Maize stomatal density regulation gene zmbhlh140, superior haplotype thereof and application

Abstract

This invention discloses a maize stomatal density regulating gene. ZmbHLH140 This invention, focusing on superior haplotypes and their applications, aims to address the technical problems of insufficient basic understanding of the genetic regulation of maize stomatal traits and the lack of superior haplotypes that can be directly used for molecular breeding. Through linkage mapping, genome-wide association analysis, and single-cell nuclear transcriptome data, this invention screens for superior haplotypes and their applications. ZmbHLH140 Gene; quantitative analysis of mutant phenotypes confirmed that this gene is a positive regulator of stomatal density, and its loss of function leads to a significant decrease in stomatal density; and a superior haplotype significantly associated with low stomatal density was identified. This invention discloses for the first time... ZmbHLH140 The gene regulates the biological function of maize stomatal density and can be used as a target to reduce stomatal density and decrease transpiration water loss. The identified superior haplotypes can be used as molecular markers for early screening of maize stomatal traits. This gene provides key gene resources and molecular markers for breeding drought-resistant maize varieties with high water use efficiency.

Description

Technical Field

[0001] This invention relates to the field of molecular-assisted breeding technology, specifically to a maize stomatal density regulating gene. ZmbHLH140 Its superior haplotypes and applications. Background Technology

[0002] Stomata are tiny pores on the epidermis of plant leaves, surrounded by a pair of guard cells. They maintain a dynamic balance between photosynthesis and transpiration by regulating carbon dioxide absorption and water loss. The density, size, and arrangement of stomata directly affect the photosynthetic efficiency, water use efficiency, and adaptability to adverse conditions such as drought and heat resistance in crops, making them important targets for crop genetic improvement.

[0003] Currently, research on stomatal development regulatory networks mainly focuses on the model plant Arabidopsis thaliana. Existing technologies have revealed a set of conserved signal transduction and transcriptional regulatory mechanisms, including EPF family signal peptides, ERECTA family receptor kinases, TMM receptors, and bHLH transcription factors SPCH, MUTE, FAMA, and their interacting proteins ICE1 / SCRM2, etc. These factors synergistically regulate the initiation, proliferation, and differentiation of stomatal lineage cells. Studies in gramineous plants such as rice and maize have shown that some Arabidopsis stomatal regulatory homologs are functionally conserved, but gramineous plants exhibit unique regulatory features, such as subsidiary cell formation and stomatal arrangement in rows, suggesting the existence of gramineous-specific regulatory factors. Existing technologies have reported genes involved in maize stomatal polarity division, such as PAN1, PAN2, ROP2 / ROP9, and the ARP2 / 3 complex, but these genes mainly focus on cytoskeleton rearrangement and cell polarity establishment.

[0004] However, current technologies still lack a systematic understanding of the genetic regulatory basis of stomatal density and arrangement patterns. Maize, as a globally important food crop and a C4 model plant, possesses rich genetic variation in its natural populations, but an integrated genetic regulatory system based on natural variation in stomatal traits has not yet been established, and stable quantitative trait loci and superior haplotypes that can be directly applied to molecular breeding are also lacking. Therefore, identifying key genes regulating stomatal density in maize and analyzing its natural variation and haplotype effects has significant theoretical value and application prospects for breeding drought-resistant maize varieties with high water use efficiency.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the background technology of this disclosure and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] This invention addresses the technical problems of insufficient basic understanding of the genetic regulation of stomatal traits in maize, and the lack of stable quantitative trait loci and superior haplotypes that can be directly used for molecular breeding. It employs a method that utilizes natural variation populations of maize to screen key candidate genes through linkage mapping, genome-wide association analysis, and single-cell nuclear transcriptome data. ZmbHLH140 Through techniques such as mutant phenotypic quantification, association analysis, haplotype analysis, and selection evolution analysis for functional verification, the study achieved its first-ever revealing... ZmbHLH140 The technical effect of gene regulation of maize stomatal density and identification of superior haplotypes significantly associated with low stomatal density.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: Provide a gene for regulating maize stomatal density ZmbHLH140 Its CDS sequence is shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 90% sequence homology and the same function.

[0008] The present invention also provides an isolated nucleic acid molecule, said nucleic acid molecule being selected from the group consisting of: (a) The nucleotide sequence described above; (b) A nucleotide sequence complementary to (a); (c) A nucleotide sequence that hybridizes with (a) or (b) under strict conditions and encodes a protein with the same function.

[0009] The present invention also provides an expression cassette or recombinant vector comprising the aforementioned nucleic acid molecule.

[0010] The present invention also provides an engineered host cell whose genome integrates the aforementioned nucleic acid molecules, or contains the aforementioned expression cassette or recombinant vector.

[0011] This invention also provides a molecular marker combinatorial structure that is significantly linked to the low stomatal density trait in maize, which is present in ZmbHLH140 Haplotypes of nonsynonymous genomic regions and 5' UTR regions, which contain the following SNP sites: SNP-23 located in the 5' UTR region, allele C or A; The SNP+2347 site, located in a non-synonymous region, has alleles G or A. The SNP+3355 site, located in a non-synonymous region, has alleles C or T. Among them, the haplotype Hap5, which consists of the A allele at SNP-23, the G allele at SNP+2347, and the T allele at SNP+3355, is significantly positively correlated with the low stomatal density phenotype in maize.

[0012] The present invention also provides a kit for detecting the molecular marker combination, comprising primer pairs, probes, or primer-probe combinations capable of specifically recognizing or amplifying the SNP-23, SNP+2347, and SNP+3355 sites.

[0013] The present invention also provides the gene. ZmbHLH140 The use of the nucleic acid molecule, the expression cassette or recombinant vector, the host cell, or the combination of molecular markers in at least one of the following: (a) Preparation of a detection reagent for identifying maize stomatal density genotypes or haplotypes; (b) Controlling the stomatal density and / or stomatal row number in maize; (c) Screening or breeding maize varieties / lines with reduced stomatal density; (d) Reduce water loss through transpiration in maize, improve water use efficiency, or cultivate drought-resistant maize varieties.

[0014] This invention also provides a non-naturally occurring maize plant material whose genome contains any of the following features: (1) Carrying the haplotype Hap5; (2) The gene ZmbHLH140 Loss-of-function mutations or suppressed expression levels result in a significant reduction in stomatal density and / or stomatal row number compared to wild-type controls; (3) The plant material was obtained through molecular marker-assisted selection, gene editing or genetic transformation technology, and its stomatal density phenotype showed a decrease.

[0015] This invention also provides a breeding method for improving the stomatal density trait of maize, comprising the following steps: (a) Genotyping of the maize germplasm to be tested using the aforementioned molecular marker combination; (b) Select individuals carrying the haplotype Hap5 as parental materials; (c) The haplotype Hap5 is introduced into the target maize variety by hybridization, backcrossing or molecular marker-assisted selection to obtain a maize line with reduced stomatal density.

[0016] This invention also provides a method for regulating maize stomatal development through genetic engineering, comprising: Constructing a gene targeting ZmbHLH140 The editing or expression medium; The vector was introduced into maize recipient cells to obtain genetically modified maize plants; Among them, when the reduction of the ZmbHLH140When gene expression activity is reduced or loss-of-function mutations are introduced, the stomatal density and / or stomatal row number of the maize plant decreases, transpiration water loss decreases, and water use efficiency increases.

[0017] One or more technical solutions provided in the embodiments of this application have at least one of the following technical effects or advantages: 1. Revealing gene function and identifying superior haplotypes: This application demonstrates through quantitative analysis of mutant phenotypes that... ZmbHLH140 Loss of gene function led to a significant decrease in stomatal density in maize, confirming for the first time that this gene is a positive regulator of stomatal density. Furthermore, haplotype analysis of 262 maize inbred lines identified five major haplotypes. Among them, the Hap5 haplotype (carrying the A allelic variant at SNP-23, the G allelic variant at SNP+2347, and the T allelic variant at SNP+3355) was significantly associated with low stomatal density. Molecular markers developed based on these SNP sites can be directly used for marker-assisted breeding.

[0018] 2. Constructing an integrated genetic regulatory system to provide breeding resources: This application comprehensively utilizes linkage mapping, genome-wide association analysis, single-cell nuclear transcriptome data, and haplotype analysis to construct an integrated genetic regulatory system for stomatal trait variation in maize. This system not only provides a generalizable technical paradigm for the genetic analysis of maize stomatal traits, but also... ZmbHLH140 The gene and its superior haplotype can be used to reduce maize stomatal density, reduce transpiration water loss, and improve water use efficiency, and have direct application value in drought-resistant maize molecular breeding. Attached Figure Description

[0019] Figure 1 This is a phenotypic variation of stomatal traits in a population according to an embodiment of this application; wherein, A: representative stomatal map of inbred lines (scale bar = 200 μm); B: correlation matrix of stomatal traits in recombinant inbred line populations; C: correlation matrix of stomatal traits in associated populations.

[0020] Figure 2 This is the genomic distribution of QTLs for stomatal traits in one embodiment of this application.

[0021] Figure 3 This is a QTL co-localized in linkage and associated populations in one embodiment of this application; where A, C, E: stomatal trait localization of linkage populations, and blue arrows indicate QTLs detected in multiple populations; B, D, F: QTL intervals co-localized in associated populations, the top row is the Manhattan plot (B, F: SAR, D: RN), the middle row is the SNP density within the interval, and the bottom row is the linkage disequilibrium mode.

[0022] Figure 4 As shown in one embodiment of this application ZmbHLH140Functional loss reduces maize stomatal density; among which, A: ZmbHLH140 Gene structure and EMS mutation and natural variation sites; B: Representative stomatal diagrams of wild-type and zmbhlh140 mutant maize leaves at the three-leaf stage (scale bar = 200 μm); CD: Comparison of stomatal density and stomatal arrangement number between wild-type and zmbhlh140 mutant; E: ZmbHLH140 Candidate gene association analysis results; F: Haplotype analysis results; G; ZmbHLH140 Nucleotide polymorphism analysis. Detailed Implementation

[0023] The following examples illustrate specific implementations of the present invention. However, these examples are merely for illustrative purposes and do not limit the scope of the invention in any way.

[0024] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; unless otherwise specified, the reagents and materials involved are all commercially available conventional products; unless otherwise specified, the test and detection methods involved are all conventional methods.

[0025] This application utilizes naturally occurring maize populations to construct an integrated genetic regulatory system for stomatal trait variation. Through linkage mapping of multiple recombinant inbred lines and genome-wide association analysis of diverse natural populations, combined with cell type-specific single-cell transcriptome data, stable quantitative trait loci (QTLs) controlling stomatal density, size, and arrangement were identified, and priority candidate genes were selected. ZmbHLH140 Preliminary functional validation, haplotype and selection analyses were conducted, and the utilization pathways of candidate genes were explored. This application reveals a novel genetic structural mechanism of stomatal arrangement patterns in maize, and lays a theoretical foundation for mechanistic research and breeding strategies to improve crop performance under drought and water scarcity conditions.

[0026] Example 1: Genetic variation of stomatal traits To elucidate the genetic structure of stomatal traits in maize, this study analyzed three recombinant inbred line (RIL) populations constructed by Professor Yan Jianbing's team at Huazhong Agricultural University: BYK (K22×By815, 163 lines), BYD (DE3×By815, 142 lines), and SZ (Z58×SK, 105 lines). Each population contained 105–163 lines. These populations were constructed by crossing superior inbred lines with significantly different genetic backgrounds, providing independent research materials for linkage mapping and enabling the identification of both population-shared and population-specific quantitative trait loci (QTLs). Simultaneously, a diverse associated population containing 262 maize inbred lines was used to conduct genome-wide association studies (GWAS) to improve mapping resolution and finely map candidate loci. This integrated experimental system, combining two complementary genetic research methods, can systematically and reliably resolve the natural variation in stomatal traits.

[0027] Statistical analysis was performed on six stomatal-related traits, including stomatal density (SD), stomatal length (SL), stomatal width (SW), stomatal area (SA), stomatal row number (RN), and stomatal arrangement pattern (SAR). Figure 1 A). In the three RIL populations, all traits showed extensive and continuous phenotypic variation, approximately conforming to a normal distribution. Stomatal density, stomatal area, and stomatal row number exhibited the highest variation, while stomatal length, stomatal width, and stomatal arrangement showed moderate variation. Correlation analysis revealed a stable and consistent pattern across all RIL populations: stomatal density was significantly positively correlated with stomatal row number, and significantly negatively correlated with stomatal length, width, and area. Figure 1 B); however, stomatal arrangement was not significantly correlated with any of the other measured traits. These characteristics indicate an inherent trade-off between stomatal density and size in maize, suggesting that stomatal development is synergistically regulated, rather than independently controlled by individual traits. The associated population also exhibited rich phenotypic variation, and the genetic correlation was completely consistent with the RIL population (B). Figure 1 (BC). The pore density is still positively correlated with the number of pore rows and negatively correlated with the pore length / width / area, while the pore arrangement remains independent.

[0028] Example 2: Identification of quantitative trait loci (QTL) for stomatal traits A total of 39 non-redundant QTLs were identified from three recombinant inbred line (RIL) populations, corresponding to 55 phenotypic QTLs for six stomatal traits. These QTLs were most abundant on chromosomes 1, 2, and 5, with only one QTL on each of chromosomes 7 and 8. A single QTL could explain 5.08%–16.87% of the phenotypic variation. Fourteen loci with a phenotypic contribution rate (PVE) greater than 10% were major-effect loci, distributed on chromosomes 1, 2, 3, 5, and 6. Figure 2Five genomic regions that were stable in at least two populations were identified: chromosome 1 (qSL1-1a / qSAR1-1b), chromosome 2 (qRN2-1b / qSA2-1a / qSL2-1a), chromosome 4 (qSW4-1b / qRN4-1a; qRN4-1b / qSA4-1b / qSL4-1c), and chromosome 9 (qSL9-2c / qSW9-1a / qSA9-1a). Furthermore, four regions (qSA3-1c / qSW3-1c, qSA3-2c / qSW3-2c, qSL5-1b / qSD5-3b, and qSL6-2a / qSA6-1a) in a single population were simultaneously associated with multiple stomatal size traits, indicating the existence of pleiotropic sites regulating stomatal morphology covariation.

[0029] GWAS identified 128 significant non-redundant single nucleotide polymorphisms (SNPs), and integration yielded 51 non-redundant associated sites. Figure 2 The locus chr2.s_214800421 on chromosome 2 was associated with both stomatal length and stomatal area. Linkage and association analyses identified seven QTL regions located on chromosomes 5 (qRN5-1a / qRN5-2a, qSD5-3b), 2 (qRN2-2b), 3 (qSL3-1a), 4 (qRN4-1b / qSA4-1b / qSL4-1c), and 6 (qSL6-1a).

[0030] Example 3: Prioritization screening of candidate genes within QTL intervals To uncover biological mechanisms from mapped stomatal trait QTLs, this study functionally annotated candidate genes within linkage and associated regions and employed an integrated screening strategy: combining known stomatal regulatory factor homology, gene functional annotation, and cell type-specific expression data for comprehensive screening. This strategy significantly narrowed the candidate gene pool, allowing for targeted analysis of key genes most likely involved in natural variations in stomatal development. Emphasis was placed on QTL regions with repeated detection across multiple populations, pleiotropic effects, linkage, and GWAS validation.

[0031] Within the pleiotropic region on chromosome 4 that simultaneously regulates stomatal row number and size traits ( Figure 2 , 3 A bHLH transcription factor was identified, homologous to SPEECHLESS, the core regulator of stomatal lineage initiation in Arabidopsis thaliana; an EPFL (epidermal pattern factor-like protein) gene was identified within the QTL region related to stomatal density and arrangement. This type of gene is a secreted signal peptide that jointly regulates stomatal spacing and density in dicotyledonous and gramineous plants. Figure 3In addition to core regulatory factors, several transcription factors involved in asymmetric cell division and epidermal pattern formation have also been identified, including SCARECROW homologous GRAS family genes that regulate stereotyped cell division and tissue formation in Arabidopsis and Poaceae. Figure 2 , 3 ).

[0032] Furthermore, genes related to cell wall synthesis and modification were significantly enriched. Multiple stable QTL regions regulating stomatal density, size, and row number were identified, including genes encoding cellulose synthase, xylan synthesis components (CESA6, IRX15A), and polysaccharide modification-related enzymes (…). Figure 2 The dumbbell-shaped guard cells unique to grasses and their coordinated development with subsidiary cells require precise regulation of cell wall deposition, rigidity, and anisotropic expansion. Therefore, allelic variations in genes regulating primary and secondary cell wall formation can simultaneously affect the number and final size of stomata, providing a molecular mechanism explanation for the pleiotropic effects of multiple QTL regions. Figure 2 The candidate genes for pleiotropic QTL regions were mostly enriched in pathways involving cell division, cell expansion, and cell structure formation, consistent with the synergistic regulation of stomatal development patterns and morphology. This result indicates that the inherent trade-off between stomatal number and size is mediated by shared developmental processes, rather than controlled by independent genetic modules.

[0033] The identification of sucrose transport proteins and calcium ion signaling pathway ion channel-related proteins related to stomatal function and guard cell physiological activities suggests that stomatal development is associated with overall plant physiological processes, and these genes are located within the QTL regions that regulate stomatal density and arrangement. Figure 2 , 3 Although its direct function in stomatal pattern formation requires further verification, it indicates a synergistic integration of developmental programs with metabolic and signaling states during epidermal differentiation. In summary, natural variation in maize stomatal traits is driven by three components: conserved developmental regulators, cell wall synthesis pathways, and pleiotropic sites that synergistically control stomatal density and size. These conclusions provide a reasonable theoretical basis for targeted functional verification of candidate genes with significant differences in stomatal developmental differentiation.

[0034] Example 4: Functional Verification of Key Genes for Stomatal Traits To determine whether the selected candidate genes are key genes regulating stomatal development, the candidate genes were analyzed. ZmbHLH140 Mutant phenotypic quantification, candidate gene association, haplotype analysis, and selection evolution analysis were performed.

[0035] ZmbHLH140Encoding a basic helical-loop-helical transcription factor (bHLH), it is a candidate gene for stomatal density and stomatal row number. Its ethyl methanesulfonate (EMS) mutagenic allele has a C-to-T mutation in exon 3, leading to premature termination of protein translation. Figure 4 A). Compared with the wild type, the homozygous bhlh140 mutant showed significantly reduced stomatal density and stomatal row number. Figure 4 BD), indicating ZmbHLH140 It is a key gene that regulates stomatal development.

[0036] Further utilization ZmbHLH140 Candidate gene association analysis was performed on nucleotide polymorphisms (SNPs) in 262 maize inbred lines, and two non-synonymous SNPs (SNP2347 and SNP3355) were identified as significantly associated with stomatal density. Figure 4 E). Consists of two non-synonymous SNPs (SNP2347: G / A, GACA) A CCATG; SNP3355: C / T, ATCT C CCTTC) and SNPs in the 5'UTR (SNP-23:C / A, ACA) C CAGGTT classified the inbred lines into 5 haplotypes (Hap), among which Hap4 showed a significantly increased stomatal density compared to Hap1. Figure 4 F). Nucleotide polymorphism analysis using HapMap3 data showed that... ZmbHLH140 Nucleotide polymorphisms in maize are significantly lower than those in its wild relative, *Tegus spp.* P =2.64E-11), indicating that this gene has been selected during the genetic improvement process. Figure 4 G). In summary, ZmbHLH140 It is a positive regulator of stomatal density and stomatal row number, and its natural variation has driven phenotypic diversity in the evolution of maize.

[0037] Although some preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0038] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this application and their equivalents, this invention is also intended to include these modifications and variations.

Claims

1. A gene regulating maize stomatal density ZmbHLH140 Its characteristics are, Its CDS sequence is shown in SEQ ID NO: 1, or a nucleotide sequence that has at least 90% sequence homology and the same function.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecules are selected from the following group: (a) The nucleotide sequence as described in claim 1; (b) A nucleotide sequence complementary to (a); (c) A nucleotide sequence that hybridizes with (a) or (b) under strict conditions and encodes a protein with the same function.

3. An expression cassette or recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 2.

4. An engineered host cell, characterized in that, Its genome integrates the nucleic acid molecule of claim 2, or contains the expression cassette or recombinant vector of claim 3.

5. A molecular marker combinatorial structure significantly linked to the low stomatal density trait in maize, characterized in that, It exists ZmbHLH140 Haplotypes of nonsynonymous genomic regions and 5' UTR regions, which contain the following SNP sites: SNP-23 located in the 5' UTR region, allele C or A; The SNP+2347 site, located in a non-synonymous region, has alleles G or A. The SNP+3355 site, located in a non-synonymous region, has alleles C or T. Among them, the haplotype Hap5, which consists of the A allele at SNP-23, the G allele at SNP+2347, and the T allele at SNP+3355, is significantly positively correlated with the low stomatal density phenotype in maize.

6. A kit for detecting the molecular marker combination of claim 5, characterized in that, It contains primer pairs, probes, or primer-probe combinations that can specifically recognize or amplify the SNP-23, SNP+2347, and SNP+3355 sites.

7. The gene according to claim 1 ZmbHLH140 The use of the nucleic acid molecule of claim 2, the expression cassette or recombinant vector of claim 3, the host cell of claim 4, or the combination of molecular markers of claim 5 in at least one of the following: (a) Preparation of a detection reagent for identifying maize stomatal density genotypes or haplotypes; (b) Controlling the stomatal density and / or stomatal row number in maize; (c) Screening or breeding maize varieties / lines with reduced stomatal density; (d) Reduce water loss through transpiration in maize, improve water use efficiency, or cultivate drought-resistant maize varieties.

8. A non-naturally occurring corn plant material, characterized in that, Its genome contains any of the following features: (1) Carrying the haplotype Hap5 as described in claim 5; (2) The gene ZmbHLH140 Loss-of-function mutations or suppressed expression levels result in a significant reduction in stomatal density and / or stomatal row number compared to wild-type controls; (3) The plant material was obtained through molecular marker-assisted selection, gene editing or genetic transformation technology, and its stomatal density phenotype showed a decrease.

9. A breeding method for improving the stomatal density trait of maize, characterized in that, Includes the following steps: (a) Genotyping of maize germplasm to be tested using the molecular marker combination described in claim 5; (b) Select individuals carrying the haplotype Hap5 as parental materials; (c) The haplotype Hap5 is introduced into the target maize variety by hybridization, backcrossing or molecular marker-assisted selection to obtain a maize line with reduced stomatal density.

10. A method for regulating maize stomatal development through genetic engineering, characterized in that, include: Constructing a target gene according to claim 1 ZmbHLH140 The editing or expression medium; The vector was introduced into maize recipient cells to obtain genetically modified maize plants; Among them, when the reduction of the ZmbHLH140 When gene expression activity is reduced or loss-of-function mutations are introduced, the stomatal density and / or stomatal row number of the maize plant decreases, transpiration water loss decreases, and water use efficiency increases.

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