Function and application of wisdom nutrient high-efficiency gene ZmGRX20 in regulation and control of corn source library organ nitrogen redistribution

Abstract

The invention discloses a function and application of a wisdom nutrient efficient gene ZmGRX20 for regulating and controlling nitrogen redistribution of a corn source library organ, and belongs to the technical field of molecular biology. Research finds that under low nitrogen stress, knockout or inhibition of the ZmGRX20 gene promotes redistribution of nitrogen in plants from old leaves to new leaves and other library organs, promotes growth of female ears and improves nitrogen utilization efficiency, so that growth and development of the plants in a low-nitrogen environment are guaranteed, and the yield of the plants is increased. The overexpressed ZmGRX20 gene inhibits redistribution of nitrogen in plant leaves from old leaves to library organs such as new leaves and the like.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically involving ZmGRX20 The function and application of genes in regulating nitrogen content and nitrogen redistribution efficiency in plant source and sink organs under low nitrogen stress. Background Technology

[0002] Nitrogen is an essential macronutrient for plant growth and development, and a key nutrient limiting maize yield. Yellowing and wilting of older leaves is one of the most typical morphological responses of maize under low nitrogen stress. This phenomenon not only provides a direct signal of nitrogen deficiency in maize but also reflects its adaptive mechanisms in nutrient allocation, physiological metabolism, and molecular regulation. Approximately 61%-77% of the nitrogen in maize kernels originates from the redistribution of nitrogen in senescent leaves; therefore, regulating nitrogen redistribution efficiency is a crucial way to improve nitrogen use efficiency and yield.

[0003] As a C4 crop with extremely high nitrogen requirements, maize exhibits a nitrogen demand pattern of "lower in the early stages, higher in the middle stages, and lower in the later stages" throughout its growth cycle, with the jointing to grain-filling stage being the critical period for nitrogen absorption. Currently, low nitrogen stress in agricultural production mainly stems from three aspects: first, low basic soil fertility, especially in fields with long-term continuous cropping and insufficient organic fertilizer input, leading to continuous depletion of the soil nitrogen pool; second, low nitrogen use efficiency, as chemical nitrogen fertilizers are easily lost through leaching, volatilization, and denitrification, resulting in an actual utilization rate of only 30%-40%; and third, extreme environmental conditions exacerbate stress, with drought and low temperatures inhibiting root absorption and translocation of nitrogen, potentially causing low nitrogen symptoms even when soil nitrogen content is sufficient. Therefore, given insufficient total nitrogen absorption, improving the redistribution efficiency of nitrogen within maize is crucial.

[0004] With the development of molecular biology and plant physiology, research on maize's response to low nitrogen stress has progressed from morphological observation to the molecular regulatory level. Currently, several gene families (such as the NRT gene family and the AREB gene family) have been identified in maize that respond to low nitrogen signals. These genes influence the timing and extent of yellowing and wilting of older leaves by regulating processes such as nitrogen uptake and transport, carbon and nitrogen metabolism balance, and hormone synthesis (such as auxin and cytokinin). However, no reports have yet been published on this topic. ZmGRX20 Genes and their mutants regulate the redistribution of nitrogen in maize under low nitrogen stress. Summary of the Invention

[0005] To address the aforementioned shortcomings of the prior art, the present invention provides a... ZmGRX20 The use of genes to regulate nitrogen content and nitrogen redistribution efficiency in plant source and sink organs under low nitrogen stress.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows: The purpose of this invention is to provide ZmGRX20 The use of genes to regulate nitrogen content and nitrogen redistribution efficiency in plant source and sink organs under low nitrogen stress.

[0007] Furthermore, the plant source and sink organs are leaves, roots, stems, or flowers, etc.

[0008] Furthermore, the plant-derived organ is the leaf.

[0009] Furthermore, under low nitrogen stress, overexpression ZmGRX20 Genes inhibit the transfer of nitrogen from older leaves to newer leaves in plant leaves.

[0010] Furthermore, under low nitrogen stress, knockout or inhibition ZmGRX20 Genes promote the transfer of nitrogen from older leaves to newer leaves in plants.

[0011] Another object of the present invention is to provide ZmGRX20 The use of gene knockout or inhibitors to promote the transfer of nitrogen from older leaves to newer leaves in plants under low nitrogen stress.

[0012] Furthermore, ZmGRX20 Gene knockout or inhibitors are at least one of nucleic acid molecules, small molecule compounds, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0013] Another object of the present invention is to provide knockout ZmGRX20 Use of genes in the development of transgenic crops that promote nitrogen allocation or female ear growth in plants.

[0014] Another object of the present invention is to provide knockout ZmGRX20 The use of genes in improving plant germplasm resources.

[0015] Furthermore, the plant is corn.

[0016] The beneficial effects of this invention are: This invention has found that under low nitrogen stress, overexpression ZmGRX20 Genes inhibit the transport of nitrogen from older leaves to sink organs such as newer leaves in plant leaves. ZmGRX20 Mutants formed by gene mutations can promote the transport of nitrogen from older leaves to newer leaves and other sink organs in plants, thereby improving the efficiency of nitrogen use in plants and promoting plant growth and development in low-nitrogen environments. Attached Figure Description

[0017] Figure 1 Under low nitrogen stress treatment grx20-EMS Phenotypic analysis diagram of mutant and B73; Figure 2 Under normal nitrogen treatment grx20-EMS Statistical graph of phenotypic analysis of mutants and B73; Figure 3 Phenotypic analysis of gene-edited lines and B104 under low nitrogen stress and normal nitrogen conditions; Figure 4 Under low nitrogen stress treatment ZmGRX20 Phenotypic analysis of gene overexpression lines and B104; Figure 5 Under normal nitrogen treatment ZmGRX20 Phenotypic analysis of gene overexpression lines and B104; Figure 6 Under low nitrogen stress grx20-EMS The development of female ears in mutants and wild types. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] Example 1 1. Mutant grx20-EMS For research ZmGRX20 The function of gene (Zm00001d023870) in maize leaves under nitrogen stress was obtained from a mutant database. ZmGRX20 mutant grx20-EMS The genetic background is maize inbred line B73, such as Figure 1 As shown in A, the mutant encodes... ZmGRX20 A mutation occurs at the 53rd base of the gene's CDS, changing from base C to base T. An amino acid mutation occurs at the 47th amino acid, changing from proline to serine, and the amino acid mutation becomes a sense mutation.

[0020] 2. Low nitrogen stress Through the grx20-EMS Mutant maize seedlings were subjected to 0.04 mM low nitrogen stress treatment. grx20-EMS The mutant's overall growth was better than that of the wild-type B73. Figure 1 B), and the dry weight of both the above-ground and underground parts is significantly greater than that of B73 (B). Figure 1 C), in addition, grx20-EMS The total nitrogen content in the aboveground parts of the mutant was also significantly higher than that of B73 ( Figure 1 D). grx20-EMS Each leaf of the mutant and wild-type B73 was separated and its phenotypic characteristics were photographed, such as... Figure 1As shown in E, under low nitrogen stress treatment, we found grx20- EMS The fourth leaf of the mutant turned more yellow and withered than that of the wild-type B73. grx20-EMS The mutant's sixth and seventh leaves grew better than those of the wild-type B73.

[0021] The results of total nitrogen content in leaves showed that ( Figure 1 F), under low nitrogen conditions grx20-EMS The nitrogen content of the fourth leaf of the mutant was significantly lower than that of the fourth leaf of the wild-type B73. grx20-EMS The nitrogen content of the 6th and 7th leaves of the mutant was significantly higher than that of the 6th and 7th leaves of the wild type.

[0022] The above results analysis shows that, ZmGRX20 When a gene mutates in a maize plant, under low nitrogen stress, it may cause the older leaves of the maize plant to transfer more nitrogen to the new leaves. As the nitrogen content of the older leaves decreases, the yellowing of the older leaves will become more severe. The nitrogen content of the new leaves increases, and the new leaves obtain more nitrogen to supply their own growth.

[0023] 3. Normal nitrogen treatment Under normal nitrogen conditions grx20-EMS The overall growth of the mutant was not significantly different from that of the wild-type B73. Figure 2 A), there was no significant difference in dry weight between the above-ground and underground parts. Figure 2 B), In addition, we measured the total nitrogen content in the aboveground and underground parts and found no significant difference in total nitrogen content. Figure 2 C). grx20-EMS Each leaf of the mutant and wild-type B73 was separated and its phenotypic characteristics were photographed, such as... Figure 2 D and E showed no significant difference in phenotype between the mutant and wild-type leaves, and no significant difference in total nitrogen content in the leaves.

[0024] 4. ZmGRX20 The effect of gene-edited materials on nitrogen allocation under different concentrations of nitrogen stress. Constructing the CPB-ZmGRX20 CRISPR gene editing vector and transforming it into maize to create... ZmGRX20 Gene knockout materials were used to obtain loss-of-function mutant lines. GRX20-CR1 and GRX20-CR2 right grx20-EMS The function of the mutant was verified, and the specific process is as follows: Will ZmGRX20Seeds of loss-of-function mutants of the polypeptide and their corresponding wild-type seeds were disinfected with 10% hydrogen peroxide for 30 minutes and soaked overnight in 2% H2O2. The seeds were then sown in seedling trays filled with perlite. Transplanting was carried out after the corn seedlings reached two leaves and a central bud. The corn was allowed to adapt for two days in a 1 / 2 Hoagland solution, followed by nitrogen treatment with 0.05 mM (low nitrogen) and 4 mM (normal nitrogen) concentrations. Phenotypic observation was conducted daily under hydroponic nitrogen stress treatment until the corn phenotype appeared. Phenotypic data were collected, photographed, and samples were taken from the corn plants. The results are shown in […]. Figure 3 .

[0025] The specific methods for measuring and sampling phenotypic indicators are as follows: Seedling biomass determination: The fresh weight of the aboveground and underground parts was weighed separately, and then the fresh samples were dried in an oven at 65℃ to constant weight. The dry weight of the aboveground and underground parts was then weighed.

[0026] Leaf length: Measured with a ruler, the length of a corn leaf is the distance from the bottom of the leaf sheath to the tip of the leaf.

[0027] Root length: Lateral root and root hair characteristics were measured using the WinRHIZO root analysis system.

[0028] like Figure 3 As shown, this invention uses CRISPR technology to create loss-of-function mutant lines. GRX20-CR1 and grx20- cr2 The two lines were deleted by 48bp and 22bp, respectively. Figure 3 A). Nitrogen stress treatment revealed that two gene-edited lines were related to... grx20-EMS The mutants exhibited consistent phenotypes, confirming that... GRX20 The gene plays an important role in improving the efficiency of nitrogen redistribution in maize. Specifically, under low nitrogen conditions (0.04 mM), the gene-edited mutant... GRX20-CR1 and GRX20-CR2 Higher nitrogen redistribution efficiency is manifested in older leaves withering faster and new leaves being larger and longer. Figure 3 B), resulting in a significantly greater aboveground fresh weight in the mutant compared to the wild type. Figure 3 C).

[0029] Under normal nitrogen (4mM) conditions, the mutant and wild type showed differences in plant growth and senescence. Figure 3 D) and fresh weight of aboveground parts ( Figure 3 There were no significant differences in traits such as E. When each leaf was separated, it was found that under low nitrogen (0.04 mM) conditions, there was no significant difference in leaf length among more mature leaves (1-5 leaves), but the younger leaves (6-7 leaves) of the mutant were significantly longer than those of the wild type. Figure 3F). Even more interestingly, the wild-type had already grown its 8th leaf at this point, while the wild-type's 8th leaf had not yet developed. Figure 3 F, G). Under normal nitrogen (4 mM) conditions, there were no significant differences in the number of leaves between the gene-edited lines and the wild type. Figure 3 H, I).

[0030] Example 2 Overexpression ZmGRX20 The influence of genes on nitrogen allocation 1. Overexpression ZmGRX20 Inhibition of nitrogen redistribution in older leaves under low nitrogen stress To further verify ZmGRX20 The function of genes is to create ZmGRX20 Gene overexpression materials OE1 and OE2 were subjected to low nitrogen stress treatment, and the results are shown in [Figure number missing]. Figure 4 .

[0031] like Figure 4 As shown in Figure A, under low nitrogen stress, the overall growth of wild-type B104 plants was significantly better than that of wild-type plants. ZmGRX20 Two overexpression lines, OE1 and OE2, were used. It can be seen that due to the low nitrogen effect, the third leaf of B104 had already withered, and the fourth leaf had begun to yellow; while the third leaf of the overexpression material had only just begun to yellow and had not yet withered, and the fourth leaf did not yet show yellowing phenotype. The wild type already showed the seventh leaf, while only a few of the overexpression materials showed the seventh leaf. The aboveground dry weight of the B104 plant was significantly greater than that of the two overexpression lines, but there was no significant difference in the underground dry weight. Figure 4 B), and the total nitrogen content in the aboveground parts of B104 plants was also significantly higher than that of the two overexpression lines (B). Figure 4 C). Each leaf of the overexpressing strain and B104 was separated for clearer observation and comparison of each leaf. Figure 4 A is consistent. Figure 4 As shown in Figure D, the third leaf of the wild-type B104 showed more pronounced yellowing than the third leaves of the two overexpression lines. Furthermore, the leaf tips of the fourth leaves of B104 also began to wither and turn yellow, while the leaf tips of the fourth leaves of the two overexpression lines did not begin to wither or turn yellow. Leaf length results also revealed that the 5th, 6th, and 7th leaves of B104 were longer than the 5th, 6th, and 7th leaves of the two overexpression lines. Figure 4 E).

[0032] The determination of total nitrogen content in its leaves revealed ( Figure 4 (F) The total nitrogen content of the third leaf of B104 was significantly lower than that of the third leaf of the two overexpression materials, while the total nitrogen content of the sixth and seventh leaves of B104 was significantly higher than that of the sixth and seventh leaves of the overexpression materials. The results indicate that ZmGRX20When this gene is overexpressed in maize plants, under low nitrogen stress, it inhibits the transport of nutrients such as nitrogen from older leaves to new leaves, leading to slower yellowing and senescence of older leaves. Since the growth of new leaves depends on nitrogen obtained from older leaves, reduced nitrogen redistribution results in slow new leaf growth, further contributing to... grx20-EMS Leaf nitrogen content results of mutants, positive and negative verification, to prove ZmGRX20 Overexpression inhibits nitrogen transport from older leaves to newer leaves in maize.

[0033] 2. Overexpression ZmGRX20 The effect of genes on nitrogen allocation under normal nitrogen treatment Further research on overexpression ZmGRX20 The effects of gene lines OE1 and OE2 on nitrogen allocation under normal nitrogen conditions are shown in the following figures. Figure 4 .

[0034] like Figure 5 As shown in Figure A, under normal nitrogen conditions, there was no significant difference in overall growth between B104 and the two overexpression lines OE1 and OE2, nor was there a significant difference in the dry weight of the aboveground and underground parts. Figure 5 B), In addition, we measured the total nitrogen content in the aboveground and underground parts and found no significant difference in total nitrogen content. Figure 5 C). B104 and... ZmGRX20 Leaf analysis of each leaf from the two overexpressing gene lines revealed no significant phenotypic differences between the B104 and OE lines. Figure 5 D, E), and there was no significant difference in total nitrogen content in leaves ( Figure 5 F).

[0035] Example 3 ZmGRX20 Effects of mutants on female ear growth To investigate grx20-EMS Whether the strong nitrogen redistribution ability of mutant seedlings has a positive impact on inflorescence development and yield at maturity will be investigated by comparing wild-type and... grx20-EMS The mutant was cultured in a complete nutrient hydroponic solution until it reached the 5-leaf stage, and then transplanted into sand culture. Nitrogen was no longer supplied in the later stages; only other nutrients were provided. The results are shown in [Figure number missing]. Figure 6 .

[0036] like Figure 6 As shown, in this environment grx20-EMS Most mutants were able to shed pollen and produce silk threads; while only a few wild-type plants shed pollen, and none produced silk threads. After removing the leaves, only the stems and inflorescences remained. Figure 6 A) It can be clearly seen grx20-EMS The mutant produced female ears in 100% of cases, while only 50% of the female ears in the wild type showed slight enlargement. Figure 6 B).

[0037] Measure the length of the female ear (at this time, the length of the bracts). grx20-EMS The mutant had an average ear length of 16.3 cm, while the wild type had an average ear length of only 3.5 cm. Figure 6 C). This experiment confirmed that... grx20-EMS The mutant's strong nitrogen redistribution capacity positively promotes inflorescence development, indicating that under low nitrogen stress... grx20-EMS The mutant female ears have stronger growth ability and have the potential to increase yield.

[0038] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. ZmGRX20 The use of genes to regulate nitrogen content and nitrogen redistribution efficiency in plant source and sink organs under low nitrogen stress.

2. The use according to claim 1, characterized in that, Plant source and reservoir organs are leaves, roots, stems or flowers.

3. The use according to claim 2, characterized in that, The plant-derived organ is the leaf.

4. The use according to claim 3, characterized in that, Under low nitrogen stress, overexpression ZmGRX20 Genes inhibit the transfer of nitrogen from older leaves to newer leaves in plant leaves.

5. The use according to claim 3, characterized in that, Knockout or inhibition under low nitrogen stress ZmGRX20 Genes promote the transfer of nitrogen from older leaves to newer leaves in plants.

6. ZmGRX20 The use of gene knockout or inhibitors to promote the transfer of nitrogen from older leaves to newer leaves in plants under low nitrogen stress.

7. The use according to claim 6, characterized in that, ZmGRX20 Gene knockout or inhibitors are at least one of nucleic acid molecules, small molecule compounds, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

8. Knockout ZmGRX20 Use of genes in the development of transgenic crops that promote nitrogen allocation or female ear growth in plants.

9. Knockout ZmGRX20 The use of genes in improving plant germplasm resources.

10. The use according to claim 8 or 9, characterized in that, The plant is corn.

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