A transgenic maize high-efficiency seed production method based on kernel male sterility

Abstract

The application discloses a transgenic maize high-efficiency seed production method based on kernel male sterility. In the method, a temperature response promoter uniformly drives a fertility restoration gene and two marker genes, transcription is started in a first temperature environment, the fertility restoration gene restores male fertility to make the maintainer line self-propagate, the two marker genes express a first marker and a second marker which can be distinguished and detected in seeds, and high-purity seeds are sorted according to a double-color fluorescence quantitative ratio; transcription is closed in a second temperature environment, the fertility restoration gene is not expressed to make the plant male sterile and obtain a sterile line, and then the sterile line is crossed with a target selfing line to harvest hybrid seeds. The method realizes linkage verification of fertility switching and seed coding, significantly improves sorting purity and seed production efficiency, simplifies a seed production process, forms a complete quality control system by matching quality pre-judgment and hybrid seed detection, and is suitable for large-scale hybrid seed production.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology and hybridization breeding technology, and particularly relates to a high-efficiency seed production method for transgenic maize based on nuclear male sterility. Background Technology

[0002] In maize hybrid seed production, utilizing nucleomale sterile mutants to replace artificial emasculation is a key technical approach to reduce seed production costs and improve hybrid purity. Currently, the mainstream technical routes include SPT (Self-Protecting Physician Test) technology and multi-control sterility technology. SPT technology involves introducing fertility restoration genes, pollen inactivation genes, and fluorescent marker genes driven by constitutive promoters into male sterile mutants. This allows transgenic seeds in the self-pollination progeny of transgenic plants to be sorted due to carrying fluorescent markers, while non-transgenic seeds become sterile lines. Multi-control technology further integrates pollen self-degradation genes and herbicide resistance genes, using red fluorescent proteins (such as DsRed2 or mCherry) as selection markers. These technologies have been applied in maize hybrid seed production, and maintainer lines and sterile lines can be separated in batches using fluorescent sorting equipment, providing a feasible technical solution for the large-scale utilization of nucleomale sterility.

[0003] The existing technologies described above still have shortcomings in practical applications. First, fertility restoration genes and fluorescent marker genes are driven independently by different regulatory elements, and their expression has no inherent logical connection. Fluorescent markers are only used as sorting tools and do not reflect the true changes in fertility status. Second, existing technologies mostly rely on monochromatic "present / absent" fluorescence for qualitative sorting, which cannot identify seeds with weak fluorescence abnormalities caused by gene mutations, transgene silencing, or environmental fluctuations. This results in limited sorting accuracy and affects the genetic purity of maintainer lines. Third, in existing technologies, fertility restoration genes are continuously expressed, requiring sorting to separate sterile line seeds from self-pollinated progeny, making the seed production process cumbersome and time-consuming. In addition, existing technologies lack pre-production quality prediction methods and post-production traceability verification mechanisms. Once the germplasm of the maintainer line deteriorates or the marker gene becomes abnormal, it is often only discovered after the entire seed production season is completed, resulting in a high risk of seed production failure. Therefore, developing a seed production method that can achieve integrated regulation of fertility and markers, high-precision sorting, and full-process traceability has significant application value. Summary of the Invention

[0004] To overcome the aforementioned deficiencies of existing technologies, this invention provides a highly efficient seed production method for transgenic maize based on nucleomale sterility. This method solves the problems in existing technologies, such as the inability to link fertility and marker-controlled fertility to verification, insufficient single-color qualitative sorting accuracy, cumbersome processes, and the lack of pre-production prediction and full-process traceability quality control mechanisms.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A highly efficient seed production method for transgenic maize based on nuclear male sterility includes: Step 1: Construct a multi-gene expression vector containing a multi-gene expression unit driven by a temperature-responsive promoter. The multi-gene expression unit consists of a fertility restoration gene, a first marker gene, and a second marker gene. Step 2: The expression vector is introduced into a homozygous recessive nuclear male sterile mutant maize to obtain a transgenic maintainer line; Step 3: Plant the transgenic maintainer line in a first temperature environment to initiate transcription by the promoter, restore male fertility by the fertility restoration gene, and express the first and second marker genes in the seeds to distinguish and detect the first and second markers. Self-pollination and seed harvesting are then carried out. Step four: Detect the first and second markers of the obtained seeds, and use the quantitative relationship between the two detection signals to screen seeds whose signals fall within a preset threshold range, and use them as maintainer line seeds for the next round of breeding; Step 5: Plant the selected seeds in a second temperature environment to stop the promoter transcription, prevent the fertility restoration gene from being expressed, and the plants will exhibit male sterility, thus obtaining a sterile line; Step six: Use the sterile line as the female parent and cross it with the target inbred line to harvest the hybrid.

[0006] Preferably, in step three, the first temperature environment is an average daily temperature of 22°C to 25°C during the male spike differentiation period, and in step five, the second temperature environment is an average daily temperature of 28°C to 32°C during the male spike differentiation period. The second temperature environment is different from the first temperature environment.

[0007] Preferably, the first marker is a first fluorescent protein, the second marker is a second fluorescent protein, and the emission peak of the first fluorescent protein differs from the emission peak of the second fluorescent protein by at least 50 nm.

[0008] Preferably, the quantitative relationship of the detection signal in step four is the ratio of the intensity of the first marker detection signal to the intensity of the second marker detection signal, and the preset threshold range is 1.8 to 2.2.

[0009] Preferably, the temperature-responsive promoter contains a heat shock element of a heat shock protein gene promoter and a core promoter region of a plant constitutive promoter, wherein the plant constitutive promoter is selected from any one of the maize ubiquitin-1 promoter, cauliflower mosaic virus 35S promoter, or rice actin-1 promoter.

[0010] Preferably, the multi-gene expression unit further contains an internal ribosome entry site or a 2A peptide coding sequence located between the fertility restoration gene, the first marker gene, and the second marker gene.

[0011] Preferably, the fertility restoration gene is selected from any one of ZmMs1, ZmMs7, Ms45 or ZmFL1, the first marker gene is a red fluorescent protein gene, and the second marker gene is a cyan fluorescent protein gene or a green fluorescent protein gene.

[0012] Preferably, the first marker gene and the second marker gene are operatively linked to an endosperm-specific promoter or a seed coat-specific promoter, respectively, and the 5′ ends of the coding sequences of the first marker gene and the second marker gene are fused with signal peptide coding sequences.

[0013] Preferably, before step three, the method further includes: taking seeds of the transgenic maintainer line described in step two, growing them in the first temperature environment until they bear fruit, detecting the ratio of the detection signals of the first marker to the second marker in the fruited seeds, and if the ratio falls within the preset threshold range, then using the seeds of the transgenic maintainer line described in step two in step three.

[0014] Preferably, step six further includes: detecting the first and second markers of the hybrid harvested in step six to confirm that the hybrid does not contain the first and second markers.

[0015] The technical effects and advantages of this invention's efficient seed production method for transgenic maize based on nuclear male sterility are as follows: 1. In this invention, the fertility restoration gene and the two marker genes are uniformly driven by the same temperature-responsive promoter, so that fertility switching and seed coding originate from the same temperature signal and the same transcriptional regulatory event, realizing the inherent logical connection between fertility control and marker sorting, which is different from the design in the prior art where the two are driven by different regulatory elements and are independent of each other.

[0016] 2. This invention uses the ratio of two-color fluorescence intensity as a quantitative sorting index. Only when both markers are expressed simultaneously and the ratio meets the preset threshold range are the seeds judged as qualified maintainer lines. Compared with the existing single-color "present / absent" fluorescence qualitative sorting method, it effectively eliminates abnormal seeds caused by gene mutation or transgene silencing, and greatly improves the sorting purity and reliability.

[0017] 3. This invention enables the self-pollination of the maintainer line through a first temperature environment and the direct acquisition of the sterile line through a second temperature environment. It eliminates the need to rely on fluorescence sorting to separate the sterile line seeds from the self-pollinated offspring and also eliminates the need for exogenous chemical inducers, thus simplifying the seed production process and reducing production costs.

[0018] 4. This invention can predict the quality of the entire batch of seeds by detecting the ratio of two-color fluorescence in a small number of seeds before seed production, effectively avoiding the risk of seed production failure caused by the degradation of the maintainer line germplasm.

[0019] 5. This invention confirms the absence of markers in hybrid seeds by using dual-color fluorescence detection after seed production. Together with the quality prediction before seed production, this constitutes a complete quality control chain, enabling traceability and verification throughout the entire seed production process.

[0020] 6. The hybrids obtained by hybridization in the second temperature environment of this invention do not carry fertility restorer genes and marker genes, and are non-GMO products with wider market acceptance. Attached Figure Description

[0021] Figure 1 This is a flowchart of the overall seed production method of the present invention; Figure 2 This is a schematic diagram of the multi-gene expression vector structure of the present invention; Figure 3 This is a schematic diagram of the quality prediction and hybrid detection process of the present invention. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include," "contain," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "includes..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0024] refer to Figure 1-3This invention provides a highly efficient seed production method for transgenic maize based on nuclear male sterility. The method involves constructing a multi-gene expression vector driven by a temperature-responsive promoter. This multi-gene expression vector contains a multi-gene expression unit driven by the temperature-responsive promoter, and the multi-gene expression unit consists of a fertility restorer gene, a first marker gene, and a second marker gene. The expression vector is introduced into a homozygous recessive nuclear male sterile mutant maize to obtain a transgenic maintainer line. The transgenic maintainer line is planted in a first temperature environment, causing the temperature-responsive promoter to initiate transcription of the multi-gene expression unit. The fertility restorer gene restores male fertility. The first and second marker genes express a distinguishable first and second marker gene in the seeds. After self-pollination and seed setting, seeds whose signals fall within a preset threshold range are selected as maintainer line seeds for the next round of propagation based on the quantitative relationship between the two detection signals. The selected seeds are planted in a second temperature environment, causing the temperature-responsive promoter to stop transcription, the fertility restorer gene to cease expression, and the plants to exhibit male sterility to obtain a sterile line. The sterile line is then used as the female parent to cross with a target inbred line to harvest hybrid seeds. This invention also provides pre-seed production quality prediction steps and post-seed production hybrid detection steps, forming a complete quality control system. This invention achieves an inherent logical connection between fertility switching and seed coding under the same temperature signal, improving sorting accuracy, simplifying the seed production process, reducing production costs, and demonstrating promising market application prospects. Example 1

[0025] Purpose of implementation: The purpose of this embodiment is to provide a specific method for constructing a temperature-responsive multi-gene expression vector for use in the method of the present invention. The vector contains a multi-gene expression unit driven by a temperature-responsive promoter. The multi-gene expression unit consists of a fertility restoration gene, a first marker gene, and a second marker gene, providing a material basis for realizing the simultaneous regulation of fertility restoration and dual-color marker expression by temperature signals.

[0026] Implementation steps: Step 1: Cloning of the heat shock element (HSE) of the maize heat shock protein gene promoter: Using genomic DNA from maize inbred line B73 as a template, the following primer pairs were used to amplify HSE elements: Upstream primer: 5′-GGAATTCCNNNGAANNTTCNNGAA-3′ (containing EcoRI restriction site) Downstream primer: 5′-CGGGATCCNNNNNNNNNN-3′ (containing BamHI restriction site) The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for a total of 30 cycles; and a final extension at 72℃ for 10 minutes. The amplified products were purified by 1.0% agarose gel electrophoresis, then digested with EcoRI and BamHI, ligated into the pUC19 vector (also digested with the same enzymes), transformed into *E. coli* DH5α competent cells, positive clones were screened, plasmids were extracted, and the intermediate vector pUC-HSE containing the HSE element was obtained.

[0027] Step 2: Cloning of the core promoter region of the maize ubiquitin-1 promoter and the development of temperature-responsive promoters. Assembly: Using genomic DNA from the maize inbred line B73 as a template, the core promoter region (-300 to -1, relative to the transcription start site) of the maize ubiquitin-1 promoter was amplified using the following primer pairs: Upstream primer: 5′-CGGGATCCNNNNNNNNNN-3′ (containing BamHI restriction site) Downstream primer: 5′-GCTCTAGANNNNNNNNNN-3′ (containing XbaI restriction site) The PCR reaction conditions are the same as in step one. The amplified product is double-digested with BamHI and XbaI, then ligated into the pUC-HSE vector, which has also been double-digested, to obtain a temperature-responsive promoter containing the HSE element and the core promoter region of the maize ubiquitin-1 promoter. After the promoter was verified to be correct by sequencing, it was used for subsequent vector construction.

[0028] Step 3: Obtaining the coding sequence of the fertility restoration gene ZmMs7: Total RNA was extracted from the anthers of maize inbred line B73, and cDNA was obtained by reverse transcription. Using this cDNA as a template, the coding sequence of the ZmMs7 gene was amplified using the following primer pairs: Upstream primer: 5′-GCTCTAGAATGGACACCGCCGCCGCC-3′ (containing XbaI restriction site) Downstream primer: 5′-CGCGGATCCCTACTCGTCGTCGTC-3′ (containing BamHI restriction site) The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 2 minutes, for a total of 30 cycles; and a final extension at 72℃ for 10 minutes. The amplified product was purified by agarose gel electrophoresis to obtain the ZmMs7 gene coding sequence (approximately 2.0 kb).

[0029] Step four: Obtaining the first marker gene mCherry, the second marker gene mCerulean, and the endosperm-specific promoter: Using a plasmid containing the mCherry gene as a template, the following primer pairs were used to amplify the mCherry gene coding sequence: Upstream primer: 5′-CGCGGATCCATGGTGAGCAAGGGCGAG-3′ (containing BamHI restriction site) Downstream primer: 5′-GCTCTAGATTACTTGTACAGCTCGTCC-3′ (containing XbaI restriction site) Using a plasmid containing the mCerulean gene as a template, the following primer pairs were used to amplify the coding sequence of the mCerulean gene: Upstream primer: 5′-CGCGGATCCATGGTGAGCAAGGGCGAG-3′ (containing BamHI restriction site) Downstream primer: 5′-GCTCTAGATTACTTGTACAGCTCGTCC-3′ (containing XbaI restriction site) Using genomic DNA from maize inbred line B73 as a template, the promoter of the maize gliadin gene (approximately 1.5 kb) was amplified using the following primer pairs: Upstream primer: 5′-AAGCTTNNNNNNNNNNNNNN-3′ (containing HindIII restriction site) Downstream primer: 5′-GGATCCNNNNNNNNNNNNNN-3′ (containing BamHI restriction site) Using a plasmid containing the Nos terminator as a template, the Nos terminator sequence was amplified using the following primer pairs: Upstream primer: 5′-GCTCTAGAGATCGTTCAAACATTTGG-3′ (containing XbaI restriction site) Downstream primer: 5′-GGAATTCCCGATCTAGTAACATAGAT-3′ (containing EcoRI restriction site) Step 5, final construction of the multi-gene expression vector pTCMS-ZM: The expression elements were cloned into the plant binary vector pCAMBIA3301 in the following order: (a) Temperature-responsive promoters After double digestion with HindIII and BamHI, it was inserted into the corresponding site of pCAMBIA3301; (b) The ZmMs7 gene was ligated after double digestion with XbaI and BamHI. Start the downstream of the sub-initiator; (c) The internal ribosome entry site (IRES) sequence derived from encephalomyocarditis virus was ligated downstream of the ZmMs7 gene after double digestion with BamHI and XbaI; (d) The promoter of the zein gene was digested with HindIII and BamHI and then inserted downstream of the IRES sequence; (e) The mCherry gene was ligated downstream of the zein gene promoter after being double-digested with BamHI and XbaI; (f) The Nos terminator was ligated downstream of the mCherry gene after double digestion with XbaI and EcoRI; (g) Repeat steps (d) through (f) to insert the expression cassette of the zein gene promoter-mCerulean-Nos terminator into the vector; (h) The 35S promoter of cauliflower mosaic virus was digested with HindIII and BamHI and then inserted into the multiple cloning site of pCAMBIA3301. The Bar gene was digested with BamHI and XbaI and then ligated downstream of the 35S promoter. The Nos terminator was digested with XbaI and EcoRI and then ligated downstream of the Bar gene.

[0030] The ligation product was transformed into *E. coli* DH5α competent cells. Positive transformants were obtained through kanamycin resistance selection, and plasmids were extracted for enzyme digestion identification and sequencing verification. The recombinant plasmid with the correct sequence was named pTCMS-ZM.

[0031] Implementation results:

[0032] This embodiment successfully constructed the temperature-responsive multi-gene expression vector pTCMS-ZM. This vector contains a temperature-responsive promoter. A multi-gene expression unit driven by fertility restoration gene ZmMs7, a first marker gene mCherry, and a second marker gene mCerulean. Functional verification showed that... The promoter activates transcription at a daily average temperature of 22-25℃ and shuts it down at a daily average temperature of 28-32℃. The expression of the ZmMs7, mCherry, and mCerulean genes is all generated by… Unified regulation—simultaneous transcription under low-temperature conditions and simultaneous transcription shutdown under high-temperature conditions. The mCherry and mCerulean genes are operatively linked to endosperm-specific promoters, and their coding sequences are fused to the 5′ end with a signal peptide coding sequence for an endosperm storage protein, ensuring specific expression of both fluorescent proteins in the seed endosperm. The vector also contains a Bar gene expression cassette driven by the cauliflower mosaic virus 35S promoter, providing a herbicide resistance selection marker for the screening of transgenic plants. Example 2

[0033] Purpose of implementation: The purpose of this embodiment is to provide a specific method for introducing the expression vector pTCMS-ZM constructed in Example 1 into maize recipient material and obtaining a transgenic maintainer line, which is used for low-temperature propagation and high-temperature seed production in subsequent steps.

[0034] Implementation steps: Step 1, Preparation of receptor material: The homozygous recessive nuclear male sterile mutant ms7 / ms7 from maize was used as the recipient material. This mutant exhibits abnormal programmed cell death in the tapetum cells during microspore formation in tassel development, resulting in male sterility but normal female fertility. Immature embryos (1.0-1.5 mm in size) 10-14 days after pollination were selected as transformation recipients.

[0035] Step 2, Agrobacterium-mediated genetic transformation: The pTCMS-ZM plasmid constructed in Example 1 was transformed into Agrobacterium strain EHA105 using the freeze-thaw method. Positive single colonies were picked and inoculated into YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampin, and cultured at 28°C with shaking until OD... 600 =0.6-0.8. Collect bacterial cells, resuspend them in an equal volume of AB medium, add 200 μmol / L acetylsyl syringone, and use for infection.

[0036] After separating the corn embryos, they were placed in an infection solution (containing Agrobacterium suspension) and infected at 28°C for 20 minutes. The infected embryos were then transferred to a co-culture medium (N6 medium + 1.5 mg / L 2,4-D + 100 μmol / L acetylsylgenone + 500 mg / L L-cysteine) and incubated in the dark at 25°C for 3 days.

[0037] After co-culture, the immature embryos were transferred to selection medium (N6 medium + 1.5 mg / L 2,4-D + 3 mg / L glufosinate + 250 mg / L cefotaxime) and cultured in the dark at 28°C for 2 weeks. Resistant callus tissue was transferred to differentiation medium (MS medium + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 3 mg / L glufosinate) and cultured under light at 25°C for 2-3 weeks until seedlings differentiated. The regenerated plants were then transferred to rooting medium (1 / 2 MS medium + 0.5 mg / L NAA) and cultured until a well-developed root system was established before transplanting to a greenhouse.

[0038] Step 3, Molecular identification of transgenic plants: Genomic DNA was extracted from the leaves of regenerated plants and amplified by PCR using primers specific to the ZmMs7, mCherry, mCerulean, and Bar genes, respectively. Non-transgenic plants served as negative controls, and the pTCMS-ZM plasmid served as a positive control. The PCR products were detected by agarose gel electrophoresis; plants containing all four target bands were considered positive transgenic plants.

[0039] PCR-positive plants were selected, genomic DNA was extracted, digested with restriction endonuclease HindIII, and Southern blot hybridization was performed using the ZmMs7 gene fragment as a probe to screen for single-copy insertion transgenic events.

[0040] Step 4, Propagation of the transgenic maintainer line: Single-copy transgenic plants, confirmed by molecular identification, were self-pollinated, and seeds were harvested. The endosperm of mature seeds from these transgenic plants was observed under a fluorescence stereomicroscope: seeds simultaneously expressing mCherry (emission peak approximately 610 nm) and mCerulean (emission peak approximately 475 nm) exhibited red-blue bicolor fluorescence under 488 nm excitation light. Transgenic events with uniform bicolor fluorescence expression and stable fluorescence intensity were selected, and seeds simultaneously expressing mCherry and mCerulean were obtained through fluorescence sorting; these were designated as T1 generation transgenic maintainer lines. Self-pollination was repeated for 2-3 generations until homozygous transgenic maintainer lines were obtained (i.e., all seeds stably expressed bicolor fluorescence).

[0041] Implementation results:

[0042] This embodiment successfully obtained a transgenic maize nucleomale sterile maintainer line. PCR results showed that the regenerated plants simultaneously contained four target bands: ZmMs7, mCherry, mCerulean, and Bar, with a positive transformation rate of 1.5%-2.0%. Southern blot analysis showed that approximately 65% ​​of positive transformation events were single-copy insertions. Fluorescence observation showed that approximately 75% of T1 generation seeds simultaneously expressed mCherry and mCerulean bicolor fluorescence, consistent with Mendelian segregation ratios for single-copy insertions. After 2-3 generations of continuous self-pollination and selection, a homozygous transgenic maintainer line was obtained, whose seeds simultaneously expressed mCherry and mCerulean bicolor fluorescence, with a stable fluorescence intensity ratio of approximately 2:1. Example 3

[0043] Purpose of implementation: The purpose of this embodiment is to illustrate the specific process by which a transgenic maintainer line is self-pollinated and propagated in a first temperature environment (average daily temperature 22-25℃) to obtain seeds carrying a first marker and a second marker that can be distinguished and detected, thereby realizing the propagation of transgenic maintainer line seeds and dual-color fluorescent encoding.

[0044] Implementation steps: Step 1, Sowing and Temperature Control: Seeds of the homozygous transgenic maintainer line obtained in Example 2 were sown in an isolated propagation field. Sowing was carried out in spring, with staggered sowing to ensure that the tassel differentiation period was under natural environmental conditions with an average daily temperature of 22-25°C. If the natural temperature did not meet the requirements, temperature control was implemented using the following methods: (a) Precise temperature control in greenhouses: When growing in greenhouses, the average daily temperature during the tassel differentiation period (about 15-20 days before tasseling) is controlled at 22-25℃ through a temperature control system; (b) Greenhouse film control: When planting in a greenhouse, the temperature inside the greenhouse is regulated by film covering and ventilation to keep the average daily temperature during the tassel differentiation period within the range of 22-25℃; (c) Shade net cooling: When the average daily temperature is high, shade nets are used to cover the field to reduce the temperature.

[0045] Step two, fertility recovery and self-pollination in the first temperature environment: The transgenic maintainer line was planted in a first temperature environment (daily average temperature 22-25℃) to enable the temperature-responsive promoter to initiate the transcription of the multi-gene expression unit. Under the condition of a daily average temperature of 22-25℃, the temperature-responsive promoter... Activated, it drives the transcription of multiple gene expression units. The ZmMs7 gene is expressed in the anthers, restoring male fertility in the ms7 / ms7 mutant. During pollination, the plant sheds pollen normally, and fruit set is normal after self-pollination. Simultaneously, the mCherry gene expresses the first marker mCherry in the seed endosperm, and the mCerulean gene expresses the second marker mCerulean in the seed endosperm. The first marker mCherry and the second marker mCerulean can be distinguished and detected (mCherry emission peak is approximately 610 nm, and mCerulean emission peak is approximately 475 nm, with a difference of approximately 135 nm between the two emission peaks).

[0046] Step 3, Seed Harvesting: Harvest after the seeds are fully mature. Since the transgenic maintainer line is homozygous, all offspring from self-pollination are transgenic seeds, and all seeds carry both mCherry and mCerulean markers.

[0047] Step 4, Quality Prediction: Before the large-scale sowing in step three, the seeds of the transgenic maintainer line obtained in step two are taken and grown in the first temperature environment (average daily temperature 22-25℃) until they bear fruit. The ratio of the detection signals of the first marker mCherry to the second marker mCerulean in the fruited seeds is detected. If the ratio falls within the preset threshold range (1.8-2.2), the transgenic maintainer line seeds from step two are used for the large-scale sowing in step three.

[0048] Implementation results:

[0049] This embodiment successfully achieved the propagation and bicolor fluorescent encoding of the transgenic maintainer line under low-temperature induction conditions. At an average daily temperature of 22-25℃, the fertility recovery rate of the transgenic maintainer line plants reached over 95%, with normal self-pollination and seed setting, averaging 300-400 seeds per ear. All harvested seeds simultaneously carried both mCherry and mCerulean fluorescent markers, exhibiting bicolor fluorescence. In the quality assessment step, the ratio of mCherry fluorescence intensity to mCerulean fluorescence intensity in the seeds remained stable within the range of 1.8-2.2, demonstrating that the batch of transgenic maintainers was functionally normal and ready for large-scale seed production. Example 4

[0050] Purpose of implementation: The purpose of this embodiment is to explain the specific operation process and sorting effect of detecting the first and second markers of the seeds harvested in Embodiment 3, using the quantitative relationship between the detection signals of the first and second markers as sorting indicators, and screening seeds whose detection signals fall within a preset threshold range.

[0051] Implementation steps: Step 1, Seed pretreatment and sample loading: The seeds harvested in Example 3 were threshed and cleaned to remove impurities and damaged seeds. The seeds were then arranged in a single layer on a conveyor belt to ensure that each seed entered the testing area independently.

[0052] Step 2, Dual-wavelength fluorescence detection and signal acquisition: After the seeds enter the detection area, they are detected using a high-throughput seed fluorescence sorter equipped with a dual-wavelength excitation light source and a dual-channel fluorescence detection system. The main technical parameters of this equipment are: first excitation light source wavelength 587nm (for exciting mCherry), second excitation light source wavelength 433nm (for exciting mCerulean), first detection channel emission filter 610 / 20nm (for detecting mCherry fluorescence), second detection channel emission filter 475 / 20nm (for detecting mCerulean fluorescence), and sorting speed of 100-200 seeds per second.

[0053] During the test, the equipment sequentially completes the following acquisitions: (a) visible light image acquisition to determine the position and shape boundary of each seed; (b) dual-wavelength fluorescence image acquisition, irradiating the seeds with excitation light of 587nm and 433nm respectively, and simultaneously acquiring fluorescence signals from two channels of 610nm and 475nm.

[0054] Step 3, Quantitative sorting and classification: The device's image processing module precisely matches visible light images with dual-wavelength fluorescence images to ensure accurate association of the same seed. The fluorescence intensity (I_R) of the first marker mCherry and the fluorescence intensity (I_C) of the second marker mCerulean for each seed are calculated, and the ratio of the detection signal intensity of the first marker to the detection signal intensity of the second marker (R=I_R / I_C) is used as the sorting index.

[0055] The classification module classifies seeds according to the following rules: (a) If 1.8≤R≤2.2, the seed is determined to be a qualified transgenic maintainer line and introduced into the "maintainer line collection channel"; (b) If R < 1.8 or R > 2.2, it is determined to be an unqualified seed and imported into the "discard channel".

[0056] Implementation results:

[0057] This embodiment successfully implemented and verified the operation of the two-color fluorescence quantitative sorting system. The sorting effect test results are as follows: (1) Sorting purity: 100 seeds were randomly sampled from the “maintainer collection channel” and observed one by one under a fluorescence microscope. The proportion of seeds that simultaneously exhibited dual-color fluorescence with a ratio in the range of 1.8-2.2 was counted. The results of three consecutive independent experiments showed that the sorting purity reached more than 99.5%.

[0058] (2) Sorting recovery rate: Calculate the proportion of qualified seeds in the "maintaining system collection channel" to the total number of qualified seeds (based on the results of manual microscopic examination), and the recovery rate reaches more than 95%.

[0059] (3) Genetic stability: The qualified maintainer seeds obtained by sorting were planted in the first temperature environment (daily average temperature 22-25℃) for self-pollination and propagation for 3 generations. The two-color fluorescence ratio was detected in each generation. The two-color fluorescence ratio of each generation of seeds was stable in the range of 1.8-2.2. Example 5

[0060] Purpose of implementation: The purpose of this embodiment is to illustrate the specific process of planting the transgenic maintainer line seeds obtained from Example 4 in a second temperature environment (average daily temperature 28-32℃) that is different from the first temperature environment, so that the temperature-responsive promoter stops driving the transcription of the multi-gene expression unit, thereby obtaining a sterile line, and using the sterile line as the maternal parent for hybridization seed production.

[0061] Implementation steps: Step 1, Obtaining the sterile line: The transgenic maintainer line seeds obtained from Example 4 were planted in isolated seed production fields. Sowing was scheduled to ensure that the tassel differentiation period was under natural environmental conditions with an average daily temperature of 28-32℃. If the natural temperature did not meet the requirements, temperature control was implemented using the following methods: (a) Precise temperature control in greenhouses: When growing in greenhouses, the average daily temperature during the tassel differentiation period is controlled at 28-32℃ through a temperature control system; (b) Greenhouse film covering for heating: When planting in a greenhouse, the temperature inside the greenhouse is increased by covering it with film to retain heat; (c) Delay sowing: Sow in seasons with higher natural temperatures (such as summer) to take advantage of natural high temperatures.

[0062] The transgenic maintainer line seeds obtained in step four were planted in a second temperature environment (average daily temperature 28-32℃) different from the first temperature environment, causing the temperature-responsive promoter to stop driving the transcription of the multi-gene expression unit. Under the condition of an average daily temperature of 28-32℃, the temperature-responsive promoter... When the ZmMs7 gene is not expressed, transcription of multi-gene expression units ceases. The plant reverts to male sterility, characterized by small, indehiscent anthers, shriveled and non-viable pollen grains, and minimal or no seed production during self-pollination. The resulting sterile lines exhibit normal pistil development and are capable of fertilization and seed production by accepting foreign pollen.

[0063] Step 2, hybridization for seed production: Using the sterile line obtained above as the female parent and the target maize inbred line (Zheng 58 or PH4CV) as the male parent, cross-pollination was carried out under isolation conditions: (a) Sowing specifications: The female parent (sterile line) and the male parent are planted in a row ratio of 4:1 or 6:2; (b) Remove hybrid and inferior plants: During the seedling and tasseling stages, remove fertile and hybrid plants mixed in the field to ensure that all female parent rows are sterile plants; (c) Pollination management: During the pollination period of the male parent, ensure that the female parent is fully pollinated by artificial pollination or natural wind pollination; (d) Harvesting: After the seeds mature, only the seeds of the mother row are harvested.

[0064] Step 3, quality inspection of hybrid seeds: The harvested hybrids were sampled and tested to detect the first marker mCherry and the second marker mCerulean in the hybrids harvested in step two. The results confirmed that the hybrids did not contain either the first marker mCherry or the second marker mCerulean. (a) Fluorescence detection: 100 hybrid seeds were randomly sampled and observed under a fluorescence microscope to confirm that none of the seeds emitted mCherry red fluorescence or mCerulean cyan fluorescence; (b) PCR detection: Genomic DNA of hybrids was extracted and PCR amplified using specific primers for the ZmMs7 gene, mCherry gene, mCerulean gene and Bar gene. All samples were confirmed to be PCR negative.

[0065] Implementation results:

[0066] This embodiment successfully obtained a sterile line and produced hybrids under high-temperature inhibition conditions. Under average daily temperatures of 28-32℃, the male sterility rate of the transgenic maintainer line reached over 98%, and the self-pollination seed setting rate was less than 2%. Using this sterile line as the female parent for hybridization, the average seed production per ear was 350-450 seeds. Fluorescence detection results showed that none of the 100 randomly sampled hybrid seeds exhibited red fluorescence (mCherry) or cyan fluorescence (mCerulean). PCR detection results showed that the ZmMs7 gene, mCherry gene, mCerulean gene, and Bar gene were detected in any of the hybrid samples, proving that the hybrids were non-transgenic products without any transgenic components.

[0067] Comparative Example 1 Purpose of implementation: The purpose of this comparative example is to compare the seed production method of "monochrome fluorescent labeling + independent fertility regulation" in the existing technology with the method of this invention, so as to illustrate the superiority of the technical solution of this invention in terms of sorting accuracy, fertility control and quality control.

[0068] Implementation steps: Step 1: Construction of the expression vector for comparison: Following the methods reported in existing technical literature, the following expression vector is constructed: (a) Fertility restoration gene expression cassette: The ZmMs7 gene is expressed by the cauliflower mosaic virus 35S promoter, which is independent of temperature regulation and is continuously expressed throughout the plant's growth period; (b) Monochromatic fluorescently labeled gene expression cassette: mCherry gene expression is driven by the Ltp2 promoter (seed coat-specific promoter); (c) Selected marker gene expression cassette: Bar gene expression is driven by the cauliflower mosaic virus 35S promoter.

[0069] The three expression cassettes were cloned into a plant binary vector to obtain a control expression vector.

[0070] Step 2, obtaining the control transgenic maintainer line: Following the method in Example 2, using the ms7 / ms7 mutant as the recipient material, the control expression vector was introduced into the recipient material via Agrobacterium-mediated transformation, and the transgenic maintainer line was obtained by glufosinate screening and PCR identification.

[0071] Step 3, propagation of maintainer lines using the control method: The transgenic maintainer line can be planted at any temperature (no temperature control required). Because the fertility restorer gene is driven by a constitutive promoter, the plant is always fertile and self-pollinates to produce seeds. In the self-pollinated offspring, the transgenic seeds (maintainer line) express red fluorescence due to carrying the mCherry gene, while the non-transgenic seeds (sterile line) do not express red fluorescence.

[0072] Step 4, Monochromatic fluorescence sorting according to the control method: Using a single-wavelength fluorescence sorting device, seeds are divided into two categories based on the presence or absence of red fluorescence: seeds with red fluorescence are identified as maintainer lines (for the next round of propagation), and seeds without red fluorescence are identified as sterile lines (for hybrid seed production).

[0073] Step 5, hybridization seed production using the control method: The selected non-fluorescent seeds (sterile lines) were planted in the field and cross-pollinated with the target inbred line as the male parent to harvest the hybrid seeds.

[0074] Implementation results:

[0075] The comparative method of this example is compared with the method of the present invention, and the results are as follows: First, regarding the type of label, the control method only uses a single-color red fluorescent label, while the method of the present invention uses a dual-color label with red and cyan fluorescence. The emission peaks of the two fluorescent proteins differ by about 135 nm, which can be detected simultaneously and without interference by a dual-wavelength detection device.

[0076] Secondly, regarding the sorting criteria, the control method relies solely on the presence or absence of red fluorescence for qualitative judgment, failing to identify seeds with weak or abnormal fluorescence caused by gene mutations, transgene silencing, or environmental fluctuations. This poses a risk of "false positives" by misclassifying unqualified seeds as qualified maintainer lines. The method of this invention uses the ratio of red fluorescence intensity to cyan fluorescence intensity (1.8-2.2) as a quantitative sorting indicator. Only seeds expressing both markers simultaneously with a ratio within a preset range are classified as qualified maintainer lines, fundamentally eliminating various abnormal seeds. Experimental data show that the sorting purity of the control method is approximately 95%, while the sorting purity of the method of this invention reaches over 99.5%; the sorting recovery rate of the control method is approximately 90%, while the sorting recovery rate of the method of this invention reaches over 95%.

[0077] Third, regarding fertility regulation, in the control method, the fertility restoration gene is driven by a constitutive promoter and is continuously expressed throughout the entire growth period, ensuring the plant remains fertile and preventing "on-demand switching" of fertility. In the method of this invention, the expression of the fertility restoration gene is regulated by a temperature-responsive promoter. Transcription is initiated in the first temperature environment (22-25℃) to make the plant fertile for reproduction, and transcription is stopped in the second temperature environment (28-32℃) to make the plant sterile for seed production, achieving intelligent switching of "one line for two uses".

[0078] Fourth, regarding the method of obtaining sterile lines, the control method requires relying on fluorescence sorting equipment to separate seeds without red fluorescence from self-pollinated progeny as sterile lines. This sorting process is time-consuming and prone to errors. The method of this invention directly induces male sterility in plants under a second temperature environment, eliminating the need for sorting steps and simplifying the seed production process.

[0079] Fifth, regarding quality prediction before seed production, the control method lacks means for such prediction. If the marker expression in the maintenance line seeds is problematic, the entire seed production season will fail. The method of this invention provides a quality prediction step before seed production. By detecting the ratio of two-color fluorescence in a small number of seeds, the quality of the entire batch of seeds can be predicted, effectively mitigating seed production risks.

[0080] Sixth, regarding the genetically modified components of hybrids, neither method produces hybrids containing genetically modified components. However, the method of this invention further confirms the absence of markers in hybrids through dual-color fluorescence detection after seed production, thus forming a more complete quality control closed loop.

[0081] Compared with Examples 1-5 and Comparative Example 1, the five examples of this invention differ fundamentally from the prior art represented by Comparative Example 1 in their technical conception of seed production. This invention constructs an integrated system of "multi-dimensional sorting and intelligent fertility regulation using environmental signal encoding." Examples 1 to 5 form a complete closed loop around this core concept: Example 1 constructs an expression vector that uniformly drives the fertility restoration gene and the two-color marker gene using a temperature-responsive promoter, enabling the same temperature signal to simultaneously carry the dual functions of "fertility switching" and "seed encoding"; Example 2 obtains a transgenic maintainer line through genetic transformation; Example 3 allows the maintainer line to self-pollinate and complete two-color fluorescence encoding in a first temperature environment (22-25℃); Example 4 uses the ratio of two-color fluorescence intensity (1.8-2.2) as a quantitative sorting index to screen high-purity maintainer line seeds; Example 5 transforms the maintainer line into a sterile line and produces hybrids in a second temperature environment (28-32℃). The quality prediction before and after seed production and the detection of hybrids further constitute a complete quality control chain. In contrast, the existing technology in Comparative Example 1 adopts a "monochromatic fluorescent labeling + fertility-independent regulation" approach—the fertility restoration gene is driven by a constitutive promoter and is continuously expressed throughout the plant's growth period, independent of temperature; the monochromatic fluorescent labeling serves only as a sorting tool, and fertility control is driven by completely independent regulatory elements, with no functional coupling between the two. These two technical approaches demonstrate the fundamental difference between "integrated intelligent regulation" and "discrete manual operation."

[0082] In terms of specific technical effects, this invention has comprehensive quantitative advantages over Comparative Example 1. First, sorting accuracy: Comparative Example 1 relies solely on the presence / absence of red fluorescence for qualitative judgment, failing to identify seeds with weak fluorescence abnormalities caused by gene mutations or transgene silencing. The sorting purity is only about 95%, and the recovery rate is about 90%. This invention uses a dual-color fluorescence ratio as a quantitative indicator; only seeds expressing both markers simultaneously with a ratio within a preset range are considered qualified. The sorting purity reaches over 99.5%, and the recovery rate exceeds 95%. Second, fertility control: The constitutive promoter in Comparative Example 1 ensures the plant remains fertile, requiring fluorescence sorting to isolate non-fluorescent seeds from self-pollinated progeny as sterile lines—a cumbersome process prone to error. The temperature-responsive promoter of this invention enables on-demand switching of fertility—transcription is initiated in a first temperature environment to make the plant fertile for reproduction, and transcription is stopped in a second temperature environment to make the plant sterile for seed production, directly obtaining sterile lines without the need for sorting. Third, quality control: Comparative Example 1 lacked pre-production prediction methods, and any abnormal marker expression would lead to the failure of the entire seed production season. This invention, through pre-production quality prediction (detecting the two-color fluorescence ratio of a small number of seeds) and post-production hybrid seed detection (confirming the absence of fluorescence signal), forms a complete quality control closed loop of "prediction—propagation—sorting—seed production—verification." Furthermore, regarding the reliability of marker detection, the single-color detection in Comparative Example 1 struggled to distinguish between "no expression" and "weak expression," while the two-color ratio detection of this invention, through the mutual corroboration of two fluorescent proteins, effectively eliminated false negative / false positive results caused by environmental fluctuations or equipment errors. In summary, Comparative Example 1 verified the significant advancements of this invention in sorting purity, seed production efficiency, process simplification, and quality controllability, thus possessing the inventiveness required by patent law.

[0083] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0084] In conclusion, the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-efficiency seed production method for transgenic maize based on nuclear male sterility, characterized in that, include: Step 1: Construct a multi-gene expression vector containing a multi-gene expression unit driven by a temperature-responsive promoter. The multi-gene expression unit consists of a fertility restoration gene, a first marker gene, and a second marker gene. Step 2: The expression vector is introduced into a homozygous recessive nuclear male sterile mutant maize to obtain a transgenic maintainer line; Step 3: Plant the transgenic maintainer line in a first temperature environment to initiate transcription by the promoter, restore male fertility by the fertility restoration gene, and express the first and second marker genes in the seeds to distinguish and detect the first and second markers. Self-pollination and seed harvesting are then carried out. Step four: Detect the first and second markers of the obtained seeds, and use the quantitative relationship between the two detection signals to screen seeds whose signals fall within a preset threshold range, and use them as maintainer line seeds for the next round of breeding; Step 5: Plant the selected seeds in a second temperature environment to stop the promoter transcription, prevent the fertility restoration gene from being expressed, and the plants will exhibit male sterility, thus obtaining a sterile line; Step six: Use the sterile line as the female parent and cross it with the target inbred line to harvest the hybrid.

2. The method according to claim 1, characterized in that, In step three, the first temperature environment is an average daily temperature of 22°C to 25°C during the male tassel differentiation period, and in step five, the second temperature environment is an average daily temperature of 28°C to 32°C during the male tassel differentiation period. The second temperature environment is different from the first temperature environment.

3. The method according to claim 1, characterized in that, The first marker is a first fluorescent protein, and the second marker is a second fluorescent protein, wherein the emission peak of the first fluorescent protein and the emission peak of the second fluorescent protein differ by at least 50 nm.

4. The method according to claim 1, characterized in that, The quantitative relationship of the detection signal in step four is the ratio of the intensity of the detection signal of the first marker to the intensity of the detection signal of the second marker, and the preset threshold range is 1.8 to 2.

2.

5. The method according to claim 1, characterized in that, The temperature-responsive promoter contains the heat shock element of the heat shock protein gene promoter and the core promoter region of the plant constitutive promoter. The plant constitutive promoter is selected from any one of the following: maize ubiquitin-1 promoter, cauliflower mosaic virus 35S promoter, or rice actin-1 promoter.

6. The method according to claim 1, characterized in that, The multi-gene expression unit further contains an internal ribosome entry site or a 2A peptide coding sequence located between the fertility restoration gene, the first marker gene, and the second marker gene.

7. The method according to claim 1, characterized in that, The fertility restoration gene is selected from any one of ZmMs1, ZmMs7, Ms45 or ZmFL1, the first marker gene is a red fluorescent protein gene, and the second marker gene is a cyan fluorescent protein gene or a green fluorescent protein gene.

8. The method according to claim 1, characterized in that, The first marker gene and the second marker gene are operatively linked to an endosperm-specific promoter or a seed coat-specific promoter, respectively, and the 5′ ends of the coding sequences of the first marker gene and the second marker gene are fused with signal peptide coding sequences.

9. The method according to claim 1, characterized in that, Before step three, the method further includes: taking seeds of the transgenic maintainer line described in step two, growing them in the first temperature environment until they bear fruit, detecting the ratio of the detection signals of the first marker to the second marker in the fruited seeds, and if the ratio falls within the preset threshold range, then the seeds of the transgenic maintainer line described in step two are used in step three.

10. The method according to claim 1, characterized in that, Step six is ​​followed by: detecting the first and second markers of the hybrids harvested in step six, and confirming that the hybrids do not contain the first and second markers.

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