A molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease

Abstract

This invention relates to the field of bio-breeding technology and discloses a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and high-altitude leaf spot disease. The method includes constructing powdery mildew-resistant target-response probes and high-altitude leaf spot-resistant target-response probes; preparing mesoporous silica nanoparticles with a surface covalently coupled to a fluorescent internal reference molecule and a plant cell-penetrating peptide; loading the powdery mildew-resistant and high-altitude leaf spot-resistant target-response probes into the mesoporous channels to obtain a self-calibrated in-situ living nanoprobe complex; infiltrating the self-calibrated in-situ living nanoprobe complex into the living tissue of a single bitter gourd plant; collecting the total fluorescence intensity and spontaneous background fluorescence intensity and calculating the standardized resistance index; comparing the standardized resistance index with a judgment threshold to screen out dual-resistant living plants. This invention eliminates the physical error caused by fluctuations in the absolute amount of vector infiltration, achieving in-situ, non-destructive, and accurate identification of dual-resistant genotypes in living plants.

Description

Technical Field

[0001] This invention relates to the field of biological breeding technology, specifically to a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot. Background Technology

[0002] Plant molecular marker-assisted breeding technology relies on differences in nucleic acid sequences at specific loci to screen for individual plants with target disease resistance traits. In vivo in situ optical detection technology delivers fluorescent probes, such as molecular beacons, into living plant tissues, causing the probes to hybridize with the target genomic DNA sequence within the cell nucleus. The optical signals released during the hybridization and unwinding process directly reflect the plant's disease-resistant single nucleotide polymorphisms or insertion / deletion mutations, thus enabling rapid genotyping without disrupting the plant's natural growth.

[0003] In existing in vivo in situ fluorescence detection applications, nanomaterials are typically used as physical delivery carriers to encapsulate or load nucleic acid probes within the carriers. Operators use mechanical pressure to penetrate the plant mesophyll tissue with a solution containing the probe carrier. The nanocarrier penetrates the plant cell wall and plasma membrane into the cell, where the probe then enters the nucleus, binds to the target sequence, and emits light. An external photoelectric acquisition device receives the absolute fluorescence intensity emitted from the infiltrated area of ​​the plant tissue. Technicians compare the absolute fluorescence intensity with a preset single fluorescence intensity threshold to determine whether the plant carries the corresponding disease-resistant gene fragment.

[0004] Current technologies rely on the absolute fluorescence intensity emitted by the target probe to determine genotype. However, in in vivo tissue infiltration operations, differences in stomatal density and cuticle thickness across different regions of plant leaves lead to uneven physical resistance to infiltration. This unevenness in physical resistance causes fluctuations in the absolute physical quantity of the probe carrier entering local mesophyll tissue. Current technologies lack an independent parametric calibration mechanism, making it impossible to distinguish whether changes in optical signal intensity stem from genotypic differences in the target sequence or from changes in the local infiltration concentration of the nanocarrier. The target optical signal and physical infiltration concentration are highly coupled; the fluctuation error in the absolute quantity of carrier infiltration caused by the spatial heterogeneity of in vivo tissue directly leads to a lack of quantitative assessment basis for photoelectric acquisition signals, reducing the accuracy and repeatability of in-situ disease resistance genotype determination. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot, thus solving the problems mentioned in the background section.

[0006] To achieve the above objectives, this invention provides the following technical solution: a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and high-altitude leaf spot disease. This method designs target response probes for powdery mildew resistance and high-altitude leaf spot resistance, respectively, targeting single nucleotide polymorphism sites for powdery mildew resistance and insertion / deletion sites for high-altitude leaf spot resistance. The 5' end of the powdery mildew resistance target response probe is covalently modified with a luminescent group (carboxyfluorescein), and the 3' end is covalently modified with a quencher group (p-dimethylaminoazobenzoic acid). The 5' end of the high-altitude leaf spot resistance target response probe is covalently modified with a luminescent group (carboxyX-rhodamine), and the 3' end is covalently modified with a quencher group (black hole quencher 2). When designing the oligonucleotide sequence of the recognition loop region of the high-altitude leaf spot resistance target response probe, the oligonucleotide sequence of the recognition loop region directly crosses the nucleotide fragment deletion site on the single-stranded target region containing the high-altitude leaf spot resistance insertion / deletion site, and is completely complementary to the flanking bases on both sides of the nucleotide fragment deletion site. When the anti-altitude leaf spot target response probe encounters a susceptible sequence with a fragment deletion, a free unpaired nucleotide convex loop structure is formed inside the susceptible sequence. Thermodynamic energy balance allows the anti-altitude leaf spot target response probe to maintain a closed stem-loop structure, thereby achieving highly specific recognition of insertion and deletion mutations.

[0007] For the physical delivery carrier, a hydrolysis-condensation reaction was performed on the mesoporous silica nanoparticle framework to limit its outer diameter to the range of 50 nm to 100 nm. The exposed silanol groups on the surface of the mesoporous silica nanoparticle framework were aminated, and a fluorescent dye Cy5 carrying a monocarboxyl functional group was covalently coupled to the outer shell surface of the aminated framework as a fluorescent internal control molecule. By controlling the concentration of Cy5, the amino groups on the surface of the mesoporous silica nanoparticle framework were kept in excess to maintain the physical openness of the mesoporous channels. Subsequently, a heterobifunctional crosslinking agent was used to covalently couple BP100 peptide, as a plant cell penetrating peptide, to the outer shell surface of the mesoporous silica nanoparticle framework. The molar concentration of BP100 peptide added to the reaction system was adjusted to ensure that the surface zeta potential of the mesoporous silica nanoparticles was greater than 15 mV. Divalent magnesium ions at a concentration of 5 to 20 mmol / L were added to the loading buffer. The internal and external pressure differences during the negative pressure evacuation and restoration to normal pressure in the vacuum drying oven were used to press the loading mixture, along with the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe, into the mesoporous channel. The anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe were fixed by the electrostatic adsorption reaction of free amino groups on the inner wall of the mesoporous channel, thus obtaining a self-calibrated in vivo in-situ nanoprobe complex.

[0008] In the field in-situ detection phase, a self-calibrated in-situ nanoprobe complex was suspended in a live plant osmotic buffer containing isotonic concentrations of 2-(N-morpholine)ethanesulfonic acid and 0.01% to 0.1% (w / w) of a nonionic surfactant. The output end of a needleless microsyringe was tightly fitted to the stomatal-dense region on the abaxial surface of the cotyledons of a single bitter gourd breeding plant. Mechanical positive pressure was applied to the plunger of the needleless microsyringe to force the live plant osmotic buffer into the cotyledons of the bitter gourd breeding plant. The self-calibrated in-situ nanoprobe complex penetrated the pores of the plant cell wall to reach the plasma membrane surface of the plant cell, and entered the cytoplasm as a probe reservoir via the plasma membrane endocytosis pathway mediated by plant cell-penetrating peptides. The target response probes for powdery mildew resistance and plateau leaf spot resistance are released free from the mesoporous channels of the self-calibrated in-situ nanoprobe complex into the cytoplasm, diffuse through the nuclear pore complex channels on the plant cell nuclear membrane into the plant cell nucleus, and undergo a base complementary pairing reaction with the single-chain target region exposed inside the decondensed euchromatin region.

[0009] The total fluorescence intensity of the powdery mildew-targeting signal, the total fluorescence intensity of the plateau leaf spot-targeting signal, the total fluorescence intensity of the fluorescent internal reference molecule, and the spontaneous background fluorescence intensity of the unpenetrated area were collected from individual plants of the bitter gourd breeding population using a multi-wavelength fluorescence detection device. Because the absolute amount of carrier penetration fluctuates due to differences in stomatal density and cuticle thickness in different regions, this invention eliminates spatial heterogeneity interference through ratiometric calculation. The specific method for calculating the standardized resistance index based on the total fluorescence intensity and spontaneous background fluorescence intensity is as follows: Subtract the difference between the total fluorescence intensity of the permeated region and the spontaneous background fluorescence intensity of the unpermeated region in the FAM channel from the total fluorescence intensity of the permeated region in the Cy5 channel. Divide this difference by the difference between the total fluorescence intensity of the permeated region and the spontaneous background fluorescence intensity of the unpermeated region in the Cy5 channel to obtain the self-calibrated fluorescence response ratio of the powdery mildew resistance site. Similarly, subtract the difference between the total fluorescence intensity of the permeated region and the spontaneous background fluorescence intensity of the unpermeated region in the ROX channel from the total fluorescence intensity of the permeated region in the ROX channel. Divide this difference by the difference between the total fluorescence intensity of the permeated region and the spontaneous background fluorescence intensity of the unpermeated region in the Cy5 channel to obtain the self-calibrated fluorescence response ratio of the plateau leaf spot resistance site.

[0010] To determine the genotype of live individual plants, positive thresholds for powdery mildew resistance and high-altitude leaf spot resistance were pre-defined. The method for defining the positive threshold for powdery mildew resistance was as follows: the statistical mean of the self-calibrated fluorescence response ratio at powdery mildew resistance sites in a homozygous susceptible control population was added to three times the standard deviation of the self-calibrated fluorescence response ratio at the same sites in the homozygous susceptible control population. Similarly, the method for defining the positive threshold for high-altitude leaf spot resistance was the same: the statistical mean of the self-calibrated fluorescence response ratio at the high-altitude leaf spot resistance sites in a homozygous susceptible control population was added to three times the standard deviation of the self-calibrated fluorescence response ratio at the same sites in the same sites in the homozygous susceptible control population. When the ratio of self-calibrated fluorescence response at the powdery mildew resistance site of a single plant in the bitter gourd breeding population is greater than or equal to the positive threshold for powdery mildew resistance, and the ratio of self-calibrated fluorescence response at the plateau leaf spot resistance site of a single plant in the bitter gourd breeding population is greater than or equal to the positive threshold for plateau leaf spot resistance, dual-resistant live plants are selected.

[0011] This invention provides a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot. It has the following beneficial effects: 1. This invention constructs a calculation logic by covalently coupling fluorescent internal reference molecules to the surface of a nanocarrier, dividing the net signal intensity of the target luminescent group by the net signal intensity of the fluorescent internal reference molecule. This eliminates the error caused by fluctuations in the absolute quantity of carrier permeation due to differences in local pore density and stratum corneum thickness in living tissue. The calculation of the standardized resistance index decouples the photoelectric acquisition signal from the physical permeation concentration, eliminates spatial heterogeneity interference, and improves the accuracy and repeatability of in-situ disease resistance genotyping in vivo.

[0012] 2. This invention establishes a physical delivery pathway across plant cell wall pores and cell membrane barriers by constructing a mesoporous silica nanoparticle framework with a specific outer diameter and covalently grafting positively charged plant cell-penetrating peptides onto the outer shell surface. The probe-loaded carrier enters the cytoplasm via electrostatic adsorption and plasma membrane endocytosis. This non-destructive micro-pressure osmosis replaces the destructive tissue sampling and nucleic acid extraction processes, enabling breeding plants to maintain in-situ growth and development in the field after genotyping.

[0013] 3. This invention establishes a conformational switching mechanism based on steric hindrance and thermodynamic equilibrium by designing response probes containing specific recognition loop regions and self-complementary stem regions targeting insertion / deletion mutation sites. The recognition loop region performs complementary pairing across nucleotide fragment deletion sites, forming an unpaired nucleotide convex loop structure when encountering susceptible sequences. The unstable energy state generated by the convex loop structure allows the probe to maintain a closed stem-loop conformation and remain optically quenched, ensuring highly specific recognition of sequence variations. Attached Figure Description

[0014] Figure 1Flowchart of molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot; Figure 2 This is a flowchart of S500-S600 of the present invention. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0016] Please see the appendix Figure 1 -Appendix Figure 2 This invention provides a molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and high-altitude leaf spot, comprising a probe construction module, a live delivery module, and an in-situ computation module. Specifically, the probe construction module, serving as the core of biochemical detection and physical carrier, includes dual molecular beacons designed for single nucleotide polymorphism sites targeting powdery mildew resistance and insertion / deletion sites targeting high-altitude leaf spot resistance, as well as mesoporous silica nanoparticles covalently coupled with fluorescent internal reference molecules and plant cell-penetrating peptides. The dual molecular beacons and mesoporous silica nanoparticles are assembled together to form a self-calibrated live in-situ nanoprobe. The live delivery module is responsible for crossing the physical barriers of plant tissues, including mechanical components or procedures for targeting the self-calibrated live in-situ nanoprobe into the mesophyll tissue plasma membrane using stomatal micropressure permeation. The in-situ computation module is responsible for acquiring live optical signals and analyzing genotypes, including a multi-wavelength fluorescence detection device and a processing unit for logical analysis based on signal ratios.

[0017] To fully present the invention screening process, the above logical modules are mapped to operational steps, and the specific steps are divided as follows: S100: A molecular beacon with specific fluorescent emitting and quenching groups was designed and synthesized targeting the powdery mildew resistance and plateau leaf spot resistance of bitter gourd. The molecular beacon maintains its fluorescence quenched state in the free, unhybridized state through fluorescence resonance energy transfer effect.

[0018] S200: Preparation of mesoporous silica nanoparticles with fixed pore sizes. An internal reference fluorescent group is covalently coupled to the outer shell surface of the mesoporous silica nanoparticles, and a plant cell-penetrating peptide is grafted onto the outer shell surface. The molecular beacon synthesized in step S100 is loaded into the mesopores to obtain a complete self-calibrating in-situ living nanoprobe complex. For the basic hydrolysis-condensation preparation process of the mesoporous silica nanoparticles, those skilled in the art can refer to the standard sol-gel method. The basic preparation process is well-known in the art and will not be described in detail in this specification.

[0019] S300: Select bitter gourd breeding populations in the cotyledon expansion stage, and use a non-destructive micro-penetration tool to permeate the suspension formed by the self-calibrated living in-situ nanoprobe complex into the interstitial space of the living leaf tissue through the cotyledon stomata with micro-pressure, and maintain the in-situ growth state of the plant.

[0020] S400: The self-calibrated in-situ nanoprobe complex, which enters the mesophyll tissue, crosses the cell wall and nuclear membrane mediated by plant cell-penetrating peptides. Within the microenvironment of the living cell nucleus, the molecular beacon undergoes specific base pairing and thermodynamic hybridization with the endogenous target sequence of bitter gourd, triggering conformational unwinding of the molecular beacon. This causes the fluorescent emitting group to separate from the quenching group, thereby releasing the corresponding target fluorescent signal.

[0021] S500: Utilizing a multi-channel photoelectric acquisition device, fluorescence signals are extracted from the permeated area to obtain the target fluorescence intensity and internal reference fluorescence intensity. Combined with the autofluorescence background value of the non-permeated area, a standardized resistance index eliminating spatial heterogeneity interference is calculated. In the calculation logic processing unit, the target fluorescence intensity after subtracting the autofluorescence background value is used as the numerator, and the internal reference fluorescence intensity after subtracting the autofluorescence background value is used as the denominator. The ratio obtained by comparing the two is used as the standardized resistance index. Through this ratio calculation mechanism between the target signal and the actual permeated internal reference signal, the deviation in the absolute uptake of nanocarriers caused by differences in cuticle and stomatal conductance in different regions of the living leaf is eliminated. Finally, by comparing the standardized resistance index with a preset threshold, a decision is made to retain or remove individual plants in the field.

[0022] Based on the probe construction module defined in the overall system architecture above, in order to achieve accurate identification of single nucleotide polymorphism sites for powdery mildew resistance in bitter gourd, the process of designing probes for powdery mildew resistance targets in step S100 is refined, including the following steps: S101: Obtain the reference and mutant sequences of the bitter gourd powdery mildew resistance-associated locus. Compare genomic fragments of homozygous resistant and homozygous susceptible lines to locate single nucleotide polymorphism (SNP) targets that determine powdery mildew resistance. Use DNA sequence fragments containing SNP targets as the target regions for probe binding.

[0023] S102: Design of the recognition loop oligonucleotide sequence for the anti-powdery mildew target response probe. The recognition loop oligonucleotide sequence is designed to be perfectly complementary to the sequence fragment containing the single nucleotide polymorphism (SNP) target. The complementary base corresponding to the SNP target is placed at the center of the recognition loop oligonucleotide sequence to increase the steric hindrance and thermodynamic instability effects caused by single base mismatches. The length of the recognition loop oligonucleotide sequence is typically set between fifteen and twenty-five bases. For the basic conversion logic of specific base sequence lengths and mismatch thermodynamic instability effects, those skilled in the art can refer to the standard nearest neighbor thermodynamic model calculation method. The nearest neighbor thermodynamic model calculation method is a well-known technique in the art and will not be elaborated further in this specification.

[0024] S103: Constructing the complementary stem region sequence of the powdery mildew resistance target response probe and validating its thermodynamic equilibrium parameters. Short nucleotide sequences capable of self-complementary pairing are spliced ​​at the 5' and 3' ends of the oligonucleotide sequence in the recognition loop region to form the stem region structure. During stem region construction, the stem region sequence must be independent of the target region to ensure that it does not undergo complementary pairing with the target DNA sequence fragment. To ensure the recognition specificity of the powdery mildew resistance target response probe in the complex microenvironment of living plant cells and avoid non-specific luminescence, a precise thermodynamic equilibrium judgment logic is constructed. The specific thermodynamic equilibrium judgment logic is designed and its parameters are verified based on the Gibbs free energy formula.

[0025] In the formula, The total amount of Gibbs free energy released when the anti-powdery mildew target response probe hybridizes and binds to a completely complementary anti-powdery mildew target sequence; The total Gibbs free energy maintained when the 5' end stem region sequence and the 3' end stem region sequence of the target response probe for powdery mildew self-complement to form a closed stem-ring structure; This represents the total Gibbs free energy released when a target response probe against powdery mildew attempts to hybridize with a susceptible polymorphic sequence containing a single base mismatch.

[0026] Based on the Gibbs free energy formula mentioned above, when the target response probe for powdery mildew enters the cell nucleus carrying the resistance gene and encounters the sequence of a homozygous resistant strain, the energy state of the probe-target binding conformation is lower than the energy state of the closed stem-ring structure, and thermodynamically drives the stem region structure to undergo a chain-breaking reaction. When it encounters a mismatch sequence of a homozygous susceptible strain, the energy state of maintaining the closed stem-ring structure is much lower than the energy state of mismatch hybridization, and the probe maintains the closed stem-ring structure and cannot hybridize with the mismatch sequence.

[0027] S104: The 5' and 3' ends of the anti-powdery mildew target response probe were covalently modified with luminescent and quenching groups, respectively. Carboxyfluorescein was selected as the luminescent group and labeled at the 5' end, while p-dimethylaminoazobenzoic acid (p-dimethylaminoazobenzoic acid) was selected as the fluorescence quenching group and labeled at the 3' end. When the anti-powdery mildew target response probe is in a free state or maintains a closed stem-loop state due to a mismatch sequence, the carboxyfluorescein at the 5' end and the p-dimethylaminoazobenzoic acid at the 3' end are spatially close, triggering a fluorescence resonance energy transfer (FRET) effect. The fluorescent photons emitted by the stimulated carboxyfluorescein are absorbed by p-dimethylaminoazobenzoic acid and dissipated as non-radiative thermal energy, resulting in an optically quenched state for the anti-powdery mildew target response probe. When specific hybridization occurs and the stem region structure dissociates, the spatial distance between the carboxyfluorescein and p-dimethylaminoazobenzoic acid increases, blocking the FRET effect, allowing the carboxyfluorescein to release an optical signal for extraction by a multi-channel photoelectric acquisition device.

[0028] After completing the design and construction process of the target response probe for powdery mildew resistance, the probe construction module continued to execute the probe design work targeting the insertion and deletion sites of bitter gourd resistance to high-altitude leaf spot disease. To achieve concurrent detection of multiple disease resistance traits in the same living cell, the design of the target response probe for high-altitude leaf spot disease resistance, while maintaining basic thermodynamic principles, specifically implemented lower-level features to address insertion and deletion mutation characteristics and spectral isolation requirements. This included the following steps: S105: Obtain the reference and variant DNA sequences of the locus associated with bitter gourd resistance to high-altitude leaf spot disease. Using sequence alignment technology, locate the specific insertion / deletion target sites that determine the high-altitude leaf spot disease resistance trait. Establish the fragments containing the specific insertion / deletion target sites and their flanking sequences as the target regions for binding to the high-altitude leaf spot disease resistance target probes.

[0029] S106: Design of the recognition loop oligonucleotide sequence for a target-response probe against high-altitude leaf spot disease. Since the high-altitude leaf spot trait is caused by insertion / deletion mutations in the sequence, the recognition loop oligonucleotide sequence is designed to be completely complementary to the sequence of the homozygous resistant strain in the target region. When the homozygous resistant strain sequence has an additional insertion nucleotide fragment compared to the homozygous susceptible strain sequence, the recognition loop oligonucleotide sequence is designed to cross and complementaryly bind to the entire insertion nucleotide fragment and the flanking bases connected to both ends of the insertion nucleotide fragment. Conversely, when the homozygous resistant strain sequence has a nucleotide deletion compared to the homozygous susceptible strain sequence, the recognition loop oligonucleotide sequence is designed to directly cross the deletion site and be completely complementary to the flanking bases on both sides of the deletion site. When the target-response probe against high-altitude leaf spot disease encounters the target region of a homozygous susceptible strain, due to the presence or absence of corresponding nucleotides within the susceptible sequence, the recognition loop oligonucleotide sequence attempts to hybridize with the susceptible sequence, forming a free, unpaired nucleotide loop structure at the deletion or insertion site. The free unpaired nucleotide loop structure can cause steric hindrance and thermodynamic instability effects.

[0030] S107: Constructing the complementary stem region sequence of the plateau leaf spot disease target response probe and verifying the thermodynamic equilibrium parameters under insertion / deletion mismatch conditions. Short nucleotide sequences capable of self-complementary pairing were spliced ​​at the 5' and 3' ends of the oligonucleotide sequence in the recognition loop region of the plateau leaf spot disease target response probe to form a closed stem region structure. During the construction of the closed stem region structure, the stem region sequence was independent of the plateau leaf spot disease target region to ensure that the stem region sequence did not undergo complementary hybridization with the target region sequence. The thermodynamic equilibrium judgment logic of the plateau leaf spot disease target response probe was verified based on the Gibbs free energy formula modified for the convex loop structure.

[0031] In the formula, The total Gibbs free energy released when the probe responding to the high altitude leaf spot disease hybridizes with a completely complementary disease-resistant target sequence. The total Gibbs free energy maintained when the 5' end stem region sequence and the 3' end stem region sequence of the probe representing the target response to plateau leaf spot disease self-complement to form a closed stem-ring structure; This represents the total Gibbs free energy released when a probe responding to the target of high-altitude leaf spot disease attempts to hybridize and bind with a susceptible sequence that has a sequence deletion or insertion, forming a free nucleotide convex loop structure.

[0032] Based on the aforementioned convex ring-corrected thermodynamic equilibrium setting, when the plateau leaf spot disease target response probe enters the cell nucleus and encounters a homozygous resistant strain sequence, thermodynamically driving the stem region structure to unwind and form a stable double strand with the resistant sequence; when the plateau leaf spot disease target response probe encounters a susceptible mutant sequence, the unstable energy state brought about by the convex ring structure causes the plateau leaf spot disease target response probe to maintain a lower-energy closed stem-ring structure, and the plateau leaf spot disease target response probe maintains a closed state and refuses to bind to the susceptible sequence.

[0033] S108: The 5' and 3' ends of the probe targeting anti-altitude leaf spot disease were covalently modified with luminescent and quenching groups, respectively. To ensure that the multi-channel photoelectric acquisition equipment could simultaneously extract powdery mildew resistance and anti-altitude leaf spot disease signals without spectral crosstalk, carboxylated Rhodamine X was specifically selected as the luminescent group and labeled at the 5' end of the probe, while Black Hole Quencher No. 2 was selected as the fluorescence quenching group and labeled at the 3' end. When the probe is in a free state or maintains a closed stem-loop state when encountering a susceptible sequence, the carboxylated Rhodamine X at the 5' end and Black Hole Quencher No. 2 at the 3' end are physically close together, producing a fluorescence resonance energy transfer effect. The optical energy generated by the stimulated carboxylated Rhodamine X is absorbed by Black Hole Quencher No. 2 and dissipated as heat, keeping the probe in an optically quenched state. When the target response probe for the resistance to high-altitude leaf spot disease specifically hybridizes with the disease-resistant sequence, causing the stem region structure to unwind, the physical distance between carboxylated Rhodamine X and the black hole quencher 2 is increased, the fluorescence resonance energy transfer channel is blocked, and carboxylated Rhodamine X releases an independent optical signal distinct from the wavelength of carboxylated fluorescein. For the high-performance liquid chromatography (HPLC) purification process at the end of molecular probe synthesis, those skilled in the art can refer to conventional nucleic acid purification and desalting procedures. HPLC purification is a well-known technique in this field and will not be described further in the specification.

[0034] After completing the specific structural design of the target-response probes for powdery mildew resistance and plateau leaf spot resistance, the probe construction module needs to further clarify the underlying unified physical mechanism of optical signal conversion and release for both probes. The optical signal response of the probes is based on the fluorescence resonance energy transfer effect and the thermodynamic conformational transformation principle, including the following physical verification and parameter confirmation steps: S109: Verification of the conditions for the occurrence of fluorescence resonance energy transfer (FRET) in the free state. When the powdery mildew-resistant and plateau leaf spot-resistant target response probes do not encounter complementary target DNA sequences, the 5' and 3' stem region sequences self-complementarily pair to form a closed stem-loop conformation. In this closed stem-loop conformation, the physical distance between the luminescent group attached to the 5' end and the quencher group attached to the 3' end is reduced to within ten nanometers. This spatial distance within ten nanometers satisfies the physical scale requirements for the occurrence of the FRET effect.

[0035] S110: Based on the fluorescence resonance energy transfer efficiency formula, the optical parameters of the quenched state are defined. The fluorescence resonance energy transfer efficiency depends on the actual spatial distance between the luminescent and quenching groups and the Foster resonance distance of the combination of the luminescent and quenching groups. The specific calculation logic of the fluorescence resonance energy transfer efficiency relies on the following physical formula:

[0036] In the formula, Represents fluorescence resonance energy transfer efficiency; The Foster resonance distance represents the combination of a specific luminescent group and its corresponding quencher group. The Foster resonance distance is defined as the spatial distance between the luminescent group and the quencher group when the fluorescence resonance energy transfer efficiency reaches 50%. r represents the actual physical spatial distance between the luminescent group and the quencher group in the closed stem-ring conformation or the hybrid unwinding conformation. Based on the above calculation logic, in the closed stem-ring conformation, the actual physical spatial distance r between the luminescent group and the quencher group is much smaller than the Foster resonance distance. This results in a fluorescence resonance energy transfer efficiency close to 100%. The target response probes for powdery mildew resistance and plateau leaf spot resistance maintain an internal energy dissipation state, while exhibiting an optical quenching state externally.

[0037] S111: Describes the conformational transition process based on target recognition. When a target-responsive probe for powdery mildew resistance or plateau leaf spot resistance enters the nucleus of a living plant cell and encounters a perfectly complementary target DNA sequence, the oligonucleotide sequence in the recognition loop region of the probe undergoes a base-pairing reaction with the target DNA sequence. To ensure that the conformational transition can proceed spontaneously, the total number of bases that pair with the target sequence in the recognition loop region is set to be greater than the total number of bases that pair with the 5' stem region sequence and the 3' stem region sequence. Based on the above structural basis, the thermodynamic binding energy released by the base-pairing reaction is greater than the system binding energy required for the 5' stem region sequence and the 3' stem region sequence to maintain a closed stem-loop conformation. This system energy difference leads to the breaking of hydrogen bonds between the 5' stem region sequence and the 3' stem region sequence, triggering the probe's physical conformation to change from a closed stem-loop conformation to a rigid double-stranded hybridization unwinding conformation.

[0038] S112: Describes the optical signal release mechanism after conformational change. In the rigid double-stranded hybridization dissociation conformation, the 5' and 3' ends of the anti-powdery mildew target response probe or the anti-altitude leaf spot target response probe are separated by a rigid DNA double-stranded structure. The actual physical spatial distance r between the luminescent group and the quencher group increases and is much greater than the Foster resonance distance of the combination of the luminescent group and the corresponding quencher group. As the spatial distance between the luminescent and quenching groups increases, the fluorescence resonance energy transfer efficiency decreases significantly and approaches zero with increasing spatial distance. After absorbing the excitation light energy from the multi-wavelength fluorescence detection device, the luminescent group no longer transfers energy to the quenching group. Instead, it emits fluorescent photons of a specific wavelength, thus converting the target sequence recognition result into an optical signal. For basic fluorescence spectral scanning and photon counting measurement methods, those skilled in the art can refer to the standard fluorescence spectrophotometer operating procedures. Basic fluorescence spectral scanning and photon counting measurement methods are well-known technologies in the field and will not be elaborated further in this specification.

[0039] After completing the sequence construction and optical parameter verification of the powdery mildew resistance probe and the plateau leaf spot resistance probe, the probe construction module entered the preparation stage of a self-calibrated in-situ nanoprobe physical delivery carrier. To non-destructively deliver the powdery mildew resistance probe and the plateau leaf spot resistance probe into the nucleus of living plant cells, a functionalized nanocarrier with an internal reference self-calibration system needs to be constructed. The underlying structure of the functionalized nanocarrier is based on a mesoporous silica nanoparticle framework. The morphological synthesis and pore size control of the mesoporous silica nanoparticle framework include the following steps: S201: A hydrolysis-condensation reaction of mesoporous silica nanoparticles was performed. In an alkaline aqueous solution, hexadecyltrimethylammonium bromide was added as a structural template agent, and tetraethyl orthosilicate (TEO) was added as a silicon source. TEO under alkaline catalytic conditions underwent hydrolysis to generate silicic acid. Silicic acid molecules then condensed around micellar structures formed by the self-assembly of hexadecyltrimethylammonium bromide in solution, forming a silica solid framework encapsulating the template agent. To enable the silica solid framework to penetrate the network pores and cell membrane structure of bitter gourd cotyledon cells, the kinetic balance of nucleation and growth was adjusted by controlling the solution temperature of the hydrolysis-condensation reaction and the dropping rate of TEO, limiting the outer diameter of the silica solid framework to the range of 50 to 100 nanometers. Specifically, lowering the solution temperature of the hydrolysis-condensation reaction or slowing down the dropping rate of TEO inhibited excessive condensation growth of silicic acid molecules, thereby reducing the particle size of the generated silica solid framework to meet the physical size requirements for penetrating the pores of plant cell walls.

[0040] S202: Regulating the internal mesoporous channel size of the mesoporous silica nanoparticle framework. The pore size formed by conventional template agents cannot meet the spatial requirements for loading molecular beacons with luminescent and quenching groups. Tris(methylbenzene) is added as a pore-expanding agent to the hydrolysis-condensation reaction system. Tris(methylbenzene) molecules enter the hydrophobic core region of hexadecyltrimethylammonium bromide micelles, increasing the physical volume of the micelles, thereby forming enlarged mesoporous channels after the condensation reaction. The pore size of the mesoporous channels needs to match the spatial size of the target response probes for powdery mildew resistance and high-altitude leaf spot resistance to ensure that the molecular beacons can smoothly enter the interior of the mesoporous channels, while ensuring that the molecular beacons maintain a closed stem-loop conformation within the mesoporous channels and do not undergo non-specific melting and luminescence due to spatial compression. The matching logic between the mesoporous channel size and the probe size is established based on the following geometric constraint formula:

[0041] In the formula, The average pore size of the mesoporous channels inside the mesoporous silica nanoparticles after the pore-expansion reaction is completed. The maximum hydrodynamic diameter of the target response probe for powdery mildew resistance or plateau leaf spot resistance in the free closed stem-ring conformation; The spatial tolerance coefficient, with a value range of 1.5 to 2.0, is used to offset the steric hindrance effect caused by the thickness of the hydration layer on the molecular beacon surface and the electrostatic repulsion of the mesoporous channel inner wall. Based on the above geometric constraint formula, by adjusting the molar ratio of mesitylene to hexadecyltrimethylammonium bromide, and given that the molar ratio of mesitylene to hexadecyltrimethylammonium bromide is positively correlated with the final pore size of the mesoporous channel, the average physical pore size of the mesoporous channel is controlled between 7 nm and 12 nm.

[0042] S203: Removal of the structural template agent and activation of the mesoporous channel inner wall. After the hydrolysis-condensation reaction, the silica solid framework containing the structural template agent was collected by centrifugation. The silica solid framework containing the structural template agent was subjected to reflux extraction using a methanol solution containing ammonium nitrate. The methanol solution containing ammonium nitrate disrupts the electrostatic interaction between hexadecyltrimethylammonium bromide and the inner wall of the silica solid framework, eluting hexadecyltrimethylammonium bromide and mesitylene from the silica solid framework, exposing hollow and interconnected mesoporous channels, and obtaining the basic mesoporous silica nanoparticle framework. Solvent extraction was used instead of the conventional high-temperature calcination method to remove the template agent, which can retain the silanol groups on the surface of the mesoporous silica nanoparticle framework. The retained silanol groups provide sufficient chemical covalent reaction active sites for subsequent coupling of internal reference fluorescent groups and plant cell penetrating peptides. For electron microscopy observation methods for characterizing the morphology and measuring the pore size of basic mesoporous silica nanoparticles, those skilled in the art can refer to the standard transmission electron microscope operating procedure. The transmission electron microscope operating procedure is a well-known technology in this field and will not be described in detail in the specification.

[0043] After obtaining the basic mesoporous silica nanoparticle framework with the structural template removed and silanol groups exposed, surface silanization covalent coupling of the constant internal control system is performed. To eliminate the interference of spatial heterogeneity of living plant tissue on the target detection signal, a reference signal layer resistant to environmental interference needs to be constructed on the outer layer of the mesoporous silica nanoparticle framework. The construction and physical parameter calibration of the constant internal control system include the following steps: S204: Aminolation modification of the exposed silanol groups on the surface of mesoporous silica nanoparticles. The mesoporous silica nanoparticle framework is dispersed in anhydrous ethanol solvent, and aminopropyltriethoxysilane is added as a silanization coupling agent. The ethoxy groups in the aminopropyltriethoxysilane molecule undergo hydrolysis in the dispersion system, and then undergo dehydration condensation with the silanol groups on the surface of the mesoporous silica nanoparticle framework to form covalently bonded siloxane-silicon bonds. The dehydration condensation reaction grafts the free amino groups onto the outer shell surface of the mesoporous silica nanoparticle framework, obtaining aminated mesoporous silica nanoparticles with a positively charged surface. For the basic heating reflux process and solvent washing steps of the silanization dehydration condensation reaction, those skilled in the art can refer to the conventional anhydrous silanization modification process, which is well known in the art and will not be described in detail in this specification.

[0044] S205: A fluorescent internal control molecule is covalently coupled to the outer shell surface of aminated mesoporous silica nanoparticles. Cy5, a fluorescent dye carrying a single carboxyl functional group (i.e., carboxylated Cy5), is selected as the fluorescent internal control molecule. Carbodiimide and hydroxysuccinimide are added as chemical condensing agents to the reaction system containing carboxylated Cy5. The chemical condensing agents convert the carboxyl group of the carboxylated Cy5 molecule into an active succinimide ester intermediate. The active succinimide ester intermediate then undergoes a nucleophilic substitution reaction with the amino groups on the surface of the aminated mesoporous silica nanoparticles, forming a stable amide covalent bond. The formation of the amide covalent bond anchors the fluorescent internal control molecule to the outer layer of the mesoporous silica, thus preparing a modified nanocarrier carrying a constant internal control system. To prevent the fluorescent internal reference molecules coupled to the surface of mesoporous silica nanoparticles from causing steric hindrance and blocking the mesoporous channel openings, it is necessary to control the feed concentration of carboxylated Cy5 molecules to ensure that the amino groups on the surface of the mesoporous silica nanoparticles are in excess. Maintaining the physical openness of the mesoporous channel openings ensures the smooth loading of subsequent molecular beacons.

[0045] S206: Physical parameters for calibrating the fluorescent internal control molecule as a spatial heterogeneity calibration benchmark. A constant internal control system possesses the physical properties of resistance to environmental interference and independent luminescence. The principle of selecting Cy5 as the fluorescent internal control molecule is that the spectral absorption and emission bands of the Cy5 molecule are physically isolated from the spectral bands of the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe loaded inside the mesoporous channel, avoiding spectral overlap and cross-excitation interference between different optical detection channels. Simultaneously, the fluorescence quantum yield of the Cy5 molecule, covalently bound to the shell by amide bonds, is unaffected by pH fluctuations within living plant cells, and the luminescence state of the Cy5 molecule is completely independent of nucleic acid hybridization and melting events occurring in the molecular beacons within the mesoporous channel. The intensity of the optical signal released by the fluorescent internal control molecule is directly proportional to the number of modified nanocarriers in the local tissue according to the following physical formula:

[0046] In the formula, The table represents the theoretical reference fluorescence intensity emitted by the fluorescent internal reference molecule at a specific excitation wavelength; The characteristic fluorescence emission coefficient represents the constant internal reference system. The characteristic fluorescence emission coefficient is determined by the inherent physical properties of the covalently coupled Cy5 molecule. This represents the molar concentration of the modified nanocarrier that has penetrated into the local tissues of a living plant. This represents the local tissue volume of the light spot during optical scanning by a multi-channel photoelectric acquisition device.

[0047] Based on the aforementioned proportional physical formula, the theoretical reference fluorescence intensity emitted by the fluorescent internal reference molecule is linearly correlated only with the concentration of the modified nanocarrier that targets and penetrates the tissue. Using the theoretical reference fluorescence intensity emitted by the constant internal reference system as the baseline denominator in the calculation offsets the interference from permeation fluctuations caused by differences in stomatal density and cuticle thickness in mesophyll tissue, providing fundamental parameter support for the calculation of the standardized resistance index.

[0048] After covalently coupling fluorescent internal control molecules to the surface of mesoporous silica nanoparticles and retaining excess free amino groups, the probe construction module enters the surface grafting stage of transmembrane dual-penetrating peptides. Plant cells are surrounded by a cell wall composed of cellulose and pectin, and have an internal cell membrane composed of a phospholipid bilayer. To enable the modified nanocarrier carrying molecular beacons to overcome the dual physical and chemical barriers of the cell wall and cell membrane to enter the cell nucleus, plant cell-penetrating peptides need to be grafted onto the surface of the modified nanocarrier. The surface grafting of plant cell-penetrating peptides includes the following steps: S207: Heterofunctional crosslinking activation of free amino groups on the surface of modified nanocarriers carrying fluorescent internal control molecules. The modified nanocarriers carrying fluorescent internal control molecules are dispersed in a buffer solution (e.g., phosphate buffer) without free amino groups, and a heterofunctional crosslinking agent containing succinimidyl ester and maleimide groups is added. The succinimidyl ester groups in the heterofunctional crosslinking agent undergo an amidation reaction with the free amino groups on the surface of the modified nanocarrier, modifying the shell surface of the modified nanocarrier with double-bonded maleimide groups, thus obtaining maleimide-activated nanoparticles. Using a buffer solution without free amino groups avoids competitive consumption reactions between the buffer components and the succinimidyl ester groups, ensuring crosslinking efficiency.

[0049] S208: Covalent coupling of plant cell-penetrating peptides. BP100 peptide was selected as the plant cell-penetrating peptide. The conventional amino acid sequence of BP100 peptide is lysine-lysine-leucine-phenylalanine-lysine-lysine-isoleucine-leucine-lysine-tyrosine-leucine. Cysteine ​​residues were introduced into the terminal sequence of BP100 peptide to provide free thiol functional groups. The BP100 peptide with free thiol functional groups was added to the reaction system containing maleimide-activated nanoparticles. The thiol functional groups at the ends of the BP100 peptide underwent a Michael addition reaction with the maleimide double bonds on the surface of the maleimide-activated nanoparticles, forming thioether covalent bonds. The formation of thioether covalent bonds anchored the BP100 peptide to the outer layer of the modified nanocarrier, thus preparing a functionalized nanocarrier with transmembrane penetration capability. For solid-phase synthesis and purification processes of peptide fragments and dialysis purification steps of Michael addition reactions, those skilled in the art can refer to standard peptide coupling specifications. Peptide coupling specifications are well-known in the field and will not be elaborated further in this specification.

[0050] S209: Testing the surface charge characteristics of functionalized nanocarriers after grafting plant cell-penetrating peptides. The BP100 peptide sequence is rich in basic amino acid residues such as lysine and arginine, which give the BP100 peptide a positive charge under the pH conditions of the plant physiological environment. This positive charge guides the functionalized nanocarriers to accumulate on the negatively charged cellulose network structure of the plant cell wall through electrostatic attraction. The overall hydrodynamic diameter of the functionalized nanocarriers is controlled to be smaller than the exclusion limit of the plant cell wall pores, allowing the functionalized nanocarriers to diffuse through the cell wall pores to reach the plasma membrane surface. Upon reaching the plasma membrane surface, the hydrophilic and hydrophobic regions of the BP100 peptides cause local lipid arrangement disturbances in the phospholipid bilayer of the plasma membrane, introducing the functionalized nanocarriers into the cytoplasm through direct penetration or endosome-mediated mechanisms. To ensure the effectiveness of the electrostatic attraction penetration mechanism, the surface Zeta potential of the functionalized nanocarriers after grafting the peptides was tested. The criteria for determining the surface Zeta potential are as follows:

[0051] In the formula, The surface zeta potential value of the functionalized nanocarrier obtained after the grafted plant cell penetrates the peptide. The initial surface Zeta potential value represents the framework of mesoporous silica nanoparticles after amination modification. The value represents the decrease in Zeta potential caused by the consumption of some amino groups by the covalently coupled fluorescent internal reference molecule; The value of the increase in zeta potential induced by grafting plant cell-penetrating peptides rich in basic amino acids; The minimum positive charge threshold represents the minimum positive charge threshold that induces effective electrostatic adsorption between the plant cell wall and plasma membrane. The minimum positive charge threshold is set to 15 millivolts.

[0052] Based on the above surface Zeta potential formula, by adjusting the molar concentration of BP100 peptide added to the reaction system, the surface Zeta potential value of the functionalized nanocarrier is ensured to be greater than 15 mV, so as to meet the parameter requirements for crossing the electrostatic barrier between plant cell walls and plasma membranes.

[0053] After completing the covalent grafting and charge parameter verification of plant cell-penetrating peptides on the surface of the functionalized nanocarrier, mesoporous loading of dual probes was performed. To integrate the powdery mildew-resistant target-response probe and the plateau leaf spot-resistant target-response probe into the functionalized nanocarrier to form a complete self-calibrated in vivo in-situ nanoprobe complex, the following steps were included: S301: Preparation of the loading mixture for the dual molecular beacons. The purified powdery mildew-resistant target response probe and the plateau leaf spot-resistant target response probe are added to the loading buffer. To ensure that the multi-channel photoelectric acquisition device can obtain a balanced fluorescence signal intensity during final detection, the initial molar ratio of the powdery mildew-resistant target response probe and the plateau leaf spot-resistant target response probe in the loading mixture is set based on the difference in relative fluorescence quantum yield between carboxyfluorescein and carboxyXrhodamine. Probes with lower fluorescence quantum yields are set with a higher molar concentration in the loading mixture; typically, the molar ratio of the powdery mildew-resistant target response probe to the plateau leaf spot-resistant target response probe is controlled between 1:1 and 1:1.5. Divalent magnesium ions are added to the loading buffer at a concentration of 5 to 20 mmol / L. Divalent magnesium ions can neutralize the negative charge on the DNA backbone of the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe, maintain the physical stability of the closed stem-loop conformation of the probe, and prevent non-specific melting and luminescence of the probe before it is loaded into the mesoporous channel.

[0054] S302: Negative-pressure driven mesoporous loading and electrostatic fixation of dual molecular beacons. Functionalized nanocarriers are ultrasonically dispersed into a loading mixture to form a solid-liquid two-phase suspension. This suspension is then placed in a vacuum drying oven for negative-pressure evacuation. The vacuum environment removes air trapped in the mesoporous channels within the functionalized nanocarriers. During the physical process of restoring normal pressure, the internal and external pressure difference forces the loading mixture, along with the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe, into the mesoporous channels. On the inner wall of the mesoporous channels, free amino groups that were not occupied by fluorescent internal control molecules and plant cell-penetrating peptides in the previous modification steps are retained. These free amino groups are positively charged in the loading buffer and undergo electrostatic adsorption with the negatively charged probe backbone. This electrostatic adsorption fixes the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe within the mesoporous channels, preventing premature leakage of the dual probes during subsequent in vivo delivery. To quantify the loading efficiency of functionalized nanocarriers, the absolute loading amount of probes was calculated using the following mass conservation formula. The total molar concentration of unloaded probes remaining in the supernatant was determined using a UV-Vis spectrophotometer combined with a standard curve method.

[0055] In the formula, The total molar amount of dual molecular beacons actually loaded inside the functionalized nanocarrier representing the unit mass. This represents the total molar concentration of the dual molecular beacons in the loading mixture prior to the loading operation. This represents the initial liquid volume of the mixture before the loading operation; This represents the total molar concentration of unloaded probes remaining in the supernatant after loading and centrifugation. This represents the actual liquid volume of the supernatant after centrifugation. This represents the total physical mass of the functionalized nanocarriers added to the reaction system.

[0056] S303: Prepare a live plant osmotic buffer and complete the final resuspension assembly of the self-calibrated live in-situ nanoprobe complex. After confirming the loading amount meets the requirements based on the above formula, separate the solid precipitate at the bottom using a high-speed centrifuge. Wash the solid precipitate multiple times with washing solution to remove free probe molecules adsorbed on the outer surface of the functionalized nanocarrier but not entering the mesoporous channels. After washing, the fully assembled self-calibrated live in-situ nanoprobe complex is obtained. Resuspend the self-calibrated live in-situ nanoprobe complex in the live plant osmotic buffer for later use. The live plant osmotic buffer contains isotonic concentration of 2-(N-morpholine)ethanesulfonic acid and a nonionic surfactant (e.g., Silwet L-77 or Tween-20) with a mass fraction of 0.01% to 0.1%. The isotonic concentration of 2-(N-morpholine)ethanesulfonic acid is used to maintain the pH of the suspension in the weakly acidic range, consistent with the physiological pH of the bitter gourd mesophyll cells, and to avoid inducing plasmolysis in plant cells. Nonionic surfactants are used to reduce the surface tension of the osmotic buffer solution for living plants, allowing droplets to wet the hydrophobic cuticle of bitter gourd leaves and smoothly enter the stomatal channels. For the basic biochemical procedures of high-speed centrifugation and liquid washing desalination, those skilled in the art can refer to standard nucleic acid purification and precipitation methods. Nucleic acid purification and precipitation methods are well-known in the field and will not be elaborated further in this specification.

[0057] After assembling the self-calibrated in-situ nanoprobe complex and resuspending it in a living plant permeation buffer, the field in-situ permeation and transmembrane delivery stage begins. To deliver the self-calibrated in-situ nanoprobe complex into the mesophyll cells of bitter gourd without disrupting the plant's growth, the following steps are included: S401: Select target tissue and perform non-destructive micro-infusion. In a field or greenhouse environment, select individual plants from the bitter gourd breeding population that have reached the cotyledon expansion stage as the target. Bitter gourd leaves at the cotyledon expansion stage have high stomatal conductance and a thin cuticle. Breeders use a needleless micro-syringe to draw up a live plant infusion buffer containing a self-calibrated live in-situ nanoprobe complex. Smoothly attach the output end of the needleless micro-syringe to the densely stomatal area on the back of the bitter gourd cotyledon, apply positive mechanical pressure to the plunger end of the needleless micro-syringe, and press the live plant infusion into the cotyledon until a uniformly distributed water-soaked diffusion area forms on the surface of the bitter gourd cotyledon. After the infusion operation, the bitter gourd plants are allowed to continue in-situ growth in the field. For the field cultivation conditions of bitter gourd and the routine holding and pressing operation of the needleless micro-syringe, those skilled in the art can refer to the standard Agrobacterium transient transformation injection specification, which is a well-known technique in the field and will not be elaborated further in this specification.

[0058] S402: The physical process of osmotic buffer passing through stomatal channels in living plants. To allow the osmotic buffer to smoothly pass through stomatal channels into the intercellular spaces of mesophyll tissue, the applied positive mechanical pressure must overcome the capillary resistance generated by the stomatal channels. The condition for overcoming the capillary resistance of the stomatal channels is established based on a fluid dynamic constraint formula modified from the Yang-Laplace equation:

[0059] In the formula, This represents the minimum osmotic mechanical positive pressure that needs to be applied to a needleless microsyringe; The surface tension of the liquid representing the osmotic buffer solution for living plants; This represents the contact angle between the living plant osmotic buffer and the epidermis of bitter gourd seed leaves; This represents the average effective physical radius of the stomatal channels in bitter gourd cotyledons. Combined with the nonionic surfactant added in the previous steps, the liquid surface tension of the living plant osmotic buffer... Contact angle The surface tension and contact angle are reduced. This decrease reduces the minimum permeation mechanical barometric pressure required. When a mechanical positive pressure satisfying the hydrodynamic constraint formula is applied, the solution containing the self-calibrated in-situ living nanoprobe complex passes through the stomatal physical barrier and forms a diffusion zone in the intercellular spaces of the spongy tissue of the mesophyll.

[0060] S403: Physical diffusion from the intercellular space to the plasma membrane surface. The self-calibrating in-situ nanoprobe complex, entering the intercellular space of spongy tissue, is located on the periphery of the network of pores in the plant cell wall, composed of cellulose microfibers, hemicellulose, and pectin. Because the outer diameter of the self-calibrating in-situ nanoprobe complex is limited to between 50 and 100 nanometers, satisfying the exclusion limit of the plant cell wall pores, the self-calibrating in-situ nanoprobe complex undergoes physical diffusion within the pores of the plant cell wall, driven by a concentration gradient. The self-calibrating in-situ nanoprobe complex then penetrates the cell wall pores and reaches the plasma membrane surface of the plant cell.

[0061] S404: Plant cell-penetrating peptide-mediated plasma membrane endocytosis and molecular beacon entry into the nucleus. Upon reaching the plasma membrane surface, the positive charge carried by the BP100 peptide grafted onto the surface of the self-calibrated in-situ nanoprobe complex interacts electrostatically with the negative charge on the surface of the plasma membrane phospholipid bilayer. This electrostatic adsorption leads to the reorganization of local lipid molecules in the plasma membrane, forming local membrane invaginations. These invaginations encapsulate and transport the self-calibrated in-situ nanoprobe complex into the cytoplasm via clathrin-mediated endocytosis. Once inside the cytoplasm, the 50-100 nm self-calibrated in-situ nanoprobe complex, exceeding the physical size exclusion limit of the nuclear pore complex channel, remains in the cytoplasm as a probe reservoir. Due to the significant difference in ion concentration between the cytoplasm and the loading buffer, and the dynamic reversible equilibrium between the electrostatic adsorption between the probe and the inner wall of the mesoporous channel, the powdery mildew-resistant and altitude-leaf spot-resistant probes are gradually released from the mesoporous channels of the self-calibrated in-situ nanoprobe complex into the cytoplasm. These free probes, detached from the carrier, have a micro-hydrodynamic diameter of less than 15 nanometers, allowing them to easily pass through the nuclear pore complex channels on the nuclear membrane and diffuse freely into the plant cell nucleus. This entry of the free probes into the nucleus provides the spatial basis for their contact with the target genome DNA sequence.

[0062] After the free powdery mildew resistance target response probes and plateau leaf spot resistance target response probes penetrate the nuclear membrane and enter the plant cell nucleus, the in-situ computing module enters the in vivo nuclear target recognition and multiple fluorescence release stage. To convert disease resistance single nucleotide polymorphism information and insertion / deletion mutation information in the plant genome sequence into quantifiable optical signals, the powdery mildew resistance target response probes and plateau leaf spot resistance target response probes undergo specific hybridization reactions in the nucleoplasmic microenvironment. In vivo nuclear target recognition and multiple fluorescence release includes the following steps: S405: Powdery mildew resistance target response probes and plateau leaf spot resistance target response probes undergo Brownian motion and target scanning in the nucleus and cytoplasm of plant cells. The deoxyribonucleic acid (DNA) within the plant cell nucleus is in a folded chromatin state. During the selected bitter gourd cotyledon unfolding stage, the powdery mildew resistance-associated loci and the plateau leaf spot resistance-associated loci are in an active basal transcriptional expression state. The chromatin regions containing the transcriptionally expressed loci exhibit a loosely structured euchromatin conformation, causing local unwinding of the double-stranded DNA, exposing single-stranded target regions containing powdery mildew resistance single nucleotide polymorphism sites and plateau leaf spot resistance insertion / deletion sites. The powdery mildew resistance target response probes and plateau leaf spot resistance target response probes, with a hydrodynamic diameter of less than 15 nanometers, penetrate into the decondensed euchromatin regions and attempt physical collisions and base pairing with the exposed single-stranded target regions.

[0063] S406: Hybridization occurs based on complete sequence complementarity. When the powdery mildew resistance target response probe collides with the target region carrying the resistance single nucleotide polymorphism site, the oligonucleotide sequence in the recognition loop region inside the powdery mildew resistance target response probe undergoes base complementarity pairing with the corresponding resistance sequence. When the plateau leaf spot resistance target response probe collides with the target region carrying the resistance insertion / deletion site, the oligonucleotide sequence in the recognition loop region inside the plateau leaf spot resistance target response probe undergoes base complementarity pairing with the corresponding resistance sequence. Because the powdery mildew resistance-associated locus and the plateau leaf spot resistance-associated locus are physically located in the bitter gourd genome, and because the recognition loop sequences of the powdery mildew resistance target response probe and the plateau leaf spot resistance target response probe are orthogonally designed and do not have cross-complementarity pairing ability, the hybridization reactions of the powdery mildew resistance target response probe and the plateau leaf spot resistance target response probe occur simultaneously in the same cell nucleus without interference.

[0064] S407: Controlled thermodynamic conformational unwinding and optical quenching release. The binding energy released by the hybridization reaction disrupts the energy balance maintaining the closed stem-ring state between the powdery mildew-resistant target response probe and the plateau leaf spot-resistant target response probe. Physical separation occurs between the 5' and 3' stem region sequences of the powdery mildew-resistant target response probe. This conformational unwinding increases the spatial distance between the carboxylated fluorescein labeled at the 5' end of the powdery mildew-resistant target response probe and the p-dimethylaminoazobenzoic acid labeled at the 3' end, and simultaneously increases the spatial distance between the carboxylated X-rhodamine labeled at the 5' end of the plateau leaf spot-resistant target response probe and the black hole quencher 2 labeled at the 3' end. This increased spatial distance blocks the fluorescence resonance energy transfer channel. The molecular proportion of probe conformational unwinding is limited by the thermodynamic equilibrium of the reaction system for specific intranuclear microenvironments. The theoretical calculation of the proportion of unchained molecules is verified based on the following Boltzmann distribution derivation formula:

[0065] In the formula, This represents the proportion of probe molecules that bind to the target DNA sequence and undergo conformational unwinding under thermodynamic equilibrium conditions, relative to the total number of corresponding probe molecules in the cell nucleus. The value of Gibbs free energy released when the probe binds to the target deoxyribonucleic acid sequence in a completely complementary manner; R represents the Gibbs free energy required to maintain the closed stem-loop structure of the probe; T represents the ideal gas constant; and T represents the absolute temperature inside the nucleus of a living plant cell.

[0066] Based on the above formula, when the probe encounters a completely complementary homozygous resistant sequence, the proportion of probe molecules undergoing conformational unwinding approaches 100%; when the probe encounters a susceptible sequence with mismatched bases or missing fragments, the proportion of probe molecules undergoing conformational unwinding approaches zero.

[0067] S408: Local Release and Background Noise Suppression of Multiple Fluorescent Signals. The powdery mildew-resistant target probe undergoing conformational unwinding exposes carboxyfluorescein to a free state without quenching barriers. The plateau leaf spot-resistant target probe undergoing conformational unwinding exposes carboxyX-rhodamine to a free state without quenching barriers. Probe molecules that do not encounter the disease-resistant sequence and those that encounter the disease-susceptible sequence maintain a closed stem-loop conformation, ensuring that the fluorophores that have not undergone targeted binding remain in an optically quenched state, avoiding non-specific optical background noise. The carboxyfluorescein and carboxyX-rhodamine that have completed the hybridization reaction are enriched in the local physical space of the plant cell nucleus, providing an optical signal source for subsequent multi-channel signal acquisition. For basic biochemical observation methods of the local discondensation state of chromatin and calculation methods of the thermal motion of nucleic acid molecules, those skilled in the art can refer to standard molecular biophysics theory, which is a well-known technique in the field and will not be elaborated further in this specification.

[0068] After the formation of a cluster of active luminescent molecules within the local physical space of the plant cell nucleus, multispectral non-destructive signal extraction is performed. To convert the photons released by carboxyfluorescein, carboxyX rhodamine, and fluorescent internal control molecules into digital electrical signals in the field under in vivo conditions, specific photoelectric conversion hardware and a band-isolated acquisition strategy are required. Multispectral non-destructive signal extraction includes the following steps: S501: Optical channels for a multi-wavelength fluorescence detection device. The multi-wavelength fluorescence detection device integrates a light-emitting diode excitation array and photomultiplier tube detectors for the corresponding wavelength bands. To extract the optical signals from the powdery mildew resistance target response probe, the plateau leaf spot resistance target response probe, and the constant internal control system, three physically isolated optical detection channels are configured within the multi-wavelength fluorescence detection device. An optical detection channel with an excitation wavelength of 490 nm and an emission wavelength of 520 nm is configured as the FAM channel for extracting the carboxyfluorescein signal; an optical detection channel with an excitation wavelength of 575 nm and an emission wavelength of 605 nm is configured as the ROX channel for extracting the carboxyX rhodamine signal; and an optical detection channel with an excitation wavelength of 650 nm and an emission wavelength of 670 nm is configured as the Cy5 channel for extracting the Cy5 signal within the constant internal control system. Physical isolation established through a narrow-bandpass filter eliminates spectral crosstalk between different luminescent groups.

[0069] S502: Optical signal scanning of the permeation area of ​​bitter gourd cotyledons. After the micro-pressure permeation operation and 2 to 4 hours of in vivo hybridization incubation, to ensure sufficient time for the self-calibrated in-situ nanoprobe complex to complete transmembrane transport and intranuclear target hybridization dissociation reaction, the operator tightly attaches the probe of the multi-wavelength fluorescence detection device to the area on the back of the bitter gourd cotyledon that underwent the micro-pressure permeation operation. The multi-wavelength fluorescence detection device sequentially illuminates the excitation light sources of the FAM channel, ROX channel, and Cy5 channel according to the time sequence. The photomultiplier tube detector collects the fluorescent photons emitted from inside the cotyledon tissue and converts them into corresponding analog voltage signals, which are then processed by the analog-to-digital converter circuit into a digitized total fluorescence intensity value. The total fluorescence intensity acquired in the FAM channel of the permeation area is recorded and denoted as _____. Record the total fluorescence intensity of the permeated region acquired in the ROX channel, denoted as . Record the total fluorescence intensity of the permeated region in the Cy5 channel, denoted as . .

[0070] S503: Obtain the autofluorescence values ​​of living plant tissues. Chlorophyll, flavonoids, and cell wall components inside plant leaves produce broad-band autofluorescence when excited by light of specific wavelengths. The total fluorescence intensity extracted by the multi-wavelength fluorescence detection device in the penetration area follows the following physical superposition formula:

[0071] In the formula, This represents the total fluorescence intensity acquired by a multi-wavelength fluorescence detection device in any specific optical channel; The intensity of the optical signal emitted by the target luminescent group; This represents the physical background fluorescence intensity produced by the plant tissue itself. Based on the aforementioned superposition rules, to extract the optical signal and internal reference signal released by the target luminescent group from the total fluorescence intensity, the physical background fluorescence intensity must be obtained as a baseline. The probe of the multi-wavelength fluorescence detection device was moved to an adjacent area of ​​the same bitter gourd cotyledon leaf that had not undergone micro-pressure permeation. Using the same excitation and detection parameters as those used to acquire the signal from the permeated area, background fluorescence data from the control area was collected. The spontaneous background fluorescence intensity acquired in the FAM channel from the unpermeated area was recorded and denoted as [insert value here]. Record the spontaneous background fluorescence intensity of the unpenetrated region in the ROX channel, denoted as . Record the spontaneous background fluorescence intensity of the unpermeable region in the Cy5 channel, denoted as . The extracted total fluorescence intensity data and spontaneous background fluorescence intensity data are used as raw input parameters and transmitted to the solution logic processing unit for subsequent self-calibration calculations. For methods to suppress dark current noise in photomultiplier tubes and the hardware layout specifications of analog-to-digital conversion circuits, those skilled in the art can refer to the standard weak photoelectric signal detection circuit design manual. The weak photoelectric signal detection circuit design manual is well-known technology in this field and will not be elaborated upon in this specification.

[0072] After the total fluorescence intensity data and spontaneous background fluorescence intensity data extracted by the multi-wavelength fluorescence detection device are transmitted to the calculation logic processing unit, the unit executes a genotype analysis algorithm and determination operation based on the internal reference ratio. In living plant tissues, due to differences in stomatal density and cuticle thickness in different regions, the absolute number of self-calibrated in-situ nanoprobe complexes penetrating into the mesophyll tissue fluctuates. This fluctuation in absolute number makes it impossible to accurately determine the genotype using the absolute fluorescence intensity of probes targeting powdery mildew resistance or plateau leaf spot resistance. To eliminate spatial heterogeneity interference caused by this fluctuation in absolute number, the calculation logic processing unit includes the following data processing and determination steps: S504: Performs physical background fluorescence subtraction and standardized resistance index calculation. The calculation logic processing unit extracts the total fluorescence intensity obtained by the permeated region in the FAM channel, the total fluorescence intensity obtained by the permeated region in the ROX channel, the total fluorescence intensity obtained by the permeated region in the Cy5 channel, and the spontaneous background fluorescence intensity obtained by the unpermeated region in the FAM channel, the ROX channel, and the Cy5 channel. The calculation logic processing unit subtracts the corresponding spontaneous background fluorescence intensity from the total fluorescence intensity to obtain the net optical signal intensity of the target luminescent group and the fluorescent internal reference molecule. The calculation logic processing unit divides the net optical signal intensity targeting powdery mildew and the net optical signal intensity targeting high-altitude leaf spot disease by the Cy5 net optical signal intensity, which represents the local permeation concentration of the self-calibrated in-situ nanoprobe complex, to calculate the standardized resistance index. The specific calculation logic is implemented based on the following mathematical formula: The standardized resistance index for powdery mildew resistance sites is calculated using the following formula:

[0073] The standardized resistance index calculation formula for the loci of high altitude leaf spot disease is as follows:

[0074] In the formula, The ratio of self-calibrated fluorescence response at the anti-powdery mildew site; The ratio of self-calibrated fluorescence response at the locus representing resistance to high altitude leaf spot disease; This represents the total fluorescence intensity acquired by the permeated region in the FAM channel; This represents the spontaneous background fluorescence intensity acquired in the FAM channel from the unpenetrated region; The total fluorescence intensity acquired by the permeated region in the ROX channel represents the total fluorescence intensity obtained in the ROX channel. Represents the spontaneous background fluorescence intensity of the unpenetrated region obtained in the ROX channel; The total fluorescence intensity acquired in the Cy5 channel represents the permeation region. This represents the spontaneous background fluorescence intensity obtained in the Cy5 channel from the unpenetrated region.

[0075] The ratio division operation cancels out the physical variable representing the permeation concentration of the self-calibrated in vivo in situ nanoprobe complex, so that the output standardized resistance index is only related to the proportion of probes that undergo hybridization and melting reactions in the cell nucleus, thus eliminating the influence of the absolute permeation of the carrier on the intensity of the optical signal.

[0076] S601: Determine the fluorescence response threshold for known genotypes. Before screening individual plants in the bitter gourd breeding population, the same self-calibrated in-situ nanoprobe complex and micro-pressure permeation procedure as for the individual plants in the bitter gourd breeding population are used to extract multispectral non-destructive signals from homozygous resistant and homozygous susceptible control lines with known genotypes. The computational logic processing unit obtains the standardized resistance index dataset of the homozygous susceptible control lines. To control the false positive probability caused by background noise to an extremely low level, the threshold is set based on the statistical distribution boundary of the homozygous susceptible control lines. The specific threshold calculation formula is as follows:

[0077] In the formula, This represents the positive threshold for powdery mildew resistance established for the anti-powdery mildew locus; The statistical mean of the self-calibrated fluorescence response ratio at the powdery mildew resistance site in a population representing a homozygous susceptible control strain; The standard deviation of the self-calibrated fluorescence response ratio at the powdery mildew resistance site represents the homozygous susceptible control population. This represents the positive threshold for resistance to high altitude leaf spot disease established for loci targeting this disease. The statistical mean of the self-calibrated fluorescence response ratio at the plateau leaf spot resistance site in a population representing a homozygous susceptible control line; This represents the standard deviation of the self-calibrated fluorescence response ratio at the plateau leaf spot resistance site in the homozygous susceptible control population. For the basic statistical significance test methods for the control dataset, those skilled in the art can refer to standard biostatistical guidelines, which are well-known techniques in the field and will not be elaborated further in this specification.

[0078] S602: Perform genotype determination and field single-plant screening. The computational logic processing unit retrieves the self-calibrated fluorescence response ratios of powdery mildew resistance sites and plateau leaf spot resistance sites for individual plants in a specific bitter gourd breeding population, and logically compares these ratios with pre-defined positive thresholds for both powdery mildew resistance and plateau leaf spot resistance. The computational logic processing unit incorporates conditional judgment logic. The unit determines that the live single plant in the detection state carries a dual-resistance genotype if and only if the input parameters satisfy both the powdery mildew resistance self-calibrated fluorescence response ratio being greater than or equal to the powdery mildew resistance positive threshold and the plateau leaf spot resistance self-calibrated fluorescence response ratio being greater than or equal to the plateau leaf spot resistance positive threshold. Based on the dual-resistance genotype instructions output by the computational logic processing unit, breeders identify and retain the dual-resistance live single plants in the field. When the input parameter does not meet either of the two threshold conditions, the solution logic processing unit determines whether the live plant is a susceptible genotype or a single-resistance genotype, and the breeder performs the removal and elimination operation on the corresponding live plant in the field according to the determination instruction.

[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot, characterized in that, Includes the following steps: S100 was used to construct target response probes for resistance to powdery mildew and target response probes for resistance to high altitude leaf spot disease; S200 preparation of mesoporous silica nanoparticles with covalently coupled fluorescent internal reference molecules and plant cell-penetrating peptides on the shell surface; S300 loads anti-powdery mildew target response probes and anti-altitude leaf spot target response probes into the mesoporous channels of mesoporous silica nanoparticles to obtain a self-calibrated in vivo in-situ nanoprobe complex. S400 was used to prepare a live plant permeation buffer. The self-calibrated live in situ nanoprobe complex was suspended in the live plant permeation buffer, and the live plant permeation buffer was then permeated into the living tissue of a single bitter gourd breeding population in the cotyledon unfolding stage. The S500 uses a multi-wavelength fluorescence detection device to collect the total fluorescence intensity of the powdery mildew signal, the total fluorescence intensity of the plateau leaf spot signal, the total fluorescence intensity of the fluorescent internal reference molecule, and the spontaneous background fluorescence intensity of the unpenetrated area of ​​a single plant in the bitter gourd breeding population. S600 calculates the standardized resistance index based on the total fluorescence intensity and the spontaneous background fluorescence intensity, and compares the standardized resistance index with the pre-defined judgment threshold to screen out dual-resistant live plants.

2. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 1, characterized in that, The S100 constructs target response probes for powdery mildew resistance and plateau leaf spot resistance, including: The recognition loop region oligonucleotide sequence and the complementary stem region sequence of the anti-powdery mildew target response probe were designed. The luminescent group carboxyfluorescein was covalently modified at the 5' end of the anti-powdery mildew target response probe, and the quenching group p-dimethylaminoazobenzoic acid was covalently modified at the 3' end of the anti-powdery mildew target response probe. The recognition loop region oligonucleotide sequence and the complementary stem region sequence of the anti-altitude leaf spot disease target response probe were designed. The luminescent group carboxylXrhodamine was covalently modified at the 5' end of the anti-altitude leaf spot disease target response probe, and the quenching group Black Hole Quencher No. 2 was covalently modified at the 3' end of the anti-altitude leaf spot disease target response probe.

3. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 2, characterized in that, The oligonucleotide sequence of the recognition loop region of the designed anti-altitude leaf spot disease target response probe includes: the oligonucleotide sequence of the recognition loop region of the anti-altitude leaf spot disease target response probe is designed to directly cross the nucleotide fragment deletion site on the single-stranded target region containing the anti-altitude leaf spot disease insertion / deletion site, and the oligonucleotide sequence of the recognition loop region of the anti-altitude leaf spot disease target response probe is completely complementary to the flanking bases on both sides of the nucleotide fragment deletion site; when the anti-altitude leaf spot disease target response probe encounters a susceptible sequence with a fragment deletion, a free unpaired nucleotide convex loop structure is formed inside the susceptible sequence, so that the anti-altitude leaf spot disease target response probe maintains a closed stem-loop structure.

4. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 1, characterized in that, The S200 method for preparing mesoporous silica nanoparticles with covalently coupled fluorescent internal reference molecules and plant cell-penetrating peptides on the outer shell surface includes: Hydrolysis and condensation reactions were carried out on the mesoporous silica nanoparticle framework to limit the outer diameter of the mesoporous silica nanoparticle framework to the range of 50 nm to 100 nm. Amide modification was performed on the exposed silanol groups on the surface of the mesoporous silica nanoparticle framework, and a fluorescent dye Cy5 carrying a monocarboxyl functional group was covalently coupled to the outer shell surface of the amide-modified mesoporous silica nanoparticle framework as a fluorescent internal reference molecule. By controlling the feeding concentration of the fluorescent dye Cy5 carrying a single carboxyl functional group, the amino groups on the surface of the mesoporous silica nanoparticle framework are in an excess state, so as to maintain the physical open state of the mesoporous channel openings.

5. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 4, characterized in that, The mesoporous silica nanoparticles prepared by S200 with covalently coupled fluorescent internal reference molecules and plant cell-penetrating peptides on the outer shell surface also include: BP100 peptide was used as a plant cell-penetrating peptide, and a heterobifunctional crosslinking agent was used to covalently couple BP100 peptide to the outer shell surface of a mesoporous silica nanoparticle framework. Adjust the molar concentration of the BP100 peptide added to the reaction system to ensure that the surface zeta potential of the mesoporous silica nanoparticles is greater than 15 mV.

6. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 1, characterized in that, The S300 includes loading the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe into the mesoporous channels of the mesoporous silica nanoparticles, including: The target response probes for powdery mildew resistance and the target response probes for high altitude leaf spot disease resistance were added to the loading buffer to form a loading mixture. The loading buffer contained divalent magnesium ions at a concentration of 5 to 20 mmol / L. Mesoporous silica nanoparticles are dispersed into a loading mixture to form a solid-liquid two-phase suspension system. The solid-liquid two-phase suspension system is subjected to negative pressure evacuation treatment in a vacuum drying oven. The internal and external pressure difference during the restoration of normal pressure is used to press the loading mixture, together with the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe, into the mesoporous channel. By utilizing the electrostatic adsorption reaction of free amino groups on the inner wall of mesoporous channels, target response probes for powdery mildew resistance and target response probes for high-altitude leaf spot disease resistance are fixed inside the mesoporous channels.

7. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 1, characterized in that, The living plant osmotic buffer contains isotonic concentration of 2-(N-morpholine)ethanesulfonic acid and a nonionic surfactant with a mass fraction of 0.01% to 0.1%. The S400 process involves permeating a living plant osmotic buffer into the living tissue of a single bitter gourd breeding plant during the cotyledon unfolding stage, including: The output end of the needleless microsyringe is placed in close contact with the densely stomata area on the back of the cotyledon of a single bitter gourd breeding plant. Mechanical positive pressure is applied to the plunger end of the needleless microsyringe to force the live plant osmotic buffer into the cotyledon of the single bitter gourd breeding plant. The self-calibrated in-situ nanoprobe complex penetrates the pores of the plant cell wall to reach the surface of the plant cell plasma membrane. The self-calibrated in-situ nanoprobe complex enters the cytoplasm through the plasma membrane endocytosis pathway mediated by plant cell-penetrating peptides, serving as a probe reservoir.

8. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 7, characterized in that, After the self-calibrated in-situ nanoprobe complex enters the cytoplasm as a probe reservoir, the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe are released free from the mesoporous channels of the self-calibrated in-situ nanoprobe complex into the cytoplasm. After being released, the anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe diffuse into the plant cell nucleus through the nuclear pore complex channel on the nuclear membrane of the plant cell. The anti-powdery mildew target response probe and the anti-altitude leaf spot target response probe undergo a base complementary pairing reaction with the single-chain target region exposed inside the decondensed euchromatin region.

9. The molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot disease according to claim 1, characterized in that, The S600 calculates the standardized resistance index based on the total fluorescence intensity and the spontaneous background fluorescence intensity, including: The difference between the total fluorescence intensity of the permeated region in the FAM channel and the spontaneous background fluorescence intensity of the non-permeated region in the FAM channel is divided by the difference between the total fluorescence intensity of the permeated region in the Cy5 channel and the spontaneous background fluorescence intensity of the non-permeated region in the Cy5 channel, to obtain the self-calibrated fluorescence response ratio of the anti-powdery mildew site for the anti-powdery mildew site. The difference between the total fluorescence intensity of the permeated region acquired in the ROX channel and the spontaneous background fluorescence intensity of the unpermeated region acquired in the ROX channel is divided by the difference between the total fluorescence intensity of the permeated region acquired in the Cy5 channel and the spontaneous background fluorescence intensity of the unpermeated region acquired in the Cy5 channel, to obtain the self-calibrated fluorescence response ratio of the anti-altitude leaf spot disease site for the anti-altitude leaf spot disease site.

10. A molecular marker-assisted breeding method for bitter gourd resistant to powdery mildew and highland leaf spot according to claim 9, characterized in that, The pre-calibrated judgment thresholds include a positive threshold for resistance to powdery mildew and a positive threshold for resistance to high altitude leaf spot disease; The method for determining the positive threshold for powdery mildew resistance is as follows: the statistical mean of the self-calibrated fluorescence response ratio at the powdery mildew resistance site of the homozygous susceptible control population is added to three times the standard deviation of the self-calibrated fluorescence response ratio at the powdery mildew resistance site of the homozygous susceptible control population to obtain the positive threshold for powdery mildew resistance. The method for determining the positive threshold for resistance to high altitude leaf spot disease is as follows: the statistical mean of the self-calibrated fluorescence response ratio of the homozygous susceptible control population at the high altitude leaf spot disease site is added to three times the standard deviation of the self-calibrated fluorescence response ratio of the homozygous susceptible control population at the high altitude leaf spot disease site to obtain the positive threshold for resistance to high altitude leaf spot disease. The S600 compares the standardized resistance index with a pre-calibrated judgment threshold to screen out dual-resistant live plants, including: when the ratio of self-calibrated fluorescence response at the powdery mildew resistance site of a single plant in the bitter gourd breeding population is greater than or equal to the positive threshold for powdery mildew resistance, and the ratio of self-calibrated fluorescence response at the plateau leaf spot resistance site of a single plant in the bitter gourd breeding population is greater than or equal to the positive threshold for plateau leaf spot resistance, dual-resistant live plants are screened out.

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