gcggatccTgttataaaatatttcaagtttctttgc(SEQ ID NO:36)

gNRT2.1-seamless-F:acttgaaatattttataacaggatccatgggtgattctactggtgagc(SEQ ID NO:37)

gNRT2.1-seamless-R:atgtttttggcatcttccattctagaaacattgttgggtgtgttctc(SEQ ID NO:38)

LUC-Xba I-F:gctctagaatggaagatgccaaaaacat(SEQ ID NO:39)

LUC-Pst I-R:ggctgcagttacacggcgatcttgccgc(SEQ ID NO:40)

the PCR reaction procedure was: (1) 98℃for 3 min, (2) 98℃for 30 s, (3) 56℃for 30 s, (4) 68℃for 3 min, (5) 68℃for 5 min, (2) - (4) 33 cycles.

The NRT2.1 promoter sequence and LUC gene sequence were ligated to pCAMBIA2300 vector using restriction enzyme ligation (DNA restriction enzyme and T4DNA ligase were purchased from NEB Co., ecoR I: #R3101S, bamH I: #R3136S, xba I: #R0145S, pst I: #R3140S, T4DNA ligase: #M0202S), and the homologous recombination seamless method was used-Uni Seamless Cloning and AThe NRT2.1 genomic sequence gNRT2.1 was ligated to a vector (Donahue et al, 2002) to construct a pNRT2.1:: gNRT2.1-LUC vector, CU101 from the full gold company. Transforming Col-0 by using an agrobacterium-mediated method, and screening and identifying to obtain a positive stable transgenic line #11-13. Further, ion beam mutagenesis was performed on #11-13 and was performed on LN (0.2 mM KNO ₃ ) Mutants with reduced LUC signal were screened on the medium (as in example 2). Designated as sine1 (sugar-insolive NRT2.1 expression 1) mutant (FIG. 1A). mRNA levels of NRT2.1 in the sine1 mutant were not affected, but protein levels were significantly reduced (FIGS. 1B, C). SINE1 gene was cloned using the MutMap method (Abe et al 2012), and gene NAR2.1 encoding a high affinity nitrate transporter (AT 5G 50200) was found to have a deletion of base G AT position 67 on its first exon, resulting in premature termination of translation to amino acid 25 (FIG. 1D).

Example 2: atNRT2.1 protein accumulation and Membrane localization was dependent on AtNAR2.1 on sugar response

CDS sequences of NRT2.1 and NAR2.1 were amplified using the following primers with the cDNA of Col-0 as a template:

NRT2.1-attB1-F:acaagtttgtacaaaaaagcaggcttcatgggtgattctactggtgagc(SEQ ID NO:41)

NRT2.1-attB2-R:accactttgtacaagaaagctgggtcaaacattgttgggtgtgttctc(SEQ ID NO:42)

NAR2.1-attB1-F:acaagtttgtacaaaaaagcaggcttcatggcgatccagaagatcctctt(SEQ ID NO:43)

NAR2.1-attB2-R:accactttgtacaagaaagctgggtctttgctttgctctatcttggcctt(SEQ ID NO:44)

the PCR reaction procedure was: (1) 98℃for 3min, (2) 98℃for 30s, (3) 56℃for 30s, (4) 68℃for 2min, (5) 68℃for 5min, (2) - (4) 33 cycles.

CDS of NRT2.1 and NAR2.1 was ligated into p35S:: GWR-GFP expression vector (kept in this laboratory) using the method of Gateway homologous recombination (Walhout et al, 2000), p 35S::: NRT2.1-GFP, p35S:: NAR2.1-GFP vector was constructed, col-0 and NAR2.1-c1 (large fragment knockout mutant obtained using CRISPR/Cas9 technique) were transformed using Agrobacterium-mediated method and positive stable transgenic lines were obtained. The Arabidopsis thaliana is contained in different formsHigh nitrogen (HN, 10mM KNO) at sucrose concentration (0%, 0.5%, 3%) ₃ ) Low nitrogen (LN, 0.2mM KNO) ₃ ) The culture medium was cultured vertically for 6 days, NAR2.1-GFP signal intensity and subcellular localization in roots of different Arabidopsis materials under different culture conditions were observed and analyzed (Wirth et al, 2007), total proteins and membrane proteins were simultaneously extracted (Belkhadir et al, 2012), and the content of NAR2.1-GFP and NRT2.1-GFP total proteins and membrane proteins was detected by Western blot. The formula of the high and low nitrogen culture medium is as follows:

0.05mM CaSO ₄ ，0.5mM MgCl ₂ ，0.1mM KH ₂ PO ₄ ，0.25mM MES，0.25μM NaFeEDTA，50μM H ₃ BO ₃ ，0.012μM MnCl ₂ ，0.001μM CuCl ₂ ，0.001μM ZnCl ₂ ，0.00003μM NH ₄ MoO ₄ ,10mM KNO ₃ Or 0.2mM KNO ₃ . Sucrose (0%: 0g/L,0.5%:5g/L,3%:30 g/L), 1% agar (10 g/L) and pH5.8 were added according to experimental requirements.

The results showed that with increasing sucrose concentration in the medium, p35S for the Col-0 background: NRT2.1-GFP, p35S: NAR2.1-GFP transgenic lines showed a significant increase in GFP signal overall, GFP signal on the cell membrane (FIGS. 2A, B), total protein and membrane protein accumulation of NRT2.1-GFP and NAR2.1-GFP (FIGS. 2D, E), indicating that sucrose enhanced protein stability of NAR2.1 and NRT2.1 and promoted localization of NAR2.1 and NRT2.1 from the cytoplasm to the cell membrane; at different sucrose concentrations, p35S of nar2.1-c1 background, the fluorescence intensity of GFP in the roots of NRT2.1-GFP transgenic lines is obviously reduced as a whole, GFP signals on cell membranes are also obviously reduced, the total protein content of NRT2.1 is obviously reduced, but a weak sugar response is also realized, and the membrane protein is almost undetectable, so that the sugar response is completely lost (FIGS. 2C and F). This suggests that the sugar response of NRT2.1 protein stability is dependent on NAR2.1, and that the sugar response of NRT2.1 protein membrane localization is also highly dependent on NAR2.1.

Example 3: phosphorylation of AtNAR2.1 protein affects its protein stability

Sugar starvation activated snrk1.1 is an energy receptor of plants, playing an important role in nitrogen metabolism and nitrate signaling. BiFC experiment using tobacco proves SnR K1.1 interacted with NAR2.1 (Hu and Kerppola, 2003), but did not interact with NRT2.1 (FIG. 3A). His-GRIK1 (Geminivirus Rep interacting kinases), his-SnRK1.1-KD (KD is Kinase domain), his-NAR2.1 protein were expressed in vitro using the E.coli prokaryotic expression system and an in vitro phosphorylation experiment was performed (Wang and Han et al, 2022). ³² The results of autoradiography showed that SnRK1.1-KD was able to phosphorylate NAR2.1 in vitro (FIG. 3B). The results of testing the protein levels of NRT2.1 and NAR2.1 in the p35S:: snRK1.1, p35S:: snRK1.1-RNAi transgenic lines show that the protein levels of NAR2.1 and NRT2.1 in the p35S:: snRK1.1 are significantly reduced compared with the wild type, while the protein levels of NAR2.1 and NRT2.1 in the p35S:: snRK1.1-RNAi are significantly increased (FIG. 3C), which indicates that SnRK1.1 can negatively regulate the protein stability of NAR2.1 and NRT2.1 through phosphorylation modification.

Co-transformation of tobacco with p35S:: NAR2.1-GFP and p35S:: snRK1.1-Flag vector (obtained using the procedure of example 2) was performed to identify the phosphorylation site of NAR2.1 in vivo using immunoprecipitation and mass spectrometry (IP-MS) (Song et al 2020). The mass spectrum detected peptide fragment covered 72% of the sequence of NAR2.1 protein, but no phosphorylation site was detected. It was speculated that the site of snrk1.1 phosphorylated NAR2.1 may be present in the remaining uncovered 28% of the sequence, six possible phosphorylation sites S10, S15, S19, S186, S189 and S209 present in this 28% of the sequence were subjected to simulated phosphorylation and simulated dephosphorylation mutations, respectively, using in vitro gene site-directed mutagenesis (Chiu et al 2004), and transient expression vectors were constructed in which protein variants fused to GFP were transformed and GFP signals were detected to find that none of the six sites S10, S15, S19, S186, S189, S209 had affected GFP fluorescence intensity and subcellular localization after mutation to alanine (a) in a simulated phosphorylated form, whereas when mutated to glutamic acid (E) in a simulated phosphorylated form, NAR2.1 ^S186E -GFP、NAR2.1 ^S189E The fluorescence intensity of GFP was significantly reduced, as was GFP signal on the membrane (fig. 4A). This suggests that the S186, S189 sites may be critical sites for phosphorylation of NAR2.1 by SnRK1.1, mediating regulation of NAR2.1 protein levels and localization by SnRK1.1. By genetic hybridization, p35S:: NAR2.1-GFP and p35S in the context of the SnRK1.1 transgenic line were obtained::NAR2.1 ^2A GFP transgenic lines. GFP detection shows that NAR2.1 compared with NAR2.1-GFP transgenic plants ^2A GFP signal and membrane localization was stronger in GFP transgenic plants (fig. 4B).

P35S:: NAR2.1-GFP and p35S:: NAR2.1 were determined by Agrobacterium-mediated methods ^2A GFP transformation of NAR2.1-c1 (CRISPR/Cas 9 technology-obtained NAR2.1 gene large fragment deletion mutant) to obtain p35S:: NAR2.1-GFP and p 35S::: NAR2.1 in the NAR2.1-c1 background ^2A GFP positive transgenic lines. NAR2.1-c1 p35S:: NAR2.1 ^2A NAR2.1-GFP (created in example 1) transgenic lines were separately applied with the photosynthesis inhibitor DCMU (dichlorophenyl dimethylurea, sigma, 45463) to the cotyledons and subjected to a time gradient treatment (0, 4, 8, 12, 24 h), and NAR2.1 was found to degrade rapidly after DCMU treatment, after 4 hours of treatment the amount of NAR2.1 protein had been significantly reduced and after 24 hours of treatment the NAR2.1 protein had been substantially completely degraded; in contrast, NAR2.1 ^2A The protein is very stable, and NAR2.1 can be detected after 24 hours of treatment with the photosynthesis inhibitor ^2A Higher accumulation of protein (fig. 4D). P35S:: NAR2.1 ^2E GFP transgenic lines were cultured vertically on HN medium with 0% sucrose for 6 days, roots were treated with MG132 (a proteasome inhibitor, sigma, M8699) for 8 hours, and NAR2.1 was detected by Western blot ^2E Amount of GFP protein. The results showed NAR2.1 ^2E The amount of protein increased significantly after MG132 treatment (fig. 4C). These results demonstrate that snrk1.1 phosphorylates the S186, S189 sites of the NAR2.1 protein to promote the degradation of the NAR2.1 protein via the ubiquitinated proteasome pathway; dephosphorylation of the S186, S189 sites improved protein stability of NAR 2.1.

P35S:: NAR2.1 ^2A GFP and p 35S::: NAR2.1 ^2E The GFP vector converts tobacco for transient expression and detects GFP signals. The results showed that NAR2.1 compared to NAR2.1-GFP ^2A GFP signal intensity is enhanced, but signal in the cytoplasm is reduced, and signal on the cell membrane is enhanced, while NAR ^2E The GFP signal was significantly reduced, the signal in the cytoplasmic spots increased, the signal on the cell membrane was reduced and no longer continuous (FIG. 5A). Meanwhile, p35S:: NAR2.1-GFP, p35S:: NAR2.1 under the Nar2.1-c1 background is constructed ^2A -GFP、p35S::NAR2.1 ^2E GFP stable transgenic Arabidopsis lines. Through RT-qPCR detection, three strains with basically consistent exogenous NAR2.1 expression quantity are selected for GFP signal analysis, and the result is consistent with the result of the tobacco transient expression experiment: NAR2.1 compared to NAR2.1-GFP ^2A Enhancement of GFP signal, enhancement of GFP signal in cell membranes, little GFP signal observed in cytoplasm; in contrast, NAR ^2E The GFP signal was attenuated and only a weak discontinuous GFP signal was observed on the cell membrane (FIG. 5B). Further, the root tissues of the three stable transgenic lines are taken to extract total protein and membrane protein, and then Western blot detection is carried out to find that NAR2.1 is compared with wild NAR2.1 ^2A Both total and membrane proteins were significantly increased, whereas NAR2.1 ^2E Both total and membrane proteins were significantly reduced (fig. 5c,5 d). These results demonstrate that phosphorylation at the S186, S189 sites not only reduces NAR2.1 protein stability, but also inhibits NAR2.1 localization to cell membranes, and that dephosphorylation at the S186, S189 sites increases NAR2.1 protein stability and potentially promotes its localization from the cytoplasm to the cell membranes. Detection of NAR2.1 using BiFC experiment system of tobacco ^2A 、NAR2.1 ^2E The interaction with NRT2.1 results indicate that NAR2.1 is compared with NAR2.1 ^2A Interaction with NRT2.1 was enhanced and more pronounced on cell membranes, whereas NAR2.1 ^2E Interaction with NRT2.1 was reduced, and fluorescent signals on cell membranes were reduced, discontinuous, and a large number of spotted interaction signals were present in the cytoplasm (fig. 5E).

Example 4: double site phosphorylation of S186, S189 affects the sugar response of the attrt 2.1 protein stability

NAR2.1-c1 p35S:: NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E GFP transgenic Arabidopsis plants were grown on LN medium of 0%, 0.5% and 3% sucrose for 5-6 days, respectively, and root GFP signals were detected, which indicated that NAR2.1-GFP signal intensity and membrane localization were enhanced with increasing sugar concentration; in contrast, NAR2.1 ^2A GFP retains a stronger GFP signal and membrane localization at different sugar concentrations, whereas NAR2.1 ^2E The GFP signal is significantly reduced at different sugar concentrations and the effect of the sugar concentration on its GFP signal and membrane localization is reduced(FIG. 6A). Thus, the dual site phosphorylation of NAR2.1 protein S186, S189 affects self protein stability and membrane localization response to sugar.

By genetic hybridization, p35S:: NAR2.1-GFP, p35S:: NAR2.1 were obtained, respectively ^2A -GFP、p35S::NAR2.1 ^2E pNRT2.1:: gNRT2.1-LUC (# 11-13) transgenic Arabidopsis plants in GFP background were cultured vertically on LN medium with 0%, 1%, 3% sucrose concentration and examined for LUC signal, and found that p 35S::: NAR2.1 was compared to #11-13 in wild type p35S:: NAR2.1-GFP background ^2A LUC signal sugar response was significantly enhanced in #11-13 in GFP background, whereas p35S:: NAR2.1 ^2E LUC signal sugar responses were significantly reduced, even almost lost in #11-13 in GFP background (fig. 6B).

Example 5: protein variant AtNAR2.1 ^2A Promoting the growth and development of root systems of arabidopsis thaliana

Col-0, NAR2.1-c1 and NAR2.1-c1 p35S:: NAR2.1-GFP, NAR2.1-c1 p 35S::: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E GFP transgenic Arabidopsis plants were grown on HN medium with 1% sucrose for 4 days and then transplanted on LN medium with 1% sucrose for 10 days. And counting the biomass of the aerial parts, the length of main roots, the number of lateral roots and the length of total lateral roots. The results show that NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1, compared to Col-0 and NAR2.1-c1 p35S:: NAR2.1 ^2A The major root length, lateral root number and total lateral root length of GFP increased by 7.8%, 34.6%, 64.9% and 12.1%, 29.4%, 36.8% (fig. 7D-F), respectively, while the aerial biomass increased by 32.4% and 21.7% (fig. 7A-C); and NAR2.1-c1 p35S:: NAR2.1 ^2E The length of main roots, the number of lateral roots and the length of total lateral roots of the GFP transgenic line are respectively reduced by 7.5%, 50.8% and 46.3% compared with that of NAR2.1-C1 p35S, the NAR2.1-GFP strain is similar to that of NAR2.1-C1 mutant (FIG. 7D-F), and the biomass of overground parts is obviously reduced by 41.3% (FIG. 7A-C). These results demonstrate that the S186, S189 sites mimic the phosphorylated form of NAR2.1 ^2E Almost no function, but to mimic the dephosphorylated form of NAR2.1 ^2A Can obviously promote the growth of main roots and lateral roots and increase the biomass of plants.

Example 6: atNAR2.1 ^2A Protein promotion of nitrate by arabidopsis thalianaIs absorbed by (a)

Detection of Col-0, NAR2.1-c1 and NAR2.1-c1p35S:: NAR2.1-GFP, NAR2.1-c1p 35S::: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E Nitrate uptake rates of GFP transgenic lines in high, low nitrogen and different sucrose concentrations. The high and low nitrogen conditions refer to 1% sucrose and 10mM KNO in the culture medium ₃ (HN) or 0.2mM KNO ₃ (LN); the different sugar concentration conditions refer to the presence of 0.2mM KNO in the medium ₃ 0% sucrose or 3% sucrose. The method comprises the following specific steps:

1. the arabidopsis seeds are firstly sown on an HN culture medium for vertical culture for 4 days, then the seedlings are transplanted on the HN culture medium and LN culture medium for vertical growth for 10 days, and then root systems are carried out ¹⁵ NO ₃ ^- Absorption rate measurement.

2. The roots were first placed in 0.1mM CaSO ₄ Soaking for 1min, and transferring to a solution containing 5mM K ¹⁵ NO ₃ Soaking in the culture solution of (2) for 5min, and finally placing in 0.1mM CaSO ₄ Soaking for 1min, wiping the liquid on the root with paper, cutting off the root, placing the root in a 2mL centrifuge tube, thoroughly drying in a 65 ℃ oven, adding small steel balls, and beating into dry powder in a sample beating device.

3. The dry powder was packed in tin cups and measured using isotope ratio mass spectrometer Delta V Advantage IRMS ¹⁵ N content, calculating ¹⁵ NO ₃ ^- Absorption rate.

The results show that, in HN (10 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) Under culture conditions, NAR2.1-c1 p35S:: NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1, compared to Col-0 and NAR2.1-c1 p35S: ^2A nitrate uptake rates of GFP increased by 15.3%, 15.9% and 21.8%, 23.8%, respectively, whereas NAR2.1-c1 p35S:: NAR2.1 ^2E Nitrate uptake rates of GFP were reduced by 18.9%, 63.4% and 14.4%, 60.9%, respectively, comparable to that of NAR2.1-c1, indicating NAR2.1 at different nitrogen concentrations ^2A Enhanced nitrate transport function, whereas NAR2.1 ^2E The function of nitrate transport was reduced (fig. 8A). Also, NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1, in comparison to Col-0 and NAR2.1-c1 p35S:: NAR2.1 under culture conditions of 0% sucrose and 3% sucrose ^2A Nitrate uptake rate of GFPThe rates increased by 14.4%, 26.7% and 18.4%, 14.2%, respectively, and NAR2.1-c1 p35S:: NAR2.1 ^2E Nitrate uptake rates of GFP were reduced by 75.4%, 66.9% and 74.5%, 70.2%, respectively, comparable to that of nar2.1-c1 (fig. 8B). These results indicate that NAR2.1 at different sugar concentrations ^2A Enhanced nitrate transport function, whereas NAR2.1 ^2E The function of transporting nitrate is weakened. NAR2.1 under different carbon and nitrogen conditions ^2A Can obviously promote the absorption of the arabidopsis to nitrate.

Example 7: atNAR2.1 ^2A Synergistic enhancement of carbon-nitrogen metabolism

Determination of Col-0, NAR2.1-c1 and NAR2.1-c p35S:: NAR2.1-GFP, NAR2.1-c p S:: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E The C/N ratio of the whole seedlings of GFP transgenic plants grown on HN and LN medium for 14 days is as follows:

1. sampling Arabidopsis materials: full seedlings were grown on HN and LN dishes for 14 days.

Drying at 2.80deg.C, and pulverizing the whole seedling on the Arabidopsis culture dish into powder with a proofing machine.

3. The dry powder is wrapped by a tin cup, the content of carbon and nitrogen elements is measured by using a Flash-2000 element analyzer of Thermo Fisher company, and the C/N ratio is calculated.

The results show that there is no difference in the C/N ratio of the whole seedlings of the different materials under HN and that both are significantly lower than the C/N ratio under LN (fig. 10); under LN, the ratio of C/N of nar2.1-C1 is 33.9% higher than that of Col-0; compared with Col, NAR2.1-C1 p35S, the ratio of C/N of NAR2.1-GFP is 10.2 percent higher, and the carbon nitrogen ratio value of the NAR2.1-C1 mutant can be partially recovered; NAR2.1-c1 p35S:: NAR2.1 ^2A The C/N ratio of GFP and Col-0 have no significant difference, and the C/N ratio of the nar2.1-C1 mutant can be completely recovered; NAR2.1-c1 p35S:: NAR2.1 ^2E There was no significant difference between the C/N ratio of GFP and nar2.1-C1, which was 27.3% higher than that of Col-0 (FIG. 9). Thus, in LN, the NAR2.1 function-deleted mutant NAR2.1-C1 had severely reduced uptake of external nitrate, resulting in reduced nitrogen content in plants, increased C/N ratio, and NAR2.1 ^2A Can recover nitrate absorption, NAR2.1 ^2A Because ofHas stronger function, so that the wild-type NAR2.1 can completely recover the normal C/N ratio, and the NAR2.1 without functions ^2E It cannot be recovered.

Example 8: NAR2.1 ^2A Promoting early ripening of Arabidopsis thaliana, and increasing biomass and seed yield

Col-0, NAR2.1-c1 and NAR2.1-c1 p35S:: NAR2.1-GFP, NAR2.1-c1 p 35S::: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E GFP transgenic lines were grown under normal greenhouse conditions (vermiculite: nutrient soil=1:1) and statistical analysis was performed on the growth development and plant type of the plants. The results show that NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1, compared to Col-0 and NAR2.1-c1 p35S:: NAR2.1 ^2A The GFP transgenic plants grew faster, plants were stronger, the bolting time was advanced, the number of rosette leaves before bolting was 21.3% and 11.4% more, respectively (FIGS. 10A, C), rosette leaves turned yellow in advance at the later stage of reproductive growth, and the fruit clips were matured faster (FIG. 10B). This suggests that NAR2.1 is overexpressed ^2A Promoting plant growth and development, and realizing early flowering and early maturing.

Col-0, NAR2.1-c1 and NAR2.1-c1 p35S:: NAR2.1-GFP, NAR2.1-c1 p 35S::: NAR2.1 ^2A -GFP、nar2.1-c1 p35S::NAR2.1 ^2E The GFP transgenic lines were grown in soil containing 15%, 30%, 60% and 100% four nitrogen concentrations, the cultivation method was as follows:

1. Preparing vermiculite in advance, namely pouring 2.5L of water from the bottom of each pot, and pouring 1L of water from the upper surface to soak the substrate after the vermiculite is fully absorbed, wherein the nutrient soil proportion is 1:2.

2. The arabidopsis seeds to be sown are soaked in water for 2 days in a refrigerator at the temperature of 4 ℃, then a plurality of seeds are sucked by a blue gun head and are beaten on a matrix soaked by water, a cover is covered, and the cover is uncovered after the seeds germinate for 4-5 days. Greenhouse culture conditions were long sunlight (16 h light/8 h dark), 22 ℃, 70% air humidity.

3. Thinning after 10 days of germination, leaving one seedling with the best growth vigor, and carrying out first watering and fertilization: the different nitrogen concentrations are 15% N, 30% N, 60% N and 100% N, and 0.3g, 0.6g, 1.2g and 2.0g of the potassium nitrate fertilizer of the Israel sea Baolifeng brand are correspondingly weighed and dissolved in 2L of water.

4. The fertilizer is applied once a week (4-5 times) for the subsequent growth, the watering quantity is adjusted according to the growth vigor of the seedlings, the flowering phase (rosette number) is counted during the period, and the phenotype is photographed.

5. Culturing until the leaves turn yellow, stopping watering, collecting the overground parts after soil is dried, drying, and weighing biomass and seed weight.

The results of the statistical analysis showed that NAR2.1-GFP, NAR2.1-c1 p35S:: NAR2.1, compared to Col-0 and NAR2.1-c1 p35S:: NAR2.1 ^2A GFP biomass was increased by 18.7%, 10.4%, 16.0%, 32.2% and 16.3%, 8.3%, 18.0%, 35.1% at 4 different nitrogen concentrations (100% N, 60% N, 30% N, 15% N), respectively, seed weights were increased by 11.6%, 8.9%, 48.3%, 70.5% and 15.3%, 15.2%, 22.7%, 27.1%, respectively. NAR2.1-c1 p35S:: NAR2.1 ^2E The biomass and seed quality of GFP were reduced by 14.5%, 19.6%, 14.8%, 73.7% and 9%, 24.6%, 30.8%, 74.8% compared to NAR2.1-c1 p35S, respectively, for NAR2.1-GFP (FIGS. 10D, E). In addition, it is notable that NAR2.1-c1 p35S:: NAR2.1 ^2A The seed weight of GFP at 60% N concentration was comparable to that of wild type at 100% N concentration (FIG. 10E), indicating the expression of NAR2.1 in plants ^2A Can reduce weight and increase yield. To sum up, NAR2.1 ^2A Can improve the nitrogen utilization efficiency and seed yield of plants.

Example 9: the S186 and S189 sites are highly conserved among NAR2.1 proteins from different sources

The inventor finds that homologous protein sequences of AtNAR2.1 exist in lower algae, mosses, ferns, gymnosperms and angiosperms through NCBI-Blast search; sequence alignment is performed by using Clustal X and DNAman, and then a phylogenetic tree is constructed by using MEGA 11. Sequence alignment showed that the S186, S189 sites on the NAR2.1 transmembrane domain are highly conserved from lower to higher plants (fig. 11A). The treeing analysis showed that NAR2.1 was not present in prokaryotic bacteria, and was originally present in eukaryotic algae capable of photosynthesis (FIG. 11B), suggesting that plant organelles with membrane coating (endomembrane system) and photosynthesis may be closely related to NAR2.1 production and function.

The inventors cloned NAR2.1 homologous genes from Chlamydomonas reinhardtii (unicellular green algae), conus (bryophytes), wheat and rice (monocotyledonous crops). Further, double point mutation was performed on the sites corresponding to S186 and S189 in Arabidopsis NAR2.1, and PCR amplification was performed using the following primers:

OsNAR2.1-attB1-F:acaagtttgtacaaaaaagcaggcttcatggcgaggctagccggcgtt(SEQ ID NO:45)

OsNAR2.1-attB1-R:accactttgtacaagaaagctgggtccttgtccttcttgcgcttctc(SEQ ID NO:46)

TaNAR2.1-attB1-F:caagtttgtacaaaaaagcaggcttcatggcacggtcggagctggccatg(SEQ ID NO:47)

TaNAR2.1-attB1-R:accactttgtacaagaaagctgggtcagttgttcttcttgcgtttctc(SEQ ID NO:48)

CrNAR2.1-attB1-F:acaagtttgtacaaaaaagcaggcttcatgaaggcatacgcgctcctctta(SEQ ID NO:49)

CrNAR2.1-attB1-R:accactttgtacaagaaagctgggtcgatgtggccgatgcgcagcgat(SEQ ID NO:50)

MpNAR2.1-attB1-F:acaagtttgtacaaaaaagcaggcttcatggcaacggtgggaaaatcga(SEQ ID NO:51)

MpNAR2.1-attB1-R:accactttgtacaagaaagctgggtccttgctgccgttcgagcgcttg(SEQ ID NO:52)

OsNAR2.1 ^2A -F:caaggtcgccgccggcgtcttcgccaccttcgccatcgccgcgctcgccttcttct(SEQ ID NO:53)

OsNAR2.1 ^2A -R:agaagaaggcgagcgcggcgatggcgaaggtggcgaagacgccggcggcgaccttg(SEQ ID NO:54)

OsNAR2.1 ^2E -F:caaggtcgccgccggcgtcttcgaaaccttcgaaatcgccgcgctcgccttcttct(SEQ ID NO:55)

OsNAR2.1 ^2E -R:agaagaaggcgagcgcggcgatttcgaaggtttcgaagacgccggcggcgaccttg(SEQ ID NO:56)

TaNAR2.1 ^2A -F:caaggttgccgccggcgtcttcgccgccttcgccgtcgcatccctcgccttcttct(SEQ ID NO:57)

TaNAR2.1 ^2A -R:agaagaaggcgagggatgcgacggcgaaggcggcgaagacgccggcggcaaccttg(SEQ ID NO:58)

TaNAR2.1 ^2E -F:caaggttgccgccggcgtcttcgaagccttcgaagtcgcatccctcgccttcttct(SEQ ID NO:59)

TaNAR2.1 ^2E -R:agaagaaggcgagggatgcgacttcgaaggcttcgaagacgccggcggcaaccttg(SEQ ID NO:60)

CrNAR2.1 ^2A -F:tgcgcgccgccgccatcgcgctcgccgtcttcgcgccgctcttcctcatcttctac(SEQ ID NO:61)

CrNAR2.1 ^2A -R:gtagaagatgaggaagagcggcgcgaagacggcgagcgcgatggcggcggcgcgca(SEQ ID NO:62)

CrNAR2.1 ^2E -F:tgcgcgccgccgccatcgcgctcgaagtcttcgaaccgctcttcctcatcttctacgc(SEQ ID NO:63)

CrNAR2.1 ^2E -R:gcgtagaagatgaggaagagcggttcgaagacttcgagcgcgatggcggcggcgcgca(SEQ ID NO:64)

MpNAR2.1 ^2A -F:tcgacgtcgctgctgccatcttcgctgcattcgccatcgggtcgttggtcttcttc(SEQ ID NO:65)

MpNAR2.1 ^2A -R:gaagaagaccaacgacccgatggcgaatgcagcgaagatggcagcagcgacgtcga(SEQ ID NO:66)

MpNAR2.1 ^2E -F:tcgacgtcgctgctgccatcttcgaagcattcgaaatcgggtcgttggtcttcttcct(SEQ ID NO:67)

MpNAR2.1 ^2E -R:aggaagaagaccaacgacccgatttcgaatgcttcgaagatggcagcagcgacgtcga(SEQ ID NO:68)

the amplification product is transferred into a p35S:: GWR-GFP expression vector by using a Gateway homologous recombination method, tobacco is transformed, and GFP signals are observed. The results show that NAR2.1 homologous protein variants derived from different species are similar to NAR2.1 protein variants of Arabidopsis thaliana, i.e.NAR2.1 compared to NAR2.1-GFP ^2A Both GFP signal and membrane localization were stronger, whereas NAR2.1 ^2E GFP signal was reduced and membrane localization was also reduced (fig. 12A). These results indicate that the effect of the corresponding sites of S186, S189 in NAR2.1 proteins of different species on NAR2.1 protein content and localization is conserved.

pNAR2.1:: mpNAR2.1-GFP, pNAR2.1:: mpNAR2.1 were constructed using NAR2.1 in Chlamydomonas reinhardtii and in liverwort, respectively ^2A -GFP、pNAR2.1::MpNAR2.1 ^2E GFP and pNAR2.1:: crNAR2.1-GFP, pNAR2.1:: crNAR2.1 ^2A -GFP、pNAR2.1::CrNAR2.1 ^2E The vector-GFP (Green fluorescent protein) is used as a vector,wherein, the gene sequences of CrNAR2.1, mpNAR2.1 and protein variants are the amplified products of the corresponding primers, the NAR2.1 promoter is an Arabidopsis NAR2.1 promoter (SEQ ID NO: 31), and the primers of the amplified promoters are as follows:

pNAR2.1-seamless-F:gctatgaccatgattacgaattcttttctgcgatttcagctcga(SEQ ID NO:69)

pNAR2.1-seamless-R:tcgactctagaggatccccgggtaccggatatatccttgaaactgaata(SEQ ID NO:70)

NAR2.1 promoter, crNAR2.1, mpNAR2.1 and double mutant gene are connected to pCAMBIA2300 vector by using homologous recombination seawill method, and mutant NAR2.1-c1 is transformed by using agrobacterium mediation method respectively to obtain positive transgenic line. Vertical culture of each transgenic line on LN medium containing 1% sucrose for 5-6 days, analysis of root phenotype found that, like Arabidopsis NAR2.1, crNAR2.1 and MpNAR2.1 were able to promote root growth and development in the NAR2.1-c1 mutant, genetically complementing the phenotype of NAR2.1-c1 mutant root; also, with NAR2.1 ^2A As such, mpNAR2.1 ^2A And CrNAR2.1 ^2A Is also a functionally enhanced mutation, which complements the root growth phenotype of the nar2.1-c1 mutant better than that of MpNAR2.1 and CrNAR2.1 (FIGS. 12B, C). Thus, from lower algae to higher plants, the sites corresponding to S186, S189 on NAR2.1 homologous proteins are functionally conserved.

Example 10: constitutive high expression TaNAR2.1 ^2A And AtNAR2.1 ^2A Improving photosynthesis, nitrogen fertilizer utilization efficiency and yield of wheat

Creating an over-expression of TaNAR2.1, taNAR2.1 in the context of wheat Fielder variety ^2A Arabidopsis NAR2.1 ^2A The specific experimental procedure for the transgenic wheat material (fig. 13A) is as follows:

1. Construction of a maize Ubiquitin promoter (SEQ ID NO: 32) driven overexpression vector as follows: pUbi:: taNAR2.1, pUbi:: taNAR2.1 ^2A 、pUbi::AtNAR2.1 ^2A 。

2. The competent cells of the agrobacterium EHA105 are transformed, the field embryo is transformed by using an agrobacterium-mediated method, a positive transgenic plant is obtained, a high-low nitrogen concentration hydroponic experiment is carried out, and the absorption rate of nitrate is measured. The method comprises the following specific steps:

1) Wheat seeds are firstly grown in clear water until two leaves are in a heart period (10 days), and then are transplanted into a normal culture solution (2 mM KNO) ₃ ) Growing for 6 days, and then carrying out root system ¹⁵ NO ₃ ^- Absorption rate measurement.

2) The roots were first placed in 0.1mM CaSO ₄ Soaking in the solution for 1min, and transferring to HN (2 mM K) ¹⁵ NO ₃ ) And LN (0.2 mM K) ¹⁵ NO ₃ ) Treating in nutrient solution for 5min, and finally placing in 0.1mM CaSO ₄ Soaking for 1min, wiping the liquid on the root with paper, cutting off the root, placing in kraft paper bag, and oven drying thoroughly at 65deg.C. Grinding to dry powder by using a ZM200 ultracentrifuge grinder of Retsch company.

3) The dry powder was packed in tin cups and measured using isotope ratio mass spectrometer Delta V Advantage IRMS ¹⁵ N content, calculating ¹⁵ NO ₃ ^- Absorption rate.

The results showed that, in HN (2 mM K) ¹⁵ NO ₃ ) And LN (0.2 mM K) ¹⁵ NO ₃ ) In the nutrient solution, pUbi:: taNAR2.1 compared with Fielder ^2A The absorption rate of the root system of the plant to nitrate is respectively improved by 19.1 percent and 29.9 percent; pUbi:: taNAR2.1 compared to pUbi:: taNAR2.1 ^2A The absorption rate of nitrate by the root system of (a) was increased by 12.7% and 14.0%, respectively (fig. 13B). In addition, atNAR2.1 ^2A Heterologous overexpression in wheat can also improve nitrate absorption rate of 8.4% and 9.3% of wheat root system.

Fielder and pUbi:: taNAR2.1, pUbi:: taNAR2.1 ^2A 、pUbi::AtNAR2.1 ^2A Seedling raising of wheat material in clear water to two leaves and one heart stage, and transplanting to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) Growing in the culture solution for 2 weeks, taking the overground part and the underground part respectively, drying at 80 ℃, weighing, and calculating the root-crown ratio. The results showed that, compared with the overexpression of TaNAR2.1, the TaNAR2.1 was overexpressed ^2A Promoting the growth of wheat root system in HN (2 mM KNO) ₃ ) The biomass of the above and below ground parts was increased by 30.4% and 11.3%, and the biomass was increased by LN (0.2 mM KNO ₃ ) The biomass of the above-ground and below-ground parts was increased by 7.3% and 19.5% (fig. 13C). In a small sizeNAR2.1 of heterologous over-expression Arabidopsis thaliana in wheat ^2A Also promote wheat growth in HN (2 mM KNO) ₃ ) The biomass of the above and below ground parts was increased by 7.2% and 0.6%, and the biomass was increased by LN (0.2 mM KNO ₃ ) Increasing biomass of the above and below ground parts by 4.8% and 4.1% (FIG. 13C), further illustrating NAR2.1 ^2A Is highly conserved among different species. In addition, taNAR2.1 or TaNAR2.1 is overexpressed ^2A Although promoting wheat growth and increasing biomass, the promotion was coordinated above and below ground without changing the root-to-crown ratio of the plants (fig. 13D).

The inventor determines the carbon and nitrogen content of the whole seedling of the water-cultured wheat material, and the method comprises the following steps:

1. germinating and growing seedlings with normal nutrient solution for 1 week, and transplanting to HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) And LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) And growing water-cultured seedlings Quan Miao in the culture solution for 4 weeks.

Oven-drying at 2.80deg.C, and grinding to powder with ZM200 ultracentrifuge pulverizer of Retsch company.

3. And (3) packaging the dry powder with a tin cup, measuring the content of carbon and nitrogen elements by using a Flash-2000 element analyzer of Thermo company, and calculating the C/N ratio.

The results show that over-expression of TaNAR2.1 increased the C/N ratio by 3.1% and 4.8% over Fielder in the wheat Fielder background, both HN and LN, over-expressed TaNAR2.1 ^2A The C/N ratio is not changed (FIG. 13E), which illustrates TaNAR2.1 ^2A The nitrogen fertilizer utilization efficiency is improved, and the carbon-nitrogen metabolism balance in the wheat can be maintained.

The inventor respectively plants a Fielder, pUbi:: taNAR2.1 and pUbi:: taNAR2.1 under the normal nitrogenous fertilizer level of the greenhouse ^2A And pUbi:: atNAR2.1 ^2A Wheat material, plant height, biomass and seed weight per pot of plant of each material in maturity are counted. The result shows that the over-expression of TaNAR2.1 can respectively increase the plant height of wheat by 24.0 percent, the biomass by 32.3 percent and the yield by 15.0 percent; overexpression of TaNAR2.1 ^2A The plant height of the wheat can be increased by 30.2%, the biomass is 53.8% and the yield is 26.3% respectively; overexpression of Arabidopsis NAR2.1 ^2A The plant height of the wheat can be increased by 19.8%, the biomass is 15.6% and the yield is 17.1% respectively. Thus TaNAR2.1 ^2A Has the strongest effect of promoting the growth and development of wheat (FIG. 13F-I).

Example 11: root-specific expression of TaNAR2.1 ^2A Improving photosynthesis, nitrogen fertilizer utilization efficiency and yield of wheat

NAR2.1 was expressed not only in roots but also in aerial parts, indicating that NAR2.1 was also functional in aerial parts. For the targeted study of root expression NAR2.1 ^2A With respect to the effect of growth of wheat and nitrogen fertilizer utilization efficiency, the present inventors created pSAD1:: taNAR2.1 using a promoter (SEQ ID NO: 33) from SAD1 gene specifically expressed in oat root ^2A And pSAD 1:TaNAR2.1 ^2E Transgenic wheat (vector construction and wheat transformation methods are as above). And carrying out water culture experiments of high and low different nitrogen concentrations, and analyzing wheat according to the method ¹⁵ NO ₃ ^- Rate of uptake and phenotype of transgenic plants. The results show that compared with Fielder, pSAD1:: taNAR2.1 ^2A In HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) The nitrate uptake rates of the plants were increased by 29.5% and 31.0%, respectively (FIG. 14B), even compared to pUbi:: taNAR2.1 ^2A The nitrate uptake rate of the plants was also 10.4% and 1.1% (fig. 13B). Meanwhile, pSAD1 is TaNAR2.1 ^2A Plants have higher biomass above and below the ground, at HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) The following increases by 107.4%, 30.1% and 40.5%, 80.1% respectively over Fielder (FIG. 14C), and pSAD1:: taNAR2.1 ^2E The nitrate absorption rate of the plants and the biomass of the overground parts and the underground parts of the plants are not different from those of the control Fielder. Likewise, tanar2.1 was driven by the SAD1 promoter ^2A And TaNAR2.1 ^2E The expression also did not change the root-to-shoot ratio of the plants (fig. 14D).

Further, the carbon and nitrogen contents of the whole seedling of the water-cultured wheat material are measured, and the C/N ratio is calculated, and the specific steps are as follows:

1. transplanting the seedling which is sprouted and grows to two leaves and one heart stage in water to HN (2 mM KNO) ₃ ) And LN (0.2 mM KNO) ₃ ) The culture solution is grown for 2 weeks,taking full seedlings.

The results show that under HN, pSAD1:: taNAR2.1 ^2A The plant C/N ratio was 5.8% lower than that of the control field, and pSAD1:: taNAR2.1 under LN ^2A The plant C/N ratio was not significantly different from Fielder (FIG. 14E), indicating root-specific TaNAR2.1 ^2A The nitrogen fertilizer utilization efficiency is improved, and the carbon-nitrogen metabolism balance of wheat under LN can be maintained.

The inventor plants Fielder and pSAD 1:TaNAR2.1 under the condition of greenhouse soil culture ^2A And pSAD 1:TaNAR2.1 ^2E Wheat material, plant height at maturity, biomass and seed weight per pot of plant were counted. The results showed that the root-specific promoter driven tanar2.1 ^2A Expression can significantly increase wheat plant height by 21.9%, biomass by 51.2% and yield by 28.2%, while root-specific promoters drive tanar2.1 ^2E Expression did not affect plant growth (FIGS. 14F-I).

Example 12: gene editing OsNAR2.1 ^S189A 、OsNAR2.1 ^2A Improving the utilization efficiency and the yield of the nitrogen fertilizer of the rice

The present inventors performed T to G single base substitution editing of TCC bases encoding serine 186 and serine 189 of OsNAR2.1 on the genome of Wuzhuang No. 7 (WYJ 7) rice cultivar (Xu and Zhang et al 2020) using an improved guided editing system (Prime Editor 3, PE 3), as follows:

1. and (3) constructing a carrier:

three rounds of PCR amplified template one and template two, and with BsaI enzyme cutting, specific amplified template as shown in FIG. 17, primers were as follows:

825F1 ttggcggtctctgccgttcgacgtcgccgggatcacgtttcagagctatgctggaaac(SEQ ID NO:71)

825R1 tccagcatagctctgaaactctccatcgccgcgctcgcctgcaccagccgggaatcgaa(SEQ ID NO:72)

825R1-2 tcgtggtctctgccttatttcaacttgctatgctgtttccagcatagctctgaaactctccatcgccgc(SEQ ID NO:73)

825F3 aaagtggcaccgagtcggtgctcgccacattcgccatcgccgaaag(SEQ ID NO:74)

825F4 gctcgccacattcgccatcgccgaaagaatacgcggttctatctagttacgc(SEQ ID NO:75)

790F2 acgacggtctctaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctt(SEQ ID NO:76)

790R2 gtcgtggtctctaaaaaacttatctttaatcatattccatagtccatac(SEQ ID NO:77)

The PE-P3 backbone was cleaved using BsaI and purified.

The T4 ligase (NEB, M0202L) ligates the vector and fragments, and the final vector is shown in FIG. 18.

2. Rice resistant callus and acquisition of positive T0 seedlings

Transforming the constructed vector Agrobacterium EHA105 competent cells into WYJ7 callus by using an agrobacterium-mediated transformation method, infecting the callus by using agrobacterium, restoring the growth of a culture medium, screening the culture medium to obtain a resistant callus, and putting the resistant callus on a differentiation culture medium to grow to obtain a T0 seedling. Extracting genome DNA of the obtained rice T0 seedling, taking the genome DNA as a template, and carrying out PCR amplification by adopting a primer pair consisting of a primer F (TACTCTCATCCACCAGTCCATC, SEQ ID NO: 78) and a primer R (GATGTTGGCGACCTCGTAT, SEQ ID NO: 79) to obtain a PCR amplification product; the PCR amplified product was subjected to agarose gel electrophoresis, and then judged as follows: if the PCR amplification product contains a DNA fragment of about 973bp, the corresponding rice T0 seedling is a rice positive T0 seedling; if the PCR amplification product does not contain a DNA fragment of about 973bp, the corresponding rice T0 seedling is not a rice positive T0 seedling.

3. Analysis of results of editing efficiency in T0 seedlings of rice

Genomic DNA from T0 seedlings was used as template and amplified using primer BY857: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTCGTCAGTCCACTGAAGCTGCGAAC (SEQ ID NO: 80), BY858: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGTGCCAGTTCAAGGTCAC (SEQ ID NO: 81). The first round of PCR products are used as templates, different forward and reverse barcodes are added to the ends of the PCR products for library construction to form mixed libraries, and the mixed libraries are sequenced by using a Miseq high-throughput sequencing platform, wherein the sequencing depth of each mixed library is more than 1000X (the engineering Co., ltd.). Analysis was performed for each pegRNA region and the editing efficiency of the guided editing system in rice T0 seedlings was calculated. Editing efficiency of the pilot editing system in rice T0 seedlings = number of positive T0 seedlings mutated at all mutation sites/total number of positive T0 seedlings analyzed x 100%.

Designing the gNAR2.1 editing target sequence of the T0 vaccine with positive mutation by the following primer amplification second generation sequencing verification:

OsNAR2.1-primer1-F:agcgtgaaggtgaagctgtgcta(SEQ ID NO:82)

OsNAR2.1-primer1-R:gaggagaacatcactgtatacat(SEQ ID NO:83)

the PCR product is subjected to first generation sequencing to obtain homozygous single amino acid site edited OsNAR2.1 ^S189A Strain, and double amino acid site edited osnar2.1 ^S186A,S189A (abbreviated as OsNAR2.1 ^2A ) Strain (fig. 15A). The homozygous lines were subjected to a hydroponic experiment under LN: rice seeds are first grown on HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ Germinating in nutrient solution for 1 week, and transplanting to LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) Growing in the culture solution for 4 weeks, taking root system to extract total protein, and detecting the content of OsNAR2.1 protein in the root by using an OsNAR2.1 antibody. The result shows that OsNAR2.1 ^S189A And OsNAR2.1 ^2A Can enhance the protein stability of the OsNAR2.1 and promote the accumulation of the OsNAR2.1 in roots (figure 15B).

By WYJ7, osnar2.1 (OsNAR2.1 function-deleted mutant) and OsNAR2.1 ^S189A 、OsNAR2.1 ^2A The gene editing material was subjected to HN and LN hydroponic experiments (FIG. 15C), and root system was measured ¹⁵ NO ₃ ^- The absorption rate is as follows:

1. rice seed first HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ Germinating in nutrient solution for 1 week, and transplanting to HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) And LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) Growth 4 in culture solutionAnd (3) week(s).

2. Root was placed in 0.1mM CaSO ₄ Soaking for 1min, and transferring to a solution containing 1.25mM ¹⁵ NH ₄ ) ₂ SO ₄ And 2.5mM K ¹⁵ NO ₃ Is treated in nutrient solution of (2) for 5min, and finally placed in 0.1mM CaSO ₄ Soaking for 1min, wiping the liquid on the root with paper, cutting off the root, placing in kraft paper bag, and oven drying thoroughly at 65deg.C. Grinding to dry powder by using a ZM200 ultracentrifuge grinder of Retsch company.

3. The dry powder was packed in tin cups and measured using isotope ratio mass spectrometer Delta V Advantage IRMS ¹⁵ N/ ¹⁴ N ratio and calculate ¹⁵ NO ₃ ^- Absorption rate.

The results show that compared with WYJ7, the nitrate absorption rate of osnar2.1 under HN is obviously reduced by 18.8 percent, and the nitrate absorption rate under LN is obviously reduced by 10.4 percent; whereas OsNAR2.1 ^S189A The nitrate absorption rate under HN is obviously increased by 20.4 percent, and the nitrate absorption rate under LN is obviously increased by 10.4 percent; osNAR2.1 ^2A The gene editing line showed a significant increase in nitrate uptake rate of 20.4% under HN and 22.4% under LN (fig. 15D). Thus, two-site edited OsNAR2.1 ^2A The nitrate uptake rate of the strain is most significantly improved.

Measurement of WYJ7, osnar2.1 and OsNAR2.1, respectively ^S189A 、OsNAR2.1 ^2A The biomass and root cap ratio of the gene editing material on the overground part and the underground part of the HN and LN lower plants are specifically as follows:

rice seeds are first grown on HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ Germinating in nutrient solution for 1 week, and transplanting to HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) And LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) Growing in the culture solution for 4 weeks, taking the overground part and the underground part respectively, drying at 80 ℃, weighing biomass of the overground part and the underground part, and measuring the root cap ratio.

The results showed that biomass of aerial parts and root systems was significantly reduced by 16.7% and 26.6% in HN under osnar2.1 compared to WYJ7, and that of LN under osnar2.1The biomass of the upper part and the root system is obviously reduced by 15.1 percent and 37.2 percent; whereas OsNAR2.1 ^S189A The biomass of the overground part and the root system under HN is obviously increased by 50.8 percent and 68.0 percent, and the biomass of the overground part and the root system under LN is obviously reduced by 36.0 percent and 21.0 percent; osNAR2.1 ^2A The biomass of the gene editing lines was significantly increased by 58.8% and 69.0% in the lower aerial part of HN and root system, respectively, and by 37.3% and 25.9% in the lower aerial part of LN and root system, respectively (fig. 15a, e). Furthermore, either osnar2.1 or osnar2.1 ^S189A And OsNAR2.1 ^2A None of the gene-edited plants changed root cap ratio (fig. 15F).

The carbon and nitrogen content of the whole seedling of the water planting rice material is measured, and the carbon-nitrogen ratio is calculated, and the specific steps are as follows:

1. HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ Germinating in nutrient solution for 1 week, transplanting to HN (1.5M NH) ₄ Cl+0.75M Ca(NO ₃ ) ₂ ) And LN (0.45M NH) ₄ Cl+0.225M Ca(NO ₃ ) ₂ ) Growing in the culture solution for 4 weeks, and taking full seedlings.

The results showed that under HN, osnar2.1, osNAR2.1 compared with WYJ7 ^S189A 、OsNAR2.1 ^2A There was no significant difference in the C/N ratio of (C/N), probably due to the fact that even if osnar2.1 was deficient in function, the low affinity nitrate transporter functions and the nitrate transported was sufficient to maintain normal nitrogen demand in the plant; however, compared with WYJ7, osnar2.1 had a significant 12.0% rise in carbon-nitrogen ratio under LN (fig. 15G), demonstrating that osnar2.1 has an important role in maintaining the carbon-nitrogen metabolic balance of rice under LN as a high-affinity nitrate transporter; whereas OsNAR2.1 ^S189A 、OsNAR2.1 ^2A The carbon-nitrogen ratio under LN was also significantly increased by 23.9% and 12.9%, respectively. (FIG. 15G), it is presumed that the root system of the gene-editing plant absorbs more nitrogen and also fixes more carbon, resulting in plantingIncrease in the carbon to nitrogen ratio under LN.

The inventor plants WYJ7 and OsNAR2.1 in Hainan field ^S189A And OsNAR2.1 ^2A Materials and agronomic traits are counted during the maturity. The results showed that compared with WYJ7, osNAR2.1 ^S189A The plant height, tillering, primary branch and stem number, secondary branch and stem number, grain number per ear and single plant seed yield are obviously increased by 11.2%, 34.7%, 27.3%, 41.9%, 11.8% and 21.0% respectively; osNAR2.1 ^2A The plant height, tillering, number of primary branches, number of secondary branches, number of grains per ear and individual seed yield of the material were significantly increased by 8.7%, 28.2%, 27.3%, 41.9%, 10.7%, 21.7%, respectively (fig. 15H-N). This indicates that the gene edited OsNAR2.1 ^S189A And OsNAR2.1 ^2A Can improve the nitrogen fertilizer utilization efficiency of rice, promote the growth and development of rice and improve the yield of rice.

Example 13: osNAR2.1 ^2A Transgenic improvement of rice nitrogen fertilizer utilization efficiency and yield

PCR amplification of OsNAR2.1 self-promoter (SEQ ID NO: 34) and OsNAR2.1, osNAR2.1 ^2A Point mutation OsNAR2.1 simulating phosphorylation form ^2E The CDS sequence and specific primers are as follows:

pOsNAR2.1-seamless-F

ccatgattacgaattcgagctcggtaccccccacctctcccacctcac(SEQ ID NO:84)

pOsNAR2.1-seamless-R

ggtcgactctagaggatccccgggtacctgctgacaaaccaaaccgact(SEQ ID NO:85)

OsNAR2.1-seamless-F

gtttgtcagcaggtacccggggatccatggcgaggctagccggcgttg(SEQ ID NO:86)

OsNAR2.1-seamless-R

gagccctggcatgcctgcaggtcgacctacttgtccttcttgcgcttc(SEQ ID NO:87)

the inventor constructs OsNAR2.1 self promoter to drive OsNAR2.1 and OsNAR2.1 by using a semless homologous recombination method ^2A 、OsNAR2.1 ^2E Expressed vectors pOsNAR2.1:OsNAR2.1, pOsNAR2.1:OsNAR2.1 ^2A 、pOsNAR2.1::OsNAR2.1 ^2E Adopts agrobacterium tumefaciens as mediumThe WYJ7 is transformed by the guiding method respectively, the transgenic rice material is obtained, and the agricultural characters are planted and counted in a normal nitrogenous fertilizer field. The results showed that compared with WYJ7, pOsNAR2.1:: osNAR2.1 and pOsNAR2.1:: osNAR2.1 ^2E The phenotype of (1) was not significantly altered (FIG. 16A), multiple agronomic traits were not significantly different (FIGS. 16B-G), and pOsNAR2.1:: osNAR2.1 ^2A The transgenic material has obviously increased plant height, tillering, secondary branch number, grain number per scion and seed yield of single plant by 8.9%, 17.4%, 27.9%, 17.2% and 16.4% except that the primary branch number is unchanged (fig. 16B-G). These results indicate that OsNAR2.1 was driven by the self promoter ^2A The expression can also improve the nitrogen fertilizer utilization efficiency and seed yield of the rice.

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The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Sequence(s)

SEQ ID NO:1 AtNAR2.1-2A-CDS

ATGGCGATCCAGAAGATCCTCTTTGCTTCACTTCTCATATGCTCACTGATCCAATCCATCCACGGGGCGGAAAAAGTAAGACTCTTCAAAGAGCTGGACAAAGGTGCACTTGATGTCACCACTAAACCCAGCCGAGAAGGACCAGGTGTTGTTTTGGATGCCGGCAAGGATACGTTGAACATTACATGGACGCTAAGCTCGATTGGGTCTAAAAGAGAGGCTGAATTTAAGATCATCAAAGTTAAGCTATGCTACGCTCCACCTAGCCAAGTTGACCGACCATGGCGCAAAACCCATGACGAGCTCTTCAAAGACAAGACCTGCCCACACAAGATCATAGCCAAGCCTTATGACAAAACACTTCAATCAACTACTTGGACTCTTGAGCGTGACATCCCCACCGGAACCTACTTCGTTCGTGCCTACGCGGTTGATGCCATTGGCCATGAAGTTGCCTATGGACAGAGCACCGACGATGCCAAGAAAACCAATCTCTTCAGCGTTCAGGCTATCAGTGGCCGCCACGCGTCCCTAGATATTGCCTCCATCTGTTTCGCAGTCTTCGCCGTCGTGGCTCTTGTCGTCTTCTTTGTCAATGAGAAGAGGAAGGCCAAGATAGAGCAAAGCAAATGA

SEQ ID NO. 2 AtNAR2.1-2A-protein

MAIQKILFASLLICSLIQSIHGAEKVRLFKELDKGALDVTTKPSREGPGVVLDAGKDTLNITWTLSSIGSKREAEFKIIKVKLCYAPPSQVDRPWRKTHDELFKDKTCPHKIIAKPYDKTLQSTTWTLERDIPTGTYFVRAYAVDAIGHEVAYGQSTDDAKKTNLFSVQAISGRHASLDIASICFAVFAVVALVVFFVNEKRKAKIEQSK

SEQ ID NO:3 AtNAR2.1-S186A-CDS

ATGGCGATCCAGAAGATCCTCTTTGCTTCACTTCTCATATGCTCACTGATCCAATCCATCCACGGGGCGGAAAAAGTAAGACTCTTCAAAGAGCTGGACAAAGGTGCACTTGATGTCACCACTAAACCCAGCCGAGAAGGACCAGGTGTTGTTTTGGATGCCGGCAAGGATACGTTGAACATTACATGGACGCTAAGCTCGATTGGGTCTAAAAGAGAGGCTGAATTTAAGATCATCAAAGTTAAGCTATGCTACGCTCCACCTAGCCAAGTTGACCGACCATGGCGCAAAACCCATGACGAGCTCTTCAAAGACAAGACCTGCCCACACAAGATCATAGCCAAGCCTTATGACAAAACACTTCAATCAACTACTTGGACTCTTGAGCGTGACATCCCCACCGGAACCTACTTCGTTCGTGCCTACGCGGTTGATGCCATTGGCCATGAAGTTGCCTATGGACAGAGCACCGACGATGCCAAGAAAACCAATCTCTTCAGCGTTCAGGCTATCAGTGGCCGCCACGCGTCCCTAGATATTGCCTCCATCTGTTTCGCAGTCTTCTCCGTCGTGGCTCTTGTCGTCTTCTTTGTCAATGAGAAGAGGAAGGCCAAGATAGAGCAAAGCAAATGA

SEQ ID NO. 4 AtNAR2.1-S186A-protein

MAIQKILFASLLICSLIQSIHGAEKVRLFKELDKGALDVTTKPSREGPGVVLDAGKDTLNITWTLSSIGSKREAEFKIIKVKLCYAPPSQVDRPWRKTHDELFKDKTCPHKIIAKPYDKTLQSTTWTLERDIPTGTYFVRAYAVDAIGHEVAYGQSTDDAKKTNLFSVQAISGRHASLDIASICFAVFSVVALVVFFVNEKRKAKIEQSK

SEQ ID NO:5 AtNAR2.1-S189A-CDS

ATGGCGATCCAGAAGATCCTCTTTGCTTCACTTCTCATATGCTCACTGATCCAATCCATCCACGGGGCGGAAAAAGTAAGACTCTTCAAAGAGCTGGACAAAGGTGCACTTGATGTCACCACTAAACCCAGCCGAGAAGGACCAGGTGTTGTTTTGGATGCCGGCAAGGATACGTTGAACATTACATGGACGCTAAGCTCGATTGGGTCTAAAAGAGAGGCTGAATTTAAGATCATCAAAGTTAAGCTATGCTACGCTCCACCTAGCCAAGTTGACCGACCATGGCGCAAAACCCATGACGAGCTCTTCAAAGACAAGACCTGCCCACACAAGATCATAGCCAAGCCTTATGACAAAACACTTCAATCAACTACTTGGACTCTTGAGCGTGACATCCCCACCGGAACCTACTTCGTTCGTGCCTACGCGGTTGATGCCATTGGCCATGAAGTTGCCTATGGACAGAGCACCGACGATGCCAAGAAAACCAATCTCTTCAGCGTTCAGGCTATCAGTGGCCGCCACGCGTCCCTAGATATTGCCTCCATCTGTTTCAGTGTCTTCGCCGTCGTGGCTCTTGTCGTCTTCTTTGTCAATGAGAAGAGGAAGGCCAAGATAGAGCAAAGCAAATGA

SEQ ID NO. 6 AtNAR2.1-S189A-protein

MAIQKILFASLLICSLIQSIHGAEKVRLFKELDKGALDVTTKPSREGPGVVLDAGKDTLNITWTLSSIGSKREAEFKIIKVKLCYAPPSQVDRPWRKTHDELFKDKTCPHKIIAKPYDKTLQSTTWTLERDIPTGTYFVRAYAVDAIGHEVAYGQSTDDAKKTNLFSVQAISGRHASLDIASICFSVFAVVALVVFFVNEKRKAKIEQSK

SEQ ID NO:7 OsNAR2.1-2A-CDS

ATGGCGAGGCTAGCCGGCGTTGCTGCTCTCTCGTTGGTGCTCGTCTTGCTCGGCGCCGGCGTGCCCCGGCCGGCGGCCGCCGCCGCGGCGAAGACGCAGGTGTTCCTCTCCAAGCTGCCCAAAGCGCTCGTCGTCGGCGTCTCGCCCAAGCACGGTGAAGTCGTGCACGCCGGCGAGAACACGGTGACGGTGACGTGGTCGCTGAACACGTCGGAGCCGGCGGGCGCCGACGCGGCGTTCAAGAGCGTGAAGGTGAAGCTGTGCTACGCGCCGGCGAGCCGGACGGACCGCGGGTGGCGCAAGGCCTCCGACGACCTGCACAAGGACAAGGCGTGCCAGTTCAAGGTCACCGTGCAGCCGTACGCCGCCGGCGCCGGCAGGTTCGACTACGTGGTGGCGCGCGACATCCCGACGGCGTCCTACTTCGTGCGCGCCTACGCGGTGGACGCGTCCGGCACGGAGGTGGCCTACGGGCAGAGCTCGCCGGACGCCGCCTTCGACGTCGCCGGGATCACCGGCATCCACGCCTCCCTCAAGGTCGCCGCCGGCGTCTTCGCCACCTTCGCCATCGCCGCGCTCGCCTTCTTCTTCGTCGTCGAGAAGCGCAAGAAGGACAAGTAG

SEQ ID NO. 8 OsNAR2.1-2A-protein

MARLAGVAALSLVLVLLGAGVPRPAAAAAAKTQVFLSKLPKALVVGVSPKHGEVVHAGENTVTVTWSLNTSEPAGADAAFKSVKVKLCYAPASRTDRGWRKASDDLHKDKACQFKVTVQPYAAGAGRFDYVVARDIPTASYFVRAYAVDASGTEVAYGQSSPDAAFDVAGITGIHASLKVAAGVFATFAIAALAFFFVVEKRKKDK

SEQ ID NO:9 OsNAR2.1-S186A-CDS

ATGGCGAGGCTAGCCGGCGTTGCTGCTCTCTCGTTGGTGCTCGTCTTGCTCGGCGCCGGCGTGCCCCGGCCGGCGGCCGCCGCCGCGGCGAAGACGCAGGTGTTCCTCTCCAAGCTGCCCAAAGCGCTCGTCGTCGGCGTCTCGCCCAAGCACGGTGAAGTCGTGCACGCCGGCGAGAACACGGTGACGGTGACGTGGTCGCTGAACACGTCGGAGCCGGCGGGCGCCGACGCGGCGTTCAAGAGCGTGAAGGTGAAGCTGTGCTACGCGCCGGCGAGCCGGACGGACCGCGGGTGGCGCAAGGCCTCCGACGACCTGCACAAGGACAAGGCGTGCCAGTTCAAGGTCACCGTGCAGCCGTACGCCGCCGGCGCCGGCAGGTTCGACTACGTGGTGGCGCGCGACATCCCGACGGCGTCCTACTTCGTGCGCGCCTACGCGGTGGACGCGTCCGGCACGGAGGTGGCCTACGGGCAGAGCTCGCCGGACGCCGCCTTCGACGTCGCCGGGATCACCGGCATCCACGCCTCCCTCAAGGTCGCCGCCGGCGTCTTCGCCACCTTCTCCATCGCCGCGCTCGCCTTCTTCTTCGTCGTCGAGAAGCGCAAGAAGGACAAGTAG

SEQ ID NO. 10 OsNAR2.1-S186A-protein

MARLAGVAALSLVLVLLGAGVPRPAAAAAAKTQVFLSKLPKALVVGVSPKHGEVVHAGENTVTVTWSLNTSEPAGADAAFKSVKVKLCYAPASRTDRGWRKASDDLHKDKACQFKVTVQPYAAGAGRFDYVVARDIPTASYFVRAYAVDASGTEVAYGQSSPDAAFDVAGITGIHASLKVAAGVFATFSIAALAFFFVVEKRKKDK

SEQ ID NO:11 OsNAR2.1-S189A-CDS

ATGGCGAGGCTAGCCGGCGTTGCTGCTCTCTCGTTGGTGCTCGTCTTGCTCGGCGCCGGCGTGCCCCGGCCGGCGGCCGCCGCCGCGGCGAAGACGCAGGTGTTCCTCTCCAAGCTGCCCAAAGCGCTCGTCGTCGGCGTCTCGCCCAAGCACGGTGAAGTCGTGCACGCCGGCGAGAACACGGTGACGGTGACGTGGTCGCTGAACACGTCGGAGCCGGCGGGCGCCGACGCGGCGTTCAAGAGCGTGAAGGTGAAGCTGTGCTACGCGCCGGCGAGCCGGACGGACCGCGGGTGGCGCAAGGCCTCCGACGACCTGCACAAGGACAAGGCGTGCCAGTTCAAGGTCACCGTGCAGCCGTACGCCGCCGGCGCCGGCAGGTTCGACTACGTGGTGGCGCGCGACATCCCGACGGCGTCCTACTTCGTGCGCGCCTACGCGGTGGACGCGTCCGGCACGGAGGTGGCCTACGGGCAGAGCTCGCCGGACGCCGCCTTCGACGTCGCCGGGATCACCGGCATCCACGCCTCCCTCAAGGTCGCCGCCGGCGTCTTCTCCACCTTCGCCATCGCCGCGCTCGCCTTCTTCTTCGTCGTCGAGAAGCGCAAGAAGGACAAGTAG

SEQ ID NO. 12 OsNAR2.1-S189A-protein

MARLAGVAALSLVLVLLGAGVPRPAAAAAAKTQVFLSKLPKALVVGVSPKHGEVVHAGENTVTVTWSLNTSEPAGADAAFKSVKVKLCYAPASRTDRGWRKASDDLHKDKACQFKVTVQPYAAGAGRFDYVVARDIPTASYFVRAYAVDASGTEVAYGQSSPDAAFDVAGITGIHASLKVAAGVFSTFAIAALAFFFVVEKRKKDK

SEQ ID NO:13 TaNAR2.1-2A-CDS

ATGGCACGGTCGGAGCTGGCCATGGCGTTGCTGGTGGTGGTCCTCGCCGCCGGCTGCTGCGCGCCGGCGGCCGCCGTGGCGTACCTCTCCAAGCTGCCTGTGACCCTCGACGTCACGGCATCCCCCAGTCCCGGCCAAGTTCTTCACGCCGGCGAGGACGTGATCACGGTGACGTGGGCCCTGAACGCGAGCCAGCCGGCCGGCAAGGACGCCGAATACAAGAACGTGAAGGTGAGCCTCTGCTACGCGCCGGTGAGCCAGAAGGAGCGCGAGTGGCGCAAGACCCACGACGACCTCAAGAAGGACAAGACCTGCCAGTTCAAGGTCACCCAGCAGGCCTACCCCGGCACCGGCAAGGTCGAGTACCGCGTCGCCCTCGACATCCCCACCGCCACCTACTACGTGCGCGCCTACGCGCTCGACGCCTCCGGCACGCAGGTCGCCTACGGCCAGACCGCGCCCGCCTCCGCCTTCAACGTCGTCAGCATCACCGGCGTCACCACCTCCATCAAGGTCGCCGCCGGCGTATTCGCCGCCTTCGCCGTCGCCTCCCTCGCCTTCTTCTTCTTCATTGAGAAACGCAAGAAGAACAACTAA

SEQ ID NO. 14 TaNAR2.1-2A-protein

MARSELAMALLVVVLAAGCCAPAAAVAYLSKLPVTLDVTASPSPGQVLHAGEDVITVTWALNASQPAGKDAEYKNVKVSLCYAPVSQKEREWRKTHDDLKKDKTCQFKVTQQAYPGTGKVEYRVALDIPTATYYVRAYALDASGTQVAYGQTAPASAFNVVSITGVTTSIKVAAGVFAAFAVASLAFFFFIEKRKKNN

SEQ ID NO:15 TaNAR2.1-S186A-CDS

ATGGCACGGTCGGAGCTGGCCATGGCGTTGCTGGTGGTGGTCCTCGCCGCCGGCTGCTGCGCGCCGGCGGCCGCCGTGGCGTACCTCTCCAAGCTGCCTGTGACCCTCGACGTCACGGCATCCCCCAGTCCCGGCCAAGTTCTTCACGCCGGCGAGGACGTGATCACGGTGACGTGGGCCCTGAACGCGAGCCAGCCGGCCGGCAAGGACGCCGAATACAAGAACGTGAAGGTGAGCCTCTGCTACGCGCCGGTGAGCCAGAAGGAGCGCGAGTGGCGCAAGACCCACGACGACCTCAAGAAGGACAAGACCTGCCAGTTCAAGGTCACCCAGCAGGCCTACCCCGGCACCGGCAAGGTCGAGTACCGCGTCGCCCTCGACATCCCCACCGCCACCTACTACGTGCGCGCCTACGCGCTCGACGCCTCCGGCACGCAGGTCGCCTACGGCCAGACCGCGCCCGCCTCCGCCTTCAACGTCGTCAGCATCACCGGCGTCACCACCTCCATCAAGGTCGCCGCCGGCGTATTCGCCGCCTTCTCCGTCGCCTCCCTCGCCTTCTTCTTCTTCATTGAGAAACGCAAGAAGAACAACTAA

SEQ ID NO. 16 TaNAR2.1-S186A-protein

MARSELAMALLVVVLAAGCCAPAAAVAYLSKLPVTLDVTASPSPGQVLHAGEDVITVTWALNASQPAGKDAEYKNVKVSLCYAPVSQKEREWRKTHDDLKKDKTCQFKVTQQAYPGTGKVEYRVALDIPTATYYVRAYALDASGTQVAYGQTAPASAFNVVSITGVTTSIKVAAGVFAAFSVASLAFFFFIEKRKKNN

SEQ ID NO:17 TaNAR2.1-S189A-CDS

ATGGCACGGTCGGAGCTGGCCATGGCGTTGCTGGTGGTGGTCCTCGCCGCCGGCTGCTGCGCGCCGGCGGCCGCCGTGGCGTACCTCTCCAAGCTGCCTGTGACCCTCGACGTCACGGCATCCCCCAGTCCCGGCCAAGTTCTTCACGCCGGCGAGGACGTGATCACGGTGACGTGGGCCCTGAACGCGAGCCAGCCGGCCGGCAAGGACGCCGAATACAAGAACGTGAAGGTGAGCCTCTGCTACGCGCCGGTGAGCCAGAAGGAGCGCGAGTGGCGCAAGACCCACGACGACCTCAAGAAGGACAAGACCTGCCAGTTCAAGGTCACCCAGCAGGCCTACCCCGGCACCGGCAAGGTCGAGTACCGCGTCGCCCTCGACATCCCCACCGCCACCTACTACGTGCGCGCCTACGCGCTCGACGCCTCCGGCACGCAGGTCGCCTACGGCCAGACCGCGCCCGCCTCCGCCTTCAACGTCGTCAGCATCACCGGCGTCACCACCTCCATCAAGGTCGCCGCCGGCGTATTCTCCGCCTTCGCCGTCGCCTCCCTCGCCTTCTTCTTCTTCATTGAGAAACGCAAGAAGAACAACTAA

SEQ ID NO. 18 TaNAR2.1-S189A-protein

MARSELAMALLVVVLAAGCCAPAAAVAYLSKLPVTLDVTASPSPGQVLHAGEDVITVTWALNASQPAGKDAEYKNVKVSLCYAPVSQKEREWRKTHDDLKKDKTCQFKVTQQAYPGTGKVEYRVALDIPTATYYVRAYALDASGTQVAYGQTAPASAFNVVSITGVTTSIKVAAGVFSAFAVASLAFFFFIEKRKKNN

SEQ ID NO:19 CrNAR2.1-2A-CDS

ATGAAGGCATACGCGCTCCTCTTAGCCTTTGCGGCCTTCCTCTTGGCCGCTCCCGGCGCGAAATCGCTGGCCGGCGAGGACATGCCCAGTGCGCCCAAGCCCACCTCTCCTCAGTACTCGCCCACCCTGGCCAACTTCACGTCGCAGTGGGCCATCAGCATGCAGCGCCGCGACTCCCTCGATGCTGCCTGGCGCCTTCACACCTGCAACGCCACCGTCATCGGCGCCCCCTGCAACCAGCCTCCCCTCGGCGGTCAGACGCCATCGCAAGTCAAGATCGTCTTTGTCCGCCGCAACGCCTCGGCCGGCCCACTTCGCACCTGGAAGGGCGGCGTGCCCACCTCCATCAAGGTCCGCCTCGACTACGGCGCCTCCGTCCAGGTCGACCGCGGCTGGCGCAAGAAGAACCAGGCTTGGCCCGGGCACGGCTGGCACGCCAAGTGGACTGTCGCCACCCTGCCCTACAACGAGACCGGCGGCGAGGCTGTGTGGGACCTGCAGACCGCGGATGAGGTGACCGACGCCATCCTCTACCCCGAAGTCTGCGTCATCTGCACCTTCCCCGACGGCTCAACCGACTACTGCCAGTGCGACCGCCGCAACGGTGCCACCAACTACCTGTCAGTCGAGACCATCGTACAGGACTCCATCACTCCCGCCATGCGCGCCGCCGCCATCGCGCTCGCCGTCTTCGCGCCGCTCTTCCTCATCTTCTACGCCACTGCCGACACGCTCTATTTCCGCAACACGGGTAAATCGCTGCGCATCGGCCACATCTAA

SEQ ID NO. 20 CrNAR2.1-2A-protein MKAYALLLAFAAFLLAAPGAKSLAGEDMPSAPKPTSPQYSPTLANFTSQWAISMQRRDSLDAAWRLHTCNATVIGAPCNQPPLGGQTPSQVKIVFVRRNASAGPLRTWKGGVPTSIKVRLDYGASVQVDRGWRKKNQAWPGHGWHAKWTVATLPYNETGGEAVWDLQTADEVTDAILYPEVCVICTFPDGSTDYCQCDRRNGATNYLSVETIVQDSITPAMRAAAIALAVFAPLFLIFYATADTLYFRNTGKSLRIGHI

SEQ ID NO:21 CrNAR2.1-S186A-CDS

ATGAAGGCATACGCGCTCCTCTTAGCCTTTGCGGCCTTCCTCTTGGCCGCTCCCGGCGCGAAATCGCTGGCCGGCGAGGACATGCCCAGTGCGCCCAAGCCCACCTCTCCTCAGTACTCGCCCACCCTGGCCAACTTCACGTCGCAGTGGGCCATCAGCATGCAGCGCCGCGACTCCCTCGATGCTGCCTGGCGCCTTCACACCTGCAACGCCACCGTCATCGGCGCCCCCTGCAACCAGCCTCCCCTCGGCGGTCAGACGCCATCGCAAGTCAAGATCGTCTTTGTCCGCCGCAACGCCTCGGCCGGCCCACTTCGCACCTGGAAGGGCGGCGTGCCCACCTCCATCAAGGTCCGCCTCGACTACGGCGCCTCCGTCCAGGTCGACCGCGGCTGGCGCAAGAAGAACCAGGCTTGGCCCGGGCACGGCTGGCACGCCAAGTGGACTGTCGCCACCCTGCCCTACAACGAGACCGGCGGCGAGGCTGTGTGGGACCTGCAGACCGCGGATGAGGTGACCGACGCCATCCTCTACCCCGAAGTCTGCGTCATCTGCACCTTCCCCGACGGCTCAACCGACTACTGCCAGTGCGACCGCCGCAACGGTGCCACCAACTACCTGTCAGTCGAGACCATCGTACAGGACTCCATCACTCCCGCCATGCGCGCCGCCGCCATCGCGCTCGCCGTCTTCTCGCCGCTCTTCCTCATCTTCTACGCCACTGCCGACACGCTCTATTTCCGCAACACGGGTAAATCGCTGCGCATCGGCCACATCTAA

SEQ ID NO. 22 CrNAR2.1-S186A-protein

MKAYALLLAFAAFLLAAPGAKSLAGEDMPSAPKPTSPQYSPTLANFTSQWAISMQRRDSLDAAWRLHTCNATVIGAPCNQPPLGGQTPSQVKIVFVRRNASAGPLRTWKGGVPTSIKVRLDYGASVQVDRGWRKKNQAWPGHGWHAKWTVATLPYNETGGEAVWDLQTADEVTDAILYPEVCVICTFPDGSTDYCQCDRRNGATNYLSVETIVQDSITPAMRAAAIALAVFSPLFLIFYATADTLYFRNTGKSLRIGHI

SEQ ID NO:23 CrNAR2.1-S189A-CDS

ATGAAGGCATACGCGCTCCTCTTAGCCTTTGCGGCCTTCCTCTTGGCCGCTCCCGGCGCGAAATCGCTGGCCGGCGAGGACATGCCCAGTGCGCCCAAGCCCACCTCTCCTCAGTACTCGCCCACCCTGGCCAACTTCACGTCGCAGTGGGCCATCAGCATGCAGCGCCGCGACTCCCTCGATGCTGCCTGGCGCCTTCACACCTGCAACGCCACCGTCATCGGCGCCCCCTGCAACCAGCCTCCCCTCGGCGGTCAGACGCCATCGCAAGTCAAGATCGTCTTTGTCCGCCGCAACGCCTCGGCCGGCCCACTTCGCACCTGGAAGGGCGGCGTGCCCACCTCCATCAAGGTCCGCCTCGACTACGGCGCCTCCGTCCAGGTCGACCGCGGCTGGCGCAAGAAGAACCAGGCTTGGCCCGGGCACGGCTGGCACGCCAAGTGGACTGTCGCCACCCTGCCCTACAACGAGACCGGCGGCGAGGCTGTGTGGGACCTGCAGACCGCGGATGAGGTGACCGACGCCATCCTCTACCCCGAAGTCTGCGTCATCTGCACCTTCCCCGACGGCTCAACCGACTACTGCCAGTGCGACCGCCGCAACGGTGCCACCAACTACCTGTCAGTCGAGACCATCGTACAGGACTCCATCACTCCCGCCATGCGCGCCGCCGCCATCGCGCTCTCCGTCTTCGCGCCGCTCTTCCTCATCTTCTACGCCACTGCCGACACGCTCTATTTCCGCAACACGGGTAAATCGCTGCGCATCGGCCACATCTAA

SEQ ID NO. 24 CrNAR2.1-S189A-protein

MKAYALLLAFAAFLLAAPGAKSLAGEDMPSAPKPTSPQYSPTLANFTSQWAISMQRRDSLDAAWRLHTCNATVIGAPCNQPPLGGQTPSQVKIVFVRRNASAGPLRTWKGGVPTSIKVRLDYGASVQVDRGWRKKNQAWPGHGWHAKWTVATLPYNETGGEAVWDLQTADEVTDAILYPEVCVICTFPDGSTDYCQCDRRNGATNYLSVETIVQDSITPAMRAAAIALSVFAPLFLIFYATADTLYFRNTGKSLRIGHI

SEQ ID NO:25 MpNAR2.1-2A-CDS

ATGGCAACGGTGGGAAAATCGATTGTTGCCGGAGCGCTCGTGCTGTTCGCTCTGCTGAGCTTCGTGGAGAGCACAGTCCTCTTCTCCTCACTGCAGAGGACATTGGTTGTGGATGCCGAGATTCAGAATCAAGTTCCCATGATGGGGATCGCCAAAGCTGGGGAAGACCACTTGGTGATCTCATGGGCATTGAACAGCACTCTCCAAGAGGGACTTCCCGGCCTCGATGCAACTTACGAGACGGTGCAGTTGAAACTCTGCTACGCACCTGTGAGCCAAGTAGAACGAGGCTGGAGGAAGAAGAATGATGACCTCCACAAGGACAAGACCTGCACCAAGGGAATCGCCAAGCAGAAGTACACTTCGGCCGGAAATTCCACCAACTGGAGAATCACCAAGGACGTCCCCGGCGCTGTGTACTTCGTCCGTGCGTACCTTTTGAACTCCAACGGGACACAAGTCGCTTACGGCCAGACCACCAACGCCGCCAGGACCACCAACTTGTTCACCGTCGTGCCCATCTCCGGAAGGCACGCTTCTATCGACGTCGCTGCTGCCATCTTCGCTGCATTCGCCATCGGGTCGTTGGTCTTCTTCCTCGCTCTCGAGAACATGAGAAGCAAGCGCTCGAACGGCAGCAAGTGA

SEQ ID NO. 26 MpNAR2.1-2A-protein

MATVGKSIVAGALVLFALLSFVESTVLFSSLQRTLVVDAEIQNQVPMMGIAKAGEDHLVISWALNSTLQEGLPGLDATYETVQLKLCYAPVSQVERGWRKKNDDLHKDKTCTKGIAKQKYTSAGNSTNWRITKDVPGAVYFVRAYLLNSNGTQVAYGQTTNAARTTNLFTVVPISGRHASIDVAAAIFAAFAIGSLVFFLALENMRSKRSNGSK

SEQ ID NO:27 MpNAR2.1-S186A-CDS

ATGGCAACGGTGGGAAAATCGATTGTTGCCGGAGCGCTCGTGCTGTTCGCTCTGCTGAGCTTCGTGGAGAGCACAGTCCTCTTCTCCTCACTGCAGAGGACATTGGTTGTGGATGCCGAGATTCAGAATCAAGTTCCCATGATGGGGATCGCCAAAGCTGGGGAAGACCACTTGGTGATCTCATGGGCATTGAACAGCACTCTCCAAGAGGGACTTCCCGGCCTCGATGCAACTTACGAGACGGTGCAGTTGAAACTCTGCTACGCACCTGTGAGCCAAGTAGAACGAGGCTGGAGGAAGAAGAATGATGACCTCCACAAGGACAAGACCTGCACCAAGGGAATCGCCAAGCAGAAGTACACTTCGGCCGGAAATTCCACCAACTGGAGAATCACCAAGGACGTCCCCGGCGCTGTGTACTTCGTCCGTGCGTACCTTTTGAACTCCAACGGGACACAAGTCGCTTACGGCCAGACCACCAACGCCGCCAGGACCACCAACTTGTTCACCGTCGTGCCCATCTCCGGAAGGCACGCTTCTATCGACGTCGCTGCTGCCATCTTCGCTGCATTCTCCATCGGGTCGTTGGTCTTCTTCCTCGCTCTCGAGAACATGAGAAGCAAGCGCTCGAACGGCAGCAAGTGA

28 MpNAR2.1-2A-protein of SEQ ID NO

MATVGKSIVAGALVLFALLSFVESTVLFSSLQRTLVVDAEIQNQVPMMGIAKAGEDHLVISWALNSTLQEGLPGLDATYETVQLKLCYAPVSQVERGWRKKNDDLHKDKTCTKGIAKQKYTSAGNSTNWRITKDVPGAVYFVRAYLLNSNGTQVAYGQTTNAARTTNLFTVVPISGRHASIDVAAAIFAAFSIGSLVFFLALENMRSKRSNGSK

SEQ ID NO:29 MpNAR2.1-S189A-CDS

ATGGCAACGGTGGGAAAATCGATTGTTGCCGGAGCGCTCGTGCTGTTCGCTCTGCTGAGCTTCGTGGAGAGCACAGTCCTCTTCTCCTCACTGCAGAGGACATTGGTTGTGGATGCCGAGATTCAGAATCAAGTTCCCATGATGGGGATCGCCAAAGCTGGGGAAGACCACTTGGTGATCTCATGGGCATTGAACAGCACTCTCCAAGAGGGACTTCCCGGCCTCGATGCAACTTACGAGACGGTGCAGTTGAAACTCTGCTACGCACCTGTGAGCCAAGTAGAACGAGGCTGGAGGAAGAAGAATGATGACCTCCACAAGGACAAGACCTGCACCAAGGGAATCGCCAAGCAGAAGTACACTTCGGCCGGAAATTCCACCAACTGGAGAATCACCAAGGACGTCCCCGGCGCTGTGTACTTCGTCCGTGCGTACCTTTTGAACTCCAACGGGACACAAGTCGCTTACGGCCAGACCACCAACGCCGCCAGGACCACCAACTTGTTCACCGTCGTGCCCATCTCCGGAAGGCACGCTTCTATCGACGTCGCTGCTGCCATCTTCTCTGCATTCGCCATCGGGTCGTTGGTCTTCTTCCTCGCTCTCGAGAACATGAGAAGCAAGCGCTCGAACGGCAGCAAGTGA

SEQ ID NO. 30 MpNAR2.1-S189A-protein

MATVGKSIVAGALVLFALLSFVESTVLFSSLQRTLVVDAEIQNQVPMMGIAKAGEDHLVISWALNSTLQEGLPGLDATYETVQLKLCYAPVSQVERGWRKKNDDLHKDKTCTKGIAKQKYTSAGNSTNWRITKDVPGAVYFVRAYLLNSNGTQVAYGQTTNAARTTNLFTVVPISGRHASIDVAAAIFSAFAIGSLVFFLALENMRSKRSNGSK

SEQ ID NO. 31 AtNAR2.1 promoter

ttttctgcgatttcagctcgagtttccacttccagacttctcttgcgttgcatttccacttcttctcgtagttcaagtatctgccaatgcagaactaaattgaggtaacaaaacaagaaggaaatacacacctcaaactacaaaacaccaacctgtttctgaagttcataagccgttccttctgcttcactggattctcagtctaaataaaaaagcatcaacagcaacaaaagtgtaaataagtttcttctatacataacaattttgaatacatgaaccaaagattaattaaaaatatcacttgaagagatccaaccacatcaaagatgatgcagcttgagtaattgttaactctttccaactttggaaagagacaaaacgtcaaagtgatttccagaaacagagaacggaagctcaaaagtctctcttgtgccaaatatattaaaccctaaaaaattcaacttctatccgaatttctcagaaacaaaacaaagacacataacttcatatgaaattccagtgaacggttacctgaggaagaaccttatttgttatcagtgtaaaataacagagaagaggtacgagtaaaagaaacaaaagtgacttaggaaacgccattgttggagactgctcactggaagatagagagtcgtgagagacagtgataaagcgtatcaagtcatatagagggtcttcttatctttttctttaacatgtgagggttgagttaattatgcgggctgattatagagtttttaaattgaatttacgattgtttttttcttacatataaatgcaatctatatttgtgttcggaataacccatctaatattactccatgtattaaactaaaatattttgcatgttttggtagatcaactttttgaatgatcacagacctacaaacatcaaccctctaattatccaattttaccataatccacgagctcttaaaattcatttttaatatatataattaaaatagttcaaacagtttaaactttgtgaccaagttaaaatatttaaaatagtttgactttgtgatcaacatatttaaatataatcaatattttcatttttaagccggaaaatcacgtcttacaaatatatttctgatagacacacctataattccaaaattttgacttttaaaacaaacaaaaaaaaatctcattaataccactacatacgtttttacaaaaataccattaaaagatattttttcaaatactaaaaaaaactaaaactaaactttaaacctataaaacactaaatcctataaagtttatattcgagtataaaccttaaaagttcaaccctaaatcctgaaagcaagaccctaaacccaaactcaaacttttaaattataaatccttaaacaaaatcatttttagtctttatggtatttttagagttaacagtttgtagttgtatttttgaaaaaaaaacactagtgataggtttttagaataaaaacttaatttagtggtattgaggatatttctctaataaaaattattcacaaaaaatatgtattaaagcaaagtgctatgcttattccatgcaatcttttttgaaaaaaaaaatattttctaaccgattagtatatcttctagaggattcatagaaaaagaggatacaattacaatatgtagagtatcttataggtgacgtaaccatgaaatatagaattctttggaatctgaaactgaattattcagttgataaatgataaaacaaatactcatatctcatcctttggcatgtttaggagccatctctcagctggacggagacaacacagacacttgtgccagagagggaacacaatcttcccaagtcttcgaaagggtaatctcagaccaaaccgttgactttgttctcattaattgtttatgctaaaccacaagatatttggatcaaaagtattaccataaatcactatataaagacataaatgtgcaccccacttttcttatcaatcataaaagtcagcaaaacacaaggcatattcctcttctcttcctcagccttatttttctgatattcagtttcaaggatatatcc

32 maize polyubiquitin gene promoter of SEQ ID NO

ctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttctgcag

SEQ ID NO. 33 SAD1 promoter

gctatgaccatgattacgaattcattatgctccaatttgtttggtaccttcagtattagtttctggacattgtacatattatgttgccgtataagctgagctagaaggatcattagtgtaattccatatatatctaaatgtacccgtggaatcacatttgaggaagttccaatgatgccctttttgccctgcacacgcatgtataagaaccctttgcccgcagcatagagctagtactagccagtatcccattgcttgttttcctcgcatacactgcccgttgtggtacccggggatcctctagagtcg

SEQ ID NO. 34 OsNAR2.1 promoter

ccccacctctcccacctcactcctacctcactcctagtcctctgccgaaagtacttcctccgtttcacaatgtaagtcattctaatatttttcacattcatattgatgtttgaatctagattgatatatatgtttagattcgttagcatcgatatgaatatgggaaatgctagaatgacttatattgtgaaacagagtgagtatcatgtaaaagttagaaggaaaaaaatagagctgtttgtgatgatatgggtgtggttgtgttgtgtgagccgatgtccattgtactgtactcattttaaatgtacgtaccgttaacttatatagttatatgcgtttgatcatttgtcaaaatttagtgaaactttaaaatttattatacttaaagtatatttaatgataaatttaaaataaaataaactttcagctgttatgttcaaaatcaacatcgtcagatattttaaattaaaggtagtacttttaaaaaaaggatttttgcggtgtgtcgtggcgaaactgctaccaagtttcaatgatcatatgccatttcataggataattactctcatcgtggtaagtaagaatcgattgcctattttcggcaggctgttgtttcaaagcatcgatctgcttggacaacttgagcaaagctagctagaactgggtcgatataattgcagcactaggcaatcaagagacggagctggccaccagctagctgagctgagctgatatgatcaacacagtgcagacttggtcgtgttcgagttcgatcgacggatggctgtcctgctcttgcgctcatgcatgtcatctcttcggaagtaggagtacagcagtacttgaggaatattattagagagtaagttgaactgttttcaatagttcagggtgtaaactaagctgaggaattgttaggaggttaaatgctgtggcaaaatagtttggaggagcgaaatgatttttttttcatatgaaaaacatctaaatttattttttgccaaaacactagtatatcatcaaattttcatccattaagaacgccttctcaatattaataattccaatgtgatatcttaatgctcaatgaacctaaaatagtttggatgagtgaaatggactctttttgagtttttttccatatgaaaacatctaaatttatttttttttgccaaaacactggtatatcatcaaattttcctccattaagaacgccttctcaacgttaataactccaatgttattatcttaatgccaaatgaacctaccatgaacgtcatgctcacaatttaattaacaacaaccgaggcactcaagatcattcgcggttgccgcttctcaccggttgcctgaacccttgggacccctccaaaagcttaattacccccaaaaccgcatgatctctctcttctcttctcttctcacacgtcgtcaaagcctctgactttggatatccccgaccccactaaacttaatcaacttgatcattacaacaattaagttgcctcttgaatccaacgaagtagctggtcaactctccgagctcgtagcctcgctctcccgcctataaattcaccgatcgatcgatcgatcgatctcagcatcagcagcagcagcagattcatttcttggtcttcgtctccgtctccgtccttgggttgatatccagaatcagtcggtttggtttgtcagca

Claims

1. A variant of a NAR2.1 protein controlling plant nitrogen use efficiency, biomass and seed yield, characterized in that said protein variant is obtained by mutating serine at positions 186, 189 of the NAR2.1 protein and its conserved serine at the homologous NAR2.1 protein in arabidopsis thaliana to an amino acid in a pseudo non-phosphorylated form selected from the group consisting of alanine and phenylalanine.

2. A variant of a NAR2.1 protein controlling plant nitrogen use efficiency, biomass and seed yield, the amino acid sequence of which is as set forth in any one of the following:

(1) 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

(2) Amino acid sequence having at least 70% identity, preferably at least 80%, more preferably at least 90% identity, especially at least 95% or 98% or 99% identity, to the amino acid sequence shown in SEQ ID No. 2, and having alanine at positions 186, 189, to the amino acid sequence shown in SEQ ID No. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

(3) Amino acid sequences which differ from the amino acid sequences shown in SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 by substitution, deletion and/or insertion of one or more amino acid residues, but which are active as proteins consisting of the amino acid sequences shown in SEQ ID NO. 2,4,6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30;

(4) An active fragment comprising any one of the amino acid sequences of (1) to (3).

3. The NAR2.1 protein variant according to claim 1 or 2 having a nucleotide sequence as set forth in any one of the following:

(1) 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29;

(2) 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, and a nucleotide sequence which has at least 70%, preferably at least 80%, more preferably at least 90% identity, in particular at least 95% or 98% or 99% identity, to the nucleotide sequence shown in SEQ ID No. 1 and which encodes alanine at codons corresponding to amino acids 186, 189;

(3) A nucleotide sequence which differs from the nucleotide sequence shown in SEQ ID NO. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 by one or more substitutions, deletions and/or insertions, but which encodes a protein having the same activity as the protein encoded by the nucleotide sequence of SEQ ID NO. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29;

(4) A nucleotide sequence which differs in sequence from SEQ ID NO. 1,3,5,7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 due to the degeneracy of the genetic code;

(5) An active fragment comprising the nucleotide sequence of any one of (1) to (4).

4. A recombinant construct expressing the NAR2.1 protein variant of any one of claims 1-3.

5. A host cell comprising the recombinant construct of claim 4, wherein the host cell is a microbial cell, preferably an e.

6. A method of increasing nitrogen fertilizer utilization efficiency, biomass and seed yield in a plant, the method comprising expressing or overexpressing the NAR2.1 protein variant of any one of claims 1-3 in a plant.

7. The method of claim 6, wherein the NAR2.1 protein variant has a regulatory sequence attached thereto selected from a constitutive promoter or a root tissue specific promoter or a self promoter of a gene encoding the NAR2.1 protein variant.

8. The method of claim 7, wherein the nucleotide sequence of the constitutive promoter is shown as SEQ ID NO. 32, the nucleotide sequence of the root tissue specific promoter is shown as SEQ ID NO. 33, and the nucleotide sequence of the self promoter of the gene encoding the NAR2.1 protein variant is shown as SEQ ID NO. 31 or SEQ ID NO. 34.

9. A method of growing plants with high nitrogen fertilizer efficiency, high biomass and high seed yield, the method comprising introducing into the plants a gene editing system targeting the coding base of serine at the 186 th and 189 th conserved sites of the atnar2.1 protein or the coding base of serine at the corresponding conserved site of the homologous NAR2.1 protein, resulting in mutation of serine to alanine, producing NAR2.1 with double site mutation to alanine ^2A Protein variants, and NAR2.1 mutated to alanine at a single site ^S186A 、NAR2.1 ^S189A Protein variants, whereby plants with high nitrogen fertilizer utilization, high biomass and high seed yield are obtained, wherein the plants are preferably arabidopsis, rice or wheat.

10. A method of increasing nitrogen uptake in a plant comprising expressing or overexpressing the NAR2.1 protein variant of any one of claims 1-3 in a plant and the recombinant construct of claim 4 or introducing a gene editing system into the plant to mutate the coding base for serine at the conserved positions 186, 189 of the atnar2.1 protein or the coding base for serine at the corresponding conserved position of the homologous NAR2.1 protein to alanine.

11. A method of modulating carbon nitrogen metabolic balance in a plant comprising expressing or overexpressing the NAR2.1 protein variant of any one of claims 1-3 in a plant.