ZmLG2 gene mutant and its application in regulating maize root architecture and nitrogen-efficient use.

Abstract

This invention discloses a ZmLG2 gene mutant and its application in regulating maize root architecture and nitrogen utilization efficiency, relating to the field of biotechnology. The ZmLG2 gene mutant has the DNA sequence shown in SEQ ID No. 3. Compared with the wild-type ZmLG2 gene SEQ ID No. 1, it contains the following mutations: a 21-base deletion at the end of the first exon CDS1; a complete deletion of the first intron intron1; a 189-base deletion at the beginning of the second exon CDS2; a T→G mutation at +2284, a T→C mutation at +2337, a G→A mutation at +2344, a C→A mutation at +2361, and a C→A mutation at +2394 downstream of the DNA start site; and a 1-base deletion at +2838. The ZmLG2 mutant plays a key role in regulating maize root architecture and improving nitrate absorption and utilization efficiency. It has significant biological functions and application value, providing new genetic resources and technical support for breeding crop varieties with high nitrogen fertilizer utilization efficiency. It also has broad application prospects in promoting green and sustainable agricultural development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the ZmLG2 gene mutant and its application in regulating maize root architecture and nitrogen utilization efficiency. Background Technology

[0002] Maize (Zea mays L.) is one of the most widely planted and highest-yielding food crops globally, and also an important food and feed crop in China. With arable land stabilizing or even decreasing, agriculture faces the dual challenges of "stable and high yields" and "efficient resource utilization." Given the current global trend of generally low nitrogen fertilizer utilization efficiency and nitrogen loss leading to environmental pollution, improving crop nitrogen use efficiency (NUE) has become a key issue in ensuring sustainable agricultural development.

[0003] Root system architecture (RSA) is fundamental to a crop's ability to acquire nitrogen, water, and other resources from the soil. Optimizing root system architecture not only enhances agronomic adaptability but also significantly improves nutrient acquisition efficiency. Recent studies have shown that genes controlling root angle, taproot length, lateral root density, and root hair development play crucial roles in regulating nitrogen uptake in crops. For example, the DEEPER ROOTING 1 (DRO1) gene in rice regulates the downward growth of roots, thereby enhancing the absorption of water and nitrogen from deeper soil layers. However, compared to model plants like rice and wheat, maize has relatively few clearly identified functional genes involved in regulating root system architecture and efficient nitrogen utilization. Currently, there is a lack of "dual-function" gene resources that simultaneously regulate root architecture and promote efficient nitrogen uptake. In-depth exploration of key genes closely related to maize root system optimization and efficient nitrogen utilization not only helps to reveal the molecular mechanisms by which maize adapts to soil nutrient environment, but also provides important genetic resource support for the breeding of high-yield and high-efficiency maize varieties. This has significant theoretical and applied value for ensuring my country's food security and the development of green agriculture. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a ZmLG2 gene mutant and its application in regulating maize root architecture and efficient nitrogen utilization.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0006] A ZmLG2 gene mutant, characterized in that the DNA sequence of the ZmLG2 mutant is shown in SEQ ID No. 3, and compared with the wild-type ZmLG2 gene SEQ ID No. 1, contains the following mutations:

[0007] The first exon CDS1 has a 21-base deletion at its end;

[0008] The first intron, intron1, is completely missing.

[0009] The second exon CDS2 has a deletion of 189 bases at the front.

[0010] Downstream of the DNA start site, there are T→G mutations at +2284, T→C mutations at +2337, G→A mutations at +2344, C→A mutations at +2361, and C→A mutations at +2394.

[0011] A single base is missing at position +2838.

[0012] Application of the above-mentioned ZmLG2 gene mutant in regulating maize root architecture under high-nitrogen or low-nitrogen conditions

[0013] More preferably, the regulation of maize root architecture specifically refers to the improvement of maize root architecture and the increase of biomass.

[0014] More preferably, the improvement in maize root architecture and the increase in biomass are manifested in the enhancement of at least one of the following traits:

[0015] Root area increases;

[0016] Increased fresh root weight;

[0017] Root trunk weight increased.

[0018] The application of the ZmLG2 gene mutant in improving nitrogen use efficiency in maize under high and low nitrogen conditions.

[0019] The improvement in nitrogen utilization efficiency is manifested in at least one of the following indicators:

[0020] The total nitrogen content of the whole plant increased;

[0021] Nitrogen uptake increases;

[0022] The nitrate content in corn is significantly increased.

[0023] The recombinant vector of the ZmLG2 gene mutant mentioned above.

[0024] A method for breeding high nitrogen-efficiency maize varieties includes the following steps:

[0025] (1) The above-mentioned ZmLG2 gene mutant was crossed with the target maize variety;

[0026] (2) Screen offspring with the above ZmLG2 gene mutant genotype;

[0027] (3) Phenotypic identification of offspring plants under high nitrogen and low nitrogen conditions was conducted to screen out individuals with significantly increased root area, biomass and nitrogen uptake.

[0028] A maize breeding material comprising the aforementioned ZmLG2 gene mutant, wherein the breeding material enables maize to exhibit at least one of the following traits under high-nitrogen and low-nitrogen conditions:

[0029] The total root area increases;

[0030] Increased biomass in the aboveground parts and roots;

[0031] Nitrogen absorption and utilization efficiency has been significantly improved.

[0032] A kit for detecting mutants of the maize ZmLG2 gene, comprising specific primer pairs for amplifying the ZmLG2 gene, said primer pairs being selected from at least one pair of SEQ ID No. 4-13.

[0033] The beneficial effects of adopting the above technical solution are as follows: This invention, through hydroponic cultivation experiments under high-nitrogen and low-nitrogen conditions, and 15 Hydroponic tracing experiments and other methods verified the important role of the ZmLG2 gene mutant in regulating maize root architecture, nitrogen absorption, and utilization. The results showed that, compared with wild-type maize, transgenic maize with the ZmLG2 gene mutant exhibited a significant increase in total root area and enhanced nitrate absorption capacity under both high-nitrogen and low-nitrogen nutrient conditions, with a significantly increased nitrate content. These findings fully demonstrate that the ZmLG2 mutant plays a crucial role in regulating maize root architecture optimization and improving nitrate absorption and utilization efficiency, possessing significant biological functions and application value. The utilization of this gene mutant provides new genetic resources and technical support for breeding crop varieties with high nitrogen fertilizer utilization efficiency, and has broad application prospects in promoting green and sustainable agricultural development. Attached Figure Description

[0034] Figure 1 This is a schematic diagram showing the tissue expression levels of the ZmLG2 gene in a public transcriptome database.

[0035] Figure 2This is a schematic diagram of the structure of the T006 transcript of the ZmLG2 gene and a schematic diagram of the location information of different allelic mutants; different numbers represent different allelic mutants, lines represent introns, and black box areas represent CDS coding regions; Figure A is a schematic diagram of the overall gene structure, and Figures B and C show the specific point mutation and deletion sequence information.

[0036] Figure 3 Phenotypic observations of wild-type B73 and ZmLG2 knockout maize at different nitrate concentrations. WT wild-type B73, lg2 is a ZmLG2 mutant with B73 as background. Scale bar is 5cm.

[0037] Figure 4 A comparison of the differences in nitrogen response of root area traits in wild-type B73 and ZmLG2 knockout maize after 10 days of growth under different nitrate concentrations.

[0038] Figure 5 The figure shows the comparison of fresh weight and dry weight biomass traits of aboveground and root parts of wild-type B73 and ZmLG2 knockout maize after 10 days of growth under different nitrate concentrations. Single-tailed t-test, **P<0.01, ***P<0.001 and ****p<0.0001 indicate very significant, extremely significant and extremely extremely significant differences, respectively.

[0039] Figure 6 The figure shows the comparison of nitrogen content and 15N uptake in the aboveground and root parts of wild-type B73 and ZmLG2 knockout maize after 10 days of growth under different nitrate concentrations. The results are shown in the single-tailed t-test. **P<0.01, ***P<0.001 and ****p<0.0001 indicate very significant, extremely significant and extremely extremely significant differences, respectively.

[0040] Figure 7 Image of a DNA extraction instrument;

[0041] Figure 8 This is a flowchart recording the hydroponics experiment process. Detailed Implementation

[0042] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0043] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0044] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0045] References to "one embodiment" or "some embodiments" as described in this specification mean that one or more embodiments of this application include a specific feature, structure, or characteristic described in connection with that embodiment. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0046] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0047] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments thereof.

[0048] However, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0049] The ZmLG2 gene (version number: Zm-B73-REFERENCE-GRAMENE-4.0: Zm00001d042777) provided by this invention has the wild-type DNA sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2. This gene exhibits high expression levels in root tissues. Figure 1 ZmLG2 gene structure and the location information of its different allelic mutants are as follows: Figure 2 As shown.

[0050] Example 1: Obtaining and Identifying the Maize ZmLG2 Mutant

[0051] In this invention, the seeds of the ZmLG2 mutant maize and its control, the wild-type WT B73 seeds, were provided by Professor Li Pinghua's team at Shandong Agricultural University. The source and detailed information of this mutant were systematically described by Wang et al. (2022) (Wang, X. et al., Plant Physiology, 190(1), 500–515). Specific references are as follows: Xiaokun Wang, Xianglan Wang, Shilei Sun, Xiaoyu Tu, Kande Lin, Lei Qin, Xingyun Wang, Gang Li, Silin Zhong, Pinghua Li, Characterization of regulatory modules controlling leaf angle in maize, Plant Physiology, Volume 190, Issue 1, September 2022, Pages 500–515. https: / / doi.org / 10.1093 / plphys / kiac308.

[0052] Sequencing analysis revealed that the ZmLG2 gene of the maize mutant exhibited a significant characteristic structural variation compared to the standard transcript (Zm00001d042777_T006) in the B73 reference genome, with one large structural deletion, five point mutations, and one site deletion in its nucleotide sequence. Specifically, the first exon (CDS1) is located in Chr3:179387864–179387985, with a normal length of 122 bp. In the mutant, 21 bases at the end of this exon are deleted, shortening its length to 101 bp. The first intron (intron1), located between the first and second CDS and with a length of approximately 1100 bp, is completely deleted in the mutant, resulting in the two exons being directly spliced ​​together. The second exon (CDS2) is located in Chr3:179389086–179389307, with a length of 222 bp. In the mutant, the first 189 bp of its front end is deleted, leaving only about 33 bp of the back end sequence. In addition to the structural variations mentioned above, the ZmLG2 mutant exhibits a T→G mutation at position +2284 downstream of the DNA start site (with A as position 0), a T→C mutation at position +2337, a G→A mutation at position +2344, a C→A mutation at position +2361, a C→A mutation at position +2394, and a 1-base deletion (del 1 bp) at position +2838. These mutations may cause changes in the amino acid composition of the coding region, thereby affecting protein structure and function. The ZmLG2 mutant sequence is shown in SEQ ID No. 3.

[0053] 1) Plant DNA extraction

[0054] Take approximately 50 mg of fresh leaves from each wild-type B73 and ZmLG2 mutant maize plant and place them in a 2 ml 96-well deep plate; use GeneExpress 3100 high-throughput automated nucleic acid extraction platform (Chengdu GeneExpress Technology Co., Ltd.) to efficiently and rapidly extract plant DNA.

[0055] 2) Sequencing primer design

[0056] Primer design: Primers were designed based on the DNA sequence Zm00001d042777 of the reference genome B73. The specific sequences are as follows:

[0057] Table 1 Primer pair sequence listing

[0058]

[0059] 3) PCR amplification

[0060] PCR amplification was performed using the designed primers. The PCR reaction system and parameters are shown in Tables 2 and 3, and the reaction cycle number was 34.

[0061] Table 2 PCR reaction system

[0062]

[0063] Table 3 PCR reaction parameters

[0064]

[0065] 4) First-generation sequencing to identify genotypes

[0066] The PCR products obtained from PCR amplification were subjected to Sanger first-generation sequencing, and sequence analysis was performed on different sites in both wild-type and mutant strains. Detailed sequence variation results are as follows: Figure 2 As shown.

[0067] Example 2: Hydroponic cultivation of maize ZmLG2 mutant material, phenotypic investigation and statistics

[0068] After germinating the seeds of the B73 wild-type control (WT) and ZmLG2 mutant in nitrogen-free sand for 9 days, the excess sand was gently washed off the roots. Healthy and undamaged seedlings of uniform size and growth were selected and transferred to a water tank for hydroponics. The basic nitrogen-free hydroponic nutrient solution formula is shown in Table 3.

[0069] Ten seedlings each from the wild type and the mutant were selected and transplanted. Five seedlings were cultured under high nitrogen (HN; 2.50 mM KNO3, 3 L) conditions, and five seedlings were cultured under low nitrogen (LN; 0.01 mM KNO3, 3 L) conditions, with potassium nitrate (KNO3) as the sole nitrogen source. To compensate for the potassium deficiency under LN conditions, 2.49 mM potassium sulfate (K2SO4) was added to the low nitrogen conditions. All macro- and micro-element compositions in the nutrient solutions were identical except for the nitrogen source. The pH of the nutrient solutions was adjusted to 6.5 by adding 1 mM KOH and 1 mM HCl, and the solutions were changed every two days.

[0070] After being cultured for 10 days in nutrient solutions containing 0.01 mM and 2.5 mM KNO3, maize seedlings were subjected to a 1-day nitrogen starvation treatment (supplying all elements except nitrogen). The seedlings treated with high and low nitrogen were then transferred to nutrient solutions containing 2.5 mM and 0.01 mM KNO3, respectively. 5 N-labeled K 15 In a fully hydroponic nutrient solution containing NO3 (99% atomic abundance, Shanghai Chemical Industry Research Institute), it was subjected to 3 hours¹ 5 N-labeled treatment. Three plants of uniform growth from each nitrogen treatment were then harvested: roots were rinsed with running water for 5 minutes, then immediately separated from the aerial parts. Root and aerial tissues were collected, inactivated at 105°C for 3 hours, dried at 85°C to constant weight, and ground into a fine powder. The total nitrogen content and¹ of the samples were then determined using a stable isotope mass spectrometer (isoprime visION, Elementar, Germany). 5 N content.

[0071] Table 3. Basic Nitrogen-Free Complete Hydroponic Nutrient Solution Formula

[0072]

[0073] All acquired phenotypic data were statistically analyzed using R-4.3.2 software. The results are shown in Tables 4 and 5. Figure 6 Show.

[0074] Table 4. Statistical table of hydroponic biomass and root area of ​​wild-type B73 and ZmLG2 mutant maize.

[0075]

[0076] Table 5. Hydroponic nitrogen content of wild-type B73 and ZmLG2 mutant maize. 15 N Absorption Statistics Table

[0077]

[0078] Table 6. Hydroponic biomass, root area, and nitrogen content of wild-type B73 and ZmLG2 mutant maize. 15 Statistical analysis results of N absorption statistics table

[0079]

[0080] From Tables 4 and 6 and Figure 4 It was found that the root area differences between the ZmLG2 mutant and the wild-type control B73 under both high and low nitrogen conditions were statistically significant (P<0.05). Among them, the root area of ​​the ZmLG2 mutant was significantly larger than that of the wild-type control B73 under both high and low nitrogen conditions.

[0081] From Tables 5 and 6 and Figure 5 , 6 It can be seen that the fresh weight, dry weight, biomass, and total nitrogen content of the ZmLG2 mutant and the wild-type control B73 under high and low nitrogen conditions are significantly different. 15 The differences in total nitrogen uptake were all statistically significant (P<0.05). Among them, the ZmLG2 mutant species had significantly greater fresh weight, dry weight biomass, total nitrogen content, and total 15N uptake than the wild-type control B73 under both high and low nitrogen conditions.

[0082] In summary, the present invention demonstrates its effectiveness through hydroponic cultivation experiments under high-nitrogen and low-nitrogen conditions, and 15 Hydroponic tracing experiments and other methods verified the important role of the ZmLG2 gene in regulating maize root architecture, nitrogen absorption, and utilization. The results showed that, compared with wild-type maize, transgenic maize with the ZmLG2 gene exhibited a significant increase in total root area and enhanced nitrate absorption capacity under both high-nitrogen and low-nitrogen nutrient conditions, with a significant increase in nitrate content. These findings fully demonstrate that ZmLG2 plays a crucial role in regulating root architecture optimization and improving nitrate absorption and utilization efficiency, possessing significant biological functions and application value. The utilization of this gene provides new genetic resources and technical support for breeding crop varieties with high nitrogen fertilizer utilization efficiency, and has broad application prospects in promoting green and sustainable agricultural development.

[0083] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0084] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0085] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A ZmLG2 gene mutant, characterized in that: The DNA sequence of the ZmLG2 mutant is shown in SEQ ID No. 3, and compared with the wild-type ZmLG2 gene SEQ ID No. 1, it contains the following mutations: The first exon CDS1 has a 21-base deletion at its end; The first intron, intron1, is completely missing; The second exon CDS2 has a deletion of 189 bases at the front. Downstream of the DNA start site, there are T→G mutations at +2284, T→C mutations at +2337, G→A mutations at +2344, C→A mutations at +2361, and C→A mutations at +2394. A single base is missing at position +2838.

2. A recombinant vector containing the ZmLG2 gene mutant as described in claim 1.

3. A method for breeding high-nitrogen-efficiency maize varieties, characterized in that, Includes the following steps: (1) The ZmLG2 gene mutant described in claim 1 is crossed with the target maize variety; (2) Screen offspring with the ZmLG2 gene mutant genotype; (3) Phenotypic identification of offspring plants under high nitrogen and low nitrogen conditions was conducted to screen out individuals with significantly increased root area, biomass and nitrogen uptake.

Images