Method for regulating and controlling ear number per plant, grain number per ear or / and yield of plant under low-nitrogen condition

Abstract

The invention discloses a method for regulating and controlling the spike number per plant, the grain number per spike or / and the yield of a plant under a low-nitrogen condition, and belongs to the technical field of biology. The method comprises the step of regulating and controlling the ear number of a single plant, the grain number per ear or / and the yield of the plant by regulating and controlling coding gene expression of protein in the plant or regulating and controlling the activity and / or the content of the protein, the protein is a protein with an amino acid sequence of SEQ ID NO: 1. Experiments prove that the yield of grains can be remarkably increased by knocking down TaNIP1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for regulating the number of spikes per plant, the number of grains per spike, and / or the yield of plants under low nitrogen conditions. Background Technology

[0002] Wheat is one of the most widely cultivated and distributed food crops globally, ranking among the top major food crops in terms of yield. Increased wheat production can effectively address the food demand pressures brought about by global population growth and reduce the occurrence of hunger. Secondly, increased production can improve the efficiency of agricultural production, bringing higher economic benefits to farmers and promoting the stability and development of the rural economy. Simultaneously, through scientific yield-increasing measures, land resources can be utilized more effectively, reducing the encroachment on forests, grasslands, and other ecological environments, thus promoting sustainable ecological development. Therefore, increasing wheat production is not only related to food supply but also to the coordinated development of society, economy, and environment. Therefore, cultivating high-yielding, superior wheat varieties through genetic engineering and other biological means is of great significance. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide a new application of TaNIP1 protein in regulating wheat yield. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: This invention provides a method for regulating the number of spikes per plant, the number of grains per spike, and / or the yield of a plant. The method includes regulating the expression of a protein-coding gene or the activity and / or content of the protein in the plant to regulate the number of spikes per plant, the number of grains per spike, and / or the yield; the protein is any one of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0005] In the above method, the yield can be the yield per plant or the yield per acre.

[0006] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0007] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein of the present invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the protein isolated in the present invention, as long as they encode and possess the aforementioned protein function, are derived from and equivalent to the nucleotide sequence of the present invention.

[0008] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0009] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, a search can be performed to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0010] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0011] In the above method, regulating the expression of the protein-coding gene or regulating the activity and / or content of the protein in the plant includes the following steps: introducing a substance into the target rice that regulates the expression level of the protein-coding gene whose amino acid sequence is SEQ ID NO:1.

[0012] In the above method, when the regulation is to upregulate, enhance, or increase the expression of the gene encoding the aforementioned protein, or the content or activity of the protein, the substance is any one of the following: B1) Nucleic acid molecules that encode the aforementioned proteins; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2).

[0013] In the above method, when the regulation is to downregulate, inhibit, or reduce the expression of the gene encoding the aforementioned protein, or the content or activity of the protein, the substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4).

[0014] In the above method, B1) the RNA molecule targets the mRNA transcribed from the gene of the aforementioned protein.

[0015] In the above-mentioned substances, B3) refers to an expression cassette containing nucleic acid molecules, which is a DNA capable of expressing the proteins described above in a host cell. The expression cassette may also include single-stranded or double-stranded nucleic acid molecules containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. The regulatory sequences, under compatible conditions, can guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory sequences are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby opening or closing gene expression. Other examples of regulatory sequences are those that can amplify genes.

[0016] This invention provides a method for preparing plants with increased panicle number per plant, number of grains per panicle, and / or yield. The method includes introducing a substance that regulates the expression of a protein-coding gene or regulates the activity and / or content of the protein into a target rice variety to obtain a plant with increased panicle number per plant, number of grains per panicle, and / or yield. The plant with increased panicle number per plant, number of grains per panicle, and / or yield has a higher number of panicles per plant, number of grains per panicle, and / or yield than the target rice variety.

[0017] In the above method, the yield can be the yield per plant or the yield per acre.

[0018] In the above method, regulating the expression of the protein-coding gene or regulating the activity and / or content of the protein in the plant includes the following steps: introducing a substance into the target rice that regulates the expression level of the protein-coding gene whose amino acid sequence is SEQ ID NO:1.

[0019] The present invention also provides the use of a protein or a substance that regulates the expression of the gene encoding said protein or a substance that regulates the activity and / or content of a protein in any of the following: M1) regulates the number of spikelets per plant; M2) to prepare products that regulate the number of spikelets in plants; M3) regulates the number of grains per spike in plants; M4) is used to prepare products that regulate the number of grains per spike in plants; M5) regulates plant yield; M6) to prepare products that regulate plant yield; The protein is any one of the following: A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

[0020] The present invention also provides the use of biomaterials related to the aforementioned proteins in any of the following: M1) regulates the number of spikelets per plant; M2) to prepare products that regulate the number of spikelets in plants; M3) regulates the number of grains per spike in plants; M4) is used to prepare products that regulate the number of grains per spike in plants; M5) regulates plant yield; M6) is used to prepare products that regulate plant yield.

[0021] The plant mentioned above is any one of the following: G1) Dicotyledons; G2) Plants of the order Poales; G3) Gramineae plants; G4) Triticum; G5) Wheat.

[0022] In the above applications, the regulation may be to upregulate, enhance, or increase the expression of the gene encoding the aforementioned protein, or the protein content or activity; alternatively, the regulation may be to downregulate, inhibit, or reduce the expression of the gene encoding the aforementioned protein, or the protein content or activity. The upregulation, enhancement, or increase of the expression level of the encoding gene of the aforementioned protein in the target plant, or the regulation of the activity or content of the aforementioned protein, can be achieved through gene overexpression. The downregulation, inhibition, or reduction of the expression level of the encoding gene of the aforementioned protein in the target plant, or the regulation of the activity or content of the aforementioned protein, can be achieved through gene knockout or gene silencing. Gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout inactivates a specific target gene by altering its DNA sequence. Gene silencing refers to the phenomenon of preventing or reducing gene expression without damaging the original DNA. Gene silencing requires no alteration of the DNA sequence to prevent or reduce gene expression. Gene silencing can occur at two levels: one is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effects; the other is post-transcriptional gene silencing, which is gene inactivation at the post-transcriptional level through specific inhibition of target RNA, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi), and microRNA (miRNA)-mediated translational repression.

[0023] The present invention also provides a method for increasing the number of spikes per plant and / or the yield of wheat under low nitrogen conditions, the method comprising introducing a substance into the target wheat that downregulates or inhibits or reduces the expression of a protein-coding gene or regulates the activity and / or content of the protein, thereby increasing the number of spikes per plant and / or the yield of the target wheat.

[0024] The low nitrogen conditions refer to planting plants in soil with an available nitrogen concentration of 74.6 mg / kg and applying fertilizer at a rate of 90 kg N / ha during the growing season.

[0025] In the above method, the yield can be the yield per plant or the yield per acre.

[0026] The proteins and biological materials mentioned above are also within the scope of protection of this invention.

[0027] This invention demonstrates through experiments that knocking down TaNIP1 can significantly increase grain yield, especially the number of effective spikes and yield of wheat under low nitrogen conditions. Attached Figure Description

[0028] Figure 1 for TaNIP1 Schematic diagram of the overexpression vector.

[0029] Figure 2 for TaNIP1 Schematic diagram of the reduced expression vector.

[0030] Figure 3 for TaNIP1 Expression levels in overexpression and underexpression systems.

[0031] Figure 4 for TaNIP1 Statistical chart of field yield traits of transgenic lines.

[0032] Figure 5 for TaNIP1 Reduced expression system yield statistics chart. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0035] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0036] KN199 (also known as KN199): It is described in the non-patent literature “Zhao Hui, Zhang Wei, Wang Jing, Ji Jun, Wang Zhiguo, Li Junming (2011) Analysis of high-yield and stable-yield characteristics of the new winter wheat variety “KN199”, Chinese Journal of Agricultural Ecology”. It can be obtained by the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes.

[0037] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used, and P < 0.05 (*) indicates that there is a significant difference.

[0038] Example 1: Method for constructing TaNIP1 transgenic material The inventors previously discovered that TaNIP1 interacts with genes that regulate photosynthetic efficiency, and that reducing the expression of this gene in wheat can increase photosynthetic rate, effective spike number, and grain yield. This indicates that it plays an important regulatory role in wheat yield composition.

[0039] I. Construction of TaNIP1 Overexpression Lines and Pulse Overexpression Lines 1. Carrier Construction Construction of the recombinant expression vector pUbi::TaNIP1 for TaNIP1 overexpression: Using wild-type wheat Kern 199 cDNA as a template, the vector was amplified using the upstream primer TaNIP1-OE-F with a BamHI restriction site and protective bases, and the downstream primer TaNIP1-OE-R with a KpnI restriction site and protective bases. TaNIP1 The full-length CDS was ligated into the pEasy-Blunt vector (purchased from Beijing TransGen Biotech Co., Ltd., catalog number CB101-01). After sequencing verification, it was double-digested with BamHI and KpnI and ligated between the Ubi promoter and Nos terminator in the plant expression vector pUbi-163. Sequencing and enzyme digestion verification yielded the recombinant expression vector pUbi::TaNIP1. The recombinant expression vector pUbi::TaNIP1 is a recombinant expression vector in which the DNA molecule with the nucleotide sequence SEQ ID NO:2 replaces the bases between the BamHI and KpnI restriction sites in the pUbi-163 vector while keeping other vectors unchanged. Figure 1 ).

[0040] 2. Preparation of TaNIP1 overexpressing plants The common wheat cultivar Kenong 199 was selected as the transformation recipient. The recombinant expression vector pUbi::TaNIP1 and the bar gene vector prepared in step 1 were transformed into the recipient common wheat cultivar Kenong 199 using a gene gun-mediated transformation method with minimal expression frame co-transformation, obtaining the T0 generation transgenic master line. The specific procedures are as follows: the pUbi::TaNIP1 and pUbi-Bar vectors were digested and the contents of the gene vectors were recovered. Ubi - NIP1-Nos The pUbi-Bar vector fragment was mixed with gold powder and transferred into wild-type wheat KN199 using a gene gun to obtain the T0 generation transgenic line.

[0041] 3. Detection of TaNIP1 overexpressing plants After obtaining the T0 generation transgenic master line, positive lines were identified by PCR using the following primers. The identification method was as follows: PCR program: 94℃, 3 min; 94℃ 30 s, 58℃ 30 s, 72℃ 40 s, 40 cycles; 72℃ 5 min. The PCR reaction system was as follows: DNA template (approximately 20 ng / μl) 2 μl; forward primer (10 μM) 0.5 μl; reverse primer (10 μM) 0.5 μl; 10× PCR amplification buffer 2 μl; dNTP Mixture 1 μl; Taq DNA polymerase 0.2 μl; ddH2O to a final volume of 20 μl. Judgment criteria: PCR products were detected by agarose gel electrophoresis and sequenced. Correct sequencing indicated a transgenic positive plant; no band at the specified location indicated a transgenic negative plant.

[0042] OE-TaNIP1-F: 5'-TTAGCCCTGCCTTTCATACGCT-3'; OE-TaNIP1-R: 5'-AGAAATTCTCCCTGCCACCAA-3'.

[0043] After positive results were obtained from both T1 and T2 generations, the progeny of the positive lines were hydroponically cultured. RNA was extracted from the aboveground parts of the hydroponic seedlings using the Trizol method. RT-PCR was performed using Thermo Fisher's K1622 reverse transcription kit to reverse transcribe the RNA into cDNA. The expression level was verified by qPCR, with TaActin as the internal control. The qPCR primers were: RT-NIP1-F: 5'-CGCTCTGCTCCAAGTCAGTATGT-3'; RT-NIP1-R: 5'-TCAAATCTGCCGGGTGTGTAC-3'; RT-TaActin-F: 5'-ACCTTCAGTTGCCCAGCAAT-3'; RT-TaActin-R: 5'-CAGAGTCGAGCACAATACCAGTTG-3'.

[0044] The identification results of TaNIP1 overexpression are shown in Figure 3 The expression level of TaNIP1 gene in the overexpression lines OE33 and OE66 was significantly higher than that in wild-type wheat KN199 ("***" indicates P<0.001).

[0045] II. Construction of TaNIP1 Reduced Expression Line 1. Carrier Construction The target interference fragment was amplified using the upstream primer TaNIP1-RNAi-F1 with a BamHI restriction site and the downstream primer TaNIP1-RNAi-R1 with an EcoRI restriction site, and the target fragment TaNIP1-F (nucleotide sequence SEQ ID NO:3) was recovered. The target interference fragment was also amplified using the upstream primer TaNIP1-RNAi-F2 with a KpnI restriction site and the downstream primer TaNIP1-RNAi-R2 with a HindIII restriction site, and the target fragment TaNIP1-R (nucleotide sequence the reverse complementary sequence of SEQ ID NO:3) was recovered. The vector intron fragment (S3) was recovered by digestion with EcoRI and HindIII, and the vector backbone (V1) was recovered by digestion with BamHI and KpnI. Then, V1 and S3 were ligated with TaNIP1-F and TaNIP1-R to construct the wheat pUbi::TaNIP1-RNAi vector. Figure 2 Sequencing and enzyme digestion verification yielded the recombinant expression vector pUbi-RNAi-TaNIP1. The recombinant vector pUbi-RNAi-TaNIP1 is obtained by replacing the bases between the BamHI and KpnI restriction sites of pUbi-RNAi with the nucleotide sequence of TaNIP1-F-S3-TaNIP1-R, while keeping other vector components unchanged. TaNIP1-F-S3-TaNIP1-R is formed by linking the inverse complementary sequences of bases 112-201 of SEQ ID NO:2, intron fragment S3 (SEQ ID NO:4), and bases 112-201 of SEQ ID NO:2 in a 5'-3' direction. pUbi-RNAi vector: described in non-patent literature "Wenjing Li, Xue He, Yi Chen, Yanfu Jing, Chuncai Shen, Junbo Yang, Wan Teng, Xueqiang Zhao, Weijuan Hu, Mengyun Hu, Hui Li, Anthony J. Miller and Yiping Tong. A wheat transcription factor positively sets seedvigor by regulating the grain nitrate signal. New Phytologist (2020) 225:1667–1680", is available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biomaterial is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0046] TaNIP1-RNAi-F1:5'-ggatccGTGTGGCACTGAACAAGAGGT-3'; TaNIP1-RNAi-R1:5'-gaattcACAAGTACTCGAAGCAGATTG-3'; TaNIP1-RNAi-F2:5'-ggtaccGTGTGGCACTGAACAAGAGGT-3'; TaNIP1-RNAi-R2:5'-aagcttACAAGTACTCGAAGCAGATTG-3'.

[0047] The lowercase letters are the enzyme cleavage site recognition sequences.

[0048] 2. Preparation of TaNIP1 Silent Plants The common wheat cultivar KN199 was selected as the transformation recipient. Using gene gun-mediated transformation, the recombinant expression vector pUbi-RNAi-TaNIP1 and the Bar gene vector containing the target gene were transformed into the recipient common wheat cultivar KN199 using the minimum expression frame method. The specific procedure was as follows: The pUbi-RNAi-TaNIP1 and pUbi-Bar vectors were digested with enzymes, and the vector fragments containing Ubi+TaNIP1-F+TaNIP1-R+Nos and pUbi-Bar were recovered and mixed with gold powder. These fragments were then transformed into wild-type wheat KN199 using a gene gun to obtain the T0 generation transgenic lines.

[0049] 3. Detection of TaNIP1-silent plants After obtaining the T0 generation transgenic master line, PCR was performed using the following primers to identify positive lines.

[0050] intron-30F: 5'-AAGCACGCCTACTAGTTCAAG-3'; NOS-R: 5'-ACCCATCTCATAAATAACGTCATGC-3'; UBI-F: 5'-TCGGAGTAGAATACTGTTTCAAACTACC-3'; intron-90R: 5'-AATGGTGATCATCCAGCTCTC-3'.

[0051] After positive identification in both T1 and T2 generations, the progeny of the positive lines were hydroponically cultured. Plants named RNAi-NIP1-4 and RNAi-NIP1-18 from these progeny lines were selected for further analysis. RNA was extracted from the aboveground parts of the hydroponic seedlings using the Trizol method. RT-PCR was performed using a Thermo Fisher K1622 reverse transcription kit to convert the RNA into cDNA. qPCR was used to verify the expression level, with [internal control information missing]. TaActin The qPCR primers are: RT-NIP1-F: 5'-CGCTCTGCTCCAAGTCAGTATGT-3'; RT-NIP1-R: 5'-TCAAATCTGCCGGGTGTGTAC-3'; RT-TaActin-F: 5'-ACCTTCAGTTGCCCAGCAAT-3'; RT-TaActin-R: 5'-CAGAGTCGAGCACAATACCAGTTG-3'.

[0052] Quantitative PCR system: DNA template (approximately 20 ng / μl) 2 μl; upstream primer (10 μM) 0.4 μl; downstream primer (10 μM) 0.4 μl; 2× mixture (light Cycler SYBR Green I master, Roche) 10 μl; ddH2O to bring the total to 20 μl.

[0053] The quantitative fluorescence reaction program was as follows: 94℃ for 5 min; 94℃ for 20 s, 60℃ for 20 s, 72℃ for 15 s, for 45 cycles.

[0054] The expression level of TaNIP1 in TaNIP1-RNAi transgenic plants was identified as follows: Figure 3 The expression level of the TaNIP1 gene in RNAi4 and RNAi18 plants was significantly lower than that in the wheat cultivar Kenong 199. Figure 3 (China CK).

[0055] Following the aforementioned method, the common wheat cultivar Kenong 199 was selected as the transformation recipient. The recombinant expression vector pUbi-RNAi containing the target gene and the Bar gene vector were transformed into the recipient common wheat cultivar Kenong 199 using the gene gun-mediated transformation method with small expression frame fragment transformation. The positive lines of the resulting plants were identified and named as empty vector control plants.

[0056] III. Phenotypic Validation Wild-type wheat Kenong 199, overexpression lines (OE33 and OE66), and RNAi silencing lines (RNAi4 and RNAi18) were planted and their grains were collected as follows: The progeny of T2 generation plants that were identified as positive were planted in the Dishang Experimental Station of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural Sciences for field experiments. The rows were 2 meters wide, with 40 grains planted in each row, and 8 rows were planted for biological replication. The plants were harvested manually and threshed individually.

[0057] The experiment was repeated three times, with each repetition as follows: High-nitrogen (HN) group: ①OE33 group: Overexpressing plants of OE33 were planted at the Dishang Experimental Station of the Grain and Oil Crops Research Institute of the Hebei Academy of Agricultural and Forestry Sciences, Gaocheng District, Shijiazhuang City, Hebei Province. Rows were 2 meters wide, with 40 seeds per row, and 8 rows were used for biological replicates. The experimental station is located in Dishang Village, Qiutou Town, Gaocheng City, 23 kilometers east of Shijiazhuang City, with geographical coordinates of 38°18′44″N, 114°58′47″E. The climate is warm temperate semi-humid continental monsoon, with an average annual temperature of 12.5℃, an average annual rainfall of 494 mm, and a frost-free period of 190 days. Two irrigations were provided after emergence during the growing season, and conventional field management was implemented. There were no serious pests, diseases, or lodging during the growing period. The soil available nitrogen concentration was 74.6 mg / kg, and fertilizer was applied at a rate of 225 kg N / ha during planting. The number of ears per plant, the number of grains per ear, the yield per plant, and the plot yield (g / m²) were recorded. 2 ). Statistical analysis was conducted on the number of ears per plant, the number of grains per ear, the yield per plant, and the yield per plot (g / m²). 2 ).

[0058] ②OE66 group: The difference between this group and the OE33 group is that the OE33 plants in the OE66 group are replaced with OE36 plants. The rest of the operation is the same as the OE33 group.

[0059] ③RNAi4 group: The difference between this group and the OE33 group is that the RNAi4 plants are used instead of the OE33 plants in the OE33 group. The rest of the operations are the same as those in the OE33 group.

[0060] ④RNAi18 group: The difference between this group and the OE33 group is that the OE33 group is replaced by RNAi18 plants. The rest of the operation is the same as the OE33 group.

[0061] ⑤ KN199 group: The difference between this group and the OE33 group is that the OE33 plants in the OE33 group are replaced by KN199 plants. The rest of the operation is the same as the OE33 group.

[0062] Low nitrogen (LN) group: ①OE33 group: The difference between this group and the high-nitrogen group OE33 group is that the fertilization of 90 kg N / ha is replaced by 225 kg N / ha of the high-nitrogen group RNAi-NIP1-4 (R4). The rest of the operation is the same as the high-nitrogen group OE33 group.

[0063] ②OE66 group: The difference between this group and the OE33 group is that the OE33 plants in the OE66 group are replaced with OE36 plants. The rest of the operation is the same as the OE33 group.

[0064] ③RNAi4 group: The difference between this group and the OE33 group is that the OE33 plants in the OE33 group are replaced by RNAi4 plants. The other operations are the same as those in the OE33 group.

[0065] ④RNAi18 group: The difference between this group and the OE33 group is that the OE33 plants in the OE33 group are replaced by RNAi18 plants. The other operations are the same as those in the OE33 group.

[0066] ⑤ KN199 group: The difference between this group and the OE33 group is that the OE33 plants in the OE33 group are replaced by KN199 plants. The rest of the operation is the same as the OE33 group.

[0067] The results showed that under high nitrogen conditions, the number of panicles per plant was reduced by 13.4% in the TaNIP1 overexpression lines OE33 and OE66 compared to the control group. p <0.01) and 25.5% ( p <0.01), the number of grains per ear decreased by 27.7% ( p <0.01) and 18.8% ( p <0.05%, and the yield per plant decreased by 53.2% ( p <0.001) and 48.8% ( p <0.001), compared with the control group, the reduced expression lines RNAi4 and RANi18 increased the number of ears per plant by 12.0% ( p <0.01) and 7.5% ( p >0.05), there was no significant difference in the number of grains per ear and the yield per plant. Figure 4 (A, B, and C) Small-plot experiments showed that reduced expression of TaNIP1 tended to increase wheat yield. Figure 5 (A), knocking out TaNIP1-3A increases production by 8% ( p <0.05), knocking out TaNIP1-3B slightly increased yield per plant but did not reach a significant level, while knocking out TaNIP1-3D increased yield by 6.6% ( p <0.01)( Figure 5 (A). Under low nitrogen conditions, the number of spikes per plant decreased by 15.0% in the TaNIP1 overexpression lines OE33 and OE66 compared to the control group.p <0.01) and 27.1% ( p <0.001), the number of grains per ear decreased by 21.2% ( p <0.001) and 23.4% ( p <0.001), the yield per plant decreased by 51.8% ( p <0.001) and 54.9% ( p <0.001), compared with the control group, the reduced expression lines RNAi4 and RANi18 increased the number of ears per plant by 14.7% ( p <0.01) and 12.4% ( p <0.05%, with no significant difference in the number of grains per ear, and the yield per plant increased by 13.0% ( p <0.05) and 12.1% ( p <0.05)( Figure 4 (D, E, and F), small-scale trials showed reduced expression TaNIP1 Increased the yield of the small cell by approximately 8.0%. p <0.05%, knocking out TaNIP1-3A increased production by 15.7% ( p <0.01, knocking out TaNIP1-3B increased cell yield by 14.7% ( p <0.05%, knocking out TaNIP1-3D increased cell yield by 23.6% ( p <0.01)( Figure 5 ).

[0068] The sequences involved in the above embodiments are as follows: The amino acid sequence of TaNIP1 (SEQ ID NO:1) MGAVQLESVAAQHAQAKLNVESLPQGPSLFDGNDTARINGSESDEYEKFDKGLMQYGCAHYRRRCRIRAPCCNEIFDCRHCHNESKNSIKIDTPTRHELPRHELQQVICTLCGTEQEVRQVCINCGVCMGKYFCEVCKLFDDDVSKQQYH CHACGICRIGGRENFFHCSKCGCCYSKVLKNSHACVEGAMHHDCPICFEYLFESRNDVSVLPCGHTIHEKCLREMKEHCQFACPLCSKSVCDMSKAWERLDAELATLSNSFDDKMVRILCNDCGAVSVEVQFHLIAHKCHNCKSYNTRQI.

[0069] The CDS sequence of TaNIP1 (SEQ ID NO:2) 5'-ATGGGTGCCGTGCAGCTCGAGTCTGTTGCTGCTCAACATGCACAAGCTAAGCTAAATGTGGAATCACTTCCTCAAGGCCCTTCGTTGTTTGATGGAAATGACACTGCCAGGATCAATGGTTCCGAGTCTGATGAGTATGAAAAATTTGACAAGGGATTAATGCAGTATGGATGTGCACATTACCGAAGGAGATGCCGCATACGAGCACCATGCTGCAATGAGATTTTTGATTGCCGGCACTGCCACAATGAATCAAAGAATTCGATCAAAATTGATACTCCTACGCGACATGAACTTCCACGCCATGAACTGCAGCAGGTCATATGCACATTGTGTGGCACTGAACAAGAGGTACGACAAGTATGCATCAACTGTGGTGTATGCATGGGAAAGTATTTCTGTGAAGTGTGCAAGCTCTTTGATGATGATGTCTCAAAACAGCAGTATCACTGCCATGCGTGTGGAATATGTAGAATTGGTGGCAGGGAGAATTTCTTTCACTGCTCAAAATGCGGATGTTGTTATTCCAAAGTGTTGAAGAACAGTCATGCATGTGTTGAAGGAGCCATGCATCATGACTGTCCAATCTGCTTCGAGTACTTGTTTGAGTCAAGAAACGATGTCTCTGTTTTGCCATGTGGTCATACAATTCATGAAAAATGCTTGAGAGAGATGAAGGAGCATTGCCAATTTGCATGCCCGCTCTGCTCCAAGTCAGTATGTGACATGTCGAAGGCATGGGAAAGACTGGACGCGGAACTAGCGACTCTGTCAAACTCCTTCGATGATAAAATGGTTCGCATATTGTGCAATGATTGTGGGGCAGTATCAGAGGTGCAGTTCCACTTAATTGCACATAAGTGCCATAATTGCAAGTCGTACAACACCCGGCAGATTTGA-3'.

[0070] Target fragment TaNIP1-F (SEQ ID NO:3) 5'-GTGTGGCACTGAACAAGAGGTACGACAAATATGCATCAACTGTGGTGTATGCATGGGAAAGTATTTCTGTGAAGTGTGCAAGCTCTTTGATGATGATGTCTCAAAACAGCAGTATCACTGCCATGCGTGTGGAATATGCAGAATTGGTGGCAGGGAGAATTTCTTCCACTGCTCAAAATGTGGATGTTGTTATTCCAAAGTGTTGAAGAACAGTCATGCATGTGTTGAAGGAGCCATGCATCATGACTGTCCAATCTGCTTCGAGTACTTGT-3'.

[0071] Intron fragment S3 (SEQ ID NO:4) 5'-AAGCTTACGTCCTCCCCTGCGCGGCGCGCAACAAGGGACGACGACGGCACCCAGATACAAAAAAAAATGGTGATCATCCAGCTCTCTCAAGAAAATATCAAGTTCTTCAGAGTTCAGATTACACACACTCTAGCTTGAACTAGTAGGCGTGCTTGATCTTGATCTTACC-3'.

[0072] Genomic sequence of TaNIP1

[0073] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for regulating the number of spikes per plant, the number of grains per spike, and / or the yield of a plant, characterized in that, The method includes regulating the number of spikes per plant, the number of grains per spike, and / or the yield of a plant by regulating the expression of genes encoding proteins in the plant or by regulating the activity and / or content of the proteins; the protein is any one of the following: A1) The amino acid sequence is that of the protein SEQ ID NO:1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

2. The method according to claim 1, characterized in that, The regulation is to upregulate or enhance or increase the expression of the gene encoding the protein in claim 1, or the content or activity of the protein.

3. The method according to claim 1, characterized in that, The regulation is to downregulate or inhibit or reduce the expression of the gene encoding the protein of claim 1, or the content or activity of the protein.

4. The method according to claim 1, characterized in that, The substance is any one of the following: B1) RNA molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1, or RNA molecules that inhibit, reduce, or downregulate the activity or content of the protein. B2) The gene encoding the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5) Recombinant microorganisms containing the gene described in B2), or recombinant microorganisms containing the expression cassette described in B3), or recombinant microorganisms containing the recombinant vector described in B4); B6) A transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7) Transgenic plant tissue containing the gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4); B8) Transgenic plant organs containing the gene described in B2), or transgenic plant organs containing the expression cassette described in B3), or transgenic plant organs containing the recombinant vector described in B4); B9) A nucleic acid molecule encoding the protein described in claim 1; B10), an expression cassette containing the nucleic acid molecule described in B9); B11), a recombinant vector containing the nucleic acid molecule described in B9), or a recombinant vector containing the expression cassette described in B10; B12) recombinant microorganisms containing the nucleic acid molecules described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B3); B13), a transgenic plant cell line containing the nucleic acid molecule described in B9), or a transgenic plant cell line containing the expression cassette described in B10; B14) transgenic plant tissue containing the nucleic acid molecules described in B9), or transgenic plant tissue containing the expression cassette described in B10; B15), transgenic plant organs containing the nucleic acid molecules described in B9), or transgenic plant organs containing the expression cassette described in B10).

5. The method according to claim 4, characterized in that, B1) The RNA molecule targets the mRNA transcribed from the gene of the protein described in claim 1.

6. A method for preparing plants with increased number of spikes per plant, number of grains per spike, and / or increased yield, characterized in that, The method includes introducing substances that regulate the expression of protein-coding genes or regulate the activity and / or content of said proteins into target rice to obtain plants with increased number of panicles per plant, number of grains per panicle, and / or yield, wherein the number of panicles per plant, number of grains per panicle, and / or yield of the plants with increased number of panicles per plant, number of grains per panicle, and / or yield are higher than those of the target rice.

7. Application, characterized in that, The use of a protein, or a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity and / or content of the protein, in any of the following: M1) regulates the number of spikelets per plant; M2) to prepare products that regulate the number of spikelets in plants; M3) regulates the number of grains per spike in plants; M4) is used to prepare products that regulate the number of grains per spike in plants; M5) regulates plant yield; M6) to prepare products that regulate plant yield; The protein is any one of the following: A1) The amino acid sequence of this protein is that of SEQ ID No. 1; A2) A protein that has more than 80% identity with and has the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in A1). A3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of any of the amino acids shown in A1) or A2).

8. Application, characterized in that, Use of the biomaterials associated with the protein of claim 7 in any of the following: M1) regulates the number of spikelets per plant; M2) to prepare products that regulate the number of spikelets in plants; M3) regulates the number of grains per spike in plants; M4) is used to prepare products that regulate the number of grains per spike in plants; M5) regulates plant yield; M6) is used to prepare products that regulate plant yield.

9. The method according to any one of claims 1-6 or the application according to claim 7 or 8, characterized in that, The plant is any one of the following: G1) Dicotyledons; G2) Plants of the order Poales; G3) Gramineae plants; G4) Triticum; G5) Wheat.

10. A method for increasing the number of spikes per plant and / or yield of wheat under low nitrogen conditions, the method comprising introducing a substance into the target wheat that downregulates or inhibits or reduces the expression of the gene encoding the protein of claim 1 or regulates the activity and / or content of the protein, thereby increasing the number of spikes per plant and / or yield of the target wheat.

Images