Application of ZmLBD19 and ZmLBD40 genes in synergistically regulating corn plant height and cultivating dwarf male sterile line

Abstract

The application relates to the field of genetic engineering and molecular breeding, and discloses ZmLBD19 and ZmLBD40 application of genes in synergistically regulating corn plant height and cultivating a dwarf male sterile line. ZmLBD19 and ZmLBD40 The application obtains mutants by knocking out genes by using CRISPR / Cas9 technology, wherein a single mutant has normal plant height and normal fertility, a double mutant has pollen sterility, significantly reduced plant height, shortened internodes, inhibited cell elongation and division, and reduced active gibberellin (GA) content, and the phenotype can be restored by exogenous GA. ZmLBD19 and ZmLBD40 The application further discloses an action mechanism of the genes in synergistically regulating corn plant height and male sterility, and provides gene resources and breeding techniques for precise improvement of corn plant height and fertility.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving ZmLBD19 and ZmLBD40 The function and application of genes in the synergistic regulation of maize plant height and pollen fertility, especially involving simultaneous editing ZmLBD19 and ZmLBD40 Methods for creating dwarf male-sterile lines through genetic engineering. Background Technology

[0002] Maize (Zea mays L.) is the world's largest food crop, and plant height is a key agronomical trait affecting its lodging resistance, planting density, and mechanized harvesting. Excessively tall plants are prone to lodging, leading to severe yield reductions; however, excessively short plants reduce biomass, limiting yield increases. Therefore, moderate dwarfing (semi-dwarfing) is one of the important goals of maize breeding. The "Green Revolution" of the 1960s significantly increased the yields of cereal crops such as wheat and rice by utilizing semi-dwarfing alleles in gibberellin metabolism or signal transduction pathways (Asami, 2023). For example, the dwarfing trait of the semi-dwarf wheat variety Norin 10 originates from... Rht-B1 and Rht-D1 Genes, among which Rht1 The DELLA protein, when mutated, renders the plant insensitive to GA signaling, resulting in dwarfing and increased harvest index (Asami, 2023); the semi-dwarf phenotype of rice IR8 "Miracle Rice" originates from... sd1 A loss-of-function mutation in the gene encoding GA20 oxidase (GA20ox), a key enzyme in the GA biosynthesis pathway, resulted in reduced GA activity and ultimately a semi-dwarf phenotype (Spielmeyer et al., 2002). In maize, several genes and QTLs related to plant height regulation have been reported. Researchers used map-based cloning to detect the semi-dwarf mutant. grmm A pleiotropic QTL was identified on the distal end of the long arm of chromosome 1. qPH1_dla This locus simultaneously controls plant height, flowering time, ear traits, and yield traits (Chen et al., 2025). Furthermore, brachytic1 ( br1 The gene regulates plant height through alternative splicing, and its mechanism is related to cell wall synthesis and cell elongation (Jiao et al., 2023; Mu et al., 2024). Although the above studies have revealed the complexity of maize plant height regulation, the resources of semi-dwarf genes that have been analyzed and can be directly applied to breeding practices are still relatively limited, and there is an urgent need to discover new plant height regulating genes and their superior allelic variations.

[0003] Maize is one of the most successful crops in utilizing heterosis, and hybrid seed production is the core link in the maize seed industry. Currently, maize hybrid seed production mainly relies on manual or mechanical emasculation, which suffers from prominent problems such as low efficiency, high cost, and difficulty in adapting to large-scale mechanized production. With the increasing shortage of rural labor, emasculation costs continue to rise, becoming a bottleneck restricting the development of the maize seed industry. The application of male-sterile lines can eliminate the manual emasculation process, saving a significant amount of labor costs, while improving seed purity and seed quality. It is an effective way to reduce production costs, improve hybrid purity, and fully utilize heterosis (Wu Suowei et al., 2024). However, the long-term application of maize sterile seed production technology is subject to many limitations. Although traditional cytoplasmic male sterility (CMS) has been applied to hybrid seed production, it suffers from drawbacks such as susceptibility to specific pathogen races, limited recovery sources, and unstable sterility traits. While nuclear male sterility (GMS) overcomes the above-mentioned defects of CMS, efficient technical means for the propagation and maintenance of its sterile lines have long been lacking. In recent years, the rapid development of gene editing technologies such as CRISPR / Cas9 has provided a powerful tool for the rapid creation of maize nuclear male sterility mutants. New engineered nuclear male sterility technology systems (such as multiple control male sterility and dominant nuclear male sterility) have been initially established and are beginning to be demonstrated and applied (Hou et al., 2026). Nevertheless, among the male sterility genes reported so far, those with both precise fertility regulation and plant architecture improvement functions remain extremely scarce.

[0004] Lateral Organ Boundaries Domain (LBD) proteins are a plant-specific family of transcription factors that play important roles in organ development, secondary growth, and stress responses (Xu et al., 2016). In Arabidopsis thaliana, LBD16 , LBD18 and LBD29 Members play a key role in the formation of lateral roots, among which LBD16 Promotes asymmetric division of lateral root initiator cells. LBD18 It is expressed in lateral root primordia and regulates the cell cycle. LBD29 It participates in the regulation of lateral root germination (Fan et al., 2012; Kim et al., 2015; Thatcher et al., 2012; Zhang et al., 2020). It is worth noting that... lbd16 and lbd18 Single mutants all showed reduced lateral root germination, while lbd16 lbd18 Double mutants exhibit a more significant reduction in the number of lateral roots, indicating functional redundancy among LBD family members (Zhang et al., 2020). In maize, two paralogous LBD genes... RTCS and RTCL They exhibit a synergistic effect in the formation of adventitious roots: RTCS It is a key regulator of coronary root initiation. RTCLBoth LBD and auxin control adventitious root elongation in early development, and while their functions are conserved in the auxin signaling pathway, they have different roles (Xu et al., 2015). Furthermore, LBD proteins, as key elements in auxin signal transduction, play a role in the initiation of maize seed roots, adventitious roots, and lateral roots (Hochholdinger et al., 2017). However, there are currently no reports on LBD genes simultaneously regulating maize plant height and male fertility.

[0005] This invention utilizes CRISPR / Cas9 gene editing technology to create... ZmLBD19 and ZmLBD40 Single gene knockout mutant Zmlbd19 and Zmlbd40 and homozygous knockout mutants of two genes Zmlbd19 / Zmlbd40 Field phenotypic analysis showed that the single mutant had no significant difference in plant height and pollen fertility compared to the wild type, while the double mutant exhibited significantly reduced plant height, shortened internodes, and complete pollen abortion. Stem cytology observation indicated that the double mutant had significantly reduced epidermal cell length and cell number compared to the wild type, suggesting inhibition of cell elongation and division. Hormone content assays showed that the double mutant had significantly lower levels of endogenous active GA compared to the wild type, and exogenous GA3 application restored its dwarfing phenotype and cell elongation defect. Molecular mechanism studies revealed that ZmLBD19 and ZmLBD40 can form a protein complex and directly bind to and activate GA synthesis genes. ZmGA20ox3 and ZmGA20ox5 The expression of ZmLBD19 and ZmLBD40 also activates transcriptional repressors. ZmRS2 The expression level of [the gene] is [high], and it interacts with the ZmRS2 protein to synergistically and indirectly inhibit GA catabolism genes. ZmGA2ox4 and ZmGA2ox10 Transcription; in addition, ZmLBD19 and ZmLBD40 also interact with the GA signaling pathway ZmD8 and promote GA catabolism metabolism genes. ZmGA2ox4 and ZmGA2ox10 Transcription. In summary, this invention discloses for the first time... ZmLBD19 and ZmLBD40 This study explores the molecular mechanisms by which GA metabolism and signaling pathways synergistically regulate maize plant height and male fertility, and provides a technical solution for creating dwarf male-sterile lines by simultaneously knocking out two genes using CRISPR / Cas9. This solution combines dwarfing breeding with sterile seed production, reducing plant height, enhancing lodging resistance, and increasing planting density while providing directly usable sterile maternal parent material for maize hybrid seed production. It has the dual value of cost reduction and efficiency improvement as well as precise improvement, providing important genetic resources and practical breeding tools for precise improvement of maize plant height and hybrid seed production. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to clarify ZmLBD19 and ZmLBD40 This study investigates the role of this gene in the synergistic regulation of maize plant height and male sterility, providing genetic resources and technical methods for the breeding of semi-dwarf and lodging-resistant maize varieties and for male-sterile hybrid seed production. The specific technical solutions are as follows: 1. Gene sequence analysis and target design: Cloning from maize inbred line B104 ZmLBD19 and ZmLBD40 Genes, the ones mentioned ZmLBD19 The nucleotide sequence of the gene is shown in SEQ ID NO.1. ZmLBD40 The nucleotide sequence of the gene is shown in SEQ ID NO.2; its encoding amino acid sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. CRISPR / Cas9 gene editing targets were designed from the conserved regions of the first exon of the two genes.

[0007] 2. Mutant material creation: Using CRISPR / Cas9 gene editing technology, mutant materials were constructed respectively. ZmLBD19 and ZmLBD40 Gene editing vectors were used, and each vector was introduced into the callus tissue of maize inbred line B104 using Agrobacterium-mediated transformation to obtain T0 generation single-gene knockout transgenic plants. ZmLBD19 and ZmLBD40 The homozygous single-gene knockout line was crossed, and the F1 generation was self-crossed. Genotyping was performed to obtain homozygous non-transgenic lines. ZmLBD19 and ZmLBD40 Double gene knockout mutant ( Zmlbd19 / Zmlbd40 ).

[0008] 3. Phenotypic Identification and Mechanism Analysis: Single mutants, double mutants, and wild-type B104 were simultaneously planted in the field. The following indicators were investigated and tested: plant height, internode length, pollen fertility, stem cell length and number, endogenous gibberellin (GA) content, and the effect of exogenous GA3 spraying on the recovery of the dwarfing phenotype. Based on this, the direct regulation of GA metabolism GA20ox and GA2ox family genes by ZmLBD19 / ZmLBD40 proteins, the interaction between ZmLBD19 / ZmLBD40 and ZmRS2 and ZmD8, and their regulatory role on GA metabolism genes were analyzed, thereby elucidating the molecular network of their synergistic regulation of gibberellin metabolism. This invention utilizes CRISPR / Cas9 technology to study maize... ZmLBD19 and ZmLBD40Site-directed gene editing yielded single and double mutants. Phenotypic identification results showed that the single mutants had no significant differences in plant height and pollen fertility compared to the wild type, while the double mutants exhibited significantly reduced plant height, shortened internodes, complete pollen abortion, inhibited cell elongation and division, and a significant decrease in endogenous active GA content. This dwarfing phenotype could be partially restored by exogenous GA3. Molecularly, both ZmLBD19 and ZmLBD40 can directly bind to and activate key genes in the GA synthesis pathway. GA20ox Family (e.g.) ZmGA20ox3 and ZmGA20ox5 Both proteins act as promoters for GA and interact with the transcriptional repressor ZmRS2, synergistically and indirectly inhibiting GA catabolism genes. GA2ox Family (e.g.) ZmGA2ox4 and ZmGA2ox10 The transcription of GA is mediated, thereby achieving dual regulation of active GA levels.

[0009] This invention reveals for the first time the molecular mechanism by which ZmLBD19 and ZmLBD40 synergistically regulate maize plant height and male fertility through the GA metabolic pathway. Utilizing the characteristic that double mutations in these two genes result in dwarfism and male sterility, this invention provides a method for simultaneous knockout... ZmLBD19 and ZmLBD40 This invention presents a technical solution for creating dwarf male-sterile lines, combining dwarfing breeding with sterile seed production. On one hand, the dwarfing characteristic reduces field management difficulty, enhances lodging resistance, and allows for higher planting density. On the other hand, the male-sterile characteristic allows it to serve as a sterile female parent in hybrid seed production, eliminating the need for manual emasculation, reducing seed production costs, and improving seed purity. This invention provides important genetic resources and practical breeding tools for precise improvement of maize plant height and hybrid seed production. Attached Figure Description

[0010] Figure 1 Maps of the maize pCas9-ZmLBD19 and pCas9-ZmLBD40 vectors. Blue sgRNA1 and yellow sgRNA2 represent the target sequences of ZmLBD19 and ZmLBD40, respectively.

[0011] Figure 2 Analysis of gene sequence variations in ZmLBD19 and ZmLBD40 knockout events. a. Gene sequence variations in ZmLBD19 knockout events (Zmlbd19#1 and Zmlbd19#2). b. Gene sequence variations in ZmLBD40 knockout events (Zmlbd40#1 and Zmlbd40#2). Base variation sites are indicated by red boxes, and deleted bases are indicated by short black lines. Zmlbd19#1 and Zmlbd40#1 were used for subsequent research (named Zmlbd19 and Zmlbd40), and double mutants Zmlbd19 / Zmlbd40 were created through genetic hybridization.

[0012] Figure 3 The double mutations of ZmLBD19 and ZmLBD40 result in complete male sterility in maize. ac. Mature tassel phenotype (Fig. a), anther phenotype (Fig. b), and pollen activity (Fig. c) of wild-type (WT) and Zmlbd19, Zmlbd40, and Zmlbd19 / Zmlbd40 mutants.

[0013] Figure 4 The double mutations ZmLBD19 and ZmLBD40 caused plant dwarfing. a, b. Phenotypic and statistical analysis of mature plant height in wild type (WT) and Zmlbd19, Zmlbd40, and Zmlbd19 / Zmlbd40 mutants. Scale bar: 50 cm; c, d. Comparison of interstem and tassel lengths between WT and Zmlbd19 / Zmlbd40 double mutants. Scale bar: 50 cm (Figure c) and 5 cm (Figure d). The p-value above the bar chart represents Student's t-test.

[0014] Figure 5 The double mutations of ZmLBD19 and ZmLBD40 resulted in decreased cell length and active GA content. a. Longitudinal cytological observation of the 8th internode of wild-type (WT) and Zmlbd19 / Zmlbd40 plants. b, c. Comparison of cell length (Fig. b) and cell number (Fig. c) in the 8th internode of WT and Zmlbd19 / Zmlbd40 plants. d. Comparison of active GA content in WT and Zmlbd19 / Zmlbd40 plants. The p-value above the bar chart represents Student's t-test.

[0015] Figure 6 Exogenous gibberellin partially restored the plant height phenotype of the ZmLBD19 and ZmLBD40 double mutants. a, b. Differences in the response of wild-type (WT) and ZmLBD19 / ZmLBD40 mutant seedlings to exogenous gibberellin. Scale bar: 10 cm. c, d. Effects of exogenous gibberellin on internode cell length in WT and ZmLBD19 / ZmLBD40. The p-value above the bar chart represents Student's t-test.

[0016] Figure 7ZmLBD19 and ZmLBD40 bind to the promoters of ZmGA20ox4 / 10 and ZmGA2ox3 / 5 and activate their transcription. a. Yeast one-hybrid assays showed that ZmLBD19 and ZmLBD40 can bind to the promoters of the ZmGA20ox4 / 10 and ZmGA2ox3 / 5 genes. b, c. Gel migration electrophoresis assays confirmed that ZmLBD19 and ZmLBD40 can bind to the promoters of the ZmGA2ox4 / 10 (Figure b) and ZmGA20ox3 / 5 (Figure c) genes. d, e. Transient protoplast expression assays showed that ZmLBD19 and ZmLBD40 activate the expression of ZmGA2ox4 / 10 (Figure d) and ZmGA20ox3 / 5 (Figure e).

[0017] Figure 8 ZmLBD19 / ZmLBD40 promotes ZmRS2 expression and forms a complex with it to inhibit the transcription of ZmGA2ox4 / 10. a. ZmLBD19 and ZmLBD40 upregulate the transcriptional expression of ZmRS2. bd. Yeast two-hybrid (Fig. b), bimolecular fluorescence complementation (Fig. c), and luciferase complementation imaging (Fig. d) experiments confirmed that ZmLBD19 and ZmLBD40 can interact with ZmRS2 protein. e. Protoplast transient expression experiments showed that ZmRS2 protein is a transcriptional repressor. f. Protoplast transient expression experiments showed that ZmRS2 interacts with ZmLBD19 / ZmLBD40 to inhibit the latter's transcription of ZmGA20ox4 / 10. Different letters above the bars indicate significant differences in Student's t-test (p<0.05).

[0018] Figure 9 ZmLBD19 / ZmLBD40 interacts with ZmD8 and promotes the transcription of ZmGA2ox4 / 10. a, b. Yeast two-hybrid (Fig. a) and in vitro protein binding (Fig. b) experiments confirm that ZmLBD19 and ZmLBD40 interact with the ZmD8 protein. c, d. Transient protoplast expression experiments show that the ZmLBD19-ZmD8 (Fig. c) and ZmLBD40-ZmD8 (Fig. d) complexes promote the transcription of ZmGA20ox4 / 10. Different letters above the bars indicate significant differences in Student's t-test (p < 0.05). Detailed Implementation

[0019] Example 1: Creation using CRISPR / Cas9 method ZmLBD19 and ZmLBD40 mutant materials To clarify ZmLBD19 and ZmLBD40The role of mutants in regulating maize plant height and pollen fertility: This invention selects maize inbred line B104 as the recipient material for gene editing, and selects... ZmLBD19 and ZmLBD40 The first exon serves as the target region for CRISPR / Cas9 gene editing, and is created using CRISPR / Cas9 technology. ZmLBD19 and ZmLBD40 Knockout mutants.

[0020] 1. ZmLBD19 and ZmLBD40 Target design and construction of knockout vectors The gene editing vector of the present invention is pBUE411-ZmLBD19-Cas9 and pBUE411-ZmLBD40-Cas9 The basic carrier of this carrier is pBUE411-Cas9 The intermediate carrier is pCBCmT1T2 Provides gRNA ( Figure 1 This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into a basic vector via enzyme digestion. The specific construction process is as follows. ZmLBD19 and ZmLBD40 The conserved gene sequence was input into the CRISPR website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR for CRISPR-Cas9 target design, and two 20nt nucleotide sequences located in the first exon were selected as knockout targets: ZmLBD19 Target sequence 1 (SEQ ID NO.5): 5'-ATCGGGGTCGGTGATCACCG-3' ZmLBD19 Target sequence 2 (SEQ ID NO.6): 5'-CATGCGCTGCGTGCAAGTTC-3' ZmLBD40 Target sequence 1 (SEQ ID NO.7): 5'-ATCGGGGTCGGTGATCGCCG-3' ZmLBD40 Target sequence 2 (SEQ ID NO.8): 5'-TTCCTCAGCAGCCTTGGCCG-3' After annealing the target oligonucleotide chain, it was ligated into the pBUN411 vector (containing maize codon-optimized Cas9 and U6 promoters), and the successful construction of the vector was verified by sequencing.

[0021] 2. Agrobacterium-mediated genetic transformation of maize Maize immature embryos were transformed using Agrobacterium-mediated transformation (An et al., 2020). B104 immature embryos (1.0-1.5 mm in size) 10-12 days after pollination were co-cultured with Agrobacterium EHA105 containing the aforementioned vector for 2-3 days. They were then transferred to selection medium containing hygromycin (50 mg / L) for resistant callus induction and differentiation, yielding T0 generation regenerated plants.

[0022] 3. Mutant screening The following steps were taken to determine the knockout mutation results in T0 generation plants: DNA was extracted from leaves of T0 generation plants, and PCR amplification was performed using primers flanking the target site, followed by sequencing detection. ZmLBD19 (Primers SEQ ID NO. 9 and SEQ ID NO. 10) and ZmLBD40 (Primers SEQ ID NO.11 and SEQ ID NO.12) Gene editing status.

[0023] ZmLBD19-cas-F (SEQ ID NO.9): 5'-GCAGGTAGGTCTTCCTCACC-3' ZmLBD19-cas-R (SEQ ID NO.10): 5'-GTTCAGCAGCTTGGTCACG-3' ZmLBD40-cas-F (SEQ ID NO.11): 5'-GCGTGCGTCCACCTCCTTG-3' ZmLBD40-cas-R (SEQ ID NO.12): 5'-GGAAGAGGACGATGCCACG-3' Individual plants exhibiting frameshift or premature termination mutations at the target site were selected and named accordingly. Zmlbd19#1 / #2 and Zmlbd40#1 / #2 ( Figure 2 a, b). Zmlbd19#1 and Zmlbd40#1 Hybridization, self-crossing of F1 generation, and sequencing identification of F2 generation yielded homozygous double mutants. Zmlbd19 Zmlbd40 .

[0024] 4. Phenotypic identification Wild-type plants, all single mutants, and double mutants were simultaneously planted at the Beijing base. Plant height (ground level to the top of the tassel lateral) and pollen viability were investigated during the pollen shedding stage. Regarding pollen fertility, the results showed no significant difference in pollen fertility between the single mutants and the wild-type, but the double mutants were completely sterile. Figure 3 ac). Comparison of plant height revealed no significant difference in plant height between the single mutant and the wild type, but the double mutant showed a significant decrease in plant height and ear height at maturity. Figure 4ac), and the length of each intersegment was significantly shortened ( Figure 4 d, e). Longitudinal sections and histological observations of the 8th internode of the plant revealed a significant reduction in cell length and number compared to the wild type. Figure 5 ac).

[0025] Example 2: Gibberellin content determination and exogenous recovery experiment

[0026] To determine the gibberellin content, the stem base tissue of plants at the jointing stage (V8) was collected, ground with liquid nitrogen, and the active GA content was determined by LC-MS / MS. The results showed: Zmlbd19 / Zmlbd40 The content of active GA in the double mutant was significantly lower than that in the wild type. Figure 5 d).

[0027] To test the effect of external application of GA on plant height, three-leaf stage seedlings were sprayed with 50 μM GA3 aqueous solution and water (control), respectively, once every two days for 14 consecutive days, for a total of 7 treatments. The results showed that after GA3 spraying, the plants... Zmlbd19 / Zmlbd40 The double mutant plant height was significantly restored, although it did not reach the level of the wild type. Figure 6 (a, b), but the intersegmental cell length was also significantly restored ( Figure 6 c, d), indicating that the lack of active GA is one of the main reasons for dwarfing.

[0028] Example 3: ZmLBD19 and ZmLBD40 Mechanism of action verification

[0029] 1. Yeast one-hybrid assay and gel migration electrophoresis assay Will ZmGA2ox4 / 10 and ZmGA20ox3 / 5 The promoter (approximately 3 kb) was cloned to pLacZi2μ The report carrier will ZmLBD19 and ZmLBD40 clone the coding region to pJG4-5 Activation vector. Yeast EGY48 was co-transformed on a medium containing galactose and X-gal, with a negative control experiment performed simultaneously. ZmGA2ox4 / 10 and ZmGA20ox3 / 5 promoter and ZmLBD19 / ZmLBD40 The co-transformed yeasts all turned blue on the culture medium, indicating that ZmLBD19 / ZmLBD40 can bind to these promoters ( Figure 7 a). Gel migration electrophoresis further verified that ZmLBD19 / ZmLBD40 can directly bind. ZmGA2ox4 / 10 and ZmGA20ox3 / 5 promoter ( Figure 7 bc).

[0030] 2. Transient activation of protoplasts

[0031] To investigate the transcriptional regulatory functions of ZmLBD19 and ZmLBD40 on gibberellin metabolism genes, effectors (35S::ZmLBD19 or 35S::ZmLBD40) and reporter ( ZmGA2ox4, ZmGA2ox10, ZmGA20ox3, ZmGA20ox5 The promoter-driven LUC and REN-based co-transformation of maize protoplasts were performed, and the LUC / REN ratio was measured. The results showed that both ZmLBD19 and ZmLBD40 significantly activated the LUC / REN promoter. mGA2ox4 and ZmGA2ox10 Transcription ( Figure 7 d); Simultaneously, it was found that both ZmLBD19 and ZmLBD40 could significantly activate Z… mGA20ox3 and ZmGA20ox5 The transcription, and there is an additive effect between ZmLBD19 and ZmLBD40 ( Figure 7 e).

[0032] 3. Regulation and protein-protein interaction of ZmLBD19 and ZmLBD40 on ZmRS2

[0033] To detect the regulation of ZmLBD19 and ZmLBD40 ZmRS2 Gene expression levels, in this invention, were obtained from wild-type WT and Z, respectively. mlbd19 / Zmlbd40 RNA was extracted from the shoot apex meristem and second internode of the double mutant and compared. ZmRS2 Transcriptional level differences were observed. The results showed that compared to the wild type, ZmRS2 Expression level at Z mlbd19 / Zmlbd40 Significantly decreased in double mutants ( Figure 8 a).

[0034] To detect whether ZmLBD19 and ZmLBD40 interact with the ZmRS2 protein, this invention will... ZmLBD19 , ZmLBD40 and ZmRS2 The coding region was amplified from cDNA and ligated into pGADT7 or pGBKT7 vectors, respectively, to construct... pGADT7-ZmRS2 and pGBKT7-ZmLBD19, pGBKT7-ZmLBD40 Recombinant plasmids were co-transformed into yeast strain AH109 using the PEG4000 method. Transformants were first screened on SD / -WL medium, then transferred to SD / -WLAH selective medium and incubated for 4-5 days to assess interactions. Results showed that ZmLBD19 and ZmLBD40 interacted with ZmRS2 (…). Figure 8 b). At the same time, construct separately pGBKT7-ZmRS2 and pGADT7-ZmLBD19, pGADT7-ZmLBD40 The vector was subjected to a yeast two-hybrid experiment again, and the results showed that ZmLBD19 and ZmLBD40 did indeed interact with ZmRS2. Figure 8 b).

[0035] This invention will also ZmLBD19 and ZmLBD40 The coding sequence was cloned to p35ScYFP In the carrier, ZmRS2 The coding sequence was cloned to p35SnYFP These constructs were co-transformed into maize protoplasts. Fluorescence was observed using a Zeiss 510 Meta confocal laser scanning microscope 48 hours after transformation. The results showed that ZmLBD19 and ZmLBD40 both interacted with ZmRS2. Figure 8 c).

[0036] This invention will also ZmLBD19 and ZmLBD40 The coding sequences were cloned into the pCAMBIA1300-cLUC vector to form cLUC-ZmLBD19 and cLUC-ZmLBD19-LBD40. ZmRS2 The coding sequence was cloned into the pCAMBIA1300-nLUC vector to form nLUC-ZmRS2. The resulting nLUC-ZmRS2, along with cLUC-ZmLBD19 and cLUC-ZmLBD19-LBD40, was transformed into Agrobacterium tumefaciens strain EHA105 and co-injected into tobacco leaves. Fluorescence was detected using a Leica fluorescence microscope. The results showed that ZmLBD19 and ZmLBD40 interact with ZmRS2 (…). Figure 8 d).

[0037] 4. Detection of ZmRS2 transcriptional activity To detect whether ZmRS2 has transcriptional repression activity, an effector vector was constructed. 35S::ZmRS2 (Will ZmRS2 Encoding region cloned to p35S A plant expression vector was constructed. Simultaneously, a reporter vector was constructed: a synthetic promoter (16×UAS) containing 16 tandem UAS (upstream activation sequence) binding sites was fused with the firefly luciferase gene (LUC) and placed in the plant expression backbone (simultaneously carrying the Renilla luciferase gene REN as an internal control). A PEG-mediated transient transformation system of maize protoplasts was used. Effectors (35S::ZmRS2 or empty vector control) were co-transformed with reporter (16×UAS::LUC) and internal control plasmid (35S::REN) into maize (e.g., B73) mesophyll protoplasts. After transformation, the plants were cultured in the dark for 24 hours, and the luminescence values ​​of LUC and REN were measured using a dual-luciferase reporter gene assay kit (Promega), calculating the LUC / REN ratio. The relative transcriptional activity was calculated using the LUC / REN ratio of the empty vector effector treatment as a baseline (set to 1). The results showed that ZmRS2 has inhibitory transcriptional activity (…). Figure 8e).

[0038] 5. Detection of transcriptional activity of the ZmLBD19 / ZmLBD40-ZmRS2 complex To investigate whether the interaction between ZmRS2 and ZmLBD19 / ZmLBD40 affects the expression of gibberellin metabolism genes by the latter, this invention constructs effector combinations: individually 35S::ZmRS2 ,alone 35S::ZmLBD19 (or 35S::ZmLBD40 ), and the combination of the two ( 35S::ZmRS2 + 35S::ZmLBD19 or 35S::ZmLBD40 The report sub-reports adopted the following methods: ZmGA2ox4 and ZmGA2ox10 The promoter of the gene (approximately 3 kb) drives LUC expression, with 35S::REN as an internal control. The above effector and reporter were co-transformed into maize protoplasts in different combinations, with three biological replicates per group. After transformation, the protoplasts were incubated in the dark for 24 hours, and the LUC / REN ratio was measured. The LUC / REN ratio for treatment with the empty vector effector was 1, and the relative transcriptional activity of each treatment group was calculated. The results showed that the ZmLBD19 / ZmLBD40-ZmRS2 complex inhibited LUC expression. ZmGA2ox4 and ZmGA2ox10 Gene transcription activity ( Figure 8 f).

[0039] 6. Interaction examination between ZmLBD19 / ZmLBD40 and ZmD8 proteins

[0040] To detect whether ZmLBD19 / ZmLBD40 interacts with the ZmD8 protein, this invention will... ZmLBD19 , ZmLBD40 and ZmD8 The coding region was amplified from cDNA and ligated into pGADT7 or pGBKT7 vectors, respectively, to construct... pGADT7-ZmLBD19, pGADT7-ZmLBD40 and pGBKT7-ZmD8 Recombinant plasmids were co-transformed into yeast strain AH109 using the PEG4000 method. Transformants were first screened on SD / -WL medium, then transferred to SD / -WLAH selective medium and cultured for 4-5 days to assess interactions. Results showed that ZmLBD19 / ZmLBD40 interacts with ZmD8 protein in (…). Figure 9 a). Furthermore, His-MBP-ZmLBD19 and GST-ZmD8 fusion protein expression vectors were constructed, and after in vitro protein expression and purification, in vitro protein binding experiments were performed. The results showed that His-MBP-ZmLBD19 and GST-ZmD8 do indeed interact ( Figure 9 b).

[0041] 7. Detection of transcriptional activity of the ZmLBD19 / ZmLBD40-ZmD8 complex

[0042] To investigate whether the interaction between ZmD8 and ZmLBD19 / ZmLBD40 affects the expression of gibberellin metabolism genes by the latter, this invention constructs effector combinations: individually 35S::ZmD8 ,alone 35S::ZmLBD19 (or 35S::ZmLBD40 ), and the combination of the two ( 35S::ZmRS2D8 + 35S::ZmLBD19 or 35S::ZmLBD40 The report sub-reports adopted the following methods: ZmGA2ox4 and ZmGA2ox10 The promoter of the gene drives LUC expression, with 35S::REN as an internal control. The above effector and reporter were co-transformed into maize protoplasts in different combinations, with three biological replicates per group. After transformation, the protoplasts were incubated in the dark for 24 hours, and the LUC / REN ratio was measured. The LUC / REN ratio for treatment with the empty vector effector was 1, and the relative transcriptional activity of each treatment group was calculated. The results showed that the ZmLBD19 / ZmLBD40-ZmRS2 complex promoted LUC expression. ZmGA2ox4 and ZmGA2ox10 Gene transcription activity ( Figure 9 c, d).

[0043] This invention reveals for the first time the key functions of ZmLBD19 and ZmLBD40 in synergistically regulating maize plant height through the gibberellin metabolic pathway. Simultaneous knockout of both genes using CRISPR / Cas9 significantly reduces plant height, and the mechanism includes: (1) direct activation of ZmLBD19 and ZmLBD40. ZmGA20ox3 / 5 (2) ZmLBD19 and ZmLBD40 upregulate the expression of the transcriptional repressor ZmRS2, which interacts with and inhibits the expression of ZmLBD19 / ZmLBD40 proteins. ZmGA2ox4 / 10 Expression to reduce gibberellin inactivation; (3) ZmLBD19 and ZmLBD40 also interact with the gibberellin signaling pathway ZmD8 to promote ZmGA2ox4 / 10 The expression of these genes maintains the level of active gibberellins in vivo. Through the aforementioned triple mechanism, ZmLBD19 and ZmLBD40 achieve precise regulation of intracellular active gibberellin levels. Compared with existing single-gene semi-dwarfing resources, this dual-gene knockout strategy exhibits significant dwarfing effects, genetic stability, and low pleiotropic negative effects, making it directly applicable to the breeding of varieties resistant to lodging, high-density planting, and mechanized harvesting. Simultaneously, the male-sterile characteristics of the double mutants combine the advantages of both a dwarf parent and a sterile maternal parent, simplifying the hybrid seed production process, reducing costs, and improving seed purity. The gene targets, editing methods, and mutant materials provided by this invention together constitute a complete technical system for the precise improvement of maize plant height and fertility, playing a significant role in promoting the green revolution in maize and the upgrading of the hybrid seed production industry.

[0044] References: Asami T. Toward the next step to the New Green Revolution. Mol Plant.2023; 16(5):802-803. Spielmeyer W, Ellis MH, Chandler PM. Semidwarf (sd-1), "green revolution" rice, contains a defective gibberellin 20-oxidase gene. Proc Natl Acad Sci U S A. 2002; 99(13):9043-8. Chen C, Yang S, Zhang R, Tian Y, Zhang H, Wang Y, Zhang H, Wang Z,Yang Q. qPH1.01, a dominant QTL that contributes to plant height and ear height in maize. Plant Genome. 2025; 18(4):e70157. Jiao S, Mamidi S, Chamberlin MA, Beatty M, Thatcher S, Simcox KD,Maina F, Wang-Nan H, Johal GS, Heetland L, Marla SR, Meeley RB, Schmutz J,Morris GP, Multani DS. Parallel tuning of semi-dwarfism via differential splicing of Brachytic1 in commercial maize and smallholder sorghum. New Phytol. 2023; 240(5):1930-1943. Mu Q, Wei J, Longest HK, Liu H, Char SN, Hinrichsen JT, Tibbs-CortesLE, Schoenbaum GR, Yang B, Li X, Yu J. A MYB transcription factor underlying plant height in sorghum qHT7.1 and maize Brachytic 1 loci. Plant J. 2024; 120(5):2172-2192. Wu Suowei, An Xueli, Wan Xiangyuan. Mechanism of male sterility in maize and its application in engineered nuclear male sterility seed production. Acta Botanica Sinica, 2024, 59 (6): 932-949. Hou Q, An X, Dong Z, et al. ZmMS1 coordinates ROS homeostasis, lipidallocation, and male fertility for maize breeding applications. J IntegrPlant Biol 2026; DOI: 10.1111 / jipb.70303. Xu C, Luo F, Hochholdinger F. LOB Domain Proteins: Beyond LateralOrgan Boundaries. Trends Plant Sci. 2016; 21(2):159-167. Fan M, Xu C, Xu K, Hu Y. LATERAL ORGAN BOUNDARIES DOMAINtranscription factors direct callus formation in Arabidopsis regeneration. Cell Res. 2012; 22(7):1169-80. Kim MJ, Kim M, Lee MR, Park SK, Kim J. LATERAL ORGAN BOUNDARIESDOMAIN (LBD)10 interacts with SIDECAR POLLEN / LBD27 to control pollendevelopment in Arabidopsis. Plant J. 2015; 81(5):794-809. Thatcher LF, Powell JJ, Aitken EA, Kazan K, Manners JM. The lateralorgan boundaries domain transcription factor LBD20 functions in Fusarium wiltSusceptibility and jasmonate signaling in Arabidopsis. Plant Physiol. 2012;160(1):407-18. Zhang F, Tao W, Sun R, Wang J, Li C, Kong X, Tian H, Ding Z. PRH1mediates ARF7-LBD dependent auxin signaling to regulate lateral rootdevelopment in Arabidopsis thaliana. PLoS Genet. 2020; 16(2):e1008044. Xu C, Tai H, Saleem M, Ludwig Y, Majer C, Berendzen KW, Nagel KA,Wojciechowski T, Meeley RB, Taramino G, Hochholdinger F. Cooperative actionof the paralogous maize lateral organ boundaries (LOB) domain proteins RTCSand RTCL in shoot-borne root formation. New Phytol. 2015; 207(4):1123-33. Hochholdinger F, Yu P, Marcon C. Genetic Control of Root SystemDevelopment in Maize. Trends Plant Sci. 2018; 23(1):79-88. An X, Ma B, Duan M, et al. Molecular regulation of ZmMs7 required formaize male fertility and development of a dominant male-sterility system inmultiple species. Proc Natl Acad Sci U S A 2020; 117(38):23499-509。

Claims

1. Knock out corn ZmLBD19 and ZmLBD40 The application of genes in the synergistic breeding of dwarf male-sterile lines is characterized by, The ZmLBD19 and ZmLBD40 The nucleotide sequences of the gene are shown in SEQ ID NO.1 and NO.2, respectively. ZmLBD19 and ZmLBD40 The amino acid sequences encoded by the gene are shown in SEQ ID NO.3 and NO.4, respectively, or the corn described. ZmLBD19 The gene comprises a nucleotide sequence in which one or more nucleotides have been substituted but which encode the same amino acids as SEQ ID NO.3, in the corn ZmLBD40 The gene consists of a nucleotide sequence in which one or more nucleotides have been substituted but which encode the same amino acid as NO.4; knockout is performed using CRISPR / Cas9 gene editing technology for targeted removal. ZmLBD19 The target sequences of the gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, targeting... ZmLBD40 The target sequences of the gene are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The application according to claim 1, characterized in that, The cultivation of dwarf male-sterile lines refers to: simultaneously knocking out ZmLBD19 and ZmLBD40 Gene or repression ZmLBD19 and ZmLBD40 When the gene is expressed, maize plants exhibit pollen sterility, significantly reduced plant height, shortened internodes, inhibited cell elongation and division, and decreased levels of active GA in the plant; when any one of these genes is knocked out or inhibited, maize plants exhibit normal pollen fertility and plant height.

3. A method for creating a dwarf male-sterile line of maize, characterized in that, Simultaneous knockout of the maize described in claim 1 using CRISPR / Cas9 gene editing technology ZmLBD19 and ZmLBD40 Genes were used to obtain homozygous biallelic mutants, which exhibited pollen sterility, significantly reduced plant height, decreased active GA content, and the dwarfing phenotype could be restored by exogenous GA application; CRISPR / Cas9 gene editing technology was used to target... ZmLBD19 The target sequences of the gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively, targeting... ZmLBD40 The target sequences of the gene are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

4. A method for breeding maize hybrids using dwarf male-sterile lines, characterized in that, Includes the following steps: (1) Obtained by the method of claim 3 Zmlbd19 / Zmlbd40 The double mutant is a dwarf male-sterile donor parent that is crossed with the target superior maize inbred line; (2) Through backcrossing and molecular marker-assisted selection, mutants are selected. ZmLBD19 and ZmLBD40 Genes were introduced into the target inbred line to obtain an improved inbred line carrying a homozygous double mutation. This improved line is characterized by dwarfism and male sterility. (3) Using the improved inbred line obtained in step (2) as the sterile maternal parent, crossbreed with other superior inbred lines to screen out hybrid combinations with yield traits that are not significantly different from the wild-type control or have higher yields than the control.

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