Rice osTKL2 protein and coding gene in regulation of plant ear grain number and nitrogen utilization efficiency

Abstract

The application discloses a rice OsTKL2 protein and an application of a coding gene in regulation of plant ear grain number and nitrogen utilization efficiency. The gene OsTKL2 (Transmembrane Kinase Like 2) can increase the ear grain number of rice after overexpression or function enhancement, and can increase the ear grain number of rice under different nitrogen levels, especially under low nitrogen conditions, so that high yield of rice and high efficient utilization of nitrogen are realized. The OsTKL2 gene has important application value for high yield and high efficient nitrogen breeding of crops.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and plant breeding technology, and more specifically, to the application of rice OsTKL2 protein and its encoding gene in regulating the number of grains per panicle and nitrogen fertilizer use efficiency. Background Technology

[0002] The number of grains per panicle in rice is a primary breeding trait for increasing yield in breeding practices. Its regulatory scope is relatively large, and it is considered key to further improving rice yield. The number of grains per panicle is a quantitative trait controlled by multiple genes and influenced by various external environmental factors (e.g., soil nitrogen fertilizer levels). Currently, through methods such as GWAS (Genome-wide association studies), QTL (Quantitative Trait Locus) analysis, and map-based cloning, several genes regulating the number of grains per panicle in rice have been identified, and it has been clarified that these genes participate in regulating meristem size and stage transitions, as well as regulating branch development and panicle type.

[0003] Nitrogen fertilizer is one of the essential nutrients for plant growth and development, and a key element determining crop yield traits (such as tiller number, grain number per panicle, and plant height). However, excessive application of nitrogen fertilizer not only increases production costs and leads to a decline in rice quality, but also causes serious environmental pollution problems. Therefore, improving the nitrogen fertilizer use efficiency of crops and reducing nitrogen fertilizer input are effective ways to achieve green and efficient agricultural production. In recent years, several key genes regulating nitrogen fertilizer use efficiency in rice have been identified, laying the foundation for breeding high-yield and nitrogen-efficient rice varieties.

[0004] DEP1 (Dense and erect panicles 1) encodes the γ subunit of a plant-specific atypical G protein. dep1-1 is a 625 bp deletion and 12 bp insertion in the fifth exon of the DEP1 gene, resulting in a protein encoding only 195 amino acids at the N-terminus. Studies have shown that, on the one hand, dep1-1 enhances apical meristem activity, promotes cell division, increases the number of branches and grains per panicle, thereby increasing yield; on the other hand, dep1-1 reduces the sensitivity of rice growth to nitrogen fertilizer, enhancing nitrogen absorption and assimilation, and increasing rice yield under appropriate nitrogen-reducing conditions. Therefore, dep1-1 is a key gene for high yield and efficient nitrogen fertilizer utilization. Analyzing the genetic regulatory network of dep1-1 in regulating rice panicle grain number and nitrogen fertilizer absorption and utilization has important guiding significance for breeding high-yielding and nitrogen-efficient rice varieties. Summary of the Invention

[0005] The purpose of this invention is to provide the application of rice OsTKL2 protein and its encoding gene in regulating the number of grains per panicle and nitrogen fertilizer use efficiency.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides any of the following applications of rice OsTKL2 protein, its encoding gene, and related biological materials: 1) Used to regulate the number of grains per spike in plants; 2) Used to regulate the nitrogen fertilizer utilization efficiency of plants.

[0007] The OsTKL2 (Transmembrane Kinase Like 2) gene is identified as LOC_Os11g26130 in the Rice Gene Database (https: / / rice.uga.edu / index.shtml).

[0008] Furthermore, the aforementioned regulation is positive regulation.

[0009] In this invention, the plant is a monocotyledonous plant or a dicotyledonous plant, preferably a grass, and more preferably rice (such as japonica rice).

[0010] Furthermore, the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0011] Secondly, the present invention provides a method for increasing the number of grains per panicle while reducing the plant's sensitivity to nitrogen fertilizer, the method comprising: introducing the rice OsTKL2 gene into the plant via plasmid or integrating it into the plant chromosome via genetic engineering.

[0012] Thirdly, the present invention provides a method for increasing the number of grains per panicle in rice under low nitrogen conditions, the method comprising: enhancing the expression of the OsTKL2 gene in rice.

[0013] Furthermore, the enhancement can be selected from the following 1) to 6), or an optional combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on the rice chromosome; 3) By altering the promoter sequences of the aforementioned genes on the rice chromosome; 4) By operatively linking a strong promoter to the gene; 5) By introducing enhancers; 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

[0014] Expression vectors carrying the target gene can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2). nd Edition).

[0015] Fourthly, the present invention provides the application of transgenic plants obtained according to the method in plant breeding.

[0016] Furthermore, breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0017] The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0018] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention is the first to discover that the rice OsTKL2 gene is associated with the number of grains per panicle and nitrogen fertilizer use efficiency. Enhancing the expression of OsTKL2 in plants can significantly improve the number of grains per panicle and nitrogen fertilizer use efficiency. The OsTKL2 gene and its applications provided by this invention can be used for the improvement of plant germplasm resources and the breeding of new rice varieties with high yield and high nitrogen fertilizer use efficiency, and have significant application value. Attached Figure Description

[0019] Figure 1 This invention provides an analysis of the ear type of the sod41 mutant provided in Example 1 of the present invention; wherein (A) is the ear type of sod41, with a scale bar of 5 cm. (BD) are statistical analyses of ear traits of sod41, namely (B) number of primary branches, (C) number of secondary branches, and (D) number of grains per ear. Values ​​are expressed as mean ± standard error (n = 8). Different letters in the figure represent significant differences (P < 0.05). The Duncan test was used to analyze significant differences.

[0020] Figure 2This is a correlation diagram of map-based cloning and genetic complementation of the SOD41 gene provided in Example 2 of the present invention; wherein (AD) represents the map-based cloning of the candidate gene SOD41, and (EG) represents the statistical analysis of ear traits in the genetically complemented lines, namely (E) number of primary branches, (F) number of secondary branches, and (G) number of grains per ear. The values ​​in (EG) are mean ± standard error (n = 8), and different letters indicate significant differences (P < 0.05). The significance analysis was performed using the Duncan test.

[0021] Figure 3 Phenotypic analysis of the OsTKL2 gene knockout and overexpression lines under the WYJ7-DEP1 background provided in Example 3 of this invention; where (A) ear type, scale bar is 5 cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear. (BD) The values ​​in the figure are mean ± standard error (n = 8), different letters indicate significant differences (P<0.05), and the significance analysis uses Duncan's test.

[0022] Figure 4 This invention provides Example 4 of the study on the response of the number of grains per ear to nitrogen in the ostkl2 mutant planted under different nitrogen fertilizer conditions (low nitrogen 60 kg / ha and high nitrogen 210 kg / ha) in the WYJ7-DEP1 background. (A) Ear type, scale bar is 5 cm. (B) Number of primary branches; (C) Number of secondary branches; (D) Number of grains per ear; (E) Nitrogen-mediated growth response (NGR) of grains per ear. (BE) The values ​​in the figures are mean ± standard error (n = 8), different letters indicate significant differences (P < 0.05), and the significance analysis used the Duncan test.

[0023] Figure 5 For the transgenic lines WYJ7-DEP1 and OsTKL2 overexpression planted under different nitrogen fertilizer conditions (low nitrogen 60 kg / ha and high nitrogen 210 kg / ha) provided in Example 5 of this invention, the nitrogen response capability of the number of grains per ear was statistically analyzed; where (A) ear type, scale bar is 5 cm; (B) number of primary branches; (C) number of secondary branches; (D) number of grains per ear; (E) nitrogen response capability of the number of grains per ear (NGR). (BE) The values ​​in the figure are mean ± standard error (n = 8), different letters indicate significant differences (P<0.05), and the significance analysis was performed using Duncan's test. Detailed Implementation

[0024] This invention provides a gene that regulates the number of grains per panicle and nitrogen fertilizer use efficiency in rice, and its application.

[0025] The inventors used ion beam radiation mutagenesis and genetic screening on Wuyunjing 7 (WYJ7) rice to obtain a mutant, sod41, with a significantly reduced number of grains per panicle. Map-based cloning and genetic complementation experiments demonstrated that this phenotype of significantly reduced grain number per panicle was due to a mutation in the OsTKL2 (Transmembrane Kinase Like 2) gene. OsTKL2 is known to encode a leucine-rich repeat receptor-like protein kinase, and existing research indicates that this gene regulates chloroplast and leaf senescence. This invention is the first to demonstrate that OsTKL2 regulates the number of grains per panicle in rice. Furthermore, this invention demonstrates that overexpression of the OsTKL2 gene in rice can increase the number of grains per panicle under low-nitrogen conditions, achieving both fertilizer reduction and efficiency improvement. This research on the OsTKL2 gene will provide a theoretical basis and a new gene resource with breeding value for high-yield and nitrogen-efficient molecular design breeding of crops.

[0026] In a first aspect, the present invention provides the application of OsTKL2 protein, or its encoding gene, or biological materials containing its encoding gene in regulating the number of grains per spike and nitrogen fertilizer use efficiency in plants.

[0027] The present invention further provides the application of OsTKL2 protein, or its encoding gene, or biological material containing its encoding gene in the cultivation of plants with high ear grain number and high nitrogen fertilizer utilization efficiency.

[0028] The present invention further provides the application of OsTKL2 protein, or its encoding gene, or biological materials containing its encoding gene in the improvement of plant germplasm resources.

[0029] Furthermore, the OsTKL2 protein comprises any one of the following amino acid sequences: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) An amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO:1.

[0030] Furthermore, the gene encoding the OsTKL2 protein includes any one of the following nucleotide sequences: (1) The nucleotide sequence shown in SEQ ID NO:2; (2) A nucleotide sequence that encodes a protein with the same function, obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO:2; (3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO:2 under strict conditions.

[0031] Furthermore, the plant is a monocotyledonous or dicotyledonous plant, preferably rice.

[0032] Furthermore, the biological material is an expression cassette, a vector, or a transgenic cell.

[0033] The transgenic cells described in this invention can be animal cells, plant cells, or microbial cells, preferably plant cells.

[0034] The transgenic cells described in this invention do not include transgenic cells that have the ability to develop independently into a complete individual, that is, they do not include plant varieties and animal varieties.

[0035] In a second aspect, the present invention provides a method for cultivating transgenic plants, comprising: Regulate the expression level of OsTKL2 protein in the plant; The amino acid sequence includes any one of the following: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) An amino acid sequence of a protein with the same function obtained by replacing, inserting or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO:1.

[0036] Furthermore, the expression level of OsTKL2 protein in the plant can be regulated using any of the following methods: Genetically modified organisms (GMOs), hybridization, backcrossing, self-pollination, or asexual reproduction.

[0037] The preferred methods for transgenic technology include: Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0039] Example 1: The sod41 mutant has a reduced number of grains per ear. 1. Wuyunjing 7 (WYJ7) carries the superior dep1-1 allele; our laboratory constructed a high-generation near-isogenic line WYJ7-DEP1 using WYJ7-dep1-1 through continuous backcrossing. This invention utilizes WYJ7-dep1-1 as parental material for ion beam radiation mutagenesis. Several mutants exhibiting altered grain number were screened from the mutagenized population and named sod (suppressor of dep1-1 grain number). Further screening revealed mutants that inhibited the increase in dep1-1 grain number. Among them, the sod41 mutant showed a significantly reduced phenotype for both primary and secondary branches compared to WYJ7, with a grain number reduction of approximately 33%. Figure 1 (AD). This indicates that the sod41 gene can reduce the number of grains per ear.

[0040] 2. In this invention, WYJ7-DEP1, WYJ7-dep1-1, and sod41 were sown in Hefei City, Anhui Province. After the plants matured, traits such as the number of primary branches, secondary branches, and grains per panicle were counted. The specific statistical method was as follows: After the rice matured in the field, plant height was measured, and the number of tillers, as well as the number of primary branches, secondary branches, and grains per panicle on the main tillers, were counted.

[0041] Example 2: Genetic complementation verification that OsTKL2 is the gene that reverts to the number of grains per ear in dep1-1. 1. Genetic analysis showed that sod41 is a recessive mutant. A mapping population was constructed by genetically crossing it with the 9311 parent. Coarse mapping of 200 exchangeable individuals in the BC1F2 population and fine mapping of 1260 exchangeable individuals in the BC2F2 population located the candidate gene within an 83 kb region. Further sequencing analysis revealed a 5 bp deletion in the first exon of OsTKL2 (Transmembrane Kinase Like 2), the gene encoding a leucine-rich repeat receptor protein kinase, causing a frameshift mutation that ultimately leads to premature termination of protein translation. Therefore, OsTKL2 was preliminarily identified as a candidate gene. Figure 2 (AD).

[0042] Table 1 Primer sequences used for SOD41 gene localization and cloning.

[0043] The molecular markers used for SOD41 gene localization and cloning mentioned above are PCR-based markers, including SSR markers (the primers and their sequences used for gene localization and cloning are shown in Table 1). The SSR markers are all derived from the microsatellite marker linkage map published by McCouch et al. (2001, 2002).

[0044] The PCR procedure was performed with slight modifications to the method of Panaud et al. (1996), as follows: Each 20 μl amplification reaction tube contained: 0.15 μM SSR primers, 200 μM dNTPs, 1×PCR reaction buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 0.01% gelatin), 50-100 ng template DNA, and 1 U Taq enzyme. The reaction program was: denaturation at 94 °C for 5 minutes; 94 °C for 1 minute, 55 °C for 1 minute, 72 °C for 1 minute, for a total of 35 cycles; extension at 72 °C for 5 minutes. The amplified PCR products were electrophoresed on a 6% polyacrylamide denaturing gel at 300 V for approximately 3 hours at room temperature. After electrophoresis, silver staining was used to record the band patterns or gel imaging was performed.

[0045] 2. To verify that OsTKL2 is the target gene that reduces the number of grains per ear in WYJ-dep1-1, this invention uses the genomic DNA of WYJ7-dep1-1 as a template to amplify the genomic DNA sequence of the OsTKL2 gene and its promoter sequence 2915 bp upstream of the start codon (sequence SEQ ID NO:3, sequence SEQ ID NO:4), and assembles it into the binary expression vector pCAMBIA2300 (purchased from Abogen (Shanghai) Trading Co., Ltd., catalog number ab275758) to obtain the genetic complementation vector pOsTKL2::gOsTKL2 of the OsTKL2 gene.

[0046] The sod41 mutant was transformed using Agrobacterium-mediated transformation to obtain the transgenic line sod41 pOsTKL2::gOsTKL2. Phenotypic analysis revealed that, compared to sod41, the transgenic line had increased plant height, comparable to that of WYJ7-dep1-1, increased number of secondary branches and grains per ear, comparable to those of WYJ7-dep1-1, and the ear phenotype of the transgenic plants was restored to the same level as WYJ7-dep1-1. Figure 2 ,EG).

[0047] This genetic complementation experiment confirmed that the OsTKL2 gene regulates the number of grains per panicle in rice, and its mutation leads to a reduction in the number of grains per panicle in WYJ7-dep1-1.

[0048] Example 3: OsTKL2 positive regulation of rice panicle grain number The inventors designed primers using the OsTKL2 gene to ligate the target sequence into an sgRNA expression cassette (sequence shown in SEQ ID NO: 5). The ligation was performed simultaneously with enzyme digestion into the insertion vector pYLCRISPR / Cas9Pubi-MH (this vector was kindly provided by the laboratory of Liu Yaoguang at South China Agricultural University; see A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Ma Xingliang et al., DOI:10.1016 / j.molp.2015.04.007). The gene knockout vector was then transformed into WYJ7-DEP1 using Agrobacterium-mediated transformation, resulting in an ostkl2 knockout line in the WYJ7-DEP1 background.

[0049] Furthermore, the inventors extracted total RNA from the leaves of WYJ7-DEP1 rice material using Trizol (reagent purchased from Invitrogen), and synthesized cDNA through reverse transcription (gDNA Removal and cDNA Synthesis SuperMix kit purchased from Tansgen). Using this cDNA as a template, OsTKL2 was amplified by PCR using Do-OsTKL2-F and Do-OsTKL2-R primers. The pActin::OsTKL2-flag vector was constructed by ligating the pCAMBIA2300-flag vector backbone (preserved in the laboratory) into the Gateway BP Clonase II Enzyme Mix and Gateway LR Clonase II Enzyme Mix kits (purchased from Invitrogen). The overexpression vector was transformed into WYJ7-dep1-1 using Agrobacterium-mediated transformation to obtain positive transgenic plants. The expression level of OsTKL2 in different transgenic lines was detected by real-time PCR. A line with an appropriate overexpression fold of OsTKL2 (pActin::OsTKL2-flag 9#) was selected and crossed with WYJ7-DEP1 to obtain homozygous overexpression lines of OsTKL2 under the background of WYJ7-DEP1.

[0050] The results showed that, compared with WYJ7-DEP1, the ostkl2 mutant exhibited a significantly reduced phenotype of primary and secondary branches, ultimately leading to a 18% decrease in grain number per panicle. Further investigation revealed that overexpression of OsTKL2 in the WYJ7-DEP1 background significantly altered the panicle phenotype. OsTKL2 overexpression lines showed increased secondary branches and a 23% increase in grain number per panicle. Figure 3 (AD).

[0051] This experiment shows that OsTKL2 is a positive regulator of grain number in rice panicles, and overexpression of this gene can increase the number of grains per panicle.

[0052] Information on the primer pairs used for OsTKL2 gene knockout and overexpression vector construction is shown in Table 2.

[0053] Table 2 Primer pairs used for OsTKL2 gene knockout and overexpression vector construction

[0054] Example 4: The number of grains per ear in the ostkl2 mutant is relatively insensitive to nitrogen fertilizer. WYJ7-DEP1, WYJ7-dep1-1, and ostkl2 were planted in fields with different nitrogen application rates (60 kg / ha for low nitrogen and 210 kg / ha for high nitrogen). Statistical analysis revealed that WYJ7-DEP1 showed a significant nitrogen response in its panicle phenotype, with a 28% decrease in grain number per panicle under low nitrogen conditions. WYJ7-dep1-1, however, only experienced a 15% decrease in grain number per panicle under low nitrogen conditions, and this was still higher than the grain number per panicle observed under high nitrogen conditions. This indicates that dep1-1 reduced the sensitivity of grain number per panicle to nitrogen fertilizer, maintaining a relatively high grain number even under low nitrogen conditions. Similar to WYJ7-dep1-1, the number of branches and grains per panicle in ostkl2 showed little change under different nitrogen application rates, with only a 16% decrease in grain number per panicle under low nitrogen conditions. Therefore, the grain number per panicle in ostkl2 showed reduced sensitivity to nitrogen fertilizer levels. Figure 4 (AD).

[0055] To describe the ability of rice panicle grain number to respond to nitrogen levels, the NGR (Nitrogen-mediated Growth Response) formula was introduced: (Number of grains per panicle under high nitrogen - Number of grains per panicle under low nitrogen) / Number of grains per panicle under low nitrogen. The NGR value can be used as a standard to measure the nitrogen sensitivity of rice panicle grain number. Compared with WYJ7-DEP1, the NGR values ​​of grain number per panicle in both WYJ7-dep1-1 and ostkl2 materials were significantly reduced. Figure 4 E).

[0056] This experiment shows that OsTKL2 is involved in regulating the rice panicle grain number response to nitrogen fertilizer.

[0057] Example 5: Overexpression of OsTKL2 increases grain number per ear under low nitrogen conditions To further analyze the effects of the OsTKL2 gene on rice panicle grain number and nitrogen fertilizer use efficiency, WYJ7-DEP1 and WYJ7-DEP1 pActin::OsTKL2-flag transgenic lines were planted in fields with different nitrogen application rates (60 kg / ha for low nitrogen and 210 kg / ha for high nitrogen). Statistical analysis of panicle phenotypes revealed that the primary and secondary branches and panicle grain number were significantly increased in OsTKL2 overexpression transgenic lines. The panicle grain number under low nitrogen conditions was close to that of the wild type under high nitrogen conditions. Figure 5 Compared with WYJ7-DEP1, the NGR value of grain number per ear in OsTKL2 overexpression lines was significantly reduced (AD). Figure 5 E). This indicates that OsTKL2 is an important new gene regulating the nitrogen response of grain number in rice panicles, and that overexpression of OsTKL2 has a more significant effect on increasing grain number under low nitrogen conditions.

[0058] In summary, overexpression of the OsTKL2 gene can increase the number of grains per panicle and nitrogen fertilizer use efficiency in rice. The experimental results above demonstrate that the OsTKL2 gene has the function of increasing the number of grains per panicle and improving nitrogen fertilizer use efficiency in rice, and has broad application prospects in high-yield and nitrogen-efficient rice breeding.

[0059] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Any of the following applications of rice OsTKL2 protein, its encoding gene, and related biological materials: 1) Used to regulate the number of grains per spike in plants; 2) Used to regulate the nitrogen fertilizer use efficiency of plants; The OsTKL2 gene is numbered LOC_Os11g26130 in the rice gene database.

2. The application according to claim 1, characterized in that, The regulation mentioned is a positive regulation.

3. The application according to claim 1 or 2, characterized in that, The plant is a monocotyledonous or dicotyledonous plant, preferably a grass, and more preferably rice.

4. The application according to any one of claims 1-3, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

5. A method for increasing the number of grains per spike while reducing the plant's sensitivity to nitrogen fertilizer, characterized in that, The method includes: introducing the rice OsTKL2 gene into a plant via a plasmid or integrating it into the plant chromosome via genetic engineering. The OsTKL2 gene is the same as described in claim 1.

6. The method according to claim 5, characterized in that, The plant is a monocotyledonous or dicotyledonous plant, preferably a grass, and more preferably rice.

7. A method for increasing the number of grains per panicle in rice under low nitrogen conditions, characterized in that, The method includes: enhancing the expression of the OsTKL2 gene in rice; The OsTKL2 gene is the same as described in claim 1.

8. The method according to claim 7, characterized in that, The enhancement method is selected from the following 1) to 6), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on the rice chromosome; 3) By altering the promoter sequences of the aforementioned genes on the rice chromosome; 4) By operatively linking a strong promoter to the gene; 5) By introducing enhancers; 6) Enhancement is achieved by using genes or alleles that encode the corresponding enzymes or proteins with high activity.

9. The use of transgenic plants obtained by the method according to any one of claims 5-8 in plant breeding.

10. The application according to claim 9, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

Images