CN111087457B - Protein NGR5 for improving nitrogen utilization rate and crop yield, and coding gene and application thereof - Google Patents
Protein NGR5 for improving nitrogen utilization rate and crop yield, and coding gene and application thereof Download PDFInfo
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- CN111087457B CN111087457B CN201911425758.9A CN201911425758A CN111087457B CN 111087457 B CN111087457 B CN 111087457B CN 201911425758 A CN201911425758 A CN 201911425758A CN 111087457 B CN111087457 B CN 111087457B
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a protein NGR5 for improving nitrogen utilization rate and crop yield, and a coding gene and application thereof. More specifically, the NGR5 gene is derived from rice, the NGR5 gene participates in the regulation and control process of nitrogen-mediated rice growth and development, the expression level of NGR5 is improved, the tillering number of the rice can be increased, the rice yield is improved under the level of reducing the nitrogen input, and the improvement of the utilization efficiency of the nitrogen in the rice is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a protein NGR5 for improving nitrogen utilization rate and crop yield, and a coding gene and application thereof.
Background
Rice (Oryza sativa L.) is an important food crop, with the total yield accounting for 1/4 of the world's total food production, and rice is the staple food for more than half of the world's population. On one hand, as the population of the world increases year by year, the demand for rigidity of the food is continuously increased; on the other hand, the problems of year-by-year decrease of the cultivated land area, shortage of water resources, frequent occurrence of natural disasters, aggravation of industrialization degree, environmental damage caused by human activities and the like are more and more serious. Therefore, how to continuously improve the yield per unit is a great challenge facing the genetic breeding of crops at present.
In agricultural production, large application of nitrogen fertilizer is one of the important measures for high yield of rice. The semi-dwarfing variety of the 'green revolution' which has always taken the leading position in the crop breeding history for more than half a century has the excellent characteristics of high fertilizer resistance, lodging resistance and high yield, but also has the limitation of low utilization efficiency (NUE) of nitrogen fertilizer, and the high yield of the semi-dwarfing variety has great dependence on high water and fertilizer input. Therefore, to increase crop yield, nitrogen fertilizers have to be used in large quantities. However, the continuous large amount of nitrogen fertilizer input not only increases the planting cost, but also causes an increasingly serious environmental pollution problem. How to improve the utilization efficiency of the nitrogen fertilizer of the rice and still keep the yield improvement under the condition of properly reducing the application amount of the nitrogen fertilizer is a bottleneck problem to be solved urgently in the current rice breeding.
Nitrogen is one of the great amount of nutritive elements essential for plant growth and development and is the important component of plant body protein, nucleic acid, phospholipid, hormone and other nitrogen containing matter. Nitrogen metabolism, one of the basic metabolic activities of plants, is related to biomass, crop yield, quality and the like of the plants. In recent years, with the rapid development of genetics, molecular biology, genomics, and other disciplines, genes involved in nitrogen uptake and assimilation have been cloned one after another, and functional verification of these genes in the regulation of nitrogen fertilizer utilization efficiency has also been greatly advanced. There is currently considerable research on the pathways by which plants take up and utilize nitrogen, such as the discovery of nitrogen transporters (Crawford and Glass 1998; Forde 2000; Howitt and Udvardi 2000; Glass et al 2001; Williams and Miller 2001), and the functional study of enzymes responsible for converting nitrogen into amino acids and other compounds (Campbell 1988; Lam et al 1996; Hirel and Lea 2001). Flooding plants such as rice mainly pass through NH4 +Transport proteins (AMTs), which absorb NH from the soil4 +.5 gene families (OsAMT1-OsAMT5) in rice code NH4+ transporter, studies showed that OsAMT1 is overexpressed; 1 NH capable of enhancing Rice root System4 +Absorption capacity, at a suitable or lower concentration of NH from the outside4 +Under the condition, the growth and development of rice are promoted; but at a high concentration of NH at the outside4 +Under these conditions, a more pronounced ammonium poisoning phenotype results (Ranathungen et al 2014). Overexpresses OsAMT 1; 3, the carbon-nitrogen balance in the rice body is disordered, the growth and development of the plants are inhibited, and the yield is reduced (Bao et al.2015). Glutamine Synthetase (GS) OsGS 1; 2 and NADH-GOGAT1 are primarily responsible for the absorption of NH by the rice roots4 +Assimilation, while GS2 and Fd-GOGAT are primarily responsible for NH in the leaf4 +And (4) assimilating. Studies showed overexpression of OsGS 1; 2 can improve rice yield under high nitrogen conditions (Brauer et al 2014), but overexpression of GS1:1 may disrupt the carbon-nitrogen metabolic balance to severely affect rice growth and reduce rice yield (Bao et al 2014). In recent years, by utilizing technologies such as GWAS analysis, QTL positioning and map-based cloning, a plurality of key genes/major QTLs participating in nitrogen absorption and metabolic regulation are successfully separated and identified. For example, in rice, many enzymes involved in nitrogen assimilation (Yamaya et al 2002; Obara et al 2001, 2004; Gao et al 2019) and candidate genes related to efficient utilization of nitrogen fertilizer (Gallais and Hirel, 2004; Martin et al, 2006; Obara et al 2001; Tabuchi et al, 2005; Sun et al 2014; Hu et al 2015; Wang et al 2018; Li et al 2018) have been detected, but there is very limited knowledge of genetic control networks for controlling the efficiency of nitrogen fertilizer utilization by plants.
Disclosure of Invention
The inventor of the invention utilizes EMS mutagenesis and genetic screening to isolate and identify a mutant ngr5(nitrogen-mediated tiller growth response5) material of rice growth and development (tillering) which loses response to nitrogen. On the basis, a key gene NGR5 for regulating and controlling the growth and development of rice by nitrogen mediated is cloned by a map-based cloning method, and the fact that the NGR5 gene expression is regulated and controlled by nitrogen is clear, and the expression quantity of the gene is positively correlated with the tillering and yield of rice; meanwhile, a batch of rice materials carrying different allelic variations of NGR5 was identified. The function of the gene is proved by phenotype analysis and genetic complementation experiment of the mutant.
The research of the inventor provides a theoretical basis for revealing a genetic regulation and control network of crop tillering (growth and development) response nitrogen from a molecular level and provides a new gene resource with breeding utilization value for the high-efficiency utilization of nitrogen fertilizer and the design and breeding of high-yield molecules of main crops including rice and wheat.
Therefore, in general, the invention provides a gene for controlling the nitrogen utilization efficiency and yield traits of rice and application thereof. In particular, the invention relates to NGR5 and its superior allelic variation NGR5Guichao2Under the condition of slightly reducing the plant height, the synergistic improvement of the utilization efficiency and the yield of the nitrogen of the rice is realized. The invention aims to provide an important functional gene capable of simultaneously improving the yield of main crops (such as rice, wheat and the like) and the utilization efficiency of nitrogen and application thereof.
Specifically, the inventor carries out EMS mutagenesis on a high-yield rice variety 9311 (China Rice research institute, or can be obtained commercially) carrying a 'green revolution' semi-dwarf gene sd1, and separates and identifies a mutant material ngr5 with growth and development traits such as tillering, plant height, spike grain number and the like which lose response to nitrogen through field phenotype screening of different nitrogen fertilizer application levels. Further, by experiments such as map-based cloning and genetic complementation, a key gene NGR5 for regulating and controlling the response of the rice tillering nitrogen is cloned. The research finds that the expression level of the NGR5 gene is induced by nitrogen, and proves that the nitrogen can also promote the high-level accumulation of the NGR5 protein. On the basis, the NGR5 gene is over-expressed under the background of a high-yield rice variety 9311, and a field test of multiple points for many years proves that the improvement of the NGR5 expression quantity can obviously increase the tillering number and the yield of rice, and can improve the utilization efficiency and the yield of the nitrogen fertilizer of the rice under the condition of properly reducing the application amount of the nitrogen fertilizer. The present inventors also found that OsGRF4 containing excellent isogenic genengr2The over-expression of NGR5 in the high-yield variety with high-efficiency utilization of nitrogen fertilizer can further improve the rice yield and the utilization efficiency of nitrogen fertilizer.
EMS mutagenesis is carried out on a high-yield rice variety 9311 carrying a green revolution gene sd1, and a nitrogen-insensitive mutant ngr5 for the growth and development of rice plants is obtained through field phenotype screening and identification under different fertilizing amount conditions (high nitrogen and low nitrogen). Specifically, the number of tillers, plant height, number of branches and number of grains per ear of the wild-type rice increased with the increase in the amount of nitrogen fertilizer applied, but the traits of the ngr5 mutant, such as the number of tillers, plant height, number of branches, number of grains per ear, and yield, lost response to nitrogen. By map-based cloning techniques we obtained the candidate gene NGR5, which encodes a transcription factor of the AP2 type. Further genetic complementation experiments prove that the NGR5 gene is a positive regulation factor of rice growth, development and yield traits in response to nitrogen.
The present invention provides a gene NGR5 for controlling the utilization efficiency and yield of nitrogen fertilizer of rice and its excellent allele, and the present inventors named the excellent allele as NGR5Guichao2。
In one embodiment, the gene NGR5 for controlling the nitrogen utilization efficiency and yield of rice and the allele thereof are provided, the coding sequence of the gene NGR5 is shown as SEQ ID NO:1, and the allele NGR5 of the gene NGR5 is providedGuichao2The gDNA sequence of (b) is shown in SEQ ID NO. 4.
The invention provides the application of protein for controlling nitrogen utilization rate and crop yield in improving nitrogen utilization rate and crop yield, wherein the protein comprises one of the following amino acid sequences:
1) an amino acid sequence shown as SEQ ID NO. 2;
2) a substitution, deletion and/or insertion of one or more (e.g., 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5,1 to 3) amino acid residues which are different from the amino acid sequence shown in SEQ ID NO. 2 but have the same activity as a protein consisting of the amino acid sequence shown in SEQ ID NO. 2;
3) an amino acid 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 with the amino acid sequence shown in SEQ ID No. 2 and whose activity is identical to that of a protein consisting of the amino acid sequence shown in SEQ ID No. 2;
4) an active fragment comprising the amino acid sequence of any one of 1) to 3).
In some embodiments of the invention, the nucleotide sequence of the gene encoding the protein for controlling nitrogen use efficiency and crop yield comprises one selected from the group consisting of:
1) 1, SEQ ID NO;
2) a nucleotide sequence in which one or more (e.g., 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5,1 to 3) nucleotide sequences are substituted, deleted and/or inserted, which is different from the nucleotide sequence shown in SEQ ID NO. 1, but which encodes a protein having the same activity as the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1;
3) and SEQ ID NO:1, and the activity of the protein encoded by the nucleotide sequence has the same activity with the protein encoded by the nucleotide sequence shown in SEQ ID NO. 1, wherein the nucleotide sequence has at least 70 percent of identity, preferably at least 80 percent of identity, more preferably at least 90 percent of identity, and especially at least 95 percent or 98 percent or 99 percent of identity;
4) a nucleotide sequence which differs in sequence from SEQ ID NO 1 due to the degeneracy of the genetic code;
5) an active fragment comprising a nucleotide sequence of any one of 1) to 4).
In other embodiments of the present invention, the nucleotide sequence of the gene encoding the protein controlling nitrogen use efficiency and crop yield comprises a nucleotide sequence that hybridizes under moderately stringent conditions, preferably high stringent hybridization conditions, to the complement of the nucleotide sequence set forth in SEQ ID NO. 1.
In some embodiments of the present invention, the nucleotide sequence of the gene encoding the protein controlling nitrogen use efficiency and crop yield comprises a nucleotide sequence complementary to any one of the nucleotide sequences 1) to 5).
In the study, the inventors named the superior allele of NGR5 as NGR5Guichao2Wherein NGR5 and NGR5Guichao2There are differences in promoter regions in genomic DNA. Specifically, NGR5 compares to the NGR5 sequence in 9311Guichao2The promoter region and gene region within 2kb of the gene have 25 SNPs differences, which are shown in SEQ ID NO:3 and SEQ ID NO: 5.
In a second aspect, the present invention provides a method of cultivating a crop with high nitrogen utilization and high yield, the method comprising: transfecting the nucleotide sequence of the gene for coding the protein for controlling the nitrogen utilization rate and the crop yield into crop cells to obtain a transgenic crop plant, so that the expression level of the gene for coding the protein for controlling the nitrogen utilization rate and the crop yield in the transgenic crop is increased, and the crop with high nitrogen utilization rate and high yield is obtained, wherein the crop is a monocotyledon, preferably rice or wheat, and more preferably rice.
In some embodiments of the invention, the gene controlling nitrogen utilization and crop yield is NGR5 or its superior allele NGR5Guichao2。
Wherein, the NGR5 gene can control the absorption and utilization efficiency of the nitrogen in the rice and the influence of the nitrogen fertilizer dosage on the biomass and yield increase of the rice. Specifically, the expression level of the NGR5 gene is increased, the absorption and assimilation of the nitrogen fertilizer of the rice can be improved, the tiller number of the rice can be increased, and the yield of the rice can be improved. Likewise, the superior allelic variation NGR5 of the geneGuichao2Can not only increase the utilization efficiency of nitrogen fertilizer of rice, but also increase the tillering number and yield of rice.
Preferably, the genes NGR5 and NGR5 related to the invention for controlling the utilization efficiency and yield of the nitrogen fertilizer of riceGuichao2The cDNA sequences of (A) and (B) are identical, the amino acid sequences encoded by the two are also identical, and the difference of the nucleotide sequences is only in a promoter region.
Specifically, NGR5 compares to the 2kb promoter of the NGR5 gene (SEQ ID NO:3)Guichao2The 2kb promoter of the gene (SEQ ID NO:5) has 21 SNPs, and further research shows that NGR5Guichao2The gene promoter region has 3 specific SNPs sites (i.e., g. -853T)>G,g.-825T>C,g.-770G>A) Associated with an increased transcription level of the gene.
The rice varieties with high nitrogen utilization rate and high yield can be identified by utilizing the specific SNPs sites. For example, if the presence of any one, two or all three specific SNPs selected from the following can be detected in the promoter region of the NGR5 gene in rice plants: g. -853T > G, g. -825T > C, g. -770G > A, then the rice variety can be judged to have the potential for high nitrogen fertilizer utilization and high yield. The SNPs difference of the promoter region or the coding region of the NGR5 gene can be detected by PCR amplification enzyme digestion or sequencing method.
In a third aspect, the present invention provides a method of cultivating a crop with high nitrogen utilization and high yield, the method comprising: co-expression of GRF4 in crop plantsngr2Genes and genes encoding said proteins controlling nitrogen utilization and crop yield or superior alleles thereof, or in a plant harboring OsGRF4ngr2The coding gene of the protein for controlling the nitrogen utilization rate and the crop yield or the excellent allele thereof is overexpressed in the crop plant of the gene, wherein the coding gene of the protein for controlling the nitrogen utilization rate and the crop yield is OsNGR5 gene, and the excellent allele thereof is OsNGR5Guichao2The gene, the crop is monocotyledon, preferably rice or wheat, and more preferably rice.
Using the gene of the invention or its allele and OsGRF4ngr2And (5) performing polymerization breeding on the genes. Co-expression of the gene of the invention or its allele and OsGRF4 in rice plantsngr2The gene can further improve the utilization rate and the yield of the nitrogen fertilizer of the rice.
Specifically, the method of pyramiding breeding may comprise: carrying excellent allele OsGRF4ngr2Over-expression of NGR5 or allele NGR5 in rice plantsGuichao2Or other forms of alleles of NGR5 engineered by gene editing techniques. In one embodiment, OsGRF4 is carried on a superior allelengr2Over-expression of NGR5 gene in rice plant. In another embodiment, OsGRF4 is carried on a superior allelengr2Over-expression of NGR5 in rice plantsGuichao2A gene. In a preferred embodiment, OsGRF4 is carried on a superior allelengr2Over-expression of NGR5 in rice plantsGuichao2A gene.
Wherein OsGRF4ngr2The gene is a gene for controlling the utilization efficiency and yield of the rice nitrogen. About OsGRF4ngr2Genes, see PCT WO2019/158911A1, which is incorporated herein by reference in its entirety.
In some embodiments of the invention, the superior allele OsNGR5Guichao2The promoter region of the gene has 3 specific SNPs sites: g. -853T>G,g.-825T>C,g.-770G>A) The specific SNPs site can increase OsNGR5Guichao2The level of transcription of the gene.
The inventionA fourth aspect provides a method of growing a crop with high nitrogen utilization and high yield, the method comprising: will contain said OsNGR5Guichao2Crossing a crop plant of the gene with another plant of the crop to produce a hybrid crop plant, such that OsNGR5 is present in the hybrid cropGuichao2The expression quantity of the gene is increased, and the crop with high nitrogen fertilizer utilization rate and high yield is obtained, wherein the crop is rice.
In a fifth aspect, the present invention provides a method of identifying a rice variety having high nitrogen utilization and high yield, the method comprising: analyzing the promoter region of the OsNGR5 gene for the presence of any one, two or all three specific SNPs selected from the group consisting of: g. -853T > G, g. -825T > C, g. -770G > A, while analyzing the gene region of OsNGR5 gene for the presence of specific SNPs: g.3326C > T, to determine if the rice variety has the potential for high nitrogen utilization and high yield.
In some embodiments of the invention, the SNPs of the promoter region and coding region of the OsNGR5 gene are detected using PCR amplification digestion or sequencing methods.
The sixth aspect of the present invention provides a gene for controlling nitrogen utilization and crop yield, the nucleotide sequence of which is shown in SEQ ID NO. 4.
The seventh aspect of the present invention provides a recombinant construct comprising the gene according to the sixth aspect.
In an eighth aspect, the invention provides a host cell comprising the gene of the sixth aspect or the recombinant construct of the seventh aspect, wherein the host cell is a microbial cell, preferably an escherichia coli cell or an agrobacterium cell.
The invention provides a promoter of the gene NGR5 for controlling the utilization efficiency and the yield of the rice nitrogen and an allele thereof, the length of the promoter is about 2kb, and the promoter comprises one of the following nucleotide sequences:
(1) SEQ ID NO:3 or SEQ ID NO: 5;
(2) a nucleotide sequence that hybridizes under moderately stringent conditions, preferably high stringent hybridization conditions, to the complement of the nucleotide sequence of (1);
(3) a nucleotide sequence having at least 70%, preferably at least 80%, more preferably at least 90%, especially at least 95% or 98% or 99% identity to the nucleotide sequence of (1).
In a preferred embodiment, the promoter sequence of NGR5 is as shown in SEQ ID NO. 3, and the allele NGR5Guichao2The promoter sequence of (1) is shown in SEQ ID NO. 5.
In a preferred embodiment, NGR5 and its allelic NGR5Guichao2The related sequences are shown in SEQ ID NOs:1-5, see Table 1 below.
TABLE 1 sequence names and sources of SEQ ID NOs 1-5
The present invention also provides mutant types produced by gene editing techniques and containing the SNPs according to the fifth aspect of the present invention or other mutant types capable of causing an increase in the expression level of the OsNGR5 gene.
The present invention provides other types of mutations that increase the stability of the OsNGR5 protein, produced using gene editing techniques. The invention provides a recombinant construct which contains the gene NGR5 or the allele NGR5 for controlling the nitrogen utilization efficiency and the yield of riceGuichao2The polynucleotide sequence of (1). Wherein the vector used for the construct may be a cloning vector or an expression vector for expressing the polynucleotide.
The invention provides a recombinant host cell, which contains the recombinant construct, or integrates the gene NGR5 or allele NGR5 for controlling the utilization efficiency and the yield of the nitrogenous fertilizer of rice into the genomeGuichao2The polynucleotide sequence of (1). 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 cell may be isolated, ex vivo, cultured or part of a plant.
The present invention provides polynucleotides of the invention (i.e.,gene NGR5 or allele NGR5 for controlling utilization efficiency and yield of rice nitrogen fertilizerGuichao2Or other forms of alleles following modification by gene editing techniques) or polypeptides or recombinant constructs of the invention or recombinant host cells of the invention for use in improving crop plant traits (e.g., increasing crop yield) and nitrogen fertilizer use efficiency.
The invention provides the NGR5 or the allele NGR5Guichao2The use of (a) for controlling nitrogen use efficiency and yield of rice, but is not limited thereto.
The present invention provides a method for breeding improved rice varieties. The method comprises the following steps: using a gene comprising NGR5 or the allele NGR5Guichao2Transfecting the rice plant with the recombinant agrobacterium cell to obtain a transgenic rice plant, or transfecting NGR5 or an allele NGR5Guichao2The rice plant with other forms of alleles after being modified by the gene editing technology is crossed with another rice plant to obtain a progeny rice plant, wherein the obtained rice plant is preferably a rice plant with improved nitrogen utilization efficiency and yield.
In a more preferred embodiment of the invention, based on more detailed experimental verification, the inventors have found that the NGR5 gene and its superior allele NGR5 can be utilizedGuichao2The breeding experiment with the function of increasing the utilization efficiency and the yield of the nitrogen fertilizer is carried out by the following three ways:
(1) alteration of NGR5 or allelic NGR5 in riceGuichao2The expression level of (a);
(2) alteration of NGR5 or allelic NGR5 in riceGuichao2The content and activity of the encoded protein;
(3) the promoter sequence or gDNA sequence of NGR5 gene in rice is changed.
The following are definitions of some terms used in the present invention. Unless otherwise indicated, terms used herein have meanings known to those of ordinary skill in the art.
In the present invention, "NGR 5 gene" and "OsNGR 5 gene" both refer to the rice NGR5 gene, "Excellent allele" and "NGR 5 geneGuichao2"and" OsNGR5Guichao2All the genes refer to rice genes having the SNPs site differences from the promoter of the NGR5 gene.
"associated"/"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 level of expression 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 that encodes mRNA or is expressed as a protein, such that the regulatory nucleic acid sequence is capable of regulating the 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 sequences as found in nature.
A "coding sequence" is a nucleic acid sequence that is transcribed into RNA, e.g., mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably, the RNA is subsequently translated in the organism to produce a protein.
"hybrid rice" is a generic term for the first generation hybrid species with heterosis produced by crossing between two rice varieties (lines) differing in genetic constitution. At present, three-line hybrid rice and two-line hybrid rice are widely used in production. The production of three-line hybrid rice seeds requires the mutual matching of a male sterile line, a male sterile maintainer line and a male sterile restorer line. The sterility of the sterile line is controlled by cytoplasm and nucleus together, and the sterile line can be obtained only by hybridizing with the maintainer line; the sterile line is hybridized with the restorer line to obtain hybrid rice seeds for field production. The production of two-line hybrid rice only needs sterile line and restoring line. The fertility of the sterile line is regulated and controlled by the recessive sterile gene in the cell nucleus and the light length and the temperature of the planting environment, and the fertility conversion from sterility to fertility is generated along with the change of the light and temperature conditions, and the fertility is unrelated to cytoplasm. The characteristics of the photo-thermo-sensitive sterile line of producing death fertility conversion along with the change of photo-thermal conditions are utilized, and the seeds can be bred by self-crossing in a proper photo-thermal period.
In the context of the present invention, "corresponding to" means that when the nucleic acid coding sequences or amino acid sequences of different NGR5 genes or proteins are compared to each other, the nucleic acids or amino acids "corresponding to" some of the enumerated positions are aligned with these positions, but not necessarily in these exact numerical positions relative to the respective nucleic acid coding sequence or amino acid sequence of a particular NGR 5. Likewise, when a particular NGR5 coding or amino acid sequence is aligned with a reference NGR5 coding or amino acid sequence, the nucleic acids or amino acids in that particular NGR5 sequence that "correspond" to some of the enumerated positions of the reference NGR5 sequence are aligned with those positions of the reference NGR5 sequence, but are not necessarily the nucleic acids or amino acids in those precise numerical positions of the respective nucleic acid coding sequence or amino acid sequence of that particular NGR5 protein.
As used herein, an "expression cassette" is intended to mean a nucleic acid sequence capable of directing the 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 comprises sequences required for proper translation of the nucleotide sequence. An expression cassette comprising a 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 a precursor of the host cell by a transformation event. Expression of the nucleotide sequence in the expression cassette may be controlled by a constitutive promoter or an inducible promoter, wherein transcription is initiated by the inducible promoter 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 to a particular tissue, or organ or developmental stage.
A "gene" is a defined region within the genome which, in addition to the aforementioned coding nucleic acid sequences, comprises other, mainly regulatory nucleic acid sequences which are responsible for the expression of the coding part, i.e.transcriptional and translational control. The gene 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 a naturally occurring nucleic acid sequence that is 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 isolated from its natural environment and is therefore not a natural product. An isolated nucleic acid molecule or protein may exist in purified form, or may exist 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 a subject that can be found in nature, as opposed to an artificially produced subject. For example, a protein or nucleotide sequence present in an organism (including viruses) that has been isolated from a natural source and that has not been intentionally artificially modified in the laboratory is "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. A "nucleic acid molecule" is also referred to as a polynucleotide molecule.
A "plant" is any plant, particularly a seed plant, at any developmental stage.
A "plant cell" is the structural and physiological unit of a plant, comprising protoplasts and a cell wall. Plant cells may be in the form of isolated individual cells or cultured cells, or as a higher organized unit such as, for example, a plant tissue, a plant organ, or a portion of a whole plant.
"plant material" refers to leaves, stems, roots, flowers or parts of flowers, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
A "plant organ" is a distinct and well-structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.
As used herein, "plant tissue" 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 or alone with any particular type of plant tissue enumerated 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 a binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also contain other elements that act as regulators of gene expression.
A "protoplast" is an isolated plant cell that has no cell wall or only a partial cell wall.
"regulatory element" refers to a sequence involved in controlling the expression of a nucleotide sequence. The regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and a termination signal. Usually they also comprise sequences required for correct translation of the nucleotide sequence.
The phrase "substantially identical" in an alignment of two nucleic acid or protein sequences 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 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 are substantially identical over at least about 150 residues. In a particularly preferred embodiment, the sequence is substantially the same throughout the length of the coding region. Moreover, substantially identical nucleic acid or protein sequences have substantially identical functions.
For sequence comparison, typically, one sequence is compared to the test sequence as a reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, the coordinates of the subsequences are specified, if necessary, and the parameters of the sequence algorithm program are specified. 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 local 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 in the Wisconsin Genetics software package, Genetics Computer Group,575Science Dr., Madison, Wis) 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-. 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 query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbor word score threshold (Altschul et al, 1990). These initial neighborhood word hits act as clues to the initial lookup to find longer HSPs containing them. These word hits will then extend as far as possible in both directions of each sequence until the cumulative alignment score no longer increases. For nucleotide sequences, cumulative scores were calculated using the parameters M (reward score for pairwise matching residues; always greater than zero) and N (penalty score for mismatching residues; always less than zero). For amino acid sequences, a scoring matrix was used to calculate the cumulative score. Word hit extension in each direction stops when the cumulative alignment score falls back by the number X of maximum achieved, the cumulative score reaches or falls below zero due to one or more negative scoring residue alignments being accumulated, or either of the two sequences reaches the endpoint. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length value (W)11, an expectation value (E)10, a cutoff value of 100, M-5, N-4 and a comparison of the two strands as defaults. For amino acid sequences, the BLASTP program uses the word length value (W)3, expectation value (E)10 and BLOSUM62 scoring matrices 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 a 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 smallest sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in 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 indication 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, forming a duplex or hybridizing under stringent conditions when the sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. "substantial binding" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and comprises fewer mismatches that can be tolerated by reducing 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 hybridizations, are sequence dependent and differ under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. A number of guidelines for Nucleic acid Hybridization can be found in the Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic acid probes, part I, Chapter 2, "Overview of principles of Hybridization and the protocol 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.
The 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 a stringent hybridization condition for hybridization of complementary nucleic acids having more than 100 complementary residues on the filter in a Southern or Northern blot is to perform the hybridization overnight at 42 ℃ in 50% formamide with 1mg heparin. An example of high stringency rinsing conditions is 72 ℃, 0.15M NaCl for about 15 minutes. An example of stringent rinse conditions is a 0.2 XSSC rinse at 65 ℃ for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Typically, a low stringency rinse is performed before a high stringency rinse to remove background probe signal. For duplexes of, for example, more than 100 nucleotides, an example of a medium stringency rinse is a 45 ℃ 1 × SSC rinse for 15 minutes. For duplexes of, for example, more than 100 nucleotides, an example of a low stringency rinse is a 40 ℃ rinse for 15 minutes in 4-6 XSSC. 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 1.0MNa 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 by the addition of destabilizing agents such as formamide. Generally, a signal-to-noise ratio 2 × (or higher) higher than that observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
The following hybridization/rinsing conditions settingsFor example, the conditions may be used to clone a homologous nucleotide sequence substantially identical to a reference nucleotide sequence of the present invention: the reference nucleotide sequence and the reference nucleotide sequence are preferably at 50 ℃ with 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO4Hybridization in 1mM EDTA, rinsing in 50 deg.C, 2 XSSC, 0.1% SDS, more desirably at 50 deg.C, 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO4Hybridization in 1mM EDTA, rinsing in 50 deg.C, 1 XSSC, 0.1% SDS, more desirably 50 deg.C, 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO4Hybridization in 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃, preferably 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO at 50 ℃4Hybridization in 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃, more preferably 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO at 50 ℃4Hybridization in 1mM EDTA, rinsing at 65 ℃ in 0.1 XSSC, 0.1% SDS.
Another indication that two nucleic acid sequences or proteins are substantially identical is that the protein encoded by the first nucleic acid immunologically cross reacts with or specifically binds 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 not found in the native sequence. For example, artificial sequences that are said to more closely resemble the G + C content and normal codon distribution of dicotyledonous and/or monocotyledonous plant genes are synthetic.
"transformation" is the process of introducing a heterologous nucleic acid into a host cell or organism, and in particular "transformation" means the stable integration of a DNA molecule into the genome of an 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 also be present as an extrachromosomal molecule. Such extrachromosomal molecules may be autonomously replicating. Transformed cells, tissues, or plants are understood to encompass not only the final product of the transformation process, but also transgenic progeny thereof. A "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, which may comprise one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences comprising exons and introns, chemically synthesized DNA and RNA sequences, as well as sense and corresponding antisense strands.
Methods for producing and manipulating the polynucleotide molecules and oligonucleotide molecules disclosed herein are known to those skilled in the art and can be accomplished according to recombinant techniques already described (see Maniatis et al, 1989, molecular cloning, A 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, A.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 (eds.), 1992, PCR technology, Oxford university Press, New York).
"plant transformation" refers to the expression of at least one foreign gene in a plant in order to confer one or more desirable phenotypic traits on the transformed plant.
In a particularly preferred embodiment, at least one gene for the rice nitrogen fertilizer use efficiency and yield traits of the invention is expressed in higher organisms such as plants. Specifically, the nucleotide sequence of the gene for rice nitrogen fertilizer use efficiency and yield traits of the present invention may be inserted into an expression cassette, which is then preferably stably integrated into the plant genome. In another preferred embodiment, the nucleotide sequence of the gene for the rice nitrogen fertilizer use efficiency and yield traits is transfected into cells or calli of a plant by including the nucleotide sequence in a non-pathogenic self-replicating virus, thereby obtaining a transformed plant, also called a transgenic plant.
Plants transformed according to the present invention may be monocotyledonous or dicotyledonous plants including, but not limited to, maize, wheat, barley, rye, sweet potato, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, zucchini, apple, pear, quince, melon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, arabidopsis and woody plants such as conifers and deciduous trees. Particularly preferred is rice, wheat, barley, corn, oats, or rye.
Once the desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or transferred using conventional breeding techniques into other varieties of the same species, including particularly commercial varieties.
Preferably, the nucleotide sequences of the present invention are expressed in transgenic plants, thereby causing the biosynthesis of proteins in transgenic plants that control nitrogen fertilizer use efficiency and yield traits, respectively. In this way, transgenic plants with improved traits can be produced. In order to express the nucleotide sequence of the present invention in transgenic plants, the nucleotide sequence of the present invention may need to be modified and optimized. All organisms have a particular preference for codon usage, which is known in the art, and the codons can be changed to conform to plant preferences while maintaining the amino acids encoded by the nucleotide sequences of the present invention. Moreover, high levels of expression in plants can best be achieved from coding sequences having at least about 35%, preferably more than about 45%, more preferably more than 50%, and most preferably more than about 60% GC content. Although preferred gene sequences can be expressed adequately in monocot and dicot species, the sequences can be modified to accommodate the specific codon preferences and GC content preferences of monocots or dicots, as these preferences have been shown to be different (Murray et al, Nucl. acids Res.17:477-498 (1989)). In addition, the nucleotide sequence can be screened for the presence of non-canonical splice sites that cause truncation of the message. All changes that need to be made in these nucleotide sequences, such as those described above, are carried out using the methods described in published patent applications EP 0385962 (Monsanto), EP 0359472 (Lubrizol) and WO 93/07278(Ciba-Geigy) using site-directed mutagenesis techniques, PCR and synthetic gene construction well known in the art.
Drawings
The invention will be described below with reference to the accompanying drawings.
FIG. 1 shows the transformation of pNGR5 against the background of wild type 9311, mutant NGR5, mutant NGR5, the transformation of pNGR5 against the background of transgenic plants of NGR5 and mutant NGR5, the phenotype of transgenic plants NGR5 under high and low nitrogen conditions (A, B) and their number of tillers under high and low nitrogen conditions (C), number of secondary branches (D), number of ears (E) and plot yield (F);
FIG. 2 shows the map-positional cloning (A) of the NGR5 gene and the changes in bases and amino acids caused by mutation (B);
FIG. 3 shows the effect of over-expressing NGR5 on tillering number (A), plant height (B), yield (C) in 9311 background;
FIG. 4 is a graph showing the results obtained in 9311-GRF4ngr2Effect of over-expression of NGR5 on yield in background;
FIG. 5 shows the effect of overexpression of NGR5 gene on the expression level of a gene involved in nitrogen uptake and assimilation;
FIG. 6 shows the presence of 5 haplotypes of the NGR5 gene (A), and the effect of the 5 haplotypes on tiller number, yield (B);
FIG. 7 shows the gene expression levels of NGR5 for different haplotypes in different rice varieties;
FIG. 8 shows NGR5 at various nitrogen levels in the Gui Dynasty No. 2 backgroundHap.1And NGR5Hap.2The level of gene expression of (a);
FIG. 9 shows the results of the yeast two-hybrid experiment of NGR5 with SLR1 (A) and NGR5 with LC2 (B);
FIG. 10 shows that NGR5 interacts with SLR1 in plants (A) and NGR5 interacts with LC2 in plants (B);
FIG. 11 shows the phenotype of NJ6, NJ6-sd1, and NJ6-gid1-10 under high and low nitrogen conditions (A), and tillering number (B) results;
FIG. 12 shows the effect of SLR1, Rht-B1a, Rht-B1B on the stability of NGR5 protein;
figure 13 shows the transcript levels of NGR5 at different nitrogen concentrations;
FIG. 14 shows the effect on NGR5-HA protein accumulation at different nitrogen concentrations;
FIG. 15 shows gene expression levels of D14 and OsSPL14 under low nitrogen and high nitrogen conditions for wild type and ngr5 mutant;
FIG. 16 shows the binding relationship of NGR5 to the promoters and gene regions of the D14 and OsSPL14 genes;
FIG. 17 shows the results of comparing phenotype (A) and tiller number (B) of WT, ngr5, D14, ngr 5D 14 double mutants and phenotype (C) and tiller number (D) of WT, ngr5, ospl 14, ngr5 ospl 14 double mutants;
FIG. 18 shows the phenotype of wild type 9311, mutant lc2 under high and low nitrogen conditions;
FIG. 19 shows the gene expression levels of D14 and OsSPL14 under low nitrogen and high nitrogen conditions (A), and the H3K27me3 modification levels at the D14 and OsSPL14 sites (B) for wild type and lc2 mutants;
FIG. 20 shows the phenotype (A) and tillering number (B) comparisons of the WT, lc2, D14, D14 lc2 double mutants and the phenotype (C) and tillering number (D) comparisons of the WT, lc2, ospl 14, ospl 14 lc2 double mutants;
FIG. 21 shows the gene expression levels of D14 and OsSPL14 under different nitrogen conditions for wild type and ngr5 mutant (A), and the H3K27me3 modification levels at D14 and OsSPL14 sites (B).
Detailed Description
The invention is further described below with reference to specific examples, but it will be understood by those skilled in the art that the invention is not limited to these specific examples.
The experimental procedures in the following examples are conventional, unless otherwise specified. Reagents, kits and experimental instruments used in the experiment can be purchased from biological instruments and reagent companies without special instructions.
Example 1: cloning of rice tillering nitrogen response insensitive gene NGR5
The present inventors performed field phenotype screening of EMS mutagenic mutant library with 9311 background at different nitrogen application rates using two different nitrogen treatments (low nitrogen treatment and normal nitrogen fertilizer control group) with 4 rows of 6 plants per row planted in each plot. And (3) respectively inspecting agronomic character indexes such as plant height, tillering number, branch number, spike grain number, cell yield and the like to find out a mutant material which is insensitive or super-sensitive to nitrogen response.
By genetic screening, we obtained a mutant ngr5 (fig. 1A) in which tiller number, branch number, spike number and cell yield were all insensitive to nitrogen response. Specifically, the ngr5 mutant showed significant increases in tiller number, branch number, ear number and cell yield under high nitrogen conditions in the 9311 compared to the wild type 9311, while the ngr5 mutant showed no significant changes in tiller number, branch number, ear number and cell yield under high nitrogen and low nitrogen conditions (fig. 1C-1F).
In order to further know the genetic control basis of promoting the growth and development of rice by nitrogen, the invention constructs a genetic population by hybridizing the ngr5 mutant and japonica rice variety lan Sheng (China Rice research institute). The gene is positioned between two SSR molecular markers RM18384 and RM19751 on chromosome 5 by a map-based cloning technology and is named NGR5 by the inventor (FIG. 2A).
In the following examples, the NGR5 candidate gene was actually determined by sequencing comparative analysis to be a known rice grain SIZE controlling SMOS1(SMALL organic SIZE1) gene encoding an AP2 type transcription factor. The inventor finds that G at the 658 th base of the NGR5 gene cDNA in the NGR5 mutant is mutated into A (figure 2B) through sequencing comparison analysis (NGR5 mutant can also obtain mutant material through the existing gene editing technology).
TABLE 2 primers and sequences for map-based cloning of the NGR5 gene
Example 2: genetic complementation of NGR5 gene and verification of response function of NGR5 gene in regulating and controlling rice tillering nitrogen element
The present inventors designed the following primers according to the promoter sequence and cDNA sequence of NGR5 gene by conventional molecular biology and genetic manipulation method:
primers for constructing the promoter of the NGR5 gene onto a vector (pCAMBIA2300, which is commercially available, for example, from Hippocampus bioengineering Co., Ltd.):
EcoRI-NGR 5-P-F: CGGAATTCTTACTATTAATCTAGTCTATTGC, as shown in SEQ ID NO: 248;
XmaI-NGR 5-P-R: ATACCCGGGGGCGCGCGCACGCAGCAGCAGC, as shown in SEQ ID NO: 249.
Primers for constructing the coding sequence of the mutated NGR5 gene in the NGR5 gene or NGR5 mutant on a vector:
XmaI-5400E 1F: ATACCCGGGATGGCGTCCCCCGGCCCCGC, as shown in SEQ ID NO: 250;
SalI-5400E 2R: CCAGTCGACTCACTCTTGACCATGGAATG, as shown in SEQ ID NO: 251.
The DNA of the 9311 and NGR5 mutants is used as a template, a pCAMBIA2300 vector skeleton is used for respectively constructing pNGR5, NGR5 and pNGR5, a NGR5 vector and an NGR5 mutant by agrobacterium-mediated transformation, and after a positive transgenic plant is obtained, the inventor plants the positive transgenic plant under the conditions of high nitrogen and low nitrogen to observe the phenotype of the transgenic plant. The results show that the NGR5 mutant material transformed pNGR5:: NGR5 restored its response to nitrogen fertilizer (FIG. 1A), while the NGR5 mutant material transformed pNGR5:: NGR5 was similar to the NGR5 mutant in that tiller number, branch number, spike number and plot yield were all unresponsive to the amount of nitrogen fertilizer applied (FIG. 1B).
Example 3: increasing the expression level of NGR5 can increase the yield of rice and the utilization efficiency of nitrogen fertilizer.
The inventor designs the following primers according to the cDNA sequence of the NGR5 gene by the conventional molecular biology and genetic operation method:
kpn1-NGR 5-F: GGGGTACCATGGCGTCCCCCGGCCCCGC, as shown in SEQ ID NO: 252;
xba1-NGR 5-R: TGCTCTAGATCACTCTTGACCATGGAATG, as shown in SEQ ID NO: 253.
A pCAMBIA2300 vector framework is utilized to construct p35S:: NGR5 vector, an agrobacterium-mediated method is utilized to transform a high-yield rice variety 9311, and after positive transgenic plants are obtained, the inventor plants wild type 9311 and p35S:: NGR5 positive transgenic lines under different nitrogen application amounts (60 kg/hectare, 120 kg/hectare, 210 kg/hectare and 300 kg/hectare). The comparison shows that the over-expression of NGR5 can slightly reduce the plant height of rice under each nitrogen application condition, but obviously increase the tiller number and can improve the rice cell yield (figures 3A-3C).
Example 4: excellent allele OsGRF4ngr2And NGR5 polymerization can further improve the rice yield and the utilization efficiency of nitrogen fertilizer.
On the basis, the inventor further uses the material 9311-GRF4 for high-efficiency utilization of nitrogen fertilizerngr2(WO2019/158911A1) transformation of p35S:: NGR5 vector described in example 3, after obtaining positive transgenic plants, the inventors planted 9311-GRF4 under different nitrogen application conditions (60 kg/ha, 120 kg/ha, 210 kg/ha, 300 kg/ha)ngr2And 9311-GRF4ngr2The field experiment result of the positive transgenic line under the background shows that the positive transgenic line is more than 9311-GRF4ngr2The yield can be further increased (fig. 4).
Example 5: NGR5 positively regulates OsGRF4 expression and improves the expression level of nitrogen absorption and assimilation related genes.
The present inventors first analyzed the expression level of OsGRF4 in wild-type 9311, NGR5 mutant, NGR5 over-expressed NGR5 transgenic material grown for 4 weeks in hydroponic conditions, and found that the expression level of OsGRF4 was reduced in NGR5 mutant compared to wild-type, while the expression level of OsGRF4 was significantly increased in over-expressed NGR5 transgenic material, which indicates that NGR5 can positively regulate OsGRF4 gene expression (fig. 5A).
Further, the present inventors analyzed the expression levels of the nitrogen uptake assimilation-associated genes (AMT1.1, GS1.2, NRT1.1b, NRT2.3a, NiR1, GS2, NADH-GoGAT2, NIA1, NPF2.4), and as a result, it was revealed that the nitrogen uptake assimilation-associated genes were expressed at a higher level in the overexpressed NGR5 transgenic material than in the wild type (FIGS. 5B to 5J).
Quantitative PCR detection primer sequence
Example 6: mining and utilization of excellent allelic variation of NGR5
The present inventors performed haplotype analysis on SNPs of the 1kb promoter region, the gene region and the 3' non-coding region of NGR5 gene of 686 rice varieties (the institute of crop science, academy of agricultural sciences, China). The results showed that there were 5 different haplotypes (Haplotype), hap.1 (e.g., Nipponbare), hap.2 (e.g., Guichao No. 2), hap.3 (e.g., Xiangzao No. 2), hap.4 (e.g., Jinghu B), and hap.5 (e.g., 9311) (FIG. 6A). Counting the tillering number and the single plant yield of the rice varieties with different haplotypes, the tillering number and the yield of the rice variety with hap.2 are found to be obviously higher than those of the rice varieties with other haplotypes (FIG. 6B). Further studies have shown that there are 3 specific SNP sites (i.e., g. -853T > G, g. -825T > C, g. -770G > A) for distinguishing between hap.2 type alleles.
On the basis, the inventor selects different varieties from 5 different haplotypes for qRT-PCR analysis (quantitative PCR primer sequence: NGR5F: ACCTGCAACTGGACATG ACACA shown as SEQ ID NO: 274; NGR5R: GAAAAGAGGGTCCGCAG AAG shown as SEQ ID NO: 275)) It was found that a rice variety carrying hap.2 haplotypes (we named their superior allelic variation NGR5Guichao2) With higher transcription level of NGR5 (fig. 7).
Further, the inventor utilizes the background of Gui Dynasty No. 2 with NGR5Hap.1The near isogenic line material of (4), detecting the gene expression level under different nitrogen levels. The results show NGR5Hap.1Is significantly lower than NGR5 at each nitrogen levelHap.2The gene expression level of (1) (FIG. 8).
The primer sequences used to identify 3 specific SNPs (G. -853T > G, G. -825T > C, G. -770G > A) were as follows:
pNGR 5-F: CGGTCCTCACATAGAGGCCA, as shown in SEQ ID NO: 276;
pNGR 5-R: GTTTCCTCGGAACGGCAAC, as shown in SEQ ID NO: 277.
Detecting the presence or absence of any one, two or all three specific SNPs in the promoter region of the NGR5 gene selected from the group consisting of: g. -853T > G, g. -825T > C, g. -770G > a, while analyzing the coding region of the OsNGR5 gene for the presence of specific SNPs: g.3326C > T, and can judge whether the rice variety has the potential of high nitrogen fertilizer utilization rate and high yield.
Example 7: identification of NGR5 interacting protein
In order to further research the function of NGR5 gene, the inventor constructs NGR5 on a yeast double-hybrid vector pGBKT7 (which can be obtained by a commercial way and is purchased from Youbao organisms for example) by a conventional molecular biology method (a primer sequence used for constructing Do-NGR 5-F1: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCGTCCCCCGGCCCCGC shown in SEQ ID NO:278 and Do-NGR 5-R1: GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCTTGACCATGGAATG shown in SEQ ID NO: 279) as a bait protein, screens a yeast library constructed by rice cDNA by a yeast double-hybrid system with the GAL4 as a reporter gene, and finds an interaction protein SLR1 (an important growth inhibitor of a rice gibberellin signal pathway) of NGR5 and an LC2 (the LC2 protein is a subunit of a PRC2 complex), and the PRC2 complex can carry out 3 methylation on the K27 position of the histone H3 so as to inhibit gene expression) (figure 9).
To further verify the interaction of NGR5 with SLR1 and LC2, the present inventors designed the following primers according to the cDNA sequences of SLR1(LOC _ Os03g49990), LC2(LOC _ Os02g05840) and NGR5 genes by conventional molecular biology manipulation methods:
Do-NGR 5-F2: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCGTCCCCCGGCCCCGC, as shown in SEQ ID NO: 280;
Do-NGR 5-R2: GGGGACCACTTTGTACAAGAAAGCTGGGTCCTCTTGACCATGGAATG, as shown in SEQ ID NO: 281;
Do-LC 2-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGATCCACCCTACGCAGGAGTA, as shown in SEQ ID NO: 282;
Do-LC 2-R: GGGGACCACTTTGTACAAGAAAGCTGGGTCATGCCAAAGTTCCATGCAGAAACC, as shown in SEQ ID NO: 283;
Do-SLR 1-F: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGAAGCGCGAGTACCAAGA, as shown in SEQ ID NO: 284;
Do-SLR 1-R: GGGGACCACTTTGTACAAGAAAGCTGGGTCCGCCGCGGCGACGCG, as shown in SEQ ID NO: 285.
Constructing nYFP-NGR5, cYFP-LC2 and cYFP-SLR1 plasmids by utilizing a pCAMBIA2300 vector skeleton, transforming agrobacterium, picking out monoclonal shake bacteria, and then using a resuspension buffer (150 mu M acetosyringone, 10mM MgCl. sub.L. sub.2pH 5.7 in 10mM MES-KOH), OD600 of the bacterial suspension of nYFP and cYFP vectors was 1.0, OD600 of p19 was 0.5. After standing for 3 hours, nYFP, cYFP and p19 were mixed in a 5:5:2 ratio and injected subcutaneously into the lower surface of 3-week-old tobacco using a 1mL syringe. After 2-3 days of injection, the interaction was observed using a confocal laser microscope. Experimental results show that NGR5 is able to interact with SLR1 and LC2, respectively (fig. 10).
Example 8: the multi-tillering character of the semi-dwarf 'green revolution' rice variety is formed depending on the existence of NGR5
The 'green revolution' characterized by semi-dwarf breeding greatly improves the rice yield, and the core is that the accumulation of DELLA proteins in rice bodies is caused by the mutation of gibberellin synthetic gene SD1(LOC _ Os01g66100), thereby reducing the plant height, improving the tillering capability of the rice, improving the lodging resistance capability and finally improving the yield.
To further clarify the function of NGR5 in DELLA protein regulation of rice tillering. The inventor designs a primer Do-GID 1-F: 52 as shown in SEQ ID NO:286, a primer Do-GID 2300-D2300-F: 35 as shown in SEQ ID NO:1 as shown in SEQ ID NO: GGGGACCACTTTGTACAAGAAAGCTGGGTGTAGTAGAGGTTAGCGTTGAG, and a primer GID-NO: GGGGACCACTTTGTACAAGAAAGCTGGGTGTAGTAGAGGTTAGCGTTGAG as shown in SEQ ID NO: GGGGACCACTTTGTACAAGAAAGCTGGGTGTAGTAGAGGTTAGCGTTGAG, and constructs a nitrogen fertilizer over-expression vector under different nitrogen fertilizer over-expression conditions by using two groups of vectors as shown in SEQ ID NO: 3646, wherein the two groups of vectors are derived from China Rice research institute, NJ6-SD1 (the near-isogenic line material obtained by introducing SD1 gene into Nanjing 6 background by genetic hybridization), NJ6-GID1-10 (Guangdong farmyard), NJ6-SD1-ngr5 (the near-isogenic line material obtained by further introducing ngr5 gene into NJ6-SD1 by genetic hybridization, and the cDNA sequence of NJ6-SD 1-573) and a transgenic material over-expressing GID1(LOC _ Os05g33730), and simultaneously treated with 1. mu.M gibberellin. The results show that SLR1 protein high-level accumulation of NJ6-SD1 and NJ6-GID1-10 tillering number is more than that of NJ6-SD1 under high nitrogen and low nitrogen conditions, while the tillering number of GID1 transgenic material over-expressing in NJ6-SD1 background and gibberellin-treated NJ6 and NJ6-SD1 material is less than that of the control group under high nitrogen and low nitrogen conditions, which indicates that SLR1 protein high-level accumulation can promote rice tillering number increase. However, NJ6-sd1-NGR5 was the less tillering phenotype under both high and low nitrogen treatment conditions, suggesting that the function of SLR1 protein in promoting rice tillering increase was dependent on NGR5 (FIG. 11).
Example 9: high-level accumulation of SLR1, Rht-B1a and Rht-B1B proteins increases the stability of NGR5 protein
Examples 3 and 8 mention that increasing both NGR5 and SLR1 promotes the production of rice tillers. The inventor utilizes in vitro protein degradation experiments to confirm the biological significance of interaction between SLR1 and NGR5, and further confirms that DELLA proteins (Rht-B1a and Rht-B1B) from wheat have functions similar to rice DELLA protein (SLR 1).
The specific method comprises the following steps: in vitro protein degradation buffer (25mM Tris-HCl, pH 7.5,10mM NaCl,10mM MgCl)24mM PMSF,5mM dithioreitol, 10mM ATP) was addedTaking the total protein of the leaf of Nanjing No. 6 rice growing for 4 weeks in the field as a reaction system. The extractive solution is subjected to allelic addition, GST-NGR5 fusion protein is added, and 50 μ M MG132, SLR1-Flag, Rht-B1a-Flag, and Rht-B1B-Flag are added respectively. The reaction was stopped at 0, 3, 6 and 9 minutes, and the change of GST-NGR5 fusion protein was detected by Western blot. The results showed that SLR1-Flag, Rht-B1a-Flag, and Rht-B1B-Flag all increased the stability of GST-NGR5 protein (FIG. 12). This also suggests that NGR5 is conserved in function in rice and wheat.
Example 10: the expression level and protein level of NGR5 gene are positively regulated by nitrogen
The inventors utilized four nitrogen concentration gradients (0N, 0.2N 0.25mM NH)4NO3,0.6N 0.75mM NH4NO3And 1N 1.25mM NH4NO3) The 9311 and ngr5 mutants were cultured in nutrient solutions. Total RNA was extracted from 9311 and NGR5 mutant leaves hydroponically cultured for 4 weeks, and the transcript level of NGR5 in the 9311 and NGR5 mutants was analyzed by quantitative PCR. The results showed that with increasing nitrogen concentration in the nutrient solution, the transcription level of NGR5 in 9311 increased with increasing nitrogen concentration, whereas little change in the expression of NGR5 gene was detectable in the NGR5 mutant (fig. 13).
The inventors utilized four nitrogen concentration gradients (0N, 0.2N 0.25mM NH)4NO3,0.6N 0.75mM NH4NO3And 1N 1.25mM NH4NO3) Culturing 9311 background over-expression NGR5-HA transgenic plants. Total protein is extracted from leaves of NGR5-HA transgenic plants with 9311 backgrounds cultured in water for 4 weeks, and the protein accumulation level of NGR5-HA is analyzed by Western-blot. The results showed that NGR5-HA protein accumulated with increasing nitrogen concentration (figure 14).
Example 11: NGR5 mediates the response of rice tillering nitrogen by regulating the expression of D14 and OsSPL14 genes.
The inventors found that the gene expression levels of D14 and OsSPL14(D14 and OsSPL14 are negative regulatory factors for rice tillering) in wild-type 9311 were inhibited by nitrogen, but were highly expressed and not inhibited by nitrogen in ngr5 mutant (FIG. 15) (the quantitative PCR primers used were D14F: ACGTGATGATCTGCGTGATGCC as shown in SEQ ID NO:288, D14R: GGCTTTGGTTTGGCCAGCTAAC as shown in SEQ ID NO:289, OsSPL14F: TGCTGCCTGAATTTGACCAAGG as shown in SEQ ID NO:290, and OsSPL14R: TTCTGAACCTGCGATGCTCACC as shown in SEQ ID NO: 291). NGR5 was shown to bind directly to D14 and OsSPL14 sites by ChIP-PCR and EMSA experiments (FIG. 16).
Furthermore, the inventor hybridizes NGR5 mutant and D14 mutant (Chinese rice research institute) to obtain double-mutant materials of NGR5 and D14 genes, and hybridizes NGR5 mutant and OsSPL14 mutant (obtained by knocking out OsSPL14 gene by conventional gene editing technology in 9311 background) to obtain double-mutant materials of NGR5 and OsSPL14 genes, and statistics of tillering number shows that the tillering number of NGR 5D 14 double-mutant is almost consistent with that of D14 mutant, and the tillering number of NGR5 OsSPL14 double-mutant is almost consistent with that of OsSPL14 mutant. This suggests that both D14 and OsSPL14 are able to inhibit the NGR5 mutant from a less tillering phenotype, i.e., D14 and ospl 14 are genetically located downstream of NGR5 (fig. 17).
Example 12: LC2 participates in the regulation of rice tillering nitrogen response by regulating the modification level of D14 and H3K27me3 at the OsSPL14 site.
The inventors planted wild-type 9311 and LC2 mutants (obtained by knocking out LC2 gene by conventional gene editing technology in the 9311 background) under high nitrogen and low nitrogen conditions, respectively, and found that nitrogen promoted the increase in 9311 tillering number, while the tillering of LC2 mutant lost response to nitrogen, which indicates that LC2 is a key gene of rice tillering response to nitrogen (fig. 18).
Further, the present inventors compared the expression changes of D14 and ospl 14 genes by quantitative PCR using 9311 and lc2 mutants planted at different nitrogen levels, and found that D14 and ospl 14 genes were inhibited by nitrogen in 9311, while D14 and ospl 14 genes were consistently highly expressed and not inhibited by nitrogen in lc2 mutants (fig. 19).
Further, the inventor hybridizes the LC2 mutant with the D14 mutant and the osppl 14 mutant respectively to obtain the double-mutant materials of LC2 and D14 and LC2 and OsSPL14, and counting the tillering number shows that the tillering number of the LC 2D 14 double-mutant is almost the same as that of the D14 mutant, and the tillering number of the LC2 ospl 14 double-mutant is almost the same as that of the ospl 14 mutant. This suggests that both D14 and OsSPL14 inhibited the LC2 mutant from a less tillering phenotype, i.e., D14 and OsSPL14 were genetically located downstream of LC2 (fig. 20).
Example 13: NGR5 regulates and controls the H3K27me3 modification level of D14 and OsSPL14 sites to realize the response of the rice tillering nitrogen.
The present inventors compared the expression changes of D14 and ospl 14 genes by quantitative PCR using 9311 and ngr5 mutants planted at different nitrogen levels, and found that D14 and ospl 14 genes were inhibited by nitrogen in 9311, while D14 and ospl 14 genes were consistently highly expressed and not inhibited by nitrogen in ngr5 mutants (fig. 21A).
Next, the present inventors compared the H3K27me3 modification levels at different nitrogen levels at D14 and OsSPL14 sites by ChIP-PCR using 9311 and ngr5 mutants planted at different nitrogen levels, and found that the H3K27me3 modification levels at D14 and OsSPL14 sites were induced by nitrogen, whereas the H3K27me3 modification levels at D14 and OsSPL14 sites were not induced by nitrogen in the ngr5 mutant. The above experiments demonstrated that nitrogen-mediated regulation of the level of modification of H3K27me3 at D14 and ospl 14 sites was dependent on NGR5 (fig. 21B).
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described in relation to an exemplary embodiment, and it is understood that the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Sequence listing
SEQ ID NO:1:
ATGGCGTCCCCCGGCCCCGCCGCGGGGATGCAGCAGAAGCTGGAGGCGGCTGCGGCGGCGGCGGGGGGAGGAGACGGGGCGGAGTGGGGCCGGGGAATGCAGAAGATGGAGGCGGTGGGAGCGGGGGGAGAGGGGGTGGGGGCGGGGGCGGAGCAGGTGGCGCCGCCGCCGAGGAGGCCCGTGGCGGCGCGGAAGGAGAGGGTGTGCACGGCCAAGGAGCGTATCAGCCGCATGCCGCCCTGCGCCGCCGGGAAGCGCAGCTCCATCTACCGCGGCGTCACCCGGCATAGGTGGACAGGCCGATATGAAGCTCACCTCTGGGATAAAAGCACATGGAATCAGAATCAGAACAAAAAAGGGAAACAAGTATATTTAGGTGCATATGATGATGAAGAGGCTGCAGCAAGAGCTTATGACCTTGCTGCATTGAAATACTGGGGAGCTGGGACACAAATAAACTTTCCTGTCTCTGATTATGCAAGAGATCTTGAGGAAATGCAGATGATCTCCAAGGAGGATTATCTTGTGTCTCTTCGGAGAAAGAGCAGTGCCTTTTCCAGGGGTTTACCAAAATATCGCGGCCTTCCTAGGCAGCTCCATAATTCCAGATGGGATGCTTCTTTGGGACACTTGCTTGGCAATGACTACATGAGCCTAGGGAAGGACATCACGCTGGATGGGAAATTTGCAGGAACCTTTGGCTTAGAGAGGAAAATTGATCTGACAAATTACATAAGGTGGTGGCTCCCAAAAAAGACACGGCAGTCAGATACATCTAAAATGGAAGAGGTTACTGATGAAATCCGTGCTATTGAAAGTTCAATGCAACGGACTGAGCCTTATAAGTTTCCTTCCCTTGGCCTCCATTCTAACTCAAAGCCCTCTTCCGTGGTCCTATCAGCATGTGATATCTTATCTCAGTCTGATGCCTTCAAAAGCTTCTCAGAAAAATCTACAAAACTATCTGAAGAATGTACTTTTAGCAAAGAAATGGATGAAGGAAAGACAGTTACCCCAGTACCTGCAACTGGACATGACACAACTGCAGTTAACATGAACGTGAATGGGTTGCTTGTGCAAAGAGCTCCATACACATTGCCCTCTGTTACTGCACAAATGAAAAATACCTGGAACCCTGCTGATCCTTCTGCGGACCCTCTTTTCTGGACCAACTTCATCCTGCCAGCAAGTCAACCTGTCACGATGGCAACAATAGCAACAACAACGTTTGCAAAGAATGAGGTGAGTTCAAGTGATCCATTCCATGGTCAAGAGTGA
SEQ ID NO:2:
MASPGPAAGMQQKLEAAAAAAGGGDGAEWGRGMQKMEAVGAGGEGVGAGAEQVAPPPRRPVAARKERVCTAKERISRMPPCAAGKRSSIYRGVTRHRWTGRYEAHLWDKSTWNQNQNKKGKQVYLGAYDDEEAAARAYDLAALKYWGAGTQINFPVSDYARDLEEMQMISKEDYLVSLRRKSSAFSRGLPKYRGLPRQLHNSRWDASLGHLLGNDYMSLGKDITLDGKFAGTFGLERKIDLTNYIRWWLPKKTRQSDTSKMEEVTDEIRAIESSMQRTEPYKFPSLGLHSNSKPSSVVLSACDILSQSDAFKSFSEKSTKLSEECTFSKEMDEGKTVTPVPATGHDTTAVNMNVNGLLVQRAPYTLPSVTAQMKNTWNPADPSADPLFWTNFILPASQPVTMATIATTTFAKNEVSSSDPFHGQE
SEQ ID NO:3:
TATTAATCTAGTCTATTGCTGAGATGTACATGTTTTATAGATAGCACCTTACTTTACCATTGCGGGTGCTCTAATAAGGCACACTCAGCGGGCGATAAGAGTGCCACGTCATCCAAATTCATTCGTGGAGGAGAGAGAAAGCTAGAGGGAGAGGGCAACGGGCGCTTCTGCAAACGCCGAGCTATAGCACAAGAAATTATGCATATTCAATATTGTGGTTGTTTTTCTCCAATAGTATAGCTATGGCCCCATTAGCAGAACACACGGGATAGGATAGAGAGTAGCAAGTTAGATTAAAAAATAGAAAAAGTTAACTACCTAGAGAAGTGTGTAGGTAGATTAGCAGCCTACTATTATACATATCATCTTTATTTTCTATCTCCGGATGACATGTACATTTTTATCGTCAGTTGATAGCGGTACTATTAAACTTGCTCTAACGGATAATCTAGCGAGCAAGTGATTCGGAAAAAAATAACTCTGTAGCCTGCACACTGGGTAATAAATGATGGTCGTCGCACCAGTTGAACTAAGCAACGGTCGGTGCGAGCAGTATCTTTACCCCCCTATTGTTACGTTGATGGCTTTTAGGCTCCTTTTAACATGTGGAGTTTACAATAATTTTATAAGAAATATTATTAGTCAAATTCAATCGCAGTTTATTTTATTGATATGCACACCCTTTAATTTAGTCGAAATTATGAATCCTAAATTAGGAAGTACGAGCGATAGTACTTTATCTACACTCAGAGAAAAAAAGAATGTCCCCTAATACACATCACTGCCGATGAACTCTATTGAAACTGGGTTATCTGTCTCCGAAAGTCACTTTCTCGGTCCTCACATAGAGGCCATTTGGTCTGTGATCTAATGTCTTTGTTGGAAGCGGTGAATACTTTTAGACATGTTTGACATGATTTTAACTTCTAAATCTAACTCTAACAGTGAAGTCTAGAGTAAAGTATGGAGCAATCCAAACCCTATTTTTCCCCTATAATTTATTTTTCATATCACTTCAGCCCACTTTATCTCCTTCAGATATTGCTTAAACATTTGGTTGGGCTACAATTTTAAGAGAGATGGAGTCAAAACCTTATCATATACTTCCTCCGTCTAAAAAAAACTTAACCTAGAAGGTCCCCTCCTATGACAACGAATCTAGATAAATGGTAGTATAAATTTGTTGTCATAAGGGTCACATCTCCTCCAAAGTTAAGTTTTTTTTTAAATGGAGGGAGTAAGCACTTAATTTATAGTTCAAGAGTTCTTTTTACACGAATAATATAGAGTATCTATATAGTGAGAATTTGAACAAGTTATGTTATGTTTTTAGTTTTTAGCTAATATACTAGTTTATTTTTTTAGATCTTATGATTACTACACAATCTATCTAATAAAAAATAAAATTAAAATGTTTAAGAGAGCAGTAGCTCCTATTGCTATACAAGCTCTTGGAATAGGACCTCAAATCCTATATCCCTAACCTAAAGAAAGAACCAAAGAAACCAAATCTATAGCGTTGTGTGTGTAAACAAATAAATAAAAAAAAAAGTCCACCGAGACAAGAAAAAACGCACCGTTGCCGTTCCGAGGAAACGACGGCGGGCAGGACAATCCCGCATCCGCCGTGTCCCTTTCCGCCCCCGGCCCGGCCAATAGCCCAGTGGGCCCCACCCCCCCTCCCCAATCCCATCGTATCCCCCCGCTTCCTCGGATTCCCCTGCCCCCTGTCCCCCCATGTGCCGGCCCACTGGGCTCCACCCCCCCATCCATCCACGCATCCACCCCCCCCATCCGTCTCCATCCCTCCCAGTCTCCCACCTCACGCACTCGATGCGATCCCCCCCATTTGAAATCCCCCCCCTCTCCTCACCGCGACCGCCTCCCCCAAACGGTCACCCCCGCTTTGGCTCTCTCTTCTCTTCTCGCCTCGCCTCGCCACCGACTCCGATCGAGTGGGGGGAGGGAGGGGGGGTTTGCTGCTGCTGCGTGCGCGCGCC
SEQ ID NO:4:
ATGGCGTCCCCCGGCCCCGCCGCGGGGATGCAGCAGAAGCTGGAGGCGGCTGCGGCGGCGGCGGGGGGAGGAGACGGGGCGGAGTGGGGCCGGGGAATGCAGAAGATGGAGGCGGTGGGAGCGGGGGGAGAGGGGGTGGGGGCGGGGGCGGAGCAGGTGGCGCCGCCGCCGAGGAGGCCCGTGGCGGCGCGGAAGGAGAGGGTGTGCACGGCCAAGGAGCGTATCAGCCGCATGCCGCCCTGCGCCGCCGGGAAGCGCAGCTCCATCTACCGCGGCGTCACCCGGTACGGCTCCTCCCCCCTTTGCCCTCTCCCTCCTCCGTGTCTCGAGTCGCGGAGAAGTTTTACTTGGATGCTTATTAGGGGATATGCAATCGTTCTCTGCTTGGATTGATCTGTGGCGCTAAATCCGTTCGTCAGGTCTCCTTGTATGCTCAAAATGGTACCGTCGATTCGGGATCTAGATTTTTCTTTTTGTTTACTTGGTTATATACGGATTAGCTGTATTCATCGGATTACCTGTTGTGTCTCCAAGGGGACGATCTGAAATAATTAGGAGCTTCGCTTGATCTAAATAAATATAAAAGCTAATGAATGATTTTCACTTGACCAGTACATCCAACATCATGCTTGATTTAAGTTGTGTCAAACTGGGTTGACAGGGTTTAGCTCTAATCTGGGTAAGAAGCAAGTTTTGGACCCTTTTCGGTCCAGACAAGTGTGGGGCTCAACTAAAAGATGAATTTTCAAACTATATGTGTATTTACATACTTAAATCTTTTATTTGGTGTTTAATCTGATCCAAAAGTAGTATTTGTTCTATATTGTTATTCAACTGTTCTTGTACCACCATATGCTACTTCTTTCTTAATTTTGTTGTAATTTGTAAGCAAGATTTTAACTTTCAGCTGTTATTATGTATGAATTTGATTTGACACTTGTTTTGTATCATCTATAGGCATAGGTGGACAGGCCGATATGAAGCTCACCTCTGGGATAAAAGCACATGGAATCAGAATCAGAACAAAAAAGGGAAACAAGGTCTGTATACCCAATGCAAATTAGCAGCTATATTTTACATGTTTTGAAAAGTTGACCAAGGGACGTTGGAAGCTAGGTGAATTTTAATTGTATTTCATAGATAATCCATGCACGTCATTTCCTTTTATATCTAGCTATAATTAATCTGATTTAATGGAATTGTCTCTTTTGCATGGGAAATTATCACCCAAAATGGCCCTGCAAGTGCAGTATATTTAGGTAAGAAATCTTTGGTGTTAACTGTAAAATGTGCAATTGTATAAACTTCTTTATCCACATCAATTCAATGCGCAAATACACAGGTGCATATGATGATGAAGAGGCTGCAGCAAGAGCTTATGACCTTGCTGCATTGAAATACTGGGGAGCTGGGACACAAATAAACTTTCCTGTGAGTCATCTATACTTGCTAATTATAATTGGTTTCTTAGCTTCCATTTAGTTCTTCTACAATTATTAACCACACACTGCCTTTTCTTCAAGACCTTCATTTGGTTAAATTTAATCATTTATATTTTGCATCACCTGAGCTCAATTTCGAAGACTGATTCTCTTAGGATGCAAATTGAAAACCAAAAGATATCTTAGAGAGTGTTGTACCAAACTTTGGATCATACTAGTCCAAACATATCCTATGCAACTGTTGTGTGATATTTGAAAAATCTGAATACTCTTGACTATTATATTATGTTCTTTTGTATGCAAAGGTTAGGGATTTATTCAGTTTACACTTGACGATTCGGAGATGGATGTTTGTGTCCTTGACAAAAAAAATCTCTTGTTTTCAGAAAATTCATCTCTGAGCATATAAGTGACATTGAAAAATCATTGTCTCAGAAGAATATTGGCAAAACAAACATATGCATACATGCCATCTCTTCAGTTTGGTATCTATATGCCTATACATCTCGATGGAAAATAATTGCGATGTTGCTTATGAATTATTGTACTATGTTTTTTCCATTCTATATTTTGCACTAGTTAATTTGATTTGTGGTAATCTTTTTCAGGCTTATGTTCATTATGTATTTGCTTATTTAGGTCTCTGATTATGCAAGAGATCTTGAGGAAATGCAGATGATCTCCAAGGAGGATTATCTTGTGTCTCTTCGGAGGTATATTTGTGAATTTATAATACGTGCATATTACTTTGTTTCTTAAGTACTAACTGTAAGACACATGCACTGCAGAAAGAGCAGTGCCTTTTCCAGGGGTTTACCAAAATATCGCGGCCTTCCTAGGTACTTATTAGCAATGAGAAATATATTTGGAAGTTAACTCTATGTTCAGTTATTATATTTGCATGCATGTTTGTGATGCTAATTCAGTAACAAGTCTCATGTCCAATTTTCTTTTCTATTGTGCACAAACTATCATTGTCCATGTTTTGTTTATTTCGGTAACATTTTCCATCTTGCTACAAATATTTATGGCAAACCAAGTAGTAGTATGTAAAGATGATGCCCTGTGGTATATTACATTAGAAATCATTTTCAGCAACTTCATGTATTTCTGCTATGAATTAAATTAGTAAGCTCAGCAGATTTTTGGAGGTAGATAGCTTAGATACTAAACTACTAGCTCTTATTCGTGTGTCATTGGTCTTCATTTAGAGAATTTTAAAATAAAGTGTCATTTAGCAAAGATGCAAACCTTCAAGTTTTGCTGAACATTAATTCTTTCCAATAATCACTCGATTGCACAGCCACAGTCCCTCAACTGTACTCGTGATGCTGAATGTGCGTCATCGAAATTCTGTGTTTCCTCACTGATTACTGTTGTCAATGGAAATTCGGCATTCAGTGCCTTCGTTCTTTGTATGGTTACAGCTATCCTGCTTTCTGTGGGATCTCAACCAACCAAAACATAATATTCTCAACAGAATATCCTTTCAGAGGAGAGTGATTACTTGTAGCTGAGATTTTGAAGAGATCGACATACTTGAAATTTCCTTCTAGGATAGGATCCCGACTTAATAGTGTCCTGAGGACCACTAGAAATTAGTTTAGGAAAGTTTATTATTTGGACATATAAAGTAAAACTATACTCCCTCCATACTCATAAAGGAAGTCGTTTAGGACAATGTTTAAGTCAAACCTTGGGAATATAAATCATTAATAACTCTTAAGTTGTTAAGTTTGACAATGCAAAAATTATATGAATAGATTTGCCTTGAAAAATACTTTCATAAAAGTATACATATATCACTTTTCAATAAATATTTTTATAGAAACAAGAAGTCAAAGTTGTGTTTTGAAGACCATCTCGCTGTCCTAAACGACTTCTTTTACGAGTACGGAGGGTGTACGTAATTCTGTTTTACTTAATTATTCACCAGATGTCAGTCTTCGACTAATCATTACAGTGCACTACACTGCTGGACACGTACAGTCACATTGATATAAATTTTATAACTGCAACCGTTGTGACATTATCATGCAGGCAGCTCCATAATTCCAGATGGGATGCTTCTTTGGGACACTTGCTTGGCAATGACTACATGAGCCTAGGTTGTGGTGTCTCCATAAAAACTTCTACACTGCTGTTTCTGGTTAACAAACTACAGAGGAGAAAGACAAATTCTATGTGGTTTTGTAACCAGTCTTTCTATTTAAATTACAGGGAAGGACATCACGCTGGATGGGAAATTTGCAGGAACCTTTGGCTTAGAGAGGAAAATTGATCTGACAAATTACATAAGGTGGTGGCTCCCAAAAAAGACACGGCAGTCAGATACATCTAAAATGGAAGAGGTTACTGATGAAATCCGTGCTATTGAAAGTTCAATGCAACGGACTGAGCCTTATAAGTTTCCTTCCCTTGGCCTCCATTCTAACTCAAAGCCCTCTTCCGTGGTCCTATCAGCATGTGATATCTTATCTCAGTCTGATGCCTTCAAAAGCTTCTCAGAAAAATCTACAAAACTATCTGAAGAATGTACTTTTAGCAAAGAAATGGATGAAGGAAAGACAGTTACCCCAGTACCTGCAACTGGACATGACACAACTGCAGTTAACATGAACGTGAATGGGTTGCTTGTGCAAAGAGCTCCATACACATTGCCCTCTGTTACTGCACAAATGAAAAATACCTGGAACCCTGCTGATCCTTCTGCGGACCCTCTTTTCTGGACCAACTTCATCCTGCCAGCAAGTCAACCTGTCACGATGGCAACAATAGCAACAACAACGGTTTGTTCCTCTTTGGCTCTACTTGACAATTTGAATTAAATCTGTAACATATTTTCAATTTTTCTTCTTGTGCTTAATTTGATGCTGATTTGTGACAGTTTGCAAAGAATGAGGTAAGTTCAAGTGATCCATTCCATGGTCAAGAGTGACCGTACGAGCTTACCAAAGTGCATGCAGGATTTGCTTTTTGGCTTGGTAAGTAGAGACTGTAGGCATAGTAAATTTTACATTTTTTCATATATTTTCTTTCTGTGTATCGCCTAAATTATCTGCTTGAAGAACTCAATGCATCACGAGATGTCCAGATTTGCATTCAAATAAGAACATGCAAGCAATGGAAATATGTTTATGTCTAACTTACAAATATCTAAGGATTTATTTTCCCTTGATTGACAGGTGCCAGTCTTGAAAAGAAAAGAATATACCTGACAATATGACACTCCGTTGAAAGAACACAATGAAAATGAACTGAGACAGTTTAAATCTAATCAATTAAACCAAACAAAAATACTGTTGTGAGATATTTATGTGTGCATAAATATTTTTATAAGGATGAAAATATGGGATTATGTTGTGTAAGTGGTGCATCGAGTATTTGAGACATGATAAATCAACATTGTTTATGTGCGTGTGCCCACGCGTGGACAGTGCTTTCAGCTTTCCCTCCGTTAAAATTTCAGTAACACTGACATGTGGGTGTCAAATTCTTAATTTACTATAGCTACCCAGGAAGA
SEQ ID NO:5:
TATTAATCTAGTCTATTGCTGAGATGTACATGTTTTATAGATAGCACCTTACTTTACCATTGCGGGTGCTCTAATAAGGCACACTCAGCGGGCGATAAGAGTGCCACGTCATCCAAATTCATTCGTGGAGGAGAGAGAAAGCTAGAGGGAGAGGGCAACGGGCGCTTCTGCAAGCGCCGAGCTACAGCACAAGAAATTATGCATATTCAATATTGTAGTTGTTTTTCTCCAATAGTATAGCTATGGCCCCATTAGCAGAACACACGGGATAGGATAGAGAGTAGCAAGTTAGATTAAAAAATAGAAAAAGTTAACTAACTAGAGAAGTGTGTAGGTAGATTAGCAGCCTACTATTGTACATATCATCTTTATTTTCTATCTCCGGATGACATGTATATTTTTATCGTCAGTTGATGGCGGTACTATTAAACTTGCTCTAACGGATAATCTAGCGAGCAAGTGATTCGGAAAAAAATAACTCTGTAGCCTGCACACTGGGTAATAAATGATGGTCGTCGCACCAGTTGAACTAAGCAACGGTCGGTGCGAGCAGTATCTTTACCCCCCTATTGTTACGTTGATGGCTTTTAGGCTCCTTTTAACATGTGGAGTTTACAATAATTTTATAAGAAATATTATTAGTCAAATTCAATCGCAGTTTATTTTATTGATATGCACACCCTTTAATTTAGTCGAAATTATGAATCCTAAATTAGGAAGTACGAGCGATAGTACTTTATCTACACTCAGAAAAAAAAAGAATGTCCCCTAATACACATCACTGCCGATAAACTCTATTGAAACTGGGTTATCTGTCTCCGAAAGTCACTTTCTCGGTCCTCACATAGAGGCCATTTGGTCTGTGATCTAATGTCTTGGTTGGAAGCGGTGAATACTTTTAGACATGTTTGACATGATTTTAATTTCTAAATCTAACTCTAACAGTGAAGTCTAGAGTAAAGTATGGAGCAATCCAAACCCTATTTTTCCCCTATAATTTATTTTTCATATCACTTCAGCCCACTTTATCTCCTTCAGATATTGCTTAAACATTTAGTTGGGCTACAATTTTAAGAGAGATGGAGTCAAAACCTTATCATATACTTCCTCCGTCTAAAAAAAACTTAACCTAGAAGGTCCCCTCCTAGCACAACGAATCTAGATAAATGGTAGTACAAATTTGTTGTCATAAGGGTCACATCTCCTCCAAAGTTAAGTTTTTTTTTAAATAGAGGGAGTAAGCACTTAATTTATAGTTCAAGAGTTCTTTTTACACGAATAATATAGAGTATCTATATAGTGAGAATTTGAACAAGTTATGTTACGTTTTTAGTTTTTAGCTAATATACTAGTTTATTTTTTTAGATCTTATGATTACTACACAATCTATCTAATAAAAAATAAAATTAAAATGTTTAAGAGAGCAGTAGCTCCTATTGCTATACAAGCTCTTGGAATAGGACCTCAAATCCTATATCCCTAACCTAAAGAAAGAACCAAAGAAACCAAATCTATAGCGTTGTGTGTGTAAACAAATAAATAAAAAAAAAAGTCCACCGAGACAAGAAAAAACGCACCGTTGCCGTTCCGAGGAAACGACGGCGGGCAGGACAATCCCGCATCCGCCGTGTCCCTTTCCGCCCCCGGCCCGGCCAATAGCCCAGTGGGCCCCACCCCCCCTCCCCAATCCCATCGTATCCCCCCGCTTCCTCGGATTCCCCTGCCCCCTGTCCCCCCATGTGCCGGCCCACTGGGCTCCACCCCCCCATCCATCCACGCATCCACCCCCCCCATCCGTCTCCATCCCTCCCAGTCTCCCACCTCACGCACTCGATGCGATCCCCCCCATTTGAAATCCCCCCCCTCTCCTCACCGCGACCGCCTCCCCCAAACGGTCACCCCCGCTTTGGCTCTCTCTTCTCTTCTCGCCTCGCCTCGCCACCGACTCCGATCGAGTGGGGGGAGGGAGGGGGGGTTTGCTGCTGCTGCGTGCGCGCGCC
Claims (16)
1. The application of the protein for controlling the nitrogen utilization rate and the crop yield in improving the nitrogen utilization rate and the crop yield, wherein the amino acid sequence of the protein is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the protein controlling nitrogen use efficiency and crop yield is as shown in SEQ ID NO 1.
3. A method of growing a crop with high nitrogen utilization and high yield, the method comprising: transfecting a nucleotide sequence of the gene encoding the protein for controlling nitrogen use efficiency and crop yield of claim 2 into a crop cell to obtain a transgenic crop plant, so that the expression level of the gene encoding the protein for controlling nitrogen use efficiency and crop yield of claim 2 in the transgenic crop plant is increased, and a crop plant having high nitrogen use efficiency and high yield is obtained, wherein the crop plant is a monocotyledon plant.
4. A method of growing crops with high nitrogen utilization and high yield as claimed in claim 3, wherein the crops are rice or wheat.
5. A method of growing crops with high nitrogen utilization and high yield as claimed in claim 3, wherein the crops are rice.
6. A method of growing a crop with high nitrogen utilization and high yield, the method comprising: co-expression of GRF4 in crop plantsngr2A gene encoding the protein for controlling nitrogen use efficiency and crop yield of claim 2 or a superior allele thereof, or a gene carrying OsGRF4ngr2A gene encoding the nitrogen use efficiency and crop yield controlling protein of claim 2, wherein the gene encoding the nitrogen use efficiency and crop yield controlling protein of claim 2 is OsNGR5 gene, and the superior allele thereof is OsNGR5 gene, or an excellent allele thereof, is overexpressed in a crop plant thereofGuichao2Gene of OsNGR5Guichao2The gDNA sequence of (a) is shown as SEQ ID NO. 4, and the crop is a monocotyledon.
7. The method of claim 6, wherein the crop is rice or wheat.
8. The method of claim 6, wherein the crop is rice.
9. The method of claim 6, wherein the superior allele OsNGR5Guichao2The promoter region of the gene has 3 specific SNPs sites: g. -853T>G,g.-825T>C,g.-770G>A, the specific SNPs site can increase OsNGR5Guichao2The level of transcription of the gene.
10. A method of growing a crop with high nitrogen utilization and high yield, the method comprising: will contain OsNGR5 as claimed in claim 9Guichao2Crossing a crop plant of the gene with another plant of the crop to produce a hybrid crop plant, such that OsNGR5 is present in the hybrid cropGuichao2The expression quantity of the gene is increased, and the crop with high nitrogen fertilizer utilization rate and high yield is obtained, wherein the crop is rice.
11. A method of identifying a rice variety with high nitrogen utilization and high yield, the method comprising: analyzing the promoter region of the OsNGR5 gene for the presence of any one, two or all three specific SNPs selected from the group consisting of: g. -853T > G, g. -825T > C, g. -770G > A, while analyzing the gene region of OsNGR5 gene for the presence of specific SNPs: g.3326C > T, to determine if the rice variety has the potential for high nitrogen utilization and high yield.
12. The method according to claim 11, wherein the SNPs of the promoter region and the coding region of the OsNGR5 gene are detected by PCR amplification digestion or sequencing.
13. The nucleotide sequence of the gene for controlling the nitrogen utilization rate and the crop yield is shown as SEQ ID NO. 4.
14. A recombinant construct comprising the gene of claim 13.
15. A host cell comprising the gene of claim 13 or the recombinant construct of claim 14, wherein the host cell is a microbial cell.
16. The host cell of claim 15, wherein the host cell is an escherichia coli cell or an agrobacterium cell.
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CN115820721B (en) * | 2022-07-26 | 2024-03-22 | 贵州大学 | Method for improving tillering and yield of Guizhou high-quality special rice and large gingko glutinous rice, promoter core sequence and application |
CN118064452B (en) * | 2024-04-17 | 2024-06-21 | 中国热带农业科学院三亚研究院 | Cyperus esculentus CeWRI gene, expression vector and application thereof in regulating and controlling vegetable oil |
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CN106554397B (en) * | 2015-09-30 | 2019-08-30 | 中国科学院遗传与发育生物学研究所 | Protein OsGRF4-M and its relevant biological material from rice are regulating and controlling the application in plant organ size |
CN107937416B (en) * | 2017-12-29 | 2020-11-03 | 中国科学院遗传与发育生物学研究所 | Gene for improving utilization efficiency and yield of rice nitrogen fertilizer and application thereof |
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