Application of GhNAP gene in regulating cotton stigma exposure rate and male fertility

Abstract

The application discloses application of a GhNAP gene in regulation of cotton stigma exposure rate and male fertility, and belongs to the technical field of plant genetic engineering and crop genetic breeding. The application identifies an important transcription factor NAC member GhNAP involved in regulation of cotton stamen development, and determines that the transcription factor is involved in the synergic regulation of cotton stigma exposure and pollen fertility by regulating the process of tapetum cell programmed death and the development of filaments. The application enriches the function research of the transcription factor NAC family in cotton stamen development, preliminarily reveals the molecular mechanism of the GhNAP in regulation of cotton stamen development, provides a research basis for further analyzing the molecular regulation network of cotton anther development, and provides potential gene resources for creation of cotton male sterile materials.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and crop genetics and breeding technology, and in particular relates to the application of the GhNAP gene in regulating the stigma exposure rate and male fertility of cotton. Background Technology

[0002] Cotton is an important economic crop in my country, and utilizing heterosis is a key approach to improving cotton yield and quality. Stigma exposure rate and male sterility directly affect hybrid seed production efficiency, and both traits are related to stamen development: stigma exposure is often accompanied by shortened filaments or abnormal anther development, while male sterility manifests as pollen abortion or anther indehiscent. The NAC transcription factor family plays an important role in plant growth and development, but its function in cotton stamen development remains unclear.

[0003] In existing technologies, Sablowski et al. (1998) reported that overexpression of NAP in Arabidopsis thaliana leads to shortening of petals and stamens, and that NAP regulates floral organ size by affecting cell expansion. Fan Kai (2015) cloned the GhNAP gene from upland cotton, confirming that it regulates leaf senescence through the ABA-mediated pathway and can serve as a marker gene for cotton leaf senescence. Cheng et al. (2020) identified an NAC transcription factor (Gh_D11G2469) as a hub gene in transcriptome analysis of cotton dominant nuclear male sterile materials, but did not perform any functional verification. Therefore, the function of GhNAP in cotton stamen development is completely unknown, and existing technologies only involve the regulation of leaf senescence. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide the application of the GhNAP gene in regulating the stigma exposure rate and male fertility in cotton.

[0005] This invention is implemented as follows: First, this invention provides the application of the GhNAP gene in regulating the stigma exposure rate of cotton, by overexpressing the GhNAP gene in cotton to increase the stigma exposure rate.

[0006] Furthermore, the relative expression level of the GhNAP gene was significantly positively correlated with the stigma exposure index. R ² = 0.769, P <0.001.

[0007] Secondly, this invention provides the application of the GhNAP gene in regulating male fertility in cotton by overexpressing the GhNAP gene in cotton to reduce pollen viability.

[0008] Furthermore, the overexpression of the GhNAP gene leads to shrunken and deformed pollen grains with increased fractal dimension.

[0009] This invention also provides the application of the GhNAP gene in regulating the stigma exposure rate and male fertility of cotton. Overexpression of GhNAP simultaneously causes cotton to exhibit a dual phenotype of increased stigma exposure rate and male sterility.

[0010] Finally, the present invention provides a method for creating male-sterile cotton materials, characterized by overexpressing the GhNAP gene in cotton, thereby inducing both male sterility and stigma exposure phenotypes.

[0011] Furthermore, the male sterility is characterized by decreased pollen viability and abnormal anther development, ultimately leading to pollen abortion due to delayed programmed cell death in the tapetum.

[0012] Furthermore, the exposed column head is characterized by an increase in the length and rate of exposed column head, and the relative expression level of GhNAP is significantly positively correlated with the comprehensive index of exposed column head.

[0013] The present invention has the following advantages: This invention identified GhNAP, an important transcription factor member of the NAC family involved in the regulation of cotton stamen development, and clarified that it participates in the synergistic regulation of stigma exposure and pollen fertility by regulating programmed cell death in the tapetum and filament development. The results obtained in this invention enrich the functional research of the NAC family of transcription factors in cotton stamen development, and also provide potential genetic resources for the creation of male-sterile cotton materials. Attached Figure Description

[0014] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0015] Figure 1 Cloning of cotton transcription factor GhNAP. (A) Amplification products of transcription factor GhNAP using whole genome DNA and cDNA as templates. Lane M is the DL 2000 DNA Marker, lane 1 is the PCR product using DNA as a template, lane 2 is the PCR product using cDNA as a template, lane 3 is the DNA blank control, and lane 4 is the cDNA blank control; (B) Gene structure analysis of cotton transcription factor GhNAP. Yellow rectangles and black lines represent exons and introns, respectively.

[0016] Figure 2Molecular identification of cotton lines overexpressing GhNAP and those with gene-edited GhNAP. (A) Schematic diagram of the GhNAP overexpression vector. (B) Genomic DNA PCR identification results of the overexpressing cotton lines. Lane M: DL2000 DNA Marker; Lane 1 is the negative control, Lane 2 is the positive control, Lanes 3-11: GhNAP overexpressing lines OE1-OE9. (C) RT-PCR identification results of the overexpressing cotton lines, lane order as described above. (D) qRT-PCR detection of the relative expression levels of GhNAP in wild-type (WT) and each overexpressing line. Data are the mean ± standard deviation (Mean ± SD) of three independent biological replicates. Different lowercase letters indicate statistically significant differences between groups. P <0.05). (E) Schematic diagram of GhNAP gene structure and CRISPR / Cas9 editing target location.

[0017] Figure 3 Effects of .GhNAP genetic modification on cotton flower organ morphology. Phenotypic images of flower buds from wild-type (WT), gene-edited lines (KO1, KO2), and overexpression lines (OE). Images are arranged in rows by genotype, clearly showing the morphological differences in pistils and stamens (especially filament length) under different genetic backgrounds. Scale bar = 1 cm.

[0018] Figure 4 Effects of cotton transcription factor GhNAP on cotton floral organ morphology. (A) Schematic diagram of cotton floral organ measurement indicators, showing parameters such as stigma exsertion length, minimum distance from anther to stigma, stamen area, and widest length of the two stamens. (B–E) Statistical analysis of floral traits in wild-type (WT), GhNAP-overexpressing lines (OE1–OE9), and gene-edited lines (KO1, KO2), showing stigma exsertion length, stigma exsertion rate, minimum distance from anther to stigma, and stamen area, respectively. (F) Statistical analysis of the widest length of the two stamens in each line. (G) Correlation analysis between relative GhNAP expression level and stigma exsertion index. Data are presented as mean ± standard error (Mean ± SE, n=10), with different lowercase letters indicating significant differences between groups. P < 0.05).

[0019] Figure 5The cotton transcription factor GhNAP affects anther development. (A) Statistical analysis of the proportion of dehiscent (MF, fertile, blue) and non-dehiscent (MS, sterile, red) flowers in each GhNAP overexpression line; (B) Box plot of male sterility rate in GhNAP high-expression and low-expression lines in GhNAP overexpression lines; (C) Correlation analysis of relative GhNAP expression level and male sterility rate, with the dashed line indicating an expression threshold of approximately 11; (D) Quantitative analysis of relative GhNAP expression level in non-dehiscent (MS, red) and dehiscent (MF, blue) flowers in GhNAP overexpression lines. (E) Phenotypic characteristics of floral organs in each strain (top: open flowers in situ in the field; middle: intact flowers harvested from the anatomical site; bottom: internal structure of the flower after removing petals); (F) Photographs of the overall morphology of flower buds in each strain; (G) Comparison of anther morphology and dehiscence in each strain under a stereomicroscope; (H) TTC staining method for detecting pollen activity in each strain (red indicates active pollen, brown indicates inactive pollen); (I) Statistical analysis of pollen activity ratio in each strain; (J) Observation of pollen grain morphology and surface structure in each strain under a scanning electron microscope; (K) Fractal dimension (FD) analysis of pollen grains in each strain. WT represents wild type, KO1 / KO2 represents gene-edited strains, and OE5 / OE7 represents GhNAP overexpression strains. * and ** indicate significant differences between groups ( P < 0.05), highly significant ( P <0.01); different lowercase letters indicate significant differences between groups ( P <0.05. Data are mean ± standard error (n ≥ 5 biological replicates).

[0020] Figure 6 Observation of anther development morphology in wild-type and GhNAP-modified materials. Paraffin sections were used to observe the tapetum morphology of wild-type (WT), mutants (KO1, KO2), and overexpression lines (OE5, OE7) at different stages of anther development (stages 1–6). Magnification was 200×.

[0021] Figure 7 The cotton transcription factor GhNAP regulates programmed cell death (PCD) in anther tapetum cells. TUNEL staining was used to detect the PCD levels in wild-type (WT), mutants (KO1, KO2), and overexpressing lines (OE5, OE7) at different stages of anther development (stages 1-6) (green fluorescence indicates TUNEL positive signal; scale bar: 100 μm). Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified in the embodiments, conditions are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0023] 1. Materials and Methods 1.1 Materials Plant materials and growing environment: This invention uses upland cotton ( Gossypium hirsutum Variety R15 (L.) was used as the wild-type (WT) for gene cloning, expression pattern analysis, physiological index determination, and as a recipient for genetic transformation. Cotton seeds were sown in nutrient soil containing 30% vermiculite and placed in a light incubator with the following parameters: 28 ℃, 60% humidity, 16 h light, 8 h dark. Seedlings were transplanted to the field after reaching the three-leaf stage for subsequent phenotypic observation and sampling. All experimental sampling was conducted under the same growth conditions to ensure data consistency.

[0024] Other biomaterials: pBI121-3×FLAG overexpression vector was purchased from Shanghai Zhishuo Biotechnology Co., Ltd.

[0025] 1.2 Extraction of genomic DNA and total RNA This invention utilizes the CTAB method to extract genomic DNA from fresh tissues of upland cotton R15. Total RNA extraction was performed using an RNA extraction kit specifically designed for polysaccharide and polyphenol plants, strictly following the instructions, and RNA purity was detected using NanoDrop. 1 μg of high-quality total RNA was used to remove genomic DNA contamination using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Japan), and the resulting first-strand cDNA was synthesized via reverse transcription. The product was diluted and stored at -20 °C for subsequent PCR amplification and quantitative fluorescence analysis.

[0026] 1.3 Cloning of members of the transcription factor GhNAP Using upland cotton R15 as the experimental material, the DNA and cDNA sequences of the GhNAP gene were amplified by PCR technology. Specific primers were designed based on the GhNAP gene sequence, and PCR amplification was performed using high-fidelity DNA polymerase. The products were detected by 1% agarose gel electrophoresis. The exon-intron structure of the GhNAP gene was analyzed using the Gene Structure Display Server 1.0 (GSDS) online tool, and a schematic diagram of the gene structure was drawn.

[0027] 1.4 Vector Construction and Plant Transformation This invention designs specific amplification primers based on the CDS sequence of the target gene, performs PCR amplification using cotton GhNAP cDNA as a template, and recovers the target fragment. The target gene is inserted into the pBI121-3×FLAG overexpression vector to obtain the pBI121-GhNAP-3×FLAG recombinant vector. After verification by PCR, enzyme digestion, and sequencing, the positive recombinant plasmid is transformed into Agrobacterium GV3101 competent cells. Cotton recipient materials are then infected using the Agrobacterium-mediated shoot tip transformation method to obtain transgenic overexpressing cotton plants.

[0028] 1.5 Pollen viability assay (TTC method) Pollen can be collected by gently shaking off cotton pollen. Prepare a 1% (w / v) 2,3,5-triphenyltetrazolium chloride (TTC) staining solution (using 0.1 mol / L pH 7.2 phosphate buffer as solvent). Suspend an appropriate amount of pollen in the staining solution and incubate at 35°C in the dark for 30 min. Remove the samples and prepare temporary slides. Observe and photograph them randomly under an optical microscope (10×). Count 5 views for each sample. Determine pollen viability based on staining: deep red or purplish-red pollen grains are considered viable, while colorless, yellow, or shrunken grains are considered inactive. The formula for calculating pollen viability is: Pollen viability (%) = (Number of red pollen grains / Total number of observed pollen grains) × 100%.

[0029] 1.6 Observation of anther paraffin sections and microstructure In this experiment, anthers of upland cotton at different developmental stages (bud length 4–5 mm to 9–10 mm) were collected and fixed in FAA fixative (50% ethanol: glacial acetic acid: formaldehyde = 18:1:1) at 4 ℃ for 24 h before being sent to Wuhan Saiweier Biotechnology Co., Ltd. The company first trimmed the anther tissue, then dehydrated it with gradient ethanol, cleared it with xylene, and impregnated it with paraffin at 65 ℃ before paraffin embedding. After cooling and solidification, the paraffin blocks were trimmed, and 8 μm thick sections were cut using a pathological sectioning machine. The sections were spread flat at 40 ℃, baked at 60 ℃, and then stored at room temperature for later use. Simultaneously, 0.1% (w / v) toluidine blue staining and high-resolution microscopic imaging were performed. Based on the obtained digital microscopic images, this invention focuses on analyzing the programmed cell death process in the tapetum, the meiotic behavior of pollen mother cells, and the developmental morphology of microspores, and performs statistical analysis.

[0030] 1.7 Detection of programmed cell death in anthers To detect programmed cell death (PCD) in the anther tapetum, fixed anther samples were collected (fixation method as described in 1.15). Paraffin sections were prepared and TUNEL in situ detection was performed by Hubei Baiaos Biotechnology Co., Ltd. The company's specific experimental procedure involved dewaxing the paraffin sections to water, followed by Proteinase K treatment, washing with phosphate-buffered saline (PBS), incubation with TUNEL Dilution Buffer, and then incubation with TUNEL reaction mixture. After incubation, the sections were washed with PBS, followed by DAPI staining of the nuclei. The sections were then washed again with PBS, mounted, and finally examined under a microscope. This invention received the stained sections and microscopic images provided by the company. Based on the obtained imaging data, the timing and intensity of PCD in the tapetum cells were further confirmed, and statistical analysis and image processing were performed on typical fields of view.

[0031] 1.8 Scanning electron microscopy observation and fractal dimension analysis of pollen Mature pollen was collected, fixed in electron microscopy (EM) solution, and stored at 4 °C. It was then sent to Wuhan Saiweier Biotechnology Co., Ltd. for processing. The company used PBS to remove surface impurities and marked the scanning planes before fixing the samples in EM fixative at room temperature for 2 hours. The samples were then transferred to 4 °C for storage. Subsequently, the samples were washed three times (15 min each) with 0.1 M PBS buffer (pH 7.4), then fixed with 1% osmium tetroxide prepared in the same buffer at room temperature in the dark for 1-2 hours. Finally, they were washed three times (15 min each) with the same concentration of PBS buffer. The samples were then subjected to a gradient dehydration process of 30%-50%-70%-80%-90%-95%-100%-100% ethanol (15 min each time), and finally treated with isoamyl acetate for 15 min. After dehydration, the samples were dried in a critical point desiccator (the preceding sampling, fixation, and dehydration steps can be omitted for drying samples and inorganic materials). After drying, the samples were tightly adhered to a conductive carbon film double-sided adhesive and placed in an ion sputtering apparatus at 15 °C. Gold was sputtered at mA current for 60 s, and then images of pollen exwall ornamentation and germination pore morphology were observed and acquired using a scanning electron microscope. The images were then imported into ImageJ software, and the surface roughness of the pollen was analyzed by fractal dimension (FD).

[0032] 1.9 Determination of floral organ phenotypic traits Under field planting conditions, after cotton (wild-type R15, overexpressing GhNAP lines, and gene-edited GhNAP lines) entered their peak flowering period, 10 naturally opened fresh flowers were randomly selected from each line (excluding insect-bitten and deformed flowers). The overall morphology of the floral organs and the structure of the stamens and pistils were photographed using a camera. The following indicators were precisely measured using ImageJ software: stigma exposure length (vertical distance of the stigma beyond the stamen cluster), stigma exposure rate (stigma exposure length / total pistil length × 100%), the widest length of the stamens, the total area of ​​the stamens, and the shortest distance between the anther and stigma.

[0033] The column head exposure rate index was normalized using Z-score standardization to eliminate dimensional differences and standardize data scale. Data are expressed as mean ± standard error (Mean ± SE) and were analyzed using SPSS 26 software using one-way ANOVA, with a significance level set to [value missing]. P <0.05; Based on the measured phenotypic indices and the relative expression levels of GhNAP, a correlation model between the two was established through linear regression fitting to clarify the regulatory effect of GhNAP expression on the phenotype of cotton flower organs.

[0034] 2 Results Analysis 2.1 Cloning and structural analysis of the cotton GhNAP gene To obtain the complete coding sequence and genomic structure information of GhNAP, a member of the transcription factor NAC in upland cotton, this invention uses whole-genome DNA and cDNA of upland cotton as templates for PCR amplification, and detects the product size by agarose gel electrophoresis. Figure 1 (A). Based on the genome sequence and coding region sequence, the genome sequence length of GhNAP is 1069 bp; while the coding region sequence length is 861 bp (GenBank: KP300008.1), indicating that this gene contains intron structures in the genome. No amplified bands were observed in the blank control (lanes 3 and 4).

[0035] Further analysis of the GhNAP gene structure ( Figure 1 The genome sequence (B) was found to contain 3 exons and 2 introns. The first exon is about 166 bp long, the second exon is about 281 bp long, and the third exon is about 414 bp long; the two introns are 109 bp and 99 bp long, respectively.

[0036] In summary, the cDNA and genome sequence of the GhNAP gene from upland cotton were successfully cloned, and its basic structural composition was clarified, laying a molecular foundation for subsequent functional verification and expression regulation research.

[0037] 2.2 Creation and Identification of Transgenic Materials Based on Cotton Transcription Factor GhNAP 2.2.1 Screening and Molecular Identification of Cotton Materials Overexpressing GhNAP To verify the function of the transcription factor GhNAP in cotton, this invention constructed a cotton overexpression vector pBI121-GhNAP-3×FLAG ( Figure 2 (A). PCR identification of the DNA of the overexpressing cotton lines showed that ( Figure 2 In both the RT-PCR and PCR-PCR lines, the overexpressing lines amplified specific bands of the same size as the positive control, while the wild-type (negative control) showed no corresponding band. The RT-PCR verification results were completely consistent with the PCR identification. Figure 2 (C). qRT-PCR quantitative analysis showed that ( Figure 2 In the overexpression lines (D), compared with the wild type, the expression level of GhNAP was significantly upregulated in all overexpression lines, with the OE7 line showing the highest expression level.

[0038] 2.2.2 Molecular identification of gene-edited GhNAP cotton lines This invention utilizes CRISPR / Cas9 technology to design dual target sites for gene editing of GhNAP based on the GhNAP gene sequence information. One of the designed target sites is located in the first exon of the GhNAP gene, and the other is located in the second exon. Figure 2 (E). Gene-edited GhNAP cotton plants were obtained using Agrobacterium-mediated transformation. Sequencing analysis of the plants successfully yielded two independent mutant lines, named KO1 and KO2, respectively.

[0039] The sequence alignment results show ( Figure 2 Compared to wild-type (WT), KO1 exhibits a single-base deletion (-C) at 110 bp at target site 1 and a single-base deletion (-G) at 379 bp at target site 2, resulting in reading frame shift; KO2 exhibits a single-base deletion (-C) at 110 bp at target site 1 and a single-base deletion (-T) at 377 bp at target site 2.

[0040] 2.3 Functional Study of Cotton Transcription Factor GhNAP in Cotton Floral Organ Development To analyze the biological functions of GhNAP, this invention divides the floral organ phenotype into two main modules: (1) filament growth and stigma exposure; (2) anther abortion and pollen fertility. The former affects hybridization through the spatial configuration of floral organs, while the latter affects hybridization through male fertility.

[0041] 2.3.1 Cotton transcription factor GhNAP regulates the stigma exhumation rate in cotton. To clarify the role of the transcription factor GhNAP in the morphology of floral organs in cotton, this invention conducted detailed observations and phenotypic analysis of flowers during full bloom in wild-type, overexpressed, and gene-edited GhNAP cotton lines. Phenotypic observations showed that ( Figure 3 Compared to the wild type, the overexpressing lines showed significantly shrunken filaments, resulting in a smaller overall stamen area and a stigma position that was significantly higher than the stamen coverage height, exhibiting a distinct "stigma exposed" phenotype.

[0042] The schematic diagram of floral organ measurement indicators clarifies the core measurement parameters such as the exposed length of the stigma, the shortest distance between the anther and the stigma, the stamen area, and the length of the widest part of the two stamens. Figure 4 (A) Compared with the wild type, there were no significant differences in stigma exposure length, stigma exposure rate, anther-stigma shortest distance, stamen area, and the length of the widest part of the two stamens in the gene-edited lines; while the stigma exposure length, stigma exposure rate, and anther-stigma shortest distance were significantly increased in the GhNAP overexpression lines. Figure 4 (BD) Meanwhile, the stamen area and the length of the widest part of the stamens on both sides of the overexpressing line were significantly smaller than those of the wild type (F in Figure 4).

[0043] To comprehensively assess the degree of stigma exposure, this invention constructs a comprehensive stigma exposure index, calculated as follows: Comprehensive Stigma Exposure Index = Z (exposed length) + Z (exposed rate) + Z (anther-stigma distance) - Z (anther area) - Z (width of anthers on both sides), where Z is the standardized trait value. Pearson correlation analysis showed that the relative expression level of GhNAP was significantly positively correlated with the comprehensive stigma exposure index. r =0.877, P <0.001, corresponding to a determination coefficient R² = 0.769, indicating that the higher the GhNAP expression level, the stronger the stigma exposure in cotton, that is, GhNAP has a positive regulatory effect on stigma exposure, and the regulatory effect is significantly dose-dependent. Linear regression analysis further verified this positive correlation ( R 2 =0.769, P <0.001, Figure 4 The regression equation for GhNAP expression level (GhNAP) is: Stigma Exposure Comprehensive Index = 0.666 × GhNAP Relative Expression Level - 7.105. This provides a key theoretical basis and predictive model for elucidating the molecular regulatory mechanism of stigma exposure in cotton and for molecular-assisted breeding.

[0044] 2.3.2 Cotton transcription factor GhNAP regulates anther development and pollen viability Besides affecting filament wrinkling, the anther dehiscence state of GhNAP-overexpressing lines was also significantly altered. To further elucidate the specific mechanism of action of the transcription factor GhNAP in anther development, this invention further systematically analyzed the anther dehiscence phenotype and related characteristics.

[0045] To elucidate the biological function of the transcription factor GhNAP in cotton anther development, this invention systematically statistically analyzed the anther dehiscence phenotype of GhNAP-overexpressing lines. Significant differences in anther dehiscence were observed among different GhNAP-overexpressing lines. Lines with high GhNAP expression had a significantly higher proportion of flowers with intact anthers, while lines with relatively low expression levels predominantly had flowers with normally dehiscent anthers. Figure 5 (A). Box plot results further confirmed that GhNAP expression level was significantly positively correlated with anther dehiscence defect; high expression lines showed a significantly increased sterility rate, while low expression lines maintained a higher fertility level. Figure 5 (B)

[0046] Based on this, an association analysis was performed on the relative expression level of GhNAP and the male infertility rate, and the results showed a significant dose-dependent relationship between the two. Figure 5 (C) Meanwhile, within the same overexpression line, the expression level of GhNAP in flower buds with undehiscent anthers was significantly higher than that in flower buds with normally dehiscent anthers, directly proving that overexpression of GhNAP is a key factor causing anther dehiscent barrier (D in Figure 5).

[0047] Based on the expression levels and phenotypic results, this invention selected two highly expressed and phenotypically significant lines, OE5 and OE7, for subsequent morphological observation. Overall phenotypic observation of the floral organs of each line revealed significant differences in the morphology of internal reproductive organs such as stamens, pistils, and anthers among the wild-type, gene-edited, and overexpression lines, while external floral organs such as petals and sepals showed no significant changes. Figure 5 (E, F). Stereoscopic observation of anther dehiscence revealed that both wild-type and mutant anthers dehiscence normally, while the anthers of the OE5 and OE7 lines exhibited a distinctly non-dehiscent phenotype, further demonstrating that overexpression of GhNAP inhibits anther dehiscence (E, F). Figure 5 (G).

[0048] To further elucidate the intrinsic mechanism of anther development, this invention compared pollen viability and structure in different strains. TTC staining results showed that the pollen viability of both wild-type and mutant strains was approximately 89%, with no significant difference between groups; however, the pollen from the overexpression strains was mostly brown, with a significantly decreased viability (H and I in Figure 5). Scanning electron microscopy revealed that the pollen grains of wild-type and mutant strains were morphologically regular with intact exine spikes; the pollen grains of the overexpression strains were noticeably shrunken and deformed, with broken surface ornamentation and impaired structural integrity. Figure 5In addition, the fractal dimension (FD) of pollen from the overexpression lines was significantly increased, indicating that the pollen grains of the overexpression lines had reduced roundness and irregular morphology. Figure 5 (Middle K).

[0049] The above results indicate that overexpression of GhNAP can lead to abnormal anther morphology, inhibited dehiscence, decreased pollen viability, and damage to the pollen exine structure. This suggests that the transcription factor GhNAP plays an important regulatory role in cotton anther development and pollen maturation, and its abnormally elevated expression level can seriously interfere with male reproductive development, ultimately leading to male infertility.

[0050] 2.3.3 Cotton transcription factor GhNAP regulates tapetum development and programmed cell death The timely programmed degradation of the tapetum is a core prerequisite for normal anther dehiscence and pollen maturation. This invention uses paraffin sectioning to conduct histological observation of the complete anther development cycle (stages 1–6) in wild-type (WT), gene-edited GhNAP lines (KO1, KO2), and GhNAP overexpression lines (OE5, OE7). Figure 6 The stages correspond to bud lengths of 4–5 mm (stage 1), 5–6 mm (stage 2), 6–7 mm (stage 3), 7–8 mm (stage 4), 8–9 mm (stage 5), and 9–10 mm (stage 6), respectively.

[0051] In the early stages of anther development (stage 1–2) and the tetrad stage (stage 3), the anther structure of all genotypes is complete, the tapetum cells are regularly morphological and tightly arranged, and the meiosis of microspore mother cells and the formation of tetrads are normal.

[0052] Entering the critical tapetal degradation stage (stage 4), WT tapetal cells have initiated programmed degradation, characterized by nuclear pyknosis, cytoplasmic degradation, and gradual disintegration of cell structure. The tapetal degradation process of the KO strain is highly synchronized with that of WT, with only a slight delay in KO1, and overall still conforms to the normal developmental sequence. However, the tapetal degradation and disintegration process of OE5 and OE7 overexpression lines is significantly inhibited: at this stage, the tapetum still maintains an intact cell structure, there is no obvious nuclear pyknosis, the degree of cytoplasmic degradation is much lower than that of WT, and at the same time, cell swelling and vacuolation are intensified, microspores are encased in tetrads and cannot be released normally, and the developmental process is completely arrested.

[0053] By stage 5, the tapetum of the WT strain had largely degraded, with very few cells remaining. Microspores successfully developed into plump pollen grains, and the anther walls gradually thinned. In the KO1 and KO2 mutants, tapetum degradation was synchronized with WT, and pollen development was normal. However, in the OE5 and OE7 strains, the tapetum still retained a large number of intact cells, the degree of disintegration was severely delayed, microspore development was abnormal, and most of them appeared shrunken and deformed, with no normal pollen grain formation observed.

[0054] During the anther maturation and dehiscence stage (stage 6), the tapetum of the WT and KO1 / KO2 mutants was almost completely degraded, the anther wall structure was intact and capable of normal dehiscence, and the microspores all developed into morphologically normal mature pollen grains. In contrast, the OE5 and OE7 overexpression lines still had a large number of undegraded cell remnants in the tapetum, the anther wall structure was intact and unable to dehiscence normally, and no mature pollen grains were formed. The microspores were mostly shrunken and deformed, completely losing their reproductive function.

[0055] In summary, GhNAP exhibits strict developmental stage specificity in regulating tapetum degradation, primarily functioning during stage 4 and subsequent anther maturation. Overexpression of GhNAP significantly delays programmed tapetum degradation, thereby blocking pollen maturation and anther dehiscence, ultimately leading to male sterility. Conversely, gene loss of function has only a slight impact on the degradation process (KO1 is slightly slower than WT), ultimately allowing for normal reproductive development. These results directly confirm that GhNAP is a key transcription factor regulating timely tapetum degradation in cotton and maintaining normal male reproductive development.

[0056] To further verify the regulatory role of GhNAP on programmed cell death (PCD) in the tapetum, this invention employed TUNEL fluorescence labeling technology to detect the progression of tapetal PCD throughout the entire anther developmental stages (stages 1–6) of wild-type (WT), gene-edited GhNAP lines (KO1, KO2), and GhNAP-overexpressing lines (OE5, OE7). Green fluorescence signals represent cells where PCD occurred. Figure 7 ).

[0057] In the early stages of anther development (stages 1–2), no obvious green TUNEL positive signal was detected in anthers of any genotype, and the nuclear structure of tapetum cells remained intact, indicating that PCD had not yet been initiated in tapetum cells at this stage. Entering the tetrad stage (stage 3), weak green fluorescence began to appear in the tapetum region of the WT and KO2 mutants, marking the formal initiation of PCD; the TUNEL signal in the KO1 mutant was extremely weak, and PCD initiation was slightly delayed compared to WT and KO2; while the OE5 and OE7 overexpression lines showed almost no green fluorescence, indicating that PCD had not been initiated, showing a significant difference from the wild type.

[0058] During the critical tapetal degradation stage (stage 4), strong green TUNEL signals appeared in the tapetal region of WT and KO2 mutants, indicating active PCD. The signal intensity of KO1 mutant was slightly weaker than that of WT, and the PCD process was still slightly delayed, but continued to advance. In contrast, only very weak or no green fluorescence was detected in OE5 and OE7 overexpression lines, PCD was almost completely halted, and the tapetal cell structure was intact, which was completely consistent with the delayed degradation phenotype observed in paraffin sections.

[0059] During the microspore development into pollen grain stage 5, the TUNEL signal of WT and KO2 mutants remained at a high level, the PCD process continued, and the tapetum gradually degraded; the signal intensity of KO1 mutant gradually approached that of WT, the lag gap narrowed, and the PCD process progressed normally; while the OE5 and OE7 overexpression lines still showed no obvious green fluorescence, the PCD was not effectively initiated, and a large number of intact cells remained in the tapetum.

[0060] By stage 6 (anther maturation and dehiscence), the tapetum of the WT and KO2 mutants was completely degraded, the TUNEL signal was significantly weakened, the PCD process was basically completed, and the anthers dehisced normally to form mature pollen grains. Although a strong green fluorescence signal could still be observed in the KO1 mutant, its tapetum had basically completed degradation, and pollen development was normal, forming mature pollen grains. This indicates that its PCD process was only delayed in time and did not block tapetum degradation; the strong fluorescence mostly came from late degradation signals of residual cells and did not affect final fertility. In contrast, the OE5 and OE7 overexpression lines showed no obvious green fluorescence throughout the process, the PCD process was continuously blocked, a large amount of tapetum remained and could not degenerate, ultimately leading to anther failure to dehisce and pollen abortion.

[0061] Therefore, the transcription factor GhNAP plays a negative regulatory role in the initiation and progression of PCD in the cotton tapetum. Overexpression of GhNAP significantly inhibits the initiation and progression of PCD in the tapetum, leading to delayed tapetum degradation. This result confirms that the transcription factor GhNAP is an important factor in regulating PCD in the cotton tapetum and maintaining normal male reproductive development.

[0062] 3. Conclusion This invention focuses on the cotton NAC family transcription factor GhNAP, systematically elucidating its function and molecular mechanism in regulating stigma exfoliation and stamen development in cotton. Overexpression of GhNAP in cotton can simultaneously induce male sterility and stigma exfoliation phenotypes: male sterility is characterized by decreased pollen viability and abnormal anther development, ultimately leading to pollen abortion due to delayed programmed cell death (PCD) in the tapetum; stigma exfoliation is characterized by increased stigma length and exfoliation rate, and the relative expression level of GhNAP is significantly positively correlated with the comprehensive index of stigma exfoliation.

[0063] In summary, this invention elucidates the molecular mechanism by which GhNAP regulates male fertility and stigma exposure rate in cotton, providing important genetic resources and theoretical support for the analysis of male sterility in cotton, germplasm innovation, and hybrid seed production.

[0064] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of the GhNAP gene in regulating the stigma exfoliation rate of cotton, characterized in that, Overexpression of the GhNAP gene in cotton increased stigma exposure.

2. The application according to claim 1, characterized in that, The relative expression level of the GhNAP gene was significantly positively correlated with the stigma exposure index. R ² = 0.769, P < 0.

001.

3. The application of the GhNAP gene in regulating male fertility in cotton, characterized by: Pollen viability was reduced by overexpressing the GhNAP gene in cotton.

4. The application according to claim 3, characterized in that, Overexpression of the GhNAP gene resulted in shrunken and deformed pollen grains with increased fractal dimension.

5. The application of the GhNAP gene in regulating stigma exposure rate and male fertility in cotton, characterized by, GhNAP overexpression simultaneously caused cotton to exhibit a dual phenotype of increased stigma exposure and male sterility.

6. A method for creating male-sterile cotton materials, characterized in that, Overexpression of the GhNAP gene in cotton induces both male sterility and stigma exposure phenotype.

7. The method according to claim 6, characterized in that, The male sterility is characterized by decreased pollen viability and abnormal anther development, ultimately leading to pollen abortion due to delayed programmed cell death in the tapetum.

8. The method according to claim 6, characterized in that, The exposed column head was characterized by an increase in the length and rate of exposed column head, and the relative expression level of GhNAP was significantly positively correlated with the comprehensive index of exposed column head.

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