Rice nitrogen efficient utilization gene OsNLP6 and its encoding protein and application

Abstract

The present invention discloses a rice nitrogen efficient utilization gene OsNLP6 and its encoding protein and application, and relates to the technical field of plant genetic engineering. The rice nitrogen efficient utilization gene OsNLP6, its nucleotide sequence is shown in SEQ ID NO.1. The polypeptide, its encoding gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The present invention adopts the above-mentioned rice nitrogen efficient utilization gene OsNLP6 and its encoding protein and application. The OsNLP6 gene can promote the absorption and utilization of nitrogen by plants, especially can improve the absorption and utilization efficiency of nitrogen by rice, can be used to cultivate new rice varieties with high nitrogen utilization efficiency, and lay the foundation for rice breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to a rice nitrogen efficient utilization gene OsNLP6 and its encoding protein and application. Background Art

[0002] Rice is one of the most important food crops in the world, and more than half of the world's population relies on rice as their staple food. Nitrogen is an important limiting factor for rice growth and yield formation, and plays a huge role in rice life activities, material production, yield formation and rice quality. The nitrogen absorbed by rice is mainly ammonium nitrogen, but due to rhizosphere nitrification, about 40% of the nitrogen is absorbed in the form of nitrate nitrogen. Although large-scale nitrogen application can increase rice yield to a certain extent, excessive use of nitrogen fertilizer not only increases costs and reduces rice quality, but also causes a series of ecological and environmental problems, limiting the full utilization of nitrogen nutritional potential. Therefore, improving nitrogen use efficiency (NUE) is conducive to high and stable rice yields, and is of great significance to ensuring national food security, protecting the ecological environment, increasing farmers' income and achieving stable and sustainable agricultural development. Exploring and utilizing excellent genetic resources and genotypes related to NUE in rice germplasm resources is the most economical and effective way to solve the above problems.

[0003] The efficient use of nitrogen is mainly determined by the absorption efficiency and utilization efficiency of nitrogen. The absorption efficiency of nitrogen refers to the ability of crops to absorb nitrogen in the soil; the utilization efficiency of nitrogen refers to the ability of crops to convert nitrogen into products. How to improve the utilization efficiency of nitrogen and reduce nitrogen input has become an urgent problem that needs to be solved. The nitrogen utilization efficiency of different crops is inconsistent. The utilization efficiency of nitrogen is not only the characteristic of a certain crop, but also the characteristic of the entire crop system. The effective nitrogen content in the soil, the field management of crops, different rotation methods and environmental conditions will affect the absorption and utilization of nitrogen by crops. Therefore, breeding crop varieties with high nitrogen utilization efficiency is the most economical and effective means to improve nitrogen utilization. Summary of the invention

[0004] The purpose of the present invention is to provide a rice nitrogen efficient utilization gene OsNLP6 and its encoding protein and application. The OsNLP6 gene can promote the absorption and utilization of nitrogen by plants, especially can improve the absorption and utilization efficiency of nitrogen by rice, and can be used to cultivate new rice varieties with high nitrogen utilization efficiency, laying the foundation for rice breeding.

[0005] To achieve the above-mentioned purpose, the present invention provides a rice nitrogen efficient utilization gene OsNLP6 and its encoded protein and application. The nitrogen efficient utilization gene OsNLP6 has a nucleotide sequence as shown in SEQ ID NO.1.

[0006] The present invention also provides a polypeptide, the coding gene of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] The present invention also provides a recombinant expression vector of a nitrogen efficient utilization gene comprising the nitrogen efficient utilization gene OsNLP6.

[0008] The present invention also provides a host cell comprising the nitrogen efficient utilization gene OsNLP6 or a recombinant expression vector, wherein the host cell is a microbial cell.

[0009] Furthermore, the microbial cells are Escherichia coli cells or Agrobacterium cells.

[0010] The present invention also provides the use of rice nitrogen efficient utilization gene OsNLP6 or a recombinant vector in cultivating plants with improved nitrogen utilization efficiency.

[0011] Further, the application environment is a low nitrogen and / or high nitrogen growth condition.

[0012] The present invention also provides a method for cultivating plants with improved nitrogen utilization efficiency, comprising the following steps: introducing the gene OsNLP6 or its active fragment or recombinant vector into plant cells to obtain transgenic plants;

[0013] Or a plant containing the OsNLP6 gene or its active fragment is hybridized with another plant to obtain a hybrid offspring plant containing the OsNLP6 gene or its active fragment.

[0014] Compared with control plants or non-hybridized plants without the introduction of the OsNLP6 gene or its active fragment or recombinant expression vector, the nitrogen utilization efficiency of the transgenic plants and hybrid offspring plants is improved.

[0015] Furthermore, the method also includes codon-optimizing the gene OsNLP6 for plant codon usage before introducing into plant cells.

[0016] Furthermore, the plant is selected from rice, corn, wheat, cotton, rapeseed, soybean, or Arabidopsis, preferably rice.

[0017] Furthermore, the gene OsNLP6 or an active fragment thereof is overexpressed in the transgenic plants.

[0018] Furthermore, the transgenic plants or hybrid offspring plants have growth advantages under low nitrogen and / or high nitrogen growth conditions.

[0019] Furthermore, the yield of the transgenic plants or hybrid progeny plants is increased.

[0020] The advantages and positive effects of the rice nitrogen efficient utilization gene OsNLP6 and its encoded protein and application described in the present invention are:

[0021] The OsNLP6 gene can promote the absorption and utilization of nitrogen by plants, especially it can improve the absorption and utilization efficiency of nitrogen by rice. It can be used to cultivate new rice varieties with high nitrogen utilization efficiency, laying the foundation for rice breeding.

[0022] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The physical map of the overexpression vector pBWA(V)HS-NLP6-OE-GLosgfp in the embodiment of the present invention;

[0024] Figure 2 A in the figure is the qRT-PCR detection of OsNLP6 gene expression in rice lines with overexpression and knockout of OsNLP6 gene, B is the CRISPR gene mutation site situation, WT represents wild-type rice, as a control, OE-1 represents a transgenic rice line with overexpression of OsNLP6 gene, and OE-2 represents another transgenic rice line with overexpression of OsNLP6 gene;

[0025] Figure 3 The subcellular localization result of the OsNLP6 gene provided in Example 1 of the present invention, wherein A is a green fluorescence image of an empty vector, B is a red fluorescence image of an empty vector, C is a bright field image of an empty vector, D is a fusion image of an empty vector, E is a green fluorescence image of an OsNLP6 fusion protein, F is a red fluorescence image of an OsNLP6 fusion protein, G is a bright field image of an OsNLP6 fusion protein, and H is a fusion image of an OsNLP6 fusion protein;

[0026] Figure 4 The growth conditions of each rice genetic material of OsNLP6 in the field under normal conditions in the present invention, LN is a low nitrogen environment, HN is a high nitrogen environment, OsNLP6 overexpression strains have obvious growth advantages, while the knockout mutant has a weaker growth trend;

[0027] Figure 5 The figures are the statistics of ear weight, effective ear number, total dry weight and single plant yield of each genetic material of OsNLP6 in the field in the present invention, LN is a low nitrogen environment, HN is a high nitrogen environment, wherein A is ear weight, B is the effective ear number, C is the total dry weight, and D is the single plant yield. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0029] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0030] Unless otherwise defined, the methods used in the present invention are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified.

[0031] In a first aspect, the present invention identifies a nitrogen efficient utilization gene OsNLP6, which encodes the amino acid sequence shown in SEQ ID NO.2.

[0032] In a specific implementation process, the nucleotide sequence of the nitrogen efficient utilization gene OsNLP6 can be selected from:

[0033] 1) DNA sequence shown in SEQ ID NO.1;

[0034] Or, 2) a nucleotide sequence that can hybridize with the DNA sequence shown in SEQ ID NO.1 under stringent conditions.

[0035] In a second aspect, the present invention provides a protein encoded by the nitrogen efficient utilization gene OsNLP6, wherein the OsNLP6 protein is the following protein (a) or (b) or (c).

[0036] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2;

[0037] (b) a protein derived from (a) in which one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2 are substituted, deleted and / or added and which has the same activity as (a);

[0038] (c) A protein encoded by another gene having more than 50% amino acid identity with the protein consisting of the amino acid sequence shown in SEQ ID NO. 2 and having the same activity as (a).

[0039] In a third aspect, the present invention relates to an expression vector comprising the nitrogen efficient utilization gene OsNLP6; a transgenic cell line comprising the nitrogen efficient utilization gene OsNLP6; and a host cell comprising the nitrogen efficient utilization gene OsNLP6. The host cell is a microbial cell, for example, a fungal cell or a bacterial cell, for example, but not limited to, Agrobacterium, yeast, Escherichia coli, etc.

[0040] The OsNLP6 gene or its active fragment or a recombinant expression vector containing the OsNLP6 gene can be introduced into plant cells through conventional transformation or transfection techniques to obtain transgenic plant cells, and further obtain transgenic plants that overexpress the OsNLP6 gene or its active fragment.

[0041] In a fourth aspect, the present invention relates to the use of the nitrogen efficient utilization gene OsNLP6 or the protein encoded by it for improving the nitrogen utilization efficiency of plants. Overexpression of the OsNLP6 gene in plants can improve the nitrogen utilization efficiency of plants. Obtaining plants overexpressing the OsNLP6 gene can be achieved through transgenic technology, for example, through Agrobacterium-mediated transfection, plasmid transformation, direct DNA transformation, microinjection and other technologies.

[0042] In a fifth aspect, the present invention provides a method for cultivating plants with improved nitrogen utilization efficiency, comprising the following steps: introducing the nitrogen efficient utilization gene OsNLP6 or its active fragment into plant cells to obtain transgenic plants with enhanced nitrogen utilization efficiency, or using a hybridization method to obtain offspring plants that overexpress the nitrogen efficient utilization gene OsNLP6 or its active fragment. In one embodiment of the present invention, plants include, but are not limited to, rice, Arabidopsis, wheat, corn, cotton, rapeseed or soybean, preferably rice. In one embodiment of the present invention, the nitrogen efficient utilization gene OsNLP6 is overexpressed in plants and plants with high nitrogen utilization efficiency are further obtained. The specific transformation and selection methods in the method can be carried out according to a variety of methods known in the art. In one embodiment of the present invention, the method can be carried out with reference to the process described in the examples and appropriately modified according to different plants.

[0043] In a preferred embodiment of the present invention, a method for cultivating plants with improved nitrogen utilization efficiency is provided, the method comprising: introducing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 or an active fragment thereof into a plant cell to obtain a transgenic plant cell. The transgenic plant expressing the nucleotide sequence or the active fragment thereof by the transgenic plant cell has improved nitrogen utilization efficiency compared with a control plant into which the nucleotide sequence or the active fragment thereof is not introduced.

[0044] In another embodiment, the nitrogen efficient utilization gene OsNLP6 is introduced into the offspring plants by hybridization. For example, a plant containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 or an active fragment thereof is hybridized with another plant to obtain offspring plants, and a stably homozygous hybrid plant containing the nucleotide sequence or an active fragment thereof is screened. Compared with the unhybridized plant, the nitrogen utilization efficiency of the obtained hybrid offspring plants is improved.

[0045] The breeding method of the present invention can be used for breeding of rice, corn, wheat, cotton, rapeseed, soybean, or Arabidopsis, preferably for rice breeding. Using this method, plant varieties with improved nitrogen utilization efficiency can be obtained. The principle of breeding is to overexpress the OsNLP6 gene in the plant, specifically, it can be achieved by overexpressing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 or its active fragment in the plant. The transgenic plants or hybrid offspring plants obtained by the breeding method have growth advantages under low nitrogen and / or high nitrogen growth conditions. In the present invention, the normal nitrogen application rate is generally 150KG nitrogen / hectare, "low nitrogen growth conditions" generally refers to the application of nitrogen fertilizer 0.1-0.5 times the normal nitrogen application rate, and "high nitrogen growth conditions" generally refers to the application of nitrogen fertilizer 1.5-2.0 times the normal nitrogen application rate.

[0046] When used to cultivate rice with improved nitrogen utilization efficiency, the cultivation method includes: introducing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 or an active fragment thereof into rice cells to obtain transgenic rice cells, and forming a transgenic rice plant expressing the nucleotide sequence or the active fragment thereof from the transgenic rice cells. Compared with a control rice plant into which the nucleotide sequence or the active fragment thereof is not introduced, the nitrogen utilization efficiency of the transgenic rice is improved. Or the method includes: hybridizing a rice plant containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO.2 or the active fragment thereof with another rice plant to obtain a progeny rice plant, and screening out a stably homozygous hybrid plant containing the nucleotide sequence or the active fragment thereof. Compared with the unhybridized rice plant, the obtained hybrid progeny plant has improved nitrogen utilization efficiency.

[0047] In general, the nitrogen efficient utilization gene OsNLP6 provided by the present invention is derived from rice and is one of the following nucleotide sequences:

[0048] 1) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO.2;

[0049] 2) the nucleotide sequence shown in SEQ ID NO.1;

[0050] 3) A nucleotide sequence that hybridizes with the DNA sequence defined in SEQ ID NO. 1 under highly stringent conditions.

[0051] High stringency conditions include hybridization and membrane washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0052] The nucleotide sequence shown in SEQ ID NO.1 consists of 2529 bases, and its coding sequence starts from the first base at the 5′ end and encodes the amino acid sequence shown in SEQ ID NO.2.

[0053] The expression vector, transgenic cell line and host bacteria containing the nitrogen efficient utilization gene OsNLP6 of the present invention are also within the protection scope of the present invention.

[0054] Primer pairs for amplifying any fragment of the nitrogen efficient utilization gene OsNLP6 are also within the protection scope of the present invention.

[0055] By using a plant expression vector, the nitrogen efficient utilization gene OsNLP6 of the present invention is introduced into plant cells to obtain transgenic cell lines and transgenic plants with enhanced nitrogen utilization efficiency.

[0056] Plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. Plant expression can also include the 3' non-translated region of the foreign gene, that is, it includes a polyadenylic acid signal and any other DNA fragment that can participate in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the rouge synthase NOS gene) and the 3' non-translated region of the plant gene (such as the soybean storage protein gene) have similar functions.

[0057] When OsNLP6 is used to construct a plant expression vector, any enhanced promoter or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the root-specific expression promoter, etc., which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of translation control signals and start codons are wide-ranging and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0058] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide Bar gene), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0059] The plant expression vector carrying the OsNLP6 gene of the present invention can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant cells or tissues can be cultivated into plants. The transformed plant host can be either monocotyledonous plants such as rice, corn, wheat, etc., or dicotyledonous plants such as Arabidopsis, soybean, rapeseed, cotton, etc.

[0060] Through the growth experiment of the transgenic rice obtained after the transformation of OsNLP6 gene in nutrient solution with different nitrogen levels and field experiment, it is proved that the gene can significantly improve the nitrogen utilization efficiency of the plant and increase the biomass of the plant after being transferred into the plant; through the growth experiment of the transgenic Arabidopsis obtained after the transformation of OsNLP6 gene in culture medium with different nitrogen levels, it is proved that the function of the gene in the model plant is conservative. The present invention provides a basis for artificially enhancing the nitrogen utilization efficiency of plants, and will play an important role in cultivating plants (especially crops such as rice, corn, wheat, rapeseed, soybean, cotton, etc.) that efficiently utilize nitrogen nutrition.

[0061] Example 1 Obtaining the Nitrogen Efficient Utilization Gene OsNLP6 and Transgenic Plants Thereof

[0062] 1. Obtaining OsNLP6 cDNA sequence:

[0063] Total RNA was extracted from 7-day-old Dongnong 427 seedlings, reverse transcribed to obtain cDNA, and then the OsNLP6 cDNA sequence was amplified by RT-PCR using cDNA as a template. The specific process was as follows: TRIzol reagent (Invitrogen, Carlsbad, CA) was used to extract rice total RNA, and 1 μg of total RNA was reverse transcribed using a reverse transcription kit produced by Dalian Bao Bioengineering Company according to the product instructions to obtain cDNA.

[0064] RT-PCR primers are as follows:

[0065] Upstream primer: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC

[0066] atggagcgcgttgttggcga-3' (SEQ ID NO.3);

[0067] Downstream primer: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTC

[0068] cctgcgctagctcagattag-3' (SEQ ID NO.4);

[0069] The uppercase sequence in the primer is the attB2 full linker, and the lowercase sequence is the sequence designed according to the OsNLP6 gene sequence (the lowercase sequence in the upstream primer is the 5' end sequence of SEQ ID NO.1, and the lowercase sequence in the downstream primer is the reverse complementary sequence of the 3' end sequence of SEQ ID NO.1).

[0070] The 50uL PCR amplification system is: 0.8uL FastPfu polymerase, 0.5uL cDNA, 10uL 5×PCR buffer, 100uM dNTPs, 25uM each of upstream and downstream primers, and then add double distilled water to the reaction system to 50uL.

[0071] The PCR program was: 95°C for 1 min, then 58°C for 30 s, and finally 72°C for 1 min, for a total of 40 cycles. After the reaction was completed, the PCR product was recovered and sequenced, and the sequencing showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO.1, encoding the protein sequence shown in SEQ ID NO.2.

[0072] 2. Construction of OsNLP6 overexpression vector:

[0073] The amplified CDS fragments of different alleles of OsNLP6 were connected into the vector pBWA(V)HS to obtain the recombinant expression vector pBWA(V)HS-NLP6-OE-GLosgfp, with the structure as shown in Figure 1 shown.

[0074] 3. Acquisition and identification of OsNLP6 overexpressing transgenic plants:

[0075] The constructed recombinant expression vector pBWA(V)HS-NLP6-OE-GLosgfp was introduced into the normal Dongnong 427 rice variety by Agrobacterium EHA105-mediated genetic transformation. When the transgenic plants grew up, the leaves were cut to extract DNA, and primers were designed for PCR. The positive plants with the OsNLP6 gene were screened out and harvested until homozygous plants were detected in the T2 generation. The homozygous lines germinated on MS medium were used to identify the expression level of the OsNLP6 gene in each line by qRT-PCR. Two lines were selected from the constructed rice lines overexpressing the OsNLP6 gene, named OE1 and OE2, for subsequent experiments.

[0076] The OsNLP6 qRT-PCR primer sequences are:

[0077] Upstream primer LP 5'-GGAGGAGCTGCAGAAACACT-3' (SEQ ID NO.5),

[0078] Downstream primer RP 5′-GCATGATCCATGGGAGGAGG-3′ (SEQ ID NO. 6).

[0079] qRT-PCR system: 10μM LP 0.25μl, 10μM RP 0.25μl, cDNA 0.3μl, double distilled water 4.2μl, SYBRGreen reagent 5μl. qRT-PCR program: 95℃, 1min; 95℃, 5s; 60℃, 30s, 40 cycles.

[0080] 4. Construction of sNLP6 gene CRISPR-cas9 overexpression vector:

[0081] First, design the sgRNA targeting the OsNLP6 gene. Select a sequence such as 5'-GGN(19)GG, 5'-GN(20)GG or 5'-N(21)GG on the OsNLP6 gene. The targeting site of the sgRNA on the OsNLP6 gene should be located on the common exons of different splicing forms. The targeting site is located in the first half of the entire gene, but not too close to ATG, and preferably in the functional domain of the gene. Secondly, the uniqueness of the sgRNA target sequence should be ensured by BLAST in the database. Then, according to the selected sgRNA, add CCGG to its 5' to obtain a forward oligonucleotide; according to the selected sgRNA, obtain the complementary chain of its corresponding DNA, and add AAAC to its 5' to obtain a reverse oligonucleotide. The above forward oligonucleotide and reverse oligonucleotide are synthesized separately, and the synthesized sgRNA oligonucleotides are denatured and annealed in pairs to form a double strand after annealing, and then connected to the yl-hu expression vector through the BsaI restriction site. Finally, it was recombinantly cloned into yl-hu-cas-Os02g0136000-DN427 through GatewayTM cloning technology.

[0082] 5. Acquisition and identification of sNLP6 gene CRISPR-cas9 transgenic plants:

[0083] The recombinant expression vector constructed by OsNLP6 gene and yl-hu-cas-Os02g0136000-DN427 was introduced into the normal Dongnong 427 rice variety by genetic transformation mediated by Agrobacterium EHA105. When the transgenic plants grow up, the leaves are cut to extract DNA, and primers are designed to sequence the target site targeted by CRISPR-cas9 to identify whether a gene mutation has occurred near the target site and what kind of mutation has occurred. Positive plants with effective mutations in the OsNLP6 gene are screened and harvested individually until homozygous plants are detected in the T2 generation. The target sequence targeted by CRISPR-cas9 is as follows: Figure 2From the multiple OsNLP6 gene mutant strains obtained, two were selected for subsequent experiments, and the two selected OsNLP6 gene mutant strains were named KO1 and KO2, respectively.

[0084] The experimental results showed that all the transgenic strains obtained were OsNLP6 overexpression strains, and the CRISPR strains produced mutations near the target site that hindered the normal function of the gene. The OsNLP6 gene of the KO1 strain had a 2-base deletion at the target site, while the OsNLP6 gene of the KO2 strain had a 1-base deletion at the target site.

[0085] 6. Subcellular localization of OsNLP6 gene:

[0086] In order to study the subcellular localization of OsNLP6, a GFP fusion vector of pBWA(V)HS-NLP6 was constructed and transiently expressed in rice protoplasts. Its subcellular localization was determined by transfecting protoplasts, and the fluorescence signal was observed by laser confocal microscopy. The green fluorescence signal of the control group sample transformed with the empty vector was present in the rice protoplasts, while the green fluorescence signal of the OsNLP6 fusion protein appeared in the nuclear region of the protoplasts, indicating that the protein encoded by the OsNLP6 gene is localized in the nucleus. Figure 3 shown.

[0087] 7. Growth status of each OsNLP6 rice genetic material under normal field conditions:

[0088] Each strain was grown in the field under normal conditions for 3 months, and the growth status of each strain was observed and photographed. Figure 4 As shown in the figure, the ear weight, effective ear number, total dry weight and single plant yield of each strain were counted. The results are as follows Figure 5 As shown in the figure, the ear weight and effective ear number of the overexpression strains increased compared with the wild type, and the total dry weight and yield were significantly improved; while the ear weight and effective ear number of the KO1 and KO2 strains decreased compared with the wild type, and the total dry weight and yield decreased significantly. The above data show that rice overexpressing OsNLP6 has obvious advantages in field growth, indicating that the gene OsNLP6 is a gene with broad application prospects. The gene OsNLP6 can improve the nitrogen utilization efficiency of crops, thereby increasing crop biomass and yield, and reducing environmental pollution.

[0089] Therefore, the present invention adopts the above-mentioned rice nitrogen efficient utilization gene OsNLP6 and its encoded protein and application. The OsNLP6 gene can promote the absorption and utilization of nitrogen by plants, especially can improve the absorption and utilization efficiency of nitrogen by rice, and can be used to cultivate new rice varieties with high nitrogen utilization efficiency, laying the foundation for rice breeding.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of rice nitrogen efficient utilization gene OsNLP6 or a recombinant vector containing rice nitrogen efficient utilization gene OsNLP6 in cultivating rice with improved nitrogen utilization efficiency, characterized in that: The nucleotide sequence of the rice nitrogen efficient utilization gene OsNLP6 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2.

2. A method for cultivating rice with improved nitrogen utilization efficiency, characterized in that: The method comprises the following steps: introducing the rice nitrogen efficient utilization gene OsNLP6 or a recombinant vector containing the rice nitrogen efficient utilization gene OsNLP6 into rice cells to obtain transgenic rice; or hybridizing the rice containing the rice nitrogen efficient utilization gene OsNLP6 with another rice to obtain hybrid offspring rice containing the gene OsNLP6; the nucleotide sequence of the rice nitrogen efficient utilization gene OsNLP6 is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that: The method also includes codon-optimizing the gene OsNLP6 according to the codon usage of rice before introducing it into rice cells.

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